Impacts of reproductive systems on grapevine genome and breeding

Domain: earth_science
Published: 2025-03-03
Editorial outcome: major_revision

Abstract
Diversified reproductive systems can be observed in the plant kingdom and applied in crop breeding; however, their impacts on crop genomic variation and breeding remain unclear. Grapevine (Vitis vinifera L.), a widely planted fruit tree, underwent a shift from dioecism to monoecism during domestication and involves crossing, self-pollination, and clonal propagation for its cultivation. In this study, we discover that the reproductive types, namely, crossing, selfing, and cloning, dramatically impact genomic landscapes and grapevine breeding based on comparative genomic and population genetics of wild grapevine and a complex pedigree of Pinot Noir. The impacts are widely divergent, which show interesting patterns of genomic purging and the Hill-Robertson interference. Selfing reduces genomic heterozygosity, while cloning increases it, resulting in a “double U-shaped” site frequency spectrum (SFS). Crossing and cloning conceal while selfing purges most deleterious and structural burdens. Moreover, the close leakage of large-effect deleterious and structural variations in repulsion phases maintains heterozygous genomic regions in 4.3% of the grapevine genome after successive selfing for nine generations. Our study provides new insights into the genetic basis of clonal propagation and genomic breeding of clonal crops by purging deleterious variants while integrating beneficial variants through various reproductive systems.

Introduction
Diversified reproductive systems can be observed in plant kingdoms and applied for crop breeding, including sexual crossing, self-pollination, and asexual clonal propagation. Crossing involves the deliberate combination of distinct varieties or even species to generate new genetic combinations. These hybrid offspring often exhibit heterosis, leading to enhanced yield and quality1. Self-pollination (selfing) includes pollination within the same plant or variety. This process promotes homozygosity and may aid in the elimination of recessive deleterious mutations through purging2,3. Nevertheless, selfing can lead to inbreeding depression, resulting in compromised growth potential, reduced yield, and diminished biotic and abiotic resistances4. The third reproductive type, clonal propagation, avoids the introduction of additional genetic material and offers the advantage of preserving favorable agronomic traits present in specific varieties5. Clonal propagated lines can, however, accrue somatic mutations. Some somatic mutations have produced new breeding resources6–8, such as bud sport varieties9, but somatic mutations can also be deleterious and negatively influence plant fitness10–12. These plant reproductive systems can shape the landscapes of genomic variation both within and among populations13, but more empirical work is necessary to characterize the magnitude of the effects of reproductive systems on genomic landscapes13. Such investigations could facilitate advancements in crop genomic breeding14,15.

Grapevine (Vitis vinifera L.) is one of the most widely planted and economically important perennial fruit trees. There are two subspecies of grapevine: domesticated V. vinifera subsp. vinifera and its wild progenitor V. vinifera subsp. sylvestris16–18. The initial steps of V. vinifera domestication may date as far back as ~15,000 years ago, and it included a shift from a dioecious, obligately outcrossing mating system to monoecy, with the potential for self-pollination18,19. Despite this shift, the cultivation of domesticated grapes has primarily been based on hybridization and on clonal propagation of highly heterozygous genotypes, in part due to substantial inbreeding depression for selfed materials20. In some cases, clonal propagation has been prolonged; some modern cultivars possess genetic information identical to grape seeds from the medieval period21. Thus, the evolutionary history and breeding of domesticated grapes have been influenced by diverse reproductive systems, significantly shaping their genomic makeup20.

Pinot Noir (PN) is a premium red wine variety popular for its flavorful, aromatic wines. With a history spanning over a millennium, PN has served as a focus of research and as a foundational parent in grapevine breeding. In its over 900 years of clonal propagation21, numerous variants have been derived from PN through bud sports, including Pinot Grigio, Pinot Meunier, Pinot Gris, and Pinot Blanc22. The initial grapevine reference genome was determined from a highly homozygous descendant of PN called PN4002423. This accession originated from successive self-pollinations of Helfensteiner (HE)24, an offspring resulting from a cross between PN and Schiava Grossa (SG) performed in 1931 (www.vivc.de). Recently, a complete telomere-to-telomere (T2T) genome assembly of PN40024 has also been made available25.

In this study, we construct a haplotype-resolved chromosomal genome of PN and characterize the haplotypic diversity present within it, including significant structural variations and gene families unique to each haplotype. Additionally, we examine both somatic and fixed variants within the PN population to enhance our understanding of the cultivar’s development. Our analysis of various grapevine germplasm samples indicates that reproductive types have a substantial impact on genomic landscapes and grapevine breeding practices. These effects differ in terms of genome heterozygosity, as well as deleterious and structural burdens. We find that selfing significantly purges heterozygous deleterious SNPs (dSNPs) and structural variants (by 62% and 65%, respectively, compared to HE) in PN40024. The close linkage of large-effect deleterious and structural variations in repulsion phases maintains 4.3% of the genomic regions in a heterozygous state even after successive selfing. Our study explores the evolutionary genomics underlying the transitions of reproductive systems in forming grapevine lineages, which sheds insights on genomic breeding of grapevine.

Results
Comparative genomics of clonal Pinot Noir and selfing PN40024
To construct the haplotype-resolved PN genome, we generated a total of 33 Gb (33,349,412,693 bp, ~65x coverage) of HiFi reads and 84 Gb (83,958,664,800 bp, ~160x coverage) of Hi-C reads (see “Methods”). Two haplotypes of PN were assembled: PN1 (496.43 Mb) and PN2 (489.78 Mb) (Fig. 1A), with a contig N50 of 23.60 Mb and 24.19 Mb, respectively. The BUSCO score of PN1 (98.3%) and PN2 (98.4%) was comparable to the complete assembly of PN40024 (PN40024_T2T, 98.5%, Supplementary Fig. 1). These statistics suggested that employing a similar methodology as PN40024_T2T25, all centromeres and most of the telomeres in the two haplotypes’ genome were identified. For genome annotation, PN1 (37,038) and PN2 (37,350) yielded a similar number of identified genes. We also identified repetitive sequences representing 66.64% and 66.21% of the PN1 and PN2 assemblies, respectively (Supplementary Table 1). Interestingly, we detected a large inversion on chromosome 19 between PN1 and PN2, which is almost 4 Mb in size (Fig. 1A and Supplementary Fig. 2). Further analysis of long reads, complemented by PCR analyses, supported the inversion between the two haplotypes at the breakpoints (Supplementary Table 2, Supplementary Figs. 3–5).

To study the impacts of reproductive systems on genomic landscapes, we also conducted comparative genomics among PN1, PN2, and PN40024_T2T (Fig. 1B). K-mer analyses estimated the genome heterozygosity to be 1.43% in PN (Supplementary Fig. 1), which is higher than PN40024_T2T (0.18%)25. The three genomes were highly diverged at the sequence level, including gene content. If no homologous genes were identified for a gene family within a genome, that gene family was considered absent from that genome. Using this approach, we identified 20,800 shared gene families among PN40024_T2T, PN1, and PN2. However, we also detected extensive variation at gene contents: 2869, 2864, and 3026 gene families were exclusive to PN1, PN2, and PN1+PN2, respectively, compared to PN40024_T2T, while smaller numbers (2243, 2214 and 581 gene families) were exclusive to PN40024_T2T compared to PN1, PN2 and PN1+PN2 (Fig. 1C), respectively. These observations could have two causes. First, it is possible that the SG, parent of HE, had fewer gene families than the PN parent. An alternative hypothesis is that selfing led to the loss of many gene families, potentially through a process that favors shorter haplotypes, as suggested for the rapid loss of genome size in selfed maize lineages3.

Selection and introgression shaped the characteristics of Pinot Noir
To assess evolutionary processes contributing to the formation of distinguishing characteristics of PN, we gathered resequencing data from 38 V. vinifera samples (Supplementary Data 1 and 2), including 18 PN clones (nine generated in this study and nine from previous publications), 20 previously published wild grapes accessions (ten from Europe (EU) and ten from the Middle East and Caucasus region (ME), respectively) and three muscadine grapes used as outgroup. These sequences constitute three groups with which to investigate introgression and selection signals in PN. After SNP and SV calling and filtering, we counted 4,687,377 SNPs and 18,469 SVs across the entire dataset. Genome-wide selection signatures were observed throughout the PN clones, especially strong peaks on chromosomes 1, 3, 4, 5, and 18, by applying the population branch statistic (PBS)26 with ME and EU populations as controls to identify genomic regions with exaggerated divergence relative to the controls (Fig. 2A). Gene set enrichment analysis (GSEA)27 revealed an enrichment of organelle assembly, glycerolipid metabolic process, regulation of protein metabolic process, response to auxin, response to abiotic stimulus and beta-glucan biosynthetic process, and so on (Fig. 2B, Supplementary Data 3). Note that the PBS is likely identifying extended lengths of the branch leading to PN, and thus it is likely that the inferred selection events occurred prior to the diversification of PN clonal lineages.

As EU wild population has a significant contribution to the origin of modern wine grapes28, we explored how such introgression events shaped the genome of PN clones using f-statistic (fd)29. Although the fd-test is primarily used in sexual populations, it focuses on shared alleles rather than allele frequencies. Therefore, it could be an efficient way for detecting introgressed fragments in clonal PN. By using ME as the sister population, we detected strong introgression signals in the PN genomes from EU, particularly on chromosomes 1, 2, 3, and 19 (Fig. 2C). The genes in the top 1% of regions with the highest fd values were enriched in pathways related to plant physiological processes, such as photosynthesis and generation of precursor metabolites and energy (Supplementary Fig. 6, Supplementary Data 4). One locus, CONSTANS-like 5 (COL5), related to plant flowering (Supplementary Fig. 7)30, stood out as one of the top outlier in introgression analysis. Both the nucleotide diversity (π) of PN clones and sequence similarity (Dxy) between PN clones and EU were significantly lower at COL5 and surrounding regions (Fig. 2D). These observations strongly suggest that both selection and introgression shaped COL5 in PN clones, conferring local adaptation to adjust flowering time in new climates after the spread of cultivars globally after domestication. We further validated the reliability of this introgression in PN by conducting a phylogenetic analysis (Supplementary Fig. 8, Supplementary Data 2). The results indicated that ME and EU were mainly grouped into two separate clades. Instead of forming an independent clade, some cultivars, including PN, clustered with the EU clade, suggesting an introgression from EU in certain wine grapevines in this region.

Germline and somatic mutations in Pinot Noir clonal lineage
To investigate the impact of clonal propagation on PN, we analyzed genetic variants among the PN clones and their genetic differentiation from other grapevine populations. In total, 3,915,920 SNP variants and 17,035 SV variants were identified in grapes (PN, EU, and ME populations) in the absence of the outgroup data (used the inferred ancestral alleles, Fig. 3A). Among them, 70,291 SNPs and 605 SVs were unique to PN (specific variants), while 2,227,889 SNPs and 8635 SVs were shared with wild EU, ME or both populations, which are primarily likely to be germline mutations (Fig. 3A). The frequency of both shared SNPs and specific SNPs in PN clones displayed a “double U-shaped” site frequency spectrum (SFS) (Fig. 3B). However, the distribution of PN-specific SNPs showed an excess of rare variants (16.1% compared to 11.1% of all PN SNPs that were observed only once in PN clones) and a likely heterozygous state (69.2% compared to 21.1% of all PN SNPs that were observed 18 times across 18 PN clones), suggesting that many of these PN-specific variants are mostly somatic mutations in heterozygous states (Fig. 3B).

Given the duration of its cultivation, there has been ample opportunity for somatic mutations to occur in different clonal lineages of PN. To assess the number and type of these mutations, we divided the PN-specific SNPs into two groups: (i) 20,328 mutations that vary among PN clones (28.9%), which reflect the accumulation of somatic mutations and (ii) mutations that were observed across PN clones (71.1%), representing germline mutations that occur during the formation of the PN cultivar prior to the diversification of the clonal lineages, or somatic mutations fixed during clonal propagation (Fig. 3C). We defined the first group as “somatic mutations” and the second as “fixed mutations (including both somatic and germline mutations)”. Previous studies identified somatic mutations across clones of other varieties31–39, but most of them failed to distinguish somatic and germline mutations. We applied control populations to detect cultivar-specific mutations, which greatly improved the precision of classifying somatic and germline mutations (Supplementary Fig. 9). Interestingly, almost all (98.6%) of these fixed mutations were maintained as heterozygous in the 18 PN clones. Additionally, 55.5% of the remaining putative somatic SNPs were unique to a single individual, suggesting the possible accumulation of somatic mutations in each clone (Fig. 3C). We randomly selected ten somatic mutation sites specific to one individual by examining the reads in IGV. The results showed that the mutation existed only in some reads of the individual predicted to have the mutation, and not in other individuals (Supplementary Figs. 10–14). The distribution of these somatic and fixed SNPs was then investigated in terms of gene structure. Somatic SNPs were found to occur more frequently in intergenic regions compared to fixed SNPs (Supplementary Fig. 15). A similar pattern has been observed across clones of the Zinfandel variety31. GO analysis showed that genes with fixed SNPs were enriched in biological processes such as cell cycle process, negative regulation of biological process, cytoskeleton organization, microtubule cytoskeleton organization, mRNA splicing via spliceosome, and so on (Supplementary Data 5).

