BACKGROUND
the wgs  approach for sequencing complex eukaryotic genomes  <cit>  has contributed to the assembling many genomes of non-model and crop species.
poplar  <cit>  and grape  <cit>  were the first plant genomes sequenced with this approach.
the advantages of wgs sequencing, as compared to the bac by bac  approach  are the speed of sequencing and the reduced cost.
however, a weakness of the wgs sequencing approach is it tends to produce a more fragmented assembly with reduced contiguity, also coupled with the risk of large-scale misassemblies.
this is especially true for complex eukaryotic genomes and the assembly process can be confounded with recent duplication events , large regions of expanded repeats , and residual heterozygosity.
the result is the production of a fragmented sequence with poor contiguity metrics such as the n <dig>  and the l <dig> .
the potential issues are greater if a highly heterozygous individual is chosen as reference  <cit> .
however, in the bac by bac approach, the use of local sequence information  mitigates the risk of large-scale misassembly.
the advent of next generation sequencing  technologies has exacerbated wgs assembly drawbacks, typically producing a more fragmented assembly.
ngs produces shorter sequence reads compared to the sanger method, making genome assembly more difficult and requiring the development of a range of dedicated bioinformatics tools and novel alignment algorithms  <cit> .
a de novo short-read ngs assembly needs high genome coverage, mainly to overcome the reduced overlap length and improve the contiguity of the resulting assembly  <cit> .
henson et al.
<cit>  calculated that by increasing the reads length from 50 bp to 1000 bp, the contig l <dig> value of the human genome can theoretically increase from 3 kb to about  <dig>  kb.
moreover, the lack of a chromosome-scale assembly, combined with putative misassemblies  precludes extensive use in evolutionary and comparative genomics studies, as well as genome wide association studies .
the availability of a chromosome-scale assembly is, therefore, crucial to maximally leverage the advantages of the wgs sequences.
the coupling of wgs genome assembly with highly saturated and high resolution molecular genetic maps has been proposed to cope with the lack of chromosome-scale wgs genomes .
genetic maps enable the reconstruction of a chromosome-scale sequence by positioning wgs scaffolds in their correct order and orientation to arrange them in long stretches of dna, representing the individual chromosomes, called “pseudomolecules” or “pseudochromosomes”.
inconsistencies between the position of markers on the map and in the assembly can highlight putative misassembled sequences that can be further resolved by breaking the chimeric scaffolds and rearranging the broken pieces in their correct order and orientation.
fragmented genome assemblies, such as those obtained with short reads ngs data, need dense genetic maps for a large fraction of the assembled sequence to be anchored on chromosomes.
the availability of wgs genome assemblies in many species combined with ngs platforms has fueled variant discovery through alignment of resequenced reads of different accessions to the reference genome.
millions of single nucleotide polymorphisms  and small insertions/deletions  dispersed throughout the genome have been discovered in different species .
this discovery, coupled with high-throughput genotyping technologies, such as snp arrays  and genotyping by sequencing  , has accelerated the construction of high-resolution genetic maps, enabling map-sequence integration of wgs scaffolds in highly fragmented de novo ngs assemblies.
medium and high-throughput genotyping tools have been developed in prunus for peach  <cit>  and cherry  <cit>  and in other rosaceae species such as apple  <cit>  and strawberry  <cit> .
to overcome the limitations of short read sequencing technologies  third generation methodologies, based on single molecule sequencing, have been recently released such as the one of pacific bioscience  <cit>  and moleculo  <cit> .
these methodologies, are capable of obtaining much longer reads, up to 50 kb  <cit> , with the trade-off of an increase in error rates of 13-15%  <cit>  in comparison to  <dig> – <dig> % in illumina short reads  <cit> .
to solve this problem, several strategies have been proposed such as the integration of long reads with more accurate ngs reads.
this hybrid sequencing strategy has been recently used in pineapple  <cit>  apple  <cit>  and arabidopsis thaliana ler  <cit> .