Similar to SNPs, we also identified 605 SVs unique to PN by comparing SVs among the three populations (Fig. 3A). The SVs were categorized into four types: deletion (DEL), tandem duplication (DUP), inversion (INV), and inter-chromosomal translocations (BND). DEL was the most prevalent type, accounting for 59.7%, followed by BND and DUP, while INV was the least common, constituting only 1.82% (Supplementary Table 3). We found that 87.4% of the SVs contained TEs. Similar to SNPs, among the 605 SVs unique to PN, 70.9% of them were shared by all 18 individuals, and the majority (91.9%) of these “fixed” SVs were present in a heterozygous state (Fig. 3C). Additionally, 176 variants were found in 1 to 17 individuals, with an average heterozygosity of 99.8%. For these fixed SVs that were retained in all 18 individuals, we annotated the genes on them and found they were enriched in biological processes related to recognition of pollen, cell recognition, ketone biosynthetic process, quinone metabolic process, quinone biosynthetic process, and cellular ketone metabolic process (Fig. 3D, Supplementary Data 6). We further zoomed in on the twelve genes associated with the pollen recognition process and found all of them belonging to the S-locus or related genes and hence probably related to self-incompatibility40 (Supplementary Data 7). Nine of the twelve genes were clustered in one SV on chromosome 10 (~4.7 Mb), which was present in PN clones but absent in EU and ME sylvestris samples. The region containing this SV was highly heterozygous in all PN lineages (Supplementary Fig. 16). Comparative genomics between the PN haplotypes and PN40024_T2T genomes indicated that both duplication and insertion contributed to the formation of this SV on PN1 (Fig. 3E).

The impact of clonal propagation on grapevine genomes
To investigate the impact of various reproductive systems on grapevine breeding, we gathered more resequencing samples from the PN lineages with different reproductive modes for analysis, including five SG clones, four Gouais Blanc (GB) clones, ten Chardonnay (CD) clones, two Gamay Noir (GN) clones, two HE clones, and four PN40024 clones (Supplementary Date 2). The phylogenetic trees showed that the wild and domesticated grapes display reciprocal monophyly (Fig. 4A). Admixture analyses indicated HE, GN, and CD with admixture components from their parents (Fig. 4A). In contrast, the PN40024 clones were inferred to be separate, non-admixed group, perhaps reflecting distinctness evolved during selfing. Kinship analysis was conducted to verify the relationship between samples in each group and to confirm the true-to-type of these samples41. As expected, PN and GB were identified as parents of CD and GN, and PN and SG were identified as the parents of HE but not PN40024 (Fig. 4B).

We assessed genetic variation within each group to elucidate the impact of reproductive modes on genetic diversity. To begin with, we calculated the nucleotide diversity (π) value and observed heterozygosity (HO) for each group. As expected, selfed PN40024 exhibited the lowest levels of genetic diversity (π=0.0003) and heterozygosity (observed heterozygosity, HO=0.01) compared to all other groups. Among the groups, the wild grape ME group had the highest nucleotide diversity (π=0.0039), while the other clonal grapes had π values ~0.002 (e.g., PN: 0.0024, GB: 0.0024, SG: 0.0019, CD: 0.0020, GN: 0.0028 and HE: 0.0029) (Supplementary Fig. 17), reflecting the effects of the domestication bottleneck. However, the average heterozygosity is slightly higher in clonal groups than in wild groups (EU: 0.20, and ME: 0.25, PN: 0.28, GB: 0.27, SG: 0.26, CD: 0.30, GN: 0.28, Supplementary Fig. 18).

The reproductive systems directly affect the efficiency of recombination, which is associated with the purging and maintenance of genetic burden42,43. To detect such effects, we identified dSNPs using SIFT44. The recessive burden for each individual was measured by the number of homozygous dSNPs; the heterozygous burden was measured by the number of heterozygous dSNPs; while the additive burden was calculated as the number of heterozygous dSNPs plus two times the number of homozygous dSNPs45. First, we examined the dSNPs shared by all individuals within each grape group and found that the clonal groups (PN, SG, GB, CD, GN, and HE) exhibited significantly higher genetic burden compared to the outcrossing wild groups (EU and ME) (Fig. 4C). The selfing PN40024 had the highest recessive burden and lowest heterozygous burden. In contrast, the wild grape groups (EU and ME) showed a significantly higher genetic burden of dSNPs that were not shared by all individuals within each grape group (Supplementary Fig. 19). A similar pattern was also observed for SV among the different grape groups (Fig. 4D and Supplementary Fig. 20).

Conserved heterozygous regions in grapevines
To understand the dynamics of alleles among cultivars during the breeding process, we assessed segregating sites that consistently occurred in each grape group. As shown in Fig. 4E, each column in the barplot represents a specific genotype combination among the grape groups, with a total of 381 combinations observed. We selected 167 combinations that include more than 30 sites for subsequent analyses (Fig. 4E). The categories with the most sites were the SNPs that were heterozygous in clonally propagated PN, SG, or HE but homozygous in PN40024 due to selfing, and the SNPs that were only heterozygous in the GB sub-lineage (including GB, CD, and GN clones). A total of 97 SNPs remained heterozygous in all grape samples during domestication and diversification (wild and cultivated grapes including PN40024) and were dispersed throughout the genome. We identified 14 genes enriched in defense response and in the tricarboxylic acid cycle with SNPs located on the genes or within 2kb up- and downstream of the genes (Supplementary Data 8). In addition, most of the homologous genes of the 14 genes were related to plant growth, and some of them are associated with recessive lethal phenotypes (Supplementary Data 9). In addition, after nine generations of selfing to generate PN40024, 99.8% of the genome was homozygous25, except for 4782 conserved heterozygous SNPs (hSNPs) (Fig. 4E). GO analysis for the 577 genes that were associated with these hSNPs was enriched in only one molecular function process called chitin binding (Supplementary Data 10).

To study the effects of recombination on genome architecture and genetic burden, we identified the specific markers (see “Methods”) between PN and SG, and inspected their distribution on HE and PN40024. As shown in Fig. 5A and Supplementary Fig. 21, most of the specific markers of PN and SG could be located on the HE genome in a syntenic region. Almost all these specific markers (98.1%) were in a heterozygous state. This result supported the fact that HE is the offspring of a PN by SG cross. After continuous selfing in PN40024, 91.2% of these specific markers became homozygous, while 8.8% of them were still heterozygous (Fig. 5A, Supplementary Fig. 22). Recombination between the PN and SG haplotypes was observed on PN40024 chromosomes. For example, on chromosome 1, the end of the long arm of the SG haplotype was replaced by PN-specific markers. Most detectable recombination events occurred at the ends of chromosomes (Supplementary Fig. 22).

Close linkage of deleterious and structural variants in repulsion phases after successive selfing
Although almost the whole PN40024 genome is homozygous, some heterozygous variants (116,199 SNPs and 1125 SVs) remained in all four PN40024 clones, and most of them were clustered in blocks (Fig. 5B, Supplementary Figs. 22 and 23)25. We identified six large heterozygous blocks (see “Methods”) (Supplementary Table 4). To mitigate the influence of potential assembly errors and structural deviations from the reference genome, we assessed the read coverage of each sample within the heterozygous blocks relative to the read coverage on the whole genome (Supplementary Data 11). We observed that the average read depth in the heterozygous block of chromosome 16 (coverage: 12,992,557–13,599,104) is 2.13 times higher than that outside this block, likely due to erroneous assembly or structural disparities with the reference genome. Therefore, this block was excluded from subsequent analysis. The remaining five blocks account for ~4.3% of the genome and contain ~93% of the hSNPs and ~90% of the SVs in the entire genome.

To find out why PN40024 retained heterozygous blocks, we compared the distribution pattern of variants and found that the distribution pattern of SVs and dSNPs in heterozygous blocks of PN40024 was inherited from HE (Fig. 5B, C, Supplementary Figs. 24–29). In addition, the heterozygous blocks overlapped with regions of low recombination (Fig. 5A, Supplementary Figs. 21 and 22). According to the heterozygous blocks on PN40024, we divided the genome into two parts: the heterozygous block regions and genomic regions outside of them. We found that there were more hSNPs located on genes and the flanking region of genes (2kb upstream and downstream) in the heterozygous block regions than in regions outside of them in HE (Supplementary Fig. 30). Previous work has shown that heterozygous blocks in selfing lineages are caused by dSNPs in repulsion in low recombination regions3,46. We assessed this phenomenon in PN40024 by assessing GERP values for dSNPs. Remarkably, dSNPs in heterozygous regions exhibited significantly rightward shifts in predicted deleterious effects relative to dSNPs outside of heterozygous regions, suggesting the presence of numerous large-effect deleterious mutations within heterozygous blocks (P < 0.001, Wilcoxon rank sum test, Fig. 5D). We then identified dSNPs in PN40024 that were inherited from both PN and SG in the heterozygous blocks, which suggested that dSNPs are probably located on both haplotypes of these blocks (Fig. 5E and Supplementary Fig. 31). Therefore, it was presumed that, due to the low recombination rate in these heterozygous regions and large-effect dSNPs in repulsion phases, the genome could not purge these variants, leading to the retention of heterozygosity in these blocks.

As an example, the ~200 kb SDR was located on the heterozygous block on chromosome 2. There are four SDR haplotypes: Male-haplotype (M), female-like haplotype (F), and hermaphrodite-haplotype (H1 and H2) with dominance effects M > H > F. H1 and H2 originated from the recombination of F and M to become hermaphroditic47,48. We found that PN, GN, HE, and PN40024 are H2/F genotypes, SG and GB are H1/F genotype, and CD is H1/H2 genotype, which indicated that HE inherited the H2 haplotype from PN and the F haplotype from SG. Interestingly, after nine generations, PN40024 maintained the heterozygous genotype (H2/F) at SDR locus, enabling it to remain hermaphroditic (Fig. 5F). The F/F genotype could not be selfing by PN40024, because the flowers would be female. However, why did the H2 haplotype not become homozygous? One possibility is the presence of strongly recessive deleterious (even recessive lethal) SNPs on the H2 haplotype. To investigate this, we screened the sites that are heterozygous (0/1 genotype) in PN40024 and cannot simultaneously exist in a homozygous state for both the reference allele (0/0 genotype) and the alternative allele (1/1 genotype) in other grape accessions. Finally, 67 SNPs were identified. To focus on dSNPs located on the H2 haplotype inherited from PN, we excluded SNPs inherited from SG, resulting in 55 SNPs found in both PN and PN40024 accession (Fig. 5G, H). All these SNPs are located on two genes (VviVI2 and VviGSM2) and their flanking regions. Six of the SNPs had a GERP score reaching the maximum value of four, indicating a strong detrimental effect on grapes (Fig. 5I). Five of the six SNPs were in the CDS region of VviVI2, along with 24 indels and an SV (87 bp) (Fig. 5J). Knocking out of the AtVI2 gene in Arabidopsis resulted in a severe reduction in pollen germination (Supplementary Fig. 32)49–52. Therefore, the VviVi2 gene, associated with recessive lethality, located on the H2 haplotype likely maintained the SDR region in a heterozygous state on chromosome 2.

Methods
Plant materials and sample collection
Fresh and healthy leaf tissue from plants of Vitis vinifera cultivar “PN” clone AGIS_01 was collected from the grapevine germplasm collection at the Agricultural Genomics Institute at Shenzhen, Shenzhen, Guangdong Province, China, and immediately frozen in liquid nitrogen. These materials were packaged for PacBio HiFi and Hi-C sequencing, respectively, and subsequently submitted to the company for genomic DNA extraction and library preparation.

Library preparation and DNA sequencing
Isolation of high-molecular-weight genomic DNAs using the DNeasy Plant Mini kit according to the manufacturer’s instructions. For PacBio HiFi sequencing, single-molecule real-time cells were sequenced on the PacBio Sequel II platform using Circular Consensus Sequencing (https://github.com/PacificBiosciences/ccs) with default parameters. For the Hi-C library, samples were digested with the restriction enzyme DpnII and constructed following a standard Hi-C protocol as described previously. These Hi-C libraries were sequenced on the Illumina HiSeq X Ten platform.

De novo haplotype-resolved genome assembly and quality assessment
Using the Hi-C Integrated Assembly mode of Hifiasm, we first generated two contig-level haplotype genomes using the HiFi and Hi-C data of PN86. Genomic heterozygosity was assessed by GenomeScope (v2.0)87, employing a k-mer-based methodology applied to raw HiFi reads. Then, we used RagTag to determine the approximate order of contigs on chromosomes by using PN40024_T2T as a reference genome25. Subsequently, the Hi-C sequencing data were harnessed to anchor all contigs via Juicer (v1.5)88. This was succeeded by employing a 3D-DNA scaffolding pipeline to further refine the structure89. Manual adjustments were conducted on the acquired outcomes using Juicebox (v1.11.08, https://github.com/aidenlab/Juicebox), followed by a secondary application of the 3D-DNA approach to achieve the genome at the scaffold level. Employing Minimap2 (v2.24)90, a comprehensive comparison was conducted between scaffold-level genomes from different Vitis species and the raw HiFi data. The outcomes were then imported into IGV (v2.12.3)91 to pinpoint the precise sequence positions of gaps. To validate the accuracy of gap filling, the identified sequences were reintegrated into the genome utilizing Minimap290.

For genome quality, we used QUAST to count the basic information of the contig level genome and the final genome (https://github.com/ablab/quast). The genome completeness was evaluated by BUSCO using the embryophyta_odb10 database92. The genome continuity was evaluated by calculating the contig N50 length. For genome accuracy, we mapped the genome with HiFi reads using Minimap2 and calculated mapping rates90.

Annotation of genes and transposable elements
We primarily used this genome-wide annotation pipeline for genome annotation (https://github.com/unavailable-2374/Genome-Wide-Annotation-Pipeline). RNA-seq datasets were collected covering a variety of tissues, including flowers, leaves, and other tissues. RNA sequences were aligned to repeat mask assemblies using Hisat2 (v2.10.2) and subsequently assembled into transcripts using StringTie (v1.3.0)93,94. Genes were first searched by using transcripts and UniProt (https://www.uniprot.org/) as evidence. In this process, we used Exonerate, Genewise, and Transfrag. In short, an initial gene model was created for the genes and further searches were performed using AUGUSTUS (v3.4.0)95. Genes involving duplicated regions, CDS regions shorter than 90 nucleotides, or without any evidence to support them were filtered out. Finally, all results were checked with a hidden Markov model downloaded from the Pfam database to obtain the final gene model.

RepeatModeler (open-2.0.3) was used to build the TE library, with the -LTRStruct96. Genome-wide TE annotation was performed using RepeatMasker (open-4.1.2, https://github.com/rmhubley/RepeatMasker), with -ermblast -lc ambig and slow model.