the peach  genome sequence was obtained by the international peach genome initiative  and is an  <dig> -fold wgs high quality draft sequence  <cit>  with long contiguity, high base accuracy, and a large portion of sequences mapped on chromosomes.
sequences were obtained using the sanger methodology and a complete homozygous reference accession, the ‘lovell’ double haploid plov2-2n.
the prunus reference map  was used to anchor the first release of the peach genome  obtaining eight pseudomolecules representing the eight prunus chromosomes.
approximately half of the markers were placed by genotyping only six seedlings using the bin mapping strategy  <cit> .
thus, even if the bin mapped markers were useful to assign scaffolds to chromosomes and check scaffold integrity, in most cases they did not provide sufficient information for anchoring the scaffolds on chromosomes.
moreover, the txe map is an interspecific map and was obtained with a limited number of individuals  resulting in a reduced recombination frequency in some regions  <cit> , providing only a rough estimation of the recombination frequency at a short physical distance.
the anchoring markers  are unevenly distributed along the linkage groups leaving portions of the genome uncovered.
later analyses of the peach genome revealed that 4% of the sequence of the peach v <dig>   was not included in the pseudomolecules; with ten scaffolds  anchored with unknown orientation and a large number of mapped scaffolds had no markers on their ends , making it difficult to detect putative chimerism  <cit> .
in this work, we describe the efforts aimed at improving the peach chromosome-scale build  using a set of linkage maps and resequencing the reference accession .
two mapping strategies were used: i) a targeted approach where markers  were targeted in specific regions of the peach genome  and mapped in two already available linkage maps  <cit> ; ii) a whole genome approach that made use of the ipsc 9 k snp array v <dig>  <cit>  to genotype a large set of progeny  <cit> .
moreover, illumina ngs resequencing of the reference accession at high coverage was performed to correct sequencing errors  and to close a number of gaps in the peach v <dig>  assembly increasing the contiguity of the final peach genome.
methods
plant material, dna extraction and quality test
four biparental mapping populations were used to refine the peach genome :i)  <dig> seedlings of the ‘texas’ x ‘earligold’ f <dig> population , an interspecific cross between almond and peach, maintained at the experimental station of crea-fru in rome, italy ;
ii)  <dig> seedlings of the peach selection if <dig> x ferganensis bc <dig> population  maintained at the experimental station of crea-fru;
iii)  <dig> seedlings of the ‘contender’ x ‘ambra’ f <dig> population  maintained in a farm belonging to the municipality of castel san pietro  leased to astra ;
iv)  <dig> seedlings of the maria dolce x sd <dig> f <dig> cross  maintained at the experimental station of crea-fru.
young leaves were collected from each seedling and lyophilized.
dna was extracted with the dneasy plant mini kit , quantified with the nanodrop spectrophotometer  and with the picogreen® assay  for samples genotyped on the ipsc 9 k snp array.
for sequenom analysis, dna was extracted from the seedlings of the cxa progeny after mercado et al.
<cit> .
target ssrs identification and mapping in txe progeny
target ssrs were selected from among the  <dig>  identified within the peach v <dig>  genome sequence  <cit>  to increase marker density in order to leverage in large unanchored scaffolds, and correct order/orientation of anchored scaffolds.
sequences of ~600 bp flanking both sides of the repeated motif were first blasted against peach v <dig> , using the blast facility available on phytozome  <cit> , and only non-repetitive regions were selected to design specific primer pairs by the primer  <dig> software  <cit> .
only single locus ssr markers were used for further analysis.
the selected ssr primers were first tested on the bin set and parents of the txe progeny.
only the most physically distant polymorphic markers in each target region were genotyped on the whole txe progeny.
to check for scaffold integrity, some ssrs were developed in the distal region of uncovered scaffold ends.