Identification of telomeres and centromeres
For telomere identification, plant telomeric sequences (CCCATTT at the 5’ end and TTTAGGG at the 3’ end) were identified, and 70 out of the expected 76 telomeres (spanning 38 chromosomes of 2 haplotypes, 35/38 for each genome) were identified using the telomere pipeline developed by the TIDK (v0.2.0)97.

TRF v4.09 was used to finish tandem repeat annotation, and then we merged the results of annotation using TRF2GFF98. To complete data visualization, we analyzed the results in IGV (v2.12.3)91. The results were compared with TE annotation and TRF using IGV to identify the centromeres.

Genome comparison between PN1, PN2 and PN40024_T2T
We aligned the PN1, PN2, PN40024_T2T genomes using Minimap2, and indexed the alignment BAM file using SAMtools (v1.4)99. Next, SyRI100 was used to find structural variants between the genomes, and the results were visualized using plotsr101.

MUMmer (v.4.0) was used to compare the genome with the reference genome PN40024_T2T using whole-genome alignments102. First, we aligned the two genome sequences using Nucmer and then filtered one-to-one alignments with a minimum alignment length of 10,000 bp (delta-filter -i 95 -l 10000).

To identify gene families in PN40024_T2T, PN1, and PN2, Orthofinder v2.5.2 was utilized103 according to a previous study104, comparing their protein-coding gene sequences. Basically, the genes that similar to each other and distinct from genes in other groups were clustered together based on the sequence similarity. If no gene was identified in a gene family for a specific genome, this gene family was counted as absent in that genome. The gene abundance in one gene family on one genome was calculated by dividing the number of the genes in this gene family on that genome by the maximum number of the genes in this gene family among the three genomes.

Comparing SVs on the Pinot Noir genomes
To identify SVs on the PN genome (between the two haplotypes), we called SVs using the Sniffles pipeline105. First, PacBio reads longer than 500 bp were mapped onto PN1 PN2 genomes using the aligner Minimap290. Variant calling was then performed with Sniffles. SV calling outputs (VCF files) were filtered by VCFtools (v0.1.16)106 to find the heterozygous SVs between the two haplotypes in PN. To further validate the existence of this inversion, we designed primers at the break points on both haplotypes, following with PCR and gel electrophoresis analyses (Supplementary Table 2 and Supplementary Fig. 4).

SNP calling and filtering
We used 68 grapevine resequencing samples, including ten wild grapes (V. vinifera subsp. sylvestris) from Europe (EU), ten wild grapes from the Middle East (ME), 18 PN, five SG, four GB, ten CD, two GN, two HE, and four PN40024, along with three muscadine grapes used as outgroup. Among these samples, nine of the clonal PN collected from Foundation Plant Services, University of California, Davis were sequenced in this study, while the remaining data was downloaded from the database on the website (Supplementary Table 4).

Using default parameters, fastp (v0.21) was used to regulate the quality of resulting raw reads107. The PN40024_T2T assembly (PRJNA882193 in NCBI)25 served as the reference genome. Quality-controlled reads were mapped to the genome using bwa (v0.7.15) with default parameters108. SAMtools (v1.4) and GATK (v4.1.8) were used for sorting and indexing the bam file with no duplicates109,110. GTX, which is based on the HaplotypeCaller of GATK, was used for SNP calling across all samples.

To reduce false positives, filtering was conducted using VCFtools (v0.1.16)106. We removed genotypes with a genotype quality <20 (option --minGQ 20). SNPs with more than two alleles were excluded (options --min-alleles 2 --max-alleles 2), as well as SNPs with more than 20% missing genotypes (option --max-missing 0.8).

The filtered SNPs were phased by Beagle (v5.4) genotype imputation method111. The results were then reversed using Model 1 in the superSFS (https://github.com/xhchauvet/superSFS) script with parameter 3 to infer ancestral alleles according to outgroup species.

Phylogeny and population structure
For the analysis related to introgression in PN, the phylogenetic tree was constructed by IQ-TREE (v2.1.4)112 based on general time reversible (GTR) model using variants from three outgroup samples, 20 wild grapes and 42 wine cultivars in the 20 kb region where COL5 is located.

For the analysis related to the impact of clonal propagation on grapevine genomes, the whole genome SNPs of the 68 previously mentioned grapevines were first thinned using VCFtools (--thin 1000). Phylogenetic tree was then constructed using IQ-TREE based on the GTR model. To construct the phylogenetic tree of the COL5 and VI2 genes among different species, the DNA sequences of these genes were analyzed using MEGAX113 with the method neighbor-joining and p-distance.

The population structure based on nuclear SNPs was estimated using Admixture (v1.3.0) with K varying from 2 to 10114. Kinship and IBS0 analyses were performed using King41. The relationship between samples were assessed according to the following criterion: identical clones (K ≥ 0.49 and IBS0 ≤ 0.001), parent-offspring (0.177 < K < 0.354 and IBS0 ≤ 0.001), highly related/sibling (0.177 < 0.354 and IBS0 ≤ 0.25)21.

Population genetic analyses
The Nucleotide diversity (π) value for each sample was calculated using VCFtools with a 100kb window size. Sequence similarity (Dxy) and f-statistic (fd) were calculated using the Python script: popgenWindows.py (https://github.com/simonhmartin/genomics_general) with every 50kb nonoverlapping windows. For fd analysis, ME were used as the sister population to PN, and the gene flow between EU and PN grapes was evaluated. The heterozygous sites were counted using VCFtools. The number of heterozygous sites in each sample was divided by the total number of SNPs to calculate the heterozygosity of each sample. These statistics were evaluated within or between all six groups (EU, n=10; ME, n=10; PN, n=18; SG, n=3; HE, n=2; and PN40024, n=4).

The heterozygosity was calculated by dividing the number of heterozygous sites in each sample by the total number of SNPs.

Selective sweep detection
PBScan (https://github.com/thamala/PBScan) was used for PBS analysis26. The PN group was designated as POP1, the ME group as POP2, and the EU group as POP3. The analysis was conducted every 50 SNPs with a step size of 50 SNPs based on sequence similarity (Dxy).

Detection of deleterious mutations
We used Sorting Intolerant From Tolerant 4G (SIFT4G) (https://github.com/pauline-ng/SIFT4G_Create_Genomic_DB) to annotate the SNP dataset in order to estimate the functional effects of mutations44. Swiss-Prot (https://www.uniprot.org/help/downloads) was utilized as a reference protein set to construct a grapevine database. Meanwhile, we used the genome and annotation files of PN40024_T2T. The GFX3 format was converted to the Ensemble GTF format. The generated database was then used to annotate the flipped SNPs. SIFT values range from 0 to 1, and any nonsynonymous position with a SIFT score of less than 0.05 was considered putatively deleterious.

ANGSD analysis
We used ANGSD software to calculate the locus spectrum (http://www.popgen.dk/angsd/index.php/ANGSD), which shows the frequencies of individual alleles at specific loci in the PN population115. First, we ran the script using the reversed VCF file, combining the reference genome to extract the sequence file of the ancestral genotype state (anc.fa) We then used the -doSaf 1 parameter to generate .saf (site allele frequency likelihood), and finally, the realSFS command was used to generate the site spectrum result file. sfs text format.

SV calling and filtering
Delly (v1.0.3)116 was used to call SVSs with PN40024_T2T as the reference genome. The “call” and “merge” functions were first used to get the .bcf files, and then the Delly “call” function was used again with “-v” parameter. Subsequently, the BCFtools (v1.13) “merge” function was used to merge the .bcf files. We removed low-quality SVs from the merged SVs that did not pass quality filters. After that, we used VCFtools to remove SV genotypes with more than 20% missing genotypes. The number of SVs containing TEs was counted based on the TE annotation of PN40024_T2T using Bedtools117.

Gene set enrichment analysis and gene ontology enrichment analysis
We downloaded the protein database (https://ftp.ncbi.nlm.nih.gov/blast/db/FASTA/swissprot.gz) from NCBI. Protein sequence alignment was then performed using Diamond118. The database was first established using the Diamond “makedb” command, and then sequence alignment was performed using the Diamond “blastp” command. Next, the GSEA and GO enrichment analyses were performed using clusterProfiler (v4.0)27 with the GO terms of the whole grapevine genome proteins as the background. First, we used “AnnotationForge” package in R to build “OrgDb” for gene to GO mapping. Then, for the GSEA analysis, the genes were ordered according to their PBS values, and the “gseGO” function of clusterProfiler was used for these genes with parameters: pAdjustMethod=“BH”, qvalueCutoff=0.05.

For the GO analysis, “enrichGO” function of clusterProfiler were used for candidate genes with the parameters: pAdjustMethod=“BH”, qvalueCutoff=0.05.

Analysis of heterozygous variants
The heterozygous blocks were detected by following the methods: if the distance between two hSNPs was less than 150,000 bp, the two SNPs were counted as continuous variation sites; if the span of the continuous variation sites was more than 500,000 bp, this region was counted as heterozygous blocks.

For analysis of different genotype combinations among cultivars, the conserved sites, which were consistent in each cultivar group, were used for counting. The combination categories with site numbers less than 30 were not included.

For the distribution analysis of SNPs on genes, 2kb upstream or downstream of the genes were counted as promotor or terminators.

The density of heterozygous SVs and dSNPs was calculated in every 200kb non-overlapping window.

Species specific marker
To select the specific marker belonging to PN or SG, we used the following criteria: the variants must exist in only one grapevine cultivar with the frequency greater than 50%, and must not be observed in all individuals of another grapevine cultivar.

The GERP score
The longest transcripts were extracted from the genomes of Arabidopsis thaliana, Populus trichocarpa, Oryza sativa, Vitis retordii, Vitis amurensis, Cissus rotundifolia, Malus domestica, Ficus macrocarpa and PN40024, then Orthofinder (v2.5.4)103 was used to generate single-copy protein sequences, which were used to construct the phylogenetic tree using IQ-TREE.

The genome of PN40024 was split into individual chromosomes, a multigene linear comparison was performed using last (https://gitlab.com/mcfrith/last), multiple .maf files were merged and converted to .fa files. Each SNP on each of PN40024’s chromosomes was assessed using GERP119.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This manuscript reassembles the haplotype-resolved genome of a world's renowned wine grape species and analyzes an important and interesting question, with statistical methods and tools that are widely accepted. However, I have some major concerns that need to be clarified, particularly about the sampling strategy and the genetic background of these samples used in this study.\n1. I would like to know how these clone populations were selected, such as the 18 PN samples. The accumulation of variations through different reproductive modes, such as crossing, cloning, and selfing, is different. For cloning reproduction, do these samples have accurate records of the time they were separated from the parent plant? The longer the time since separation, the more likely mutations have accumulated. Alternatively, what is the somatic mutation rate in grapes, and can this mutation rate be estimated using these clone samples, or vice versa? If these questions cannot be answered, then there is missing information, and it would be difficult to assess the representativeness of the 18 clone samples. This information should be detailed in the Materials and Methods section.\n2. For the selfing population, it would be highly informative to compare the differences between the samples chosen from different selfing generations, even if only a few generations out of all 9 generations are available. Whether we can completely rule out the possibility that the heterozygous sites in PN40024 are randomly retained.\n3. In each GO enrichment analysis Figure, please state the number of genes included in the analyzed gene set. Due to the limitations of the statistical method used, I believe that if the number of genes is less than 20, it would result in a significant bias.\n4. Fig. 2A selects some key genes, and the method used to determine the function of these genes should be detailed in the Materials and Methods section. Providing the phylogenetic relationship of these genes with the source genes described in the literature in the supplementary material is necessary to help readers better understand the functional annotation of these genes.\n5. To distinguish somatic variations from germline ones, the authors identified variations unique to PN population. I don't fully understand the underlying assumptions of this strategy. Why wouldn't somatic mutations found in the clone population also be present in the wild population? Are 10 EU + 10 ME representative enough? If I include more wild populations, will the results change?\n6. Line 283, the authors claim that \"Similar patterns were also found on structure variation and deleterious SNPs in each group. (Fig. 4C-D) \". What exactly is this pattern? Is “the heterozygosity of PN, SG, and HE is higher than wild grapes”? Is it statistically significant in Fig. 4C-D to claim that?\n7. The “protein phosphorylation pathway” was under selection in PN genome (Fig. 2B) and also keep heterozygous in PN40024 and HE but homozygous in wild grapes (Fig. 5B). I am curious if there could be any connection, either biologically or computationally.\n8. The data is presented twice in the same sentence (lines 281 and 283).\n9. What do the different colors represent in Fig. S11?\n10. Line 39. The “cossing” should be “crossing”.", "reviewer_2": "This work of Wang et al provides new data leading to a chromosome-scale assembly of an important grapevine cultivar and to an inventory of somatic variations in its clonally propagated plant material. In the version in which it is currently presented, the manuscript is missing a clearly defined biological question.\nTitle, abstract, and a large part of the Introduction section anticipate that this manuscript is going to focus on the different modes of propagation in grapevine (vegetative, selfing, outcrossing) and their effect on genome properties and variation in the cultivated germplasm. However, a large part of the results and discussion sections deal with the genome assembly of a single genotype (Pinot Noir) and its comparison with the genome assembly of one descendant of Pinot Noir (PN40024, an experimental genotype forcibly obtained from reiterated selfing starting from an offspring of Pinot Noir). As for the parts of the manuscript that deal more specifically with the topics anticipated in the introduction, the authors move from observations in a few cultivated genotypes and draw generalizations for the crop germplasm.\nThe manuscript has elements of potential interest for the grapevine scientific community that require, however, both a deeper and a wider investigation to yield robust and conclusive evidence, but also several aspects of weakness that should be more carefully considered (see below for specific points).\nSpecific points:\nLine 68: transformation from dioecy (obligate outcrossing) to monoecy (potential self-pollination and cleistogamy).\nLines 69-70: history of outcrossing and clonal propagation of highly heterozygous genotypes.\nLines 113-120 are inappropriately placed in the Results Section.\nLine 128: it should be made more explicit at first citation and in figure legends that the acronym PN_T2T refers to the T2T genome assembly of PN40024, an individual that was obtained by repeated rounds of selfing, starting from Helfensteiner.\nLines 146-155: the 4-Mbp inversion on chromosome 19 should be experimentally validated using a PCR-based assay. This assay could also be used for assessing the origin and frequency in the grapevine germplasm of the haplotype carrying the inverted region.\nIn the paragraph “Introgression and selection shaped the Pinot Noir genome”, the authors explained that they “used resequencing data from 38 samples (Table S2), including 18 Pinot Noir (PN) clones, 10 EU wild grapes, and 10 ME wild grapes, to explore the introgression and selection signals in PN”. It is unclear how they conducted this analysis to identify introgressed regions (I assume from the EU wild grape population) into domesticated lineages deriving from ME wild grape population (then leading to Pinot Noir), selective sweeps and candidate genes under selection, using a group of Pinot Noir clonal replicates with software that normally applies to comparisons among populations. As the resulting candidate genes (deriving from adaptive introgression ? from selection of ME ancestral diversity ?) are then described in detail and discussed extensively in Lines 162-204, their identification should be supported by stronger evidence or accompanied by a more detailed description of the analytical procedure.\nLine 206: what is Pinot Noir (PN) population? The group of Pinot Noir bud sports, the offspring Helfensteiner and PN40024, as described in Lines 555-556 of the Methods section?\nParagraph “Somatic mutations in Pinot Noir clonal population” and Lines 553-565\nIt makes little sense using the reference genome assembly of a different genotype (derived from selfing of Helfensteiner) to realign short-reads of Pinot Noir bud sports and call somatic variants, when a haplotype-resolved T2T assembly of Pinot Noir has been generated in the present paper. A preprint that has recently appeared in bioRxiv shows the power of the use of a haplotype-resolved T2T assembly of the very same diploid genotype in the identification of somatic variants among Malbec bud sports (https://doi.org/10.1101/2023.11.30.569420). Regardless of the reference used, experimental validation of a set of candidate somatic variants needs to be provided in order to estimate false discovery rate.\nLine 254: wording would suggest that the authors used 4 independent lines obtained from selfing of Pinot Noir. Is it correct ?\nOr are they clonal replicates of PN40024 ? PN40024 is a single seedling obtained from several rounds of selfing starting from selfing of Helfensteiner.\nParagraph “Effect of various mating systems on grape genomes”.\nThe authors used “groups” of cultivated varieties that were formed by one single genotype each, and three “groups” of these consisted of a parent-offspring trio. The forth “group” consisted of one single experimental highly inbred line (PN40024), showing severe inbreeding depression, that has no parallel in the grapevine cultivated germplasm. While π is largely independent from the number of sampled chromosomes and among-groups comparisons would make sense even using one diploid individual per group, data and inferences reported in Lines 279-294 refer to individual genomes and should be used with caution for inferring generalization on the species and on crop germplasm, as those discussed in Lines 377-434.\nParagraph “Conserved heterozygous regions in cultivated grapes”.\nLines 296-300. These sentences need rephrasing.\nLines 303-305. Why intriguingly ? Heterozygous regions in PN40024 must be heterozygous in Helfensteiner by definition, as PN40024 derived from selfing of Helfensteiner.\nLines 333-334. Again. Why SVs and hSNPs should differ in heterozygous regions between Helfensteiner and PN40024, if both PN40024 homologs are identical by descent with respect to Helfensteiner ?\nParagraph “Close leakage of deleterious variants in repulsion phases”\nI understand that this paragraph deals with the possible reasons (i.e. linked heterozygous deleterious variants in repulsion on the two homologs), other than chance alone, explaining why residual heterozygosity was found in specific chromosome blocks of PN40024. Figs. S13-S14 do not seem to suggest that residual heterozygous regions in PN40024 contain more deleterious variants than other heterozygous regions in Helfensteinter, which later became homozygous in the descendant upon selfing. In supporting their claim, the authors also should provide evidence that what is shown in Fig. S15 is not simply the result of incorrect variant phasing across heterozygous regions, because hap1 and hap2 are both identical by descent between Helfensteiner and PN40024 and do not appear as such in the graphical representation of Fig. S15.\nLines 385-387: This is a generalization that is inferred from observations in only three individuals.\nMethodology for the assembly of the Pinot Noir genome in the Methods Section\nLines 481-489: in the generation of the HiFi-based assembly, Hi-C reads were repeatedly used at the stage of contig generation (using Hifiasm) and scaffolding (using Juicer) as well as for other intermediate steps (also in combination with RagTag-based contig ordering) that are not clearly described.\nLines 495-500: the procedure used for integrating and merging the HiFi-based partial assembly and the ONT-based partial assembly into a haplotype-resolved T2T final assembly needs a more clear and detailed explanation. Adding Supplementary Tables that report for each chromosome contig-level and scaffold-level metrics obtained for the HiFi-based assembly and for the ONT-based assembly would help follow procedure and the results described in the main text.\nLine 538: structural annotation should read structural variation\nReferences to the additional homologs illustrated in Fig. 3D should be added.", "reviewer_3": "Wang et al's manuscript, \"Various mating systems shape grapevine genome evolution\" uses genomic resources to investigate evolutionary processes in grapes. Overall, the authors seem to aim to \"To investigate the genomic effects of mating systems during clonal, crossing, and selfing\" (L351; first line of Discussion, and this phrase is consistent with the message of the Abstract). It is noteworthy that previous studies have addressed these same questions; e.g., the effect of clonal growth on somatic mutations and the effect of self-fertilization on genetic diversity, so the ideas presented in the manuscript are not very novel, although the data are new and useful. (Frustratingly, the authors fail to point out that a large body of work has addressed the effect of mating system and clonality on genetic diversity and plant fitness, which makes the current manuscript appear more novel than it really is. With respect to clonal growth, see this review from 2015; much other work examines the effect of selfing - https://www.pnas.org/doi/abs/10.1073/pnas.1501712112) The authors do present data that suggest an increase in somatic mutations curing clonal growth (which is consistent with previous work see link provided above), and these data are interesting. The authors also present results that suggest that some genome regions remain exceptionally heterozygous and suggest that this heterozygosity is maintained by deleterious alleles in repulsion phase. This latter result might be exciting, but I maintain healthy skepticism as it would require very strong selection (e.g., akin to recessive lethal effects) to maintain the patterns of genetic diversity documented here (assuming I understood the paper).\nOverall, the authors present a new genome with some analysis, and provide some results that are potentially exciting. However, my sense is that the ideas tested here (and some of the main results) are not as novel as the manuscript leads the reader to believe.\nGOALS (L93-110):\n1) Look at Pinot Noir genome... no real goal stated other than \"look at intricacies\";\n2) Look at processes to generate Pniot Noir\n3) Examine structural variants and deleterious variants and their changes\n4) Why do some regions remain heterozygous in PN40024 -\nOUTPUT 1 (L121 - 155) Genome of (heterozygous) Pinot Noir - Does this meet their goal 1? They highlight an inversion.\nOUTPUT 2 (L157 - 203) - Introgression analysis of Pinot Noir Genome - IS THIS GOAL 2?\n- This section provides evidence for selection on a few genes, but says little (if anything?) about the role for introgression in the evolution of the Pinot Noir genome, despite the title of this section (L156) indicating that introgressions is a process of interest. It is unclear to me whether the authors even examined for evidence of introgression - if they did, how much evidence for introgression did they detect (and in which direction between regions)?.\nSOMATIC MUTATIONS IN PINOT NOIR CLONAL POPULATION (L204)\n- The authors state, \"To distinguish somatic variations from germline ones, we identified variations unique to PN\". Is is unclear to me how this approach can reliably detect somatic mutations because it assumes that the mutations that have been identified in the small number of samples of European and Middle Eastern samples (20 genomes, total) will include *all* possible mutations that could have been present in the ancestor of the Pinot Noir clonal population. i.e., with only 20 EU and ME samples the authors have sampled only 40 alleles from the wild populations (2 alleles for each of the 20 samples, with 10 from EU and 10 from ME). Therefore, the authors are unlikely to identify variants at relatively low frequency in wild populations that can be mistaken as somatic mutations. Notably, the authors use patterns Fig 3B to support arguments that 18 mutations arose somatically. However, it is unclear to me how the authors distinguish between whether these mutations arose somatically *after* clonal propagation began vs. were simply unique to Pinot Noir and present in the genotype that gave rise to the clonal genotype *before* clonal propagation began. The authors may be correct in their logic, but they need to clarify their presentation to eliminate this alternative explanation to their data. For example, it would be useful to include a figure that illustrated the evolutionary process that the authors imagine that yields the pattern observed in their data.\nL251: EFFECT OF MATING SYSTEMS ON GRAPE GENOMES - GOAL 3(?)\nL265 - the authors state, \"Due to nine generations of selfing, PN40024 was not counted as parent-offspring relationship with the other three cultivars.\" What evidence do the authors have to conclude that it was selfing, per se, that caused the analysis to fail to indicate that PN40024 was not counted as an offspring in the analysis? Selfing, per se, should only reduce genetic diversity by increasing homozygosity - therefore, it is unclear to me how the \"Kinship\" analysis would be so affected by increased homozygosity. The authors need to either further justify their assertion or consider either (1) that PN40024 does not derive from Helfensteiner (although this is unlikely) or (2) there are issues with the Kinship analysis, more generally, which should make the reader question *any* conclusions from this analysis.\nL275: When comparing genetic diversity of inbred vs. outcrossed varieties, the authors conclude \"the selfing PN40024 exhibited the lowest values for both diversity and heterozygosity when compared to all other groups\". This is not a surprise, as the impact of selfing on genetic diversity is understood well in theoretical terms and is well documented empirically. Therefore, while this result adds to current data on the effect of self-fertilization on genetic diversity, it does not expand our understanding.\nL280: The authors also find that the genetic diversity of colonal propagated lines exceeds that of outbred populations; \"the heterozygosity of PN, SG, and HE is higher than wild grapes, ME, and EU\". They authors conclude that clonal propogation increases genomic diversity and this this occurs via (in part, at least) deleterious mutations. These data are very useful, and are consistent with previous findings (see a review in 2015 here: https://www.pnas.org/doi/epdf/10.1073/pnas.1501712112) that also suggest that clonal reproduction increases opportunity for somatic mutation. The authors need to highlight that previous work also points to the accumulation of deleterious, somatic mutations during clonal growth; if the authors wish to claim that their results are somehow novel, they need to clarify which aspects are indeed novel.