in the presence of suspected chimeric regions, further markers spaced approximately every 100 kb were identified, developed, and mapped to restrict the size of misassembly containing regions.
primer sequences and features are listed in additional file 1: table s <dig>
all pcr reactions were carried out in a 10 μl volume with a final concentration of 1x pcr buffer,  <dig>  mm mgcl <dig>  200 μm of each dntp and  <dig>  μm of each primer, 10 ng genomic dna, and  <dig> u of platinum®taq dna polymerase .
the amplification profile was: one cycle at 94 °c for 5 min, followed by  <dig> touchdown cycles, with a decrement of the annealing step temperature of  <dig>  °c/cycle, starting with a denaturation at 94 °c for 30 s, an annealing step five degrees above the primer-specific annealing temperature  for 30 s, and an elongation at 72 °c for 30 s, followed by 25 cycles at 94 °c for 30 s, ta °c for 30 s, 72 °c for 30 s, and a final elongation of 30 min at 72 °c.
pcr products were then separated on a 3% high-resolution agarose gel  in tbe 1x with a voltage of ∼ 5 v/cm and stained with the gelred™ .
markers that could not be easily scored on agarose gel were separated by capillary electrophoresis on a ceq  <dig> genetic analyzer .
ssrs genotyped in the whole txe progeny were integrated with the previous txe dataset  <cit>  and mapped using the software mapmaker  <cit> , grouping them at a lod score higher than  <dig>  they were located by using the try and ripple commands.
after mapping, the error detection command of mapmaker was used and putative double recombinants were manually checked.
snp identification and mapping in the cxa progeny
the f <dig> parent of the cxa progeny was resequenced with an illumina platform .
the clc genomics workbench  <dig>   was used for read alignment and snp calling.
only reads aligning to a single location with at least 92% identity over at least 90% of their length were considered.
for snp calling, variants were retained when: i) the coverage ranged between  <dig>  and  <dig> x of the average coverage ; ii) minor allele frequency > 30%; iii) the polymorphic nucleotide phred-scaled quality score ≥  <dig> and the average quality ≥  <dig> for the 11 bp surrounding the putative snp.
snps were manually selected based on their distribution on the peach genome.
snps surrounded by repetitive sequences and/or located within a stretch of bases identical to that of the snp itself  were avoided.
the surrounding sequences  were obtained from the peach gbrowse available on the iga website  <cit>  and blasted against the v <dig>  peach genome at gdr  <cit>  to verify their uniqueness.
only unique snp-surrounding sequences were used to design the assays , and combine them in multiplex reactions  by the software mass array design  <dig> .
all locus-specific pcr primers and probes were blasted against the peach genome to further verify their specificity.
only the snps that passed all these quality checks were retained for further analyses.
a total of ten iplex were designed to attain the optimal genome coverage, including two iplex  with snps selected in specific uncovered regions, or where snps previously tested were not useful.
information about the primers used for the genotyping is listed in additional file 1: table s <dig>
genotyping in the cxa progeny was performed using iplex gold technology  <cit>  and mass array high-throughput dna analysis mass spectrometry  at the centre for applied biomedical research  of bologna.
the sequenom data for each snp were first verified by checking the heterozygosity of the cxa f <dig> parent and its consistency with the two grandparents ‘contender’ and ‘ambra’.
all the snp data were integrated with the dataset of  <dig> ssr markers genotyped following eduardo et al.