\nCONSERVED HETEROZYGOUS REGIOUS IN CULTIVATED GRAPES (GOAL 4?)\nL300: The authors \"identified 379 alleles that remain heterozygous in all grape samples (wild and cultivated).\" The authors further remark, \"Remarkably, we identified a total of 59,944 alleles that retained their heterozygous status within PN40024. Intriguingly, these heterozygous genes exhibit consistency between PN40024 and HE, and they also remain in heterozygous states in at least one PN and one SG sample.\" In reading this, my first reaction was that this result can arise due to errors in genome assembly; the authors examine read coverage to address this possibility (L318 - 327 - this is good) and remove several cases where this explanation likely is true. The authors conclude that heterozygosity is maintained due to deleterious mutations in repulsion-phase (also termed pseudo-overdominance; see Charlesworth, Deborah, and John H. Willis. \"The genetics of inbreeding depression.\" Nature reviews genetics 10.11 (2009): 783-796.) [[Please note that what I say next relies on my understanding of the author's phrase, \"alleles that remain heterozygous in all grape samples\", as this implies to me that all individuals that were genotyped were heterozygous at the focal loci - if I mis-understood the author's meaning, then my following comments need to be adjusted.]] If the authors' explanation is true, that multiple loci remain heterozygous in **all** genotypes individuals, then this implies *very* strong natural selection in order to maintain heterozygosity in all sampled individuals. Personally, I am surprised by (and therefore have healthy skepticism for) this finding and suggestion; I do find it interesting, but encourage authors to emphasize the requirement for very strong selection to maintain this pattern of genetic variation, and I encourage the authors to point out that this finding does require further investigation to confirm the suggestions made in the current manuscript. I can illustrate my skepticism mathematically. Take, for example, a single locus that remains highly heterozygous due to very strong selection: in this case, self-fertilization (which is how PN40024 reproduces) will lead to homozygous offspring at this locus for 50% of its offspring and many of them will experience a large fitness cost due to selection at this single locus. Now, the authors have implied that multiple loci remain heterozugous due to strong selection - if this is correct, then the fitness cost I just described for a single locus will multiple across other genome regions. Overall, we expect that most offspring that derive from self-pollination of PN40024 to have low fitness if the authors' explanation for maintaining heterozygosity is correct (and if I've understood their manuscript correctly); is this seen empirically? If not, then my sense is that the authors need to either (i) clarify their presentation (because I may have mis-understood aspects I highlight above), or (ii) amend or clarify their arguments / re-evaluate their findings. Are genome assembly or read-mapping errors possible reasons for the observed results?"}, "author_response": "Authors response to reviewers’ comments\nReviewer #1 (Remarks to the Author):\nThis manuscript reassembles the haplotype-resolved genome of a world's renowned\nwine grape species and analyzes an important and interesting question, with statistical\nmethods and tools that are widely accepted. However, I have some major concerns that\nneed to be clarified, particularly about the sampling strategy and the genetic background\nof these samples used in this study.\nResponse: We are grateful for your positive views and constructive comments for our\nmanuscript. Following your advice, we have clarified the information of the sampled\nused in this study. We also addressed all minor concerns, please read the manuscript\nwith major revisions highlighted for details. We hope the revised version will be more\nsuitable for publication on NC.\n1. I would like to know how these clone populations were selected, such as the 18 PN\nsamples. The accumulation of variations through different reproductive modes, such as\ncrossing, cloning, and selfing, is different. For cloning reproduction, do these samples\nhave accurate records of the time they were separated from the parent plant? The longer\nthe time since separation, the more likely mutations have accumulated. Alternatively,\nwhat is the somatic mutation rate in grapes, and can this mutation rate be estimated\nusing these clone samples, or vice versa? If these questions cannot be answered, then\nthere is missing information, and it would be difficult to assess the representativeness\nof the 18 clone samples. This information should be detailed in the Materials and\nMethods section.\nResponse: Thank you for concerning this issue. We have revised the manuscript based\non your advice. As shown in Supplementary Table 3, we included information about\nthe separated time from the parent plant and resource for the clone population samples\nin the revised version. The geographically widespread distribution enhances the\nrepresentativeness of these 18 clone samples, albeit at the expense of missing records\nregarding the separated time from the parent plant in some samples.\n2. For the selfing population, it would be highly informative to compare the differences\nbetween the samples chosen from different selfing generations, even if only a few\ngenerations out of all 9 generations are available. Whether we can completely rule out\nthe possibility that the heterozygous sites in PN40024 are randomly retained.\nResponse: Thanks for your suggestion. PN40024 was generated before 2007 and was\nfirst used for the first grape genome assembly (Jaillon et al. 2007 Nature). This 9-\ngeneration selfing material has been spread to and cultivated in many labs for further\nstudies. However, there are no other materials available out of 9 generations. In this\nstudy, we focused on the large heterozygous fragments, and there is only a 50%\npossibility of keeping heterozygous in each generation. We think there is an extremely\nlow probability for them to retain heterozygous randomly after 9 selfing generations\nbecause there is a 50% chance to turn to homozygous during selfing except undergo\nstrong selection to maintain this pattern. In addition, almost all the retained\nheterozygous sites (90.1%) in PN40024 are included in five heterozygous blocks\ninstead of randomly distributed on the grape genome (5.2% of the PN40024 genome).\nThus, it is unlikely these heterozygous sites are randomly retained. Thanks for the\nsuggestion from the reviewer, we also added some discussion about this in the\nmanuscript Lines 482-508.\nReference:\nJaillon, O. et al. The grapevine genome sequence suggests ancestral hexaploidization\nin major angiosperm phyla. Nature 449, 463–467; 10.1038/nature06148 (2007).\n3. In each GO enrichment analysis Figure, please state the number of genes included in\nthe analyzed gene set. Due to the limitations of the statistical method used, I believe\nthat if the number of genes is less than 20, it would result in a significant bias.\nResponse: Thanks for the comment. We have included the number of genes in each GO\nterm, updated the figures (Fig.2B, Fig. 3D, and Supplementary Figure 15) and data\nsheet (Supplementary Data). Upon examining the number of genes enriched in these\npathways, we found that some pathways had fewer than 20 enriched genes. However,\nwe also found that most of the Gene Ontology (GO) databases inherently contain a\nlimited number of genes. Therefore, in most cases, the available data could serve as a\nreference.\n4. Fig. 2A selects some key genes, and the method used to determine the function of\nthese genes should be detailed in the Materials and Methods section. Providing the\nphylogenetic relationship of these genes with the source genes described in the\nliterature in the supplementary material is necessary to help readers better understand\nthe functional annotation of these genes.\nResponse: We appreciate your suggestions. We have provided the phylogenetic trees\nof the candidate genes with the source gene and other homologous genes in\nSupplementary Figures 6 and 7 according to your suggestion.\n4. To distinguish somatic variations from germline ones, the authors identified\nvariations unique to PN population. I don't fully understand the underlying assumptions\nof this strategy. Why wouldn't somatic mutations found in the clone population also be\npresent in the wild population? Are 10 EU + 10 ME representative enough? If I include\nmore wild populations, will the results change?\nResponse: We apologize for the unclear description. According to the definition of\nsomatic variants, they are genetic mutations that occur in the body cells instead of the\ngerm cells, which cannot be passed on to the next generation through sexual\nreproduction. Thus, to detect the somatic variations, we considered these mutations to\nbe specific to the PN population and not shared by all PN individuals. The variants\nshared by all PN individuals are counted as germ-line mutations as these 18 individuals\nwere collected worldwide (Supplementary Table 3), and not possible the clones of the\nsame PN sample. As to the somatic mutation in PN presented in the wild population,\nthis could happen when PN lost the variation in wild grapes, and then mutation\nhappened in the same site with the same type (A, T, C, or G) in PN as wild grapes.\nHowever, the possibility is extremely low. We can make a rough estimation, the size of\nthe grape genome is around 500M, the total variations in the wild population is around\n2.2M, and there are four types of nucleotide (ATCG), so a new mutation is the same as\nthe old one is:\n2.2/500/3=0.15%\nThus, we think the effect of this situation is negligible.\nThe number of wild populations used could affect the results of somatic variants\ndetection but is limited. We use wild grapes to detect PN-specific mutations and somatic\nmutations are counted as the mutations of PN-specific mutations only existed in part of\nthe PN samples. If the mutations originated from wild grapes, they should be shared by\nall PN samples as they were clonal propagation. Of course, we didn’t consider the small\nprobability event, such as back mutation, as mentioned above, which has a very limited\nimpact on the result. We estimated the effect of different numbers of wild grapes used.\nAs we can see (Response Figure 1), when the number of wild grapes increased the\nnumber of somatic variants detected tended to be stable quickly. More wild grapes\nincluded can but in a very limited impact on the result. Thus, we think the result using\n20 wild grapes in this study is credible.\nResponse Figure 1. Effects of the number of wild grapes used on mutation detection.\nThe blue line indicates the number of the mutations shared by PN and wild grapes;\nthe\norange line indicates the number of PN-specific mutations; the yellow line indicates the\nnumber of predicted somatic mutations.\n5. Line 283, the authors claim that \"Similar patterns were also found on structure\nvariation and deleterious SNPs in each group. (Fig. 4C-D) \". What exactly is this pattern?\nIs “the heterozygosity of PN, SG, and HE is higher than wild grapes”? Is it\nstatistically significant in Fig. 4C-D to claim that?\nResponse: We apologize for the unclear description. We have revised these sentences\n(Lines 275-285) and made a statistical analysis according to the reviewer’s comment\nwhich shown on Figure 4D&4E. We found that different mating systems could affect\nthe accumulation of deleterious SNPs and SVs, especially on the variants in\nheterozygous states. We have made a detailed discussion in Discussion section (Lines\n468-479).\n6. The “protein phosphorylation pathway” was under selection in PN genome (Fig. 2B)\nand also keep heterozygous in PN40024 and HE but homozygous in wild grapes (Fig.\n5B). I am curious if there could be any connection, either biologically or\ncomputationally.\nResponse: We greatly appreciate your suggestions. However, we redid the GO analyses\nwith another tool to confirm the reliability of our results. It seems there are many\ndifference between the new results and the old one, and we have updated the manuscript\nwith the new results. The previous analysis was based on Arabidopsis protein database,\nwhich is different from grapes. In the revised manuscript, we constructed the database\nusing grape genome annotation first, and conducted GO analysis using clusterProfiler\n(Wu, T. et al., 2021 Innovation (Camb)) as shown in Material and Methods section\n(Lines 718-728). We think this updated results is more reliable.\nReference:\nWu, T. et al. clusterProfiler 4.0: A universal enrichment tool for interpreting omics data.\nInnovation (Cambridge (Mass.)) 2, 100141; 10.1016/j.xinn.2021.100141 (2021)\n8. The data is presented twice in the same sentence (lines 281 and 283).\nResponse: Thanks you. We have corrected it.\n9. What do the different colors represent in Fig. S11?\nResponse: We apologize for the unclear presentation of the figures. We have corrected\nthis figure. The gray color indicates the sites with 0/0 genotype, the red color indicates\nthe sites with 0/1 genotype and the blue indicates the sites with 1/1 genotype. It is\nSupplementary Figure 14 in the revised manuscript.\n10. Line 39. The “cossing” should be “crossing”.\nResponse: Thank you. We have corrected it.\nReviewer #2 (Remarks to the Author):\nThis work of Wang et al provides new data leading to a chromosome-scale assembly of\nan important grapevine cultivar and to an inventory of somatic variations in its clonally\npropagated plant material. In the version in which it is currently presented, the\nmanuscript is missing a clearly defined biological question.\nResponse: Thank you for your time and constructive comments. We have weakened\nthe parts of genome assembly and paid more attention to the effect of the mating system\non grapes to make our research focus on the effects of mating systems on grape genome\nand breeding. In previous studies, the effects of outcrossing, cloning, and selfing have\nbeen discussed. However, all of them were independent research. Here, we tried to\nobserve this in a continuous process. Fortunately, we found the grape materials\nincluding various mating systems in a lineage, which could help us further understand\nthe interaction effect on the grape genome between different mating systems that can’t\nbe studied in an independent system. We think this is significant for future grape\nbreeding. We had specified the research questions in the Introduction part. We hope the\nrevised version is more suitable for publication on NC.\nTitle, abstract, and a large part of the Introduction section anticipate that this manuscript\nis going to focus on the different modes of propagation in grapevine (vegetative, selfing,\noutcrossing) and their effect on genome properties and variation in the cultivated\ngermplasm. However, a large part of the results and discussion sections deal with the\ngenome assembly of a single genotype (Pinot Noir) and its comparison with the genome\nassembly of one descendant of Pinot Noir (PN40024, an experimental genotype\nforcibly obtained from reiterated selfing starting from an offspring of Pinot Noir). As\nfor the parts of the manuscript that deal more specifically with the topics anticipated in\nthe introduction, the authors move from observations in a few cultivated genotypes and\ndraw generalizations for the crop germplasm.\nResponse: Thank you for this suggestion. We modified the manuscript and significantly\nshortened the genome assembly part, and paid more attention to the effect of the mating\nsystem on grapes. The mating system transition might have more significant effects on\ngenomic variation than previously focused evolutionary factors, such as genetic drift\nand selection. According to the reviewer, we have modified the results and discussion\nto give cautious conclusions from our results. Although these results are drawn from\ngrape data, we think they also provide valuable information on other crops.\nThe manuscript has elements of potential interest for the grapevine scientific\ncommunity that require, however, both a deeper and a wider investigation to yield\nrobust and conclusive evidence, but also several aspects of weakness that should be\nmore carefully considered (see below for specific points).\nResponse: Thanks for the comment. We have revised the manuscript to yield a deeper\nand a wider influence of this study. We also added more analyses and modified the\nmanuscript to give cautious conclusions from our results.\nSpecific points:\nLine 68: transformation from dioecy (obligate outcrossing) to monoecy (potential self-\npollination and cleistogamy).\nResponse: Thanks. We have modified the sentence.\nLines 69-70: history of outcrossing and clonal propagation of highly heterozygous\ngenotypes.\nResponse: Thanks. We have modified the sentence.\nLines 113-120 are inappropriately placed in the Results Section.\nResponse: Thanks for the comment. We rewrote this paragraph (Lines 109-114) and\nmoved some of the content into the introduction (Lines 82-84)\nLine 128: it should be made more explicit at first citation and in figure legends that the\nacronym PN_T2T refers to the T2T genome assembly of PN40024, an individual that\nwas obtained by repeated rounds of selfing, starting from Helfensteiner.\nResponse: Thanks for your suggestion. We have modified these sentences (Lines 122-\n124).\nLines 146-155: the 4-Mbp inversion on chromosome 19 should be experimentally\nvalidated using a PCR-based assay. This assay could also be used for assessing the\norigin and frequency in the grapevine germplasm of the haplotype carrying the inverted\nregion.\nResponse: Thank you for the suggestion. We have validated the 4-Mbp inversion using\nPCR according to your advice (Supplementary Figure 4). We also examined the\nfrequency of this inversion and found it only existed in heterozygous Pinot Noir, but\nlost in PN40024. In the future, with more available haplotype resolved assemblies for\ngrapes, we could conduct a population genomic analyses of this INV and other ones.\nIn the paragraph “Introgression and selection shaped the Pinot Noir genome”, the\nauthors explained that they “used resequencing data from 38 samples (Table S2),\nincluding 18 Pinot Noir (PN) clones, 10 EU wild grapes, and 10 ME wild grapes,\nto explore the introgression and selection signals in PN”. It is unclear how they conducted\nthis analysis to identify introgressed regions (I assume from the EU wild grape\npopulation) into domesticated lineages deriving from ME wild grape population (then\nleading to Pinot Noir), selective sweeps and candidate genes under selection, using a\ngroup of Pinot Noir clonal replicates with software that normally applies to\ncomparisons among populations. As the resulting candidate genes (deriving from\nadaptive introgression ? from selection of ME ancestral diversity ?) are then described\nin detail and discussed extensively in Lines 162-204, their identification should be\nsupported by stronger evidence or accompanied by a more detailed description of the\nanalytical procedure.\nResponse: We apologize for the unclear description. We have modified the sentences\nin the Results (Lines 157-191) and Material and Methods (Lines 664-665) section. For\nintrogression detection, we used fd analysis which is based on the classical ABBA\nmethod. In this analysis, it used 4 pops (P1, P2, P3 and outgroup). It can detect the\nintrogression between P2 and P3 using P1 and outgroup as control. In our study, we\nused ME as P1, PN as P2 and EU as P3. For selection analysis, we make CLR analysis\non the data of the PN group using “Sweed”, a widely used software for selection region\ndetection.\nLine 206: what is Pinot Noir (PN) population? The group of Pinot Noir bud sports, the\noffspring Helfensteiner and PN40024, as described in Lines 555-556 of the Methods\nsection?\nResponse: We apologize for any confusion. PN population is 18 clonal Pinot Noir\nsamples that we download or sequenced by ourselves (Supplementary Table 3 & 4).\nSorry, we make a mistake in the description of the PN40024 in the Material and\nMethods section. PN40024 is not Pinot Noir and is not included in the PN group. We\nmodified this sentence in Lines 634-635 of this updated manuscript and also presented\nthe grouping information in Supplementary Table 3 & 4.\nParagraph “Somatic mutations in Pinot Noir clonal population” and Lines 553-565\nIt makes little sense using the reference genome assembly of a different genotype\n(derived from selfing of Helfensteiner) to realign short-reads of Pinot Noir bud sports\nand call somatic variants, when a haplotype-resolved T2T assembly of Pinot Noir has\nbeen generated in the present paper. A preprint that has recently appeared in bioRxiv\nshows the power of the use of a haplotype-resolved T2T assembly of the very same\ndiploid genotype in the identification of somatic variants among Malbec bud sports\n(https://doi.org/10.1101/2023.11.30.569420). Regardless of the reference used,\nexperimental validation of a set of candidate somatic variants needs to be provided in\norder to estimate false discovery rate.\nResponse: We appreciate your constructive comments. We have read this paper and\ncited it in our manuscript. We agree with you that it might be a better idea to use hap1\nand hap2 assemblies of Pinot Noir as reference genome in somatic mutation detection.\nHowever, to compare our results with previous population genetic studies, we chose to\nuse PN40024_T2T assembly as a reference genome for somatic variants identification\nsince it has better genome annotations.\nBased on our previous investigations, most of the somatic mutations are heterozygous.\nIt is impossible to distinguish the somatic mutation with germline mutation. So we take\nthe comparative population genetics approach using the wild grapes (ME and EU) as a\ncontrol. It turns out to be very efficient showed in our forward simulations (SLIM).\nAs shown in the response Figure 2, using wild grapes as a control could decrease the\nfalse positive of our detection and the accuracy of our somatic mutation detection is more\nthan 95%, and the recall rate is more than 85%.\nResponse Figure 2. Estimation of the detection efficiency using simulated data by SLIM.\nLine 254: wording would suggest that the authors used 4 independent lines obtained\nfrom selfing of Pinot Noir. Is it correct? Or are they clonal replicates of PN40024?\nPN40024 is a single seedling obtained from several rounds of selfing starting from\nselfing of Helfensteiner.\nResponse: Thanks for your comment. PN40024 is a single seedling from nine rounds\nof selfing starting from Helfensteiner. All the 4 PN40024 lines are clones. Sorry for the\nconfusion due to our unclear description. We have modified the sentence (Line 246).\nParagraph “Effect of various mating systems on grape genomes”.\nThe authors used “groups” of cultivated varieties that were formed by one single\ngenotype each, and three “groups” of these consisted of a parent-offspring trio. The\nforth “group” consisted of one single experimental highly inbred line (PN40024),\nshowing severe inbreeding depression, that has no parallel in the grapevine cultivated\ngermplasm. While π is largely independent from the number of sampled chromosomes\nand among-groups comparisons would make sense even using one diploid individual\nper group, data and inferences reported in Lines 279-294 refer to individual genomes\nand should be used with caution for inferring generalization on the species and on crop\ngermplasm, as those discussed in Lines 377-434.\nResponse: Thank you for your insightful comments. We have rewrote the whole\nDiscussion section, drawn most of the conclusion on the cultivar we focus and make\ngeneral discussion with caution.\nParagraph “Conserved heterozygous regions in cultivated grapes”.\nLines 296-300. These sentences need rephrasing.\nResponse: Thanks. We have modified the sentence (Lines 299-303).\nLines 303-305. Why intriguingly ? Heterozygous regions in PN40024 must be\nheterozygous in Helfensteiner by definition, as PN40024 derived from selfing of\nHelfensteiner.\nResponse: Thank you. We have modified the sentence (Lines 306-307).\nLines 333-334. Again. Why SVs and hSNPs should differ in heterozygous regions\nbetween Helfensteiner and PN40024, if both PN40024 homologs are identical by\ndescent with respect to Helfensteiner ?\nResponse: Thanks for your suggestions. We understand that all the heterozygous\nvariants in heterozygous regions should be also heterozygous in Helfensteiner.\nHowever, it different from the comment above involved Lines 303-305, we assumed\nthat the number of these variants (SVs, hSNPs) should be less than Helfensteiner due\nto the selfing process, as shown in other regions, the heterozygous hSNPs and SVs\nalmost disappeared. However, we observed that these variants are almost no change in\nPN40024 compared to Helfensteiner. That’s why we said “interestingly”. Sorry for our\nunclear description. We have modified the sentence to make it clearer (Lines 354-356).\nParagraph “Close leakage of deleterious variants in repulsion phases”\nI understand that this paragraph deals with the possible reasons (i.e. linked\nheterozygous deleterious variants in repulsion on the two homologs), other than chance\nalone, explaining why residual heterozygosity was found in specific chromosome\nblocks of PN40024. Figs. S13-S14 do not seem to suggest that residual heterozygous\nregions in PN40024 contain more deleterious variants than other heterozygous regions\nin Helfensteinter, which later became homozygous in the descendant upon selfing. In\nsupporting their claim, the authors also should provide evidence that what is shown in\nFig. S15 is not simply the result of incorrect variant phasing across heterozygous\nregions, because hap1 and hap2 are both identical by descent between Helfensteiner\nand PN40024 and do not appear as such in the graphical representation of Fig. S15.\nResponse: We appreciate your suggestion. We used BEAGLE to phase our VCF data.\nThe result of BEAGLE has been verified as reliable and widely used in other studies.\nIn the revised manuscript, we made more analyses and restructured the latter part of the\nresults (Lines 299-383). We used the population specific marker to make a discussion\non recombination on the PN40024 genome during selfing, and focused on the\nheterozygous region on Chr2 which includes SDR regions to explain what we find in\nthis study. We hope this revised manuscript presents more persuasive results.\nLines 385-387: This is a generalization that is inferred from observations in only three\nindividuals.\nResponse: Thank you for the comment. We have modified the sentence and the whole\nDiscussion section.\nMethodology for the assembly of the Pinot Noir genome in the Methods Section\nLines 481-489: in the generation of the HiFi-based assembly, Hi-C reads were\nrepeatedly used at the stage of contig generation (using Hifiasm) and scaffolding (using\nJuicer) as well as for other intermediate steps (also in combination with RagTag-based\ncontig ordering) that are not clearly described.\nResponse: We apologize for the previous unclear description. HiC reads are used in\ntwo places in the assembly process, one at HiFiasm to assemble the raw HiFi data to\nthe contig level, and the other is to divide the contig segments to the chromosome using\nHiC to the scaffold level. In HiFiasm, the HiC data are simply used for typing into two\nsets of haplotypes, and in the subsequent Juicer stage, the HiC data is further utilized in\ndepth for contig segmentation and assembly to the scaffold level. Meanwhile, we used\nRagTag just to perform a simple chromosome localization using the reference genome\nPN40024_T2T.\nLines 495-500: the procedure used for integrating and merging the HiFi-based partial\nassembly and the ONT-based partial assembly into a haplotype-resolved T2T final\nassembly needs a more clear and detailed explanation. Adding Supplementary Tables\nthat report for each chromosome contig-level and scaffold-level metrics obtained for\nthe HiFi-based assembly and for the ONT-based assembly would help follow procedure\nand the results described in the main text.\nResponse: Thank you for your suggestion We are sorry for the unclearness. We actually\nassmebled the haplotype resolved genome using a combination of HiFi and HiC reads.\nThe ONT reads were noisy with low quality, so that we didn’t include it in the revision.\nWe have modified the methods part (Lines 567-570) and the differences in N50 values\nfor contig and scaffold levels are presented in Supplementary Table S1\nLine 538: structural annotation should read structural variation\nResponse: Thanks. We have corrected it.\nReferences to the additional homologs illustrated in Fig. 3D should be added.\nResponse: Thank you for your comment. We presented the detailed results of GO\nanalysis in Supplementary Data.\nReviewer #3 (Remarks to the Author):\nWang et al's manuscript, \"Various mating systems shape grapevine genome evolution\"\nuses genomic resources to investigate evolutionary processes in grapes. Overall, the\nauthors seem to aim to \"To investigate the genomic effects of mating systems during\nclonal, crossing, and selfing\" (L351; first line of Discussion, and this phrase is\nconsistent with the message of the Abstract). It is noteworthy that previous studies have\naddressed these same questions; e.g., the effect of clonal growth on somatic mutations\nand the effect of self-fertilization on genetic diversity, so the ideas presented in the\nmanuscript are not very novel, although the data are new and useful. (Frustratingly, the\nauthors fail to point out that a large body of work has addressed the effect of mating\nsystem and clonality on genetic diversity and plant fitness, which makes the current\nmanuscript appear more novel than it really is. With respect to clonal growth, see this\nreview from 2015; much other work examines the effect of selfing -\nhttps://www.pnas.org/doi/abs/10.1073/pnas.1501712112) The authors do present data\nthat suggest an increase in somatic mutations curing clonal growth (which is consistent\nwith previous work see link provided above), and these data are interesting. The authors\nalso present results that suggest that some genome regions remain exceptionally\nheterozygous and suggest that this heterozygosity is maintained by deleterious alleles\nin repulsion phase. This latter result might be exciting, but I maintain healthy skepticism\nas it would require very strong selection (e.g., akin to recessive lethal effects) to\nmaintain the patterns of genetic diversity documented here (assuming I understood the\npaper). Overall, the authors present a new genome with some analysis, and provide\nsome results that are potentially exciting. However, my sense is that the ideas tested\nhere (and some of the main results) are not as novel as the manuscript leads the reader\nto believe.\nResponse: We are grateful for your positive views and constructive comments for our\nmanuscript. We have read the related papers and cited them in appropriate places. In\nprevious work, some research could have been done on clonal and selfing population\nindependently. However, in this study, we discussed the crossing, clonal, and selfing in\none family. We observed the effects of different mating systems in a successive process.\nIn the revised manuscript, we reshaped the introduction and discussion parts and added\nsome new analyses, such as discussing recombination based on specific markers, and\nrestructured the latter part of results and the whole discussion section. We hope this new\nversion is more suitable for publication on NC.\nGOALS (L93-110):\n1) Look at Pinot Noir genome... no real goal stated other than \"look at intricacies\";\n2) Look at processes to generate Pniot Noir\n3) Examine structural variants and deleterious variants and their changes\n4) Why do some regions remain heterozygous in PN40024 -\nOUTPUT 1 (L121 - 155) Genome of (heterozygous) Pinot Noir - Does this meet their\ngoal 1? They highlight an inversion.\nResponse: Thanks for your comment. The haplotype-resolved chromosomal Pinot Noir\ngenome was assembled to compare it with PN40024_T2T (its offspring associated with\ncrossing and inbreeding generations), which was used to detect the effects on mating\nsystems on genome structure and contents. According to your suggestion and other\nreviewers, we shortened this part and put more attention on the results of the effect of\nthe mating system. We also removed the verification of inversion to supplementary files.\nWe added the results of the comparative genomics to detect the effects of mating\nsystems on genome structure and contents in clonal grape (PN) and selfing grape\n(PN40024).\nOUTPUT 2 (L157 - 203) - Introgression analysis of Pinot Noir Genome - IS THIS\nGOAL 2?\n- This section provides evidence for selection on a few genes, but says little (if\nanything?) about the role for introgression in the evolution of the Pinot Noir genome,\ndespite the title of this section (L156) indicating that introgressions is a process of\ninterest. It is unclear to me whether the authors even examined for evidence of\nintrogression - if they did, how much evidence for introgression did they detect (and in\nwhich direction between regions)?.\nResponse: We appreciate your input. We modified the sentences in this part (Lines 149-\n191). There are many studies showed that a strong introgression in Wine grapes from\nEU wild grapes. But most of them are performed on population level that involved\nmany cultivars. In this study, we tried to confirm this introgression signal based on Pinot\nNoir population to make a deeper understand of this famous wine cultivar. We used f\nd\nstatistic method, one of the most popular methods for introgression detection, to\nevaluated the gene flow between PN and EU grapes. As shown in Fig. 2C & 2D, the\nPN genome has affected by EU significantly. However, we didn’t pay much attention\non this part, as we focused on the effect of mating system on the PN lineage grapes.\nSOMATIC MUTATIONS IN PINOT NOIR CLONAL POPULATION (L204)\n- The authors state, \"To distinguish somatic variations from germline ones, we\nidentified variations unique to PN\". Is is unclear to me how this approach can reliably\ndetect somatic mutations because it assumes that the mutations that have been identified\nin the small number of samples of European and Middle Eastern samples (20 genomes,\ntotal) will include *all* possible mutations that could have been present in the ancestor\nof the Pinot Noir clonal population. i.e., with only 20 EU and ME samples the authors\nhave sampled only 40 alleles from the wild populations (2 alleles for each of the 20\nsamples, with 10 from EU and 10 from ME). Therefore, the authors are unlikely to\nidentify variants at relatively low frequency in wild populations that can be mistaken\nas somatic mutations. Notably, the authors use patterns Fig 3B to support arguments\nthat 18 mutations arose somatically. However, it is unclear to me how the authors\ndistinguish between whether these mutations arose somatically *after* clonal\npropagation began vs. were simply unique to Pinot Noir and present in the genotype\nthat gave rise to the clonal genotype *before* clonal propagation began. The authors\nmay be correct in their logic, but they need to clarify their presentation to eliminate this\nalternative explanation to their data. For example, it would be useful to include a figure\nthat illustrated the evolutionary process that the authors imagine that yields the pattern\nobserved in their data.\nResponse: We greatly appreciate your suggestions. For the first question, the number\nof wild populations used could affect the results of somatic variants detection. Thus, we\nestimated the effect of different numbers of wild grapes used. As shown in Response\nFigure 1, when the number of wild grapes increased the number of somatic variants\ndetected tended to be stable. More wild grapes included can but in a very limited impact\non the result. Thus, we think the result using 20 wild grapes in this study is credible.\nFor the second question, to detect the somatic variations, we considered these mutations\nto be specific to the PN population and not shared by all PN individuals as they are\ngenetic mutations that occur in the body cells instead of the germ cells. The variants\nshared by all PN individuals are counted as germ-line mutations as these 18 individuals\nwere collected from worldwide, and not possible the clones of the same one PN sample.\nWe are sorry not to describe clearly that the PN-specific SNPs shown in Fig. 3B are not\nonly somatic mutations and also include germ-lime mutations. As shown in Fig. 3C, the\nmutations only shared by less than 18 PN samples were counted as somatic mutations.\nWe have modified these sentences to make it more clearly (Lines 193-210)\nWe also used SLIM to simulated data to estimate the accuracy of our methods. As\nshown in response Figure 2, using wild grapes as a control could decrease the false\npositive of our detection and the accuracy of our somatic mutation detection is more\nthan 95%, and the recall rate is more than 85%.\nL251: EFFECT OF MATING SYSTEMS ON GRAPE GENOMES - GOAL 3(?)\nL265 - the authors state, \"Due to nine generations of selfing, PN40024 was not counted\nas parent-offspring relationship with the other three cultivars.\" What evidence do the\nauthors have to conclude that it was selfing, per se, that caused the analysis to fail to\nindicate that PN40024 was not counted as an offspring in the analysis? Selfing, per se,\nshould only reduce genetic diversity by increasing homozygosity - therefore, it is\nunclear to me how the \"Kinship\" analysis would be so affected by increased\nhomozygosity. The authors need to either further justify their assertion or consider\neither (1) that PN40024 does not derive from Helfensteiner (although this is unlikely)\nor (2) there are issues with the Kinship analysis, more generally, which should make\nthe reader question *any* conclusions from this analysis.\nResponse: Thanks for the comment. The relationship between PN, SG, HE and\nPN40024 is already known. The reason we make kinship here is to confirm that the\nsamples we used are true to type and present some characteristics between these 4\ngrapes based on Kinship. The standard for judging the relationship between grapes\nbased on Kinship has been done in previous studies (Roach M et al., PloS genetics,\n2018; Jazmín, R et al, 2018, Nature Plants) and has been proven reliable in other studies.\nWe have revised the sentence to make it clearer (Lines 255-260).\nReference:\nRoach, M. J. et al. Population sequencing reveals clonal diversity and ancestral\ninbreeding in the grapevine cultivar Chardonnay. PLoS genetics 14, e1007807;\n10.1371/journal.pgen.1007807 (2018).\nRamos-Madrigal, J. et al. Palaeogenomic insights into the origins of French grapevine\ndiversity. Nature plants 5, 595–603; 10.1038/s41477-019-0437-5 (2019).\nL275: When comparing genetic diversity of inbred vs. outcrossed varieties, the authors\nconclude \"the selfing PN40024 exhibited the lowest values for both diversity and\nheterozygosity when compared to all other groups\". This is not a surprise, as the impact\nof selfing on genetic diversity is understood well in theoretical terms and is well\ndocumented empirically. Therefore, while this result adds to current data on the effect\nof self-fertilization on genetic diversity, it does not expand our understanding.\nResponse: Thanks for your comment. We have made more analyses on other aspects of\nslefing PN40024. We restructured the latter part of the results (Lines 261-383). We used\nthe population specific marker to make a discussion on recombination on the PN40024\ngenome during selfing, and focused on the heterozygous region on Chr2 which includes\nSDR regions to explain what we find in this study. We hope this revised manuscript\npresents more persuasive results. We have also rewrote the discussion to make a deeper\nand a wider presentation of the significant effects of various mating systems on crop\ngenome and breeding.\nL280: The authors also find that the genetic diversity of colonal propagated lines\nexceeds that of outbred populations; \"the heterozygosity of PN, SG, and HE is higher\nthan wild grapes, ME, and EU\". They authors conclude that clonal propogation\nincreases genomic diversity and this this occurs via (in part, at least) deleterious\nmutations. These data are very useful, and are consistent with previous findings (see a\nreview in 2015 here: https://www.pnas.org/doi/epdf/10.1073/pnas.1501712112) that\nalso suggest that clonal reproduction increases opportunity for somatic mutation. The\nauthors need to highlight that previous work also points to the accumulation of\ndeleterious, somatic mutations during clonal growth; if the authors wish to claim that\ntheir results are somehow novel, they need to clarify which aspects are indeed novel.\nResponse: Thank you for your thoughtful recommendation. We have carefully cited the\nreferences you provided. We also modified the latter part of the results to provide a\ncomprehensive view of the manuscript.\nCONSERVED HETEROZYGOUS REGIOUS IN CULTIVATED GRAPES (GOAL 4?)\nL300: The authors \"identified 379 alleles that remain heterozygous in all grape samples\n(wild and cultivated).\" The authors further remark, \"Remarkably, we identified a total\nof 59,944 alleles that retained their heterozygous status within PN40024. Intriguingly,\nthese heterozygous genes exhibit consistency between PN40024 and HE, and they also\nremain in heterozygous states in at least one PN and one SG sample.\" In reading this,\nmy first reaction was that this result can arise due to errors in genome assembly;\nthe authors examine read coverage to address this possibility (L318 - 327 - this is good)\nand remove several cases where this explanation likely is true. The authors conclude\nthat heterozygosity is maintained due to deleterious mutations in repulsion-phase\n(also termed pseudo-overdominance; see Charlesworth, Deborah, and John H.\nWillis. \"The genetics of inbreeding depression.\" Nature reviews genetics 10.11 (2009): 783-796.)\n[[Please note that what I say next relies on my understanding of the author's phrase,\n\"alleles that remain heterozygous in all grape samples\", as this implies to me that all\nindividuals that were genotyped were heterozygous at the focal loci - if I mis-\nunderstood the author's meaning, then my following comments need to be adjusted.]]\nIf the authors' explanation is true, that multiple loci remain heterozygous in **all**\ngenotypes individuals, then this implies *very* strong natural selection in order to\nmaintain heterozygosity in all sampled individuals. Personally, I am surprised by (and\ntherefore have healthy skepticism for) this finding and suggestion; I do find it\ninteresting, but encourage authors to emphasize the requirement for very strong\nselection to maintain this pattern of genetic variation, and I encourage the authors to\npoint out that this finding does require further investigation to confirm the suggestions\nmade in the current manuscript. I can illustrate my skepticism mathematically. Take,\nfor example, a single locus that remains highly heterozygous due to very strong\nselection: in this case, self-fertilization (which is how PN40024 reproduces) will lead\nto homozygous offspring at this locus for 50% of its offspring and many of them will\nexperience a large fitness cost due to selection at this single locus. Now, the authors\nhave implied that multiple loci remain heterozugous due to strong selection - if this is\ncorrect, then the fitness cost I just described for a single locus will multiple across other\ngenome regions. Overall, we expect that most offspring that derive from self-pollination\nof PN40024 to have low fitness if the authors' explanation for maintaining\nheterozygosity is correct (and if I've understood their manuscript correctly); is this seen\nempirically? If not, then my sense is that the authors need to either (i) clarify their\npresentation (because I may have mis-understood aspects I highlight above), or (ii)\namend or clarify their arguments / re-evaluate their findings. Are genome assembly or\nread-mapping errors possible reasons for the observed results?\nResponse: Thank you for this great question. First of all, we has reevaluated our\nanalyses that our results based on population samples (n ≥ 4) were solid and reliable.\nWe do see a strong inbreeding depression in PN40024, it barely reproductive, since\nreproductive were selected during each sdlfing generations. In the five heterozygous\nregions, we also detected genes enriched in the biological functions including\nreproduction and other important biological process for plant life. We also added the\nliterature you suggested and reshaped the main text, please see the revised manuscript\nfor details."}, {"round": 2, "reviewer_comments": {"reviewer_1": "I have read the revised MS, which has been improved a lot based on the comments and suggestions of reviewers including mine.\n1. I suggest not using exaggerated expressions, like I propose the following modifications： change “We found very interestingly that the mating systems, namely, crossing, clonal, and selfing have dramatic effects on grape genomic variation and breeding based on comparative genomics and population genetics.” to “We found that the mating systems, namely, crossing, clonal, and selfing have significant effects on grape genomic variation and breeding based on comparative genomics and population genetics.”", "reviewer_2": "The revised version R1 is mostly text editing, with two exceptions: the requested wet-lab validation of a large inversion in Pinot Noir and a new analysis that focused on the seemingly forced maintenance of the heterozygous state at the flower sex locus upon selfing of a F/H2 individual, which could theoretically become H2/H2 homozygous at any selfing generation, yet maintaining an hermaphrodite phenotype for the next round of selfing, but it never did so until the S9 generation.\nIn my previous remarks, I pointed out that a deeper and a wider investigation to yield robust and conclusive evidence on the effects of mating systems on genetic variation was required to support the authors’ claims. From my point of view, there is not additional data in the revised version that makes the overall manuscript robust enough for being accepted for publication in a high impact journal such as Nature Communications. Most of the remarks were not addressed convincingly or exhaustively.\nWhile some inaccuracy in the terms used in the original version of the manuscript for referring to populations, reproduction systems, mutation types seemed to be at that stage simply a matter of semantics, it seems now from their revised text and from their replies to the reviewers’ remarks that it actually reflects some misconception.\nAn illustrative yet not exhaustive list includes: vegetative propagation is not a mating system, a group of phenotypically divergent clonal lines is not a population, heterozygosity and chimerism of somatic mutations are not treated separately, chimerism is entirely ignored in the analysis.\nSome misconception has not only theoretical implications but also important consequences on the adopted methods of analysis.\nAs an example, the authors state in their response to previous remarks and therefore assume in their analyses “that somatic variants, they are genetic mutations that occur in the body cells instead of the germ cells, which cannot be passed on to the next generation through sexual reproduction”, which is not true. Novel somatic variation can be sexually transmitted if the mutation has occurred in somatic cells belonging to the cell layer of the shoot apical meristem that is going to generate the germ line.\nThe authors also state and therefore assume that “to detect the somatic variations, we considered these mutations to be specific to the PN population and not shared by all PN individuals. The variants shared by all PN individuals are counted as germ-line mutations as these 18 individuals were collected worldwide (Supplementary Table 3), and not possible the clones of the same PN sample.” The condition mentioned by the authors to be verified in the group of the 18 stocks that were derived from the historical vegetative propagation of the original seedling of Pinot Noir is not sufficient for distinguishing somatic mutations and germline mutations. Part of the somatic mutations might also be private to Pinot Noir, yet shared by all analyzed stocks, if they occurred before clonal lineages with distinctive phenotypic variation were intentionally selected by viticulturists and nurseries and if the model of clonal lineage evolution was tree-shaped instead of star-shaped.\nBelow is a detailed list of critical points that were not completely fixed or have newly emerged with the R1 changes.\nLine 32 and elsewhere: clonal propagation is not a mating system. Mating systems always involve crossing and if this occurs between unrelated individuals it is defined as outcrossing, between related individuals is defined as inbreeding and with itself it is defined as selfing. Clonal propagation is a mean to propagate the same genotype and does not involve mating between a male and female gamete, so it cannot be considered as a type of mating system.\nLines 33-35: There is no statistics in the results to support the claim that “The mating systems have more significant effects than traditionally focused factors such as genetic drift and natural/artificial selection.”\nLine 37: “cloning increases genomic heterozygosity”. In terms of relative numbers, somatic mutations lead to a minimal increase of heterozygosity cultivar-wise, as shown by this manuscript and by previous research. Furtherly considering that a substantial part of this novel heterozygosity is expected to be chimerical within each individual, its population-wise contribution through subsequent sexual reproduction is even less impactful than cultivar-wise. Overall, this sentence is misleading for novel point mutations and it does not consider the opposite effect of somatic deletions. It has been shown exactly in Pinot Noir that vegetative propagation leads to local loss of heterozygosity in the case of nonchimeric deletions (i.e. white-skinned bud sport) and local reduction of heterozygosity in the case of chimeric somatic deletions (i.e. gray-skinned bud sport).\nLines 99-100: “Second, we embarked on an exploration of the processes that underly clonal diversity among Pinot Noir clones, including the identification of somatic variants and genomic regions that may contribute to phenotypic traits.”\nActually, this paper only reports the identification of putative somatic SNPs and the list of affected genes. Association with phenotypes is not reported experimetally.\nLines 109-114: This knowledge was well-established prior to the present work.\nLines 115-126: In the revised version and in the response to reviewers’ comments letter, the authors explain that the new Pinot Noir assembly in the latest version of the manuscript is built by omitting the step of integrating the HiFi-based assembly and the ONT-based assembly, compared to the original version of the manuscript. In spite of this, the statistics of the assembly, in particular chromosome pseudomolecule sizes have not changed between versions. Overall, this adds new uncertainty and concern about how the assembly procedure was performed and how carefully it is described, rather than easing previous concerns.\nLines 139-140: I acknowledge that the authors have experimentally validated the inversion with a wet-lab assay, as requested.\nLines 141-147: How gene families and the boundaries between gene families were defined ? Do the numbers refer to the present or absence of entire gene families or individual gene family members ? The closing comment is inappropriate: what the authors observed might be due to pre-existing presence/absence variation between the Pinot Noir and Schiava Grossa haplotypes that were donated to Helfensteiner and then to PN40024.\nLines 148-191: My previous remark was not address either in the revised text or in the response letter. I do understand how SweeD and ABBA-BABA work, but how can this software do the job for which they were designed if fed with two populations that might be approximated to natural populations and another population that is not a population but a group of clonal replicates with no germline ancestral variation ? The group of 18 Pinot Noir clones contains only novel somatic variation and collectively captures only one or two ancestral haplotypes over each genomic region. Both software deal with allele frequencies within and between populations. In particular for the statistically supported detection of selective sweeps, it is totally unclear to me how this was applied to the specific case of this manuscript in which allele frequency in Pinot Noir that may only be 0, 0.5, 1 for germline SNPs, while somatic SNPs in Pinot Noir have by definition allele frequency = 0 in the ancestral populations.\nSee my previous comment on the original submission in detail. In the paragraph “Introgression and selection shaped the Pinot Noir genome”, the authors explained that they “used resequencing data from 38 samples (Table S2), including 18 Pinot Noir (PN) clones, 10 EU wild grapes, and 10 ME wild grapes, to explore the introgression and selection signals in PN”. It is unclear how they conducted this analysis to identify introgressed regions, selective sweeps and candidate genes under selection, using a group of Pinot Noir clonal replicates with software that normally applies to comparisons among populations.\nFor supporting the authors’ claim that most of Pinot Noir private SNPs are somatic mutations (approximately 50,000 out of 70,291), the plot in Fig.3B should report on the x-axis the read count ratio supporting the ancestral and the derived allele.\nThe direction of the mutation can be defined using the ME and EU populations the define the ancestral state. In the present form, this distribution is only showing graphically a subsampling bias or suggesting that many of the putative somatic SNPs may be false variant sites.\nLines 211-212: As most of the putative somatic SNPs were predicted to be private to individual clones, this may be either indicative of a star-shaped model of clonal differentiation or, more likely, suggestive of a high incidence of false variant sites.\nIn the absence of an experimental wet-lab validation of at least a random subset of candidate somatic SNPs to assess the false discovery rate, this part of the manuscript reporting on somatic SNP identification is weakly supported.\nLines 215-216: The fact that putative somatic SNPs are more frequently associated with intergenic regions is another indication that many of them might be false variant sites associated with partial read misalignments in repetitive regions.\nLines 199-201: Here and in general across this section, it seems that the issue of the chimerical state of somatic mutations has not even been considered in the analyses, which may provide alternative explanations to the data compared to those given by authors.\nLines 222-244: only germline structural variation in the cultivar Pinot Noir is reported here. If the authors detected structural variation among clones, it should be reported in this paragraph. If not, they should state that they did not detect structural variation among clones.\nLines 255-260: This part has already been shown and better explained by Velt et al 2023 10.1093/g3journal/jkad067\nLine 263: The He symbol in population genetics is used for expected heterozygosity under Hardy Weinberg equilibrium while the heterozigosity measured here is rather observed heterozygosity. So it would be better to list it as either Ho or just H.\nFigure 4B: PN is erroneously labelled by HE in two instances.\nLines 266-272: These interpretations are questionable and not supported by the data presented in this manuscript. If one considers the number of somatic SNPs identified e.g. among the Pinot Noir clones, it is clear that in comparison to the total number of germinal SNPs that number cannot justify such a large difference in He. This difference could instead be attributed to the mating system because cultivated varieties are the result of crosses that often combine different genetic materials while wild populations may suffer from a certain level of inbreeding. It is commonly observed and generally accepted that heterozygosity is higher in grape cultivars than in the present-day individuals of the wild progenitor, but this is commonly explained by the fact that heterozygosity is exceptionally low in wild population due to small population effective size and geographical isolation leading to forced inbreeding rather than heterozygosity is exceptionally high in the cultivars due to other reasons.\nLine 276: It is rather obvious that the dSNPs are seen less frequently in heterozygous condition in PN40024 where >90% of the genome is in homozygous state. So the comparison should not be with highly heterozygous accessions such as PN or SG or HE but rather it should be evaluated in comparison with the frequency of heterozygosity of non deleterious or neutral SNPs in PN40024\nLine 277: This should not be indicated as recessive because there is no information on the effect of the deleterious alleles in terms of recessive or dominant, so it should be indicated as homozygous.\nLines 279-280: this could be simply explained by the fact that selfing generations of Helfensteiner leading to PN40024 and PN40024 itself are experimental genotypes that were not exposed to natural selection and were, on the contrary, nurtured with extreme care for their survival and reproduction. This is an extreme artificial situation that has no parallel in grapevine evolution and viticulture. Artificial selection during the generation of PN40024 has only targeted hermaphroditism in order to proceed with the next round of selfing.\nLines 289-296: Putative somatic mutations in Schiava Grossa, Helfensteiner and PN20024 are first mentioned here. If the procedure of identification was the same as in Pinot Noir, the numbers are not comparable among cultivars as they depend on the number of individuals that are compared within each cultivar.\nLine 295: truncated sentence\nLines 329-339: This section is not very relevant because there is no quantitative analysis of the local recombination rates in this manuscript.\nLines 374-386. This part is an interesting addition with respect to the original R1 version. In the end and overall, this is the only really exciting part of the manuscript. However, as it occurred to other parts, it only scratches the surface of the biological issue and would require extended data.\nLines 376-377: If I am not wrong, H1 derived from a recombination between F and M haplotypes. H2 has then derived from a recombination between H1 and F, shortening the M derived region in the H haplotype.\nLines 435-438: Any conclusion and comment on the effects of domestication and improvement on diversity by comparing populations of the wild ancestor against single individual genomes of cultivated varieties makes no sense.\nLines 509-534: The entire paragraph “Lessons and implications for grapevine genomic breeding” contains general considerations and personal interpretations that are inappropriate in this context.\nSupplementary Table 4. Taxonomic assignment in the column “species” is not accurate.\nThe issue with former Figure 5 panel F and Supplementary Figure S15 has been simply removed, and not solved.", "reviewer_4": "This is a genome paper for Pinot noir. The primary data contribution is a phased outbred whole genome assembly of a Pinot Noir plant. The authors present many bioinformatic analyzes of the two chromosome sets (PN1 and PN2) in relation to the “reference genome” – a chromosome level assembly on one highly inbred line of this species (PN40024_T2T) – plus lots of resequencing data from other grapes. The latter is (apparently) Illumina data published previously, although this is not entirely clear from the paper. I was not a referee on the first submission and was asked to comment on mating system aspects and particularly on the claims (lines 103-104 and elsewhere) about residual heterozygosity in the 9th generation inbred line.\nIt is plausible for a locus to be heterozygous after 9 generations of selfing. With no selection, the probability is only 0.002, but with preferential preservation of heterozygotes during line formation, the probability increases. For example, if heterozygotes are twice as likely to survive as homozygotes, the probability increases to 0.075, which is still not a large number, but if numerous loci across the genome are experiencing this selection, a few might remain after 9 generations. For the loci on chromosomes 2 and 7 (Fig 5), the authors have the correct data signal – many heterozygous SNPs within a localized ‘block’ combined with the observation of normal read depths when Illumina short reads are mapped back onto the genome. The authors absolutely need to be much more explicit on one key point: How many heterozygous blocks do they believe are real residual heterozygosity? On lines 484-485, they say “lots of heterozygous regions are real and can’t be ignore” – what number corresponds to “lots”? For how many regions can the authors make the picture they have for chroms 2 and 7 (Fig 5a)? The heterotic selection argument (that heterozygote advantage is responsible for residual heterozygosity) becomes less and less plausible as you add more and more loci.\nOf course, the most compelling relevant data would be to directly measure the fitness effects of alternative genotypes. If you generate and genotype a few hundred selfed progeny from the line, differential fitness should be estimable because fitness differences must be large (else the line would not be heterozygous). The GERP stuff (line 364) is fine, but very indirect.\nApparently, information about genotype to phenotype/fitness is available for the SDR locus on Chrom 2 (lines 374 onward). There are no citations here, but it seems that one homozygote (f/f) cannot self (has fitness = 0 during inbred line formation) because it makes only female plants. It is not clear what is going on with the alternative homozygote (H2/H2) – are these plants hermaphrodites or not? These are the essential details to make the residual heterozygosity interesting.\nFor the broader mating system arguments, I have no doubt that the frequencies of outcrossing/selfing/clonal propagation were important for grape genomes. However, the text requires revision because it does not make sense to say “The mating systems have more significant effects than traditionally focused factors such as genetic drift and natural/artificial selection” (lines 33-35, similar on lines 418-419 and elsewhere). Mating system does not change allele frequencies on its own. It alters the way that genetic drift, gene flow, and natural/artificial selection act on variation. There are numerous good reviews to guide these arguments – see papers by Stephen Wright and references therein.\nOther comments:\nLine 148: I could not make sense of the arguments on introgression. For most biologists, introgression is an explanation for shared variation between populations or species—often, it is posited as an alternative to shared ancestral variation. Here, we need a much better sense of what the different lineages actually are, and what alleles moved between lineages, and how we can be certain that these variants were not present in the common ancestor of the (now) distinct lineages. Are EU and PN (lines 179-180) sister lineages currently isolated from each other?\nLine 202 onward: The general audience will need an explanation of how the authors are using the term “somatic mutation.” Plants do not have a segregated germline, so all mutations start out as somatic. Are the somatic mutations identified here those that have never been transmitted through gametes? If so, the main paper could use a sentence or two to explain how that is established from sequence data.\nLines 273-274: “Then we compared the genetic burden in each group by identifying deleterious SNPs (dSNPs)…” The very important term “deleterious SNPs” comes out of nowhere without any explanation or citation. What does this mean? Does it mean that the SNP has two alleles, one that is good and one that is always bad? If so, how is this determined (the paper has no fitness assays that I can see). There are bioinformatic classifications of alternative alleles (e.g. synonymous versus non-synonymous) but these do not identify the fitness effects of particular loci. They make groups where the distribution of selection coefficients is likely be different by some (always unknown) amount between groups. This can be useful (e.g. if the fraction of non-syn SNPs is much higher in selfing species than outcrossing), but the reader will need much more explanation to make any sense of the downstream analyses on ‘dSNPs’.\nLines 321-323: “… after nine generations of selfing to generate PN40024, most of the genome was homozygous, except for a 59,944 heterozygous SNPs.”\nWhat fraction of 60k SNPs are within the heterozygous blocks that the authors believe to be residual heterozygosity? After only 9 generations, real het blocks should be quite large (e.g. Chrom 2 and 7 in Fig 5). After excluding all the SNPs that are false hets due to mapping, how many loci are there in this inbred line? Perhaps the authors just need to explain the results in Supplementary Table 10."}, "author_response": "Response: We are grateful for the insightful feedback. Based on your comments, we have made the following revisions: 1) We included additional grape cultivars related to Pinot Noir and expanded the sample sizes for previously analyzed cultivars; 2) We clarified the definitions of key terms, including “somatic mutation” and “reproductive system”; 3) We reanalyzed the selective sweep using the PBS analysis instead of the CLRanalysis;4)Weperformedfurtheranalysisonthesexdeterminationregionwithinthe heterozygous blocks of selfing PN40024, which further clarified the Hill-Robertson effect. We identified a candidate lethal recessive gene that may maintain the SDR region in a heterozygous state in PN40024; 5) We enhanced the presentation of the Results and Discussion sections. Wehave provided detailed explanations in response to each of your comments,we believe theserevisionshave substantiallystrengthenedthe manuscript."}, {"round": 3, "reviewer_comments": {"reviewer_1": "I have no more comments.", "reviewer_2": "In the newest version, the addition of an extra focus in one individual on the effect of experimentally-induced multiple rounds of selfing on the flower sex locus provides substantially novel and relevant biological information in comparison to the rest of the manuscript. I appreciated this extended part.\nThe other methodological concerns that I expressed in my previous rounds of reviewing remain unaddressed in this new version. In particular, those regarding the issues related to the identification of reliable sequence variation among clonal lines, to the identification of introgressed regions in Pinot Noir, and to the consequences brought about by selfing.\nAs to the entire parts of the manuscript regarding the impacts of vegetative propagation on the genetic composition of crop germplasm, all of this is elaborated upon a critical set of variant sites. As explained by the authors in one point of their rebuttal letter, it is commonplace to acknowledge that from 1% to 5% SNP calls in reference-based analyses using short-read alignments are likely to be false variant sites. If they called 3,915,920 SNPs in their analysis of different grapevine genotypes (Line 176), it follows that they consider a reasonable number between 39,000 and 195,000 variant sites to be possible errors. If they had analysed 18 clonal lines that are genetically identical with no sequence variation, under the same experimental and analytical conditions as above, in the absence of sequence variation in the biological samples they would have therefore expected to call a similar absolute number of false variant sites due to the experimental procedure alone, which are to be considered possible errors. Among 18 Pinot Noir clones that might have biological sequence variation, the authors called a number of variant sites comprised between 20,328 and 70,291, which largely overlap with the absolute numbers of false variant sites that may result from the experimental procedure alone, as above. This situation is nearly identical for SVs. I do not believe that all of these variant sites among Pinot Noir clonal lineages are false, but in the absence of an experimental validation and in the absence of an independent assessment of false discovery rate in this experiment, I cannot take this dataset on trust either. While up to 200 K false SNPs out of 4 M true SNPs have no significant impacts on the results of population genetics analyses, the absolute number of called SNPs among clonal lineages compared to the expected absolute number of possible errors might lead to what the authors called in the point I mentioned above “horrible incorrect studies” or conclusions.\nAs to the entire parts of the manuscript regarding the impacts of selfing on the genetic composition of crop germplasm, I do not have doubts on the data extracted from the genome composition of the PN40024 line. However, part of the observations of the characteristics of the genome in a single individual might be due to the effect of chance alone and drawing generalizations on the consequences of selfing at population-wise level is unsound.", "reviewer_4": "The authors have improved their paper in response to my previous comments. My review focused on the issue of residual heterozygosity within a putatively highly inbred line. They have resolved the real heterozygosity to five loci that cover about 4% of the genome. Given strong selection against homozygotes in certain portions of the genome, this number is reasonable even after 9 generations of selfing. The authors did not collect additional data to explore the nature of heterotic selection, but that may be beyond the scope of this work. The overall narrative still needs a great deal of work, but I am satisfied with their treatment of the heterozygosity issue."}, "author_response": "Response: We appreciate your time and constructive comments on our manuscript. We had revised the manuscript and we hope the current version is more suitable for publication."}, {"round": 4, "reviewer_comments": {"reviewer_2": "Regarding the specific question I am asked, whether my remaining concerns have been addressed in the last round of revision of the manuscript, the answer is no."}, "author_response": "Response: Thanks for your comments. In this new version, we did more analyses and revised the related sections to enhance clarity and rigor."}, {"round": 5, "reviewer_comments": {"reviewer_2": "The manuscript has been improved to an adequate degree. The results and discussion sections include sufficient explanations about the limitations of this study."}, "author_response": "Response: Thank you very much for your time, patience, and constructive comments! The manuscript has been greatly improved after revision following your suggestions."}]

Decision letter
[Editorial Note: See attachment]