<cit> , and then analyzed by joinmap  <dig>  software  <cit>  with the default parameters and the kosambi  <cit>  mapping function.
linkage groups were established at lod value  higher than  <dig>
all these snps were also tested on the bin set of the txe mapping progeny.
ipsc 9 k snp array genotyping and mapping
the ipsc 9 k snp array  <cit>  was used to genotype  <dig> individuals of the pxf and  <dig> of the mdxsd progenies, using the illumina infinium ii design probes, and the dual color channel assay .
snp genotypes were scored with the genotyping module of the genome studio data analysis software .
snps with a genetrain score ≥ <dig>  and less than 10% missing data were retained.
allele segregation was also checked and snps showing unexpected segregations  or unexpected genotype classes , were inspected using genome studio.
if possible they were re-clustered using the “define cluster” function.
those still showing missing or unexpected classes were discarded.
linkage analysis and map construction were performed with joinmap  <dig>   <cit>  using the cp and bc <dig> population types for pxf and mdxsd, respectively.
linkage groups were established at lod value  higher than  <dig> as described above.
the multipoint maximum likelihood mapping algorithm was used with the default parameters.
kosambi units  <cit>  were used; for the cp population type, haldane map distances were manually converted in kosambi units using the formula provided in joinmap  <dig>  manual.
map-sequence integration
the original raw version of the peach v <dig>  assembly, post filtering organelle, repetitive, and small scaffolds  sequences, was used for the new map-sequence integration.
markers were placed on the wgs scaffolds using two methods as reported in verde et al.
<cit> .
ssr and snp markers having primer sequences  were placed using three successive rounds of electronic pcr  with n =  <dig>  n =  <dig> and n =  <dig>  markers with a known sequence, including rflp  and snp markers or ssrs whose primers had not been found with the three rounds of epcr, were placed with blastn.
the additional breaks were made in regions of low bac/fosmid coverage and the broken sequences reordered according to the new maps.
the mapped wgs scaffolds were joined as described in verde et al  <cit>  to form  <dig> pseudomolecules .
each map join is denoted by  <dig>  n bps.
in this work, to avoid confusion between v <dig>  and v <dig>  releases, scaffolds composing the peach v <dig>  assembly were named and are hereafter referred to as “scaffold_##”.
the  <dig> wgs scaffolds included in the  <dig> v <dig>  pseudomolecules were named as “scf_##”.
the wgs scaffolds composing the v <dig>  pseudomolecules  were named as “super_##”.
lovell dh resequencing
resequencing of the ‘lovell’ double haploid  was performed using the miseq illumina platform.
paired-end reads  were assembled with the abyss software  <cit>  after quality checking and filtering.
the resulting contigs were used to patch gaps in the peach v <dig>  assembly after the new breaks and joins described above were applied .
contigs were aligned to the repeat masked modified v <dig>  assembly using blat  <cit> .
contigs whose ends aligned to either side of a gap, with at least 1/ <dig> of the contig length anchoring to the edges of the gap at ≥95% identity, were used to patch the gap.
sequence and quality scores were then integrated into the v <dig>  modified assembly.
finally, homozygous snps and indels were corrected using ~43x illumina reads.
reads were aligned using bwa  <cit>  and variants  called using the standard gatk pipeline  <cit>  including base quality score recalibration, indel realignment, and duplicate removal.
physical vs genetic distance comparison and identification of centromeric regions
mareymaps were obtained, for each mapping progeny, by plotting the genetic positions of molecular markers  against their physical position on the peach v <dig>    <cit> .
cumulative recombination curves for each chromosome were estimated using the cubic spline interpolation method with default parameters and the cross-validation type present in the mareymap package.
the recombination value per position was obtained calculating the slope per markers and their curves were plotted for each chromosome.
in order to identify the putative centromeric region of each chromosome, regions displaying the lowest recombination rate, as highlighted by the mareymaps, were manually checked on the peach v <dig>  using the jbrowse available on phytozome  <cit>  for the absence of transcripts and the abundance of repeated elements associated with centromeric and pericentromeric regions.
sequences retrieved from neumann et al.
<cit> , representing a catalog of plant repeated elements associated with centromeric regions, were aligned with blastn  <cit>  to the peach genome assembly.
sequences aligning within the putative peach centromeric regions , with at least 63% of identity and an e-value greater than  <dig> x 10− <dig>  were retained.
recombination frequency was compared among all progenies by multiple comparison statistics implemented in past  <dig>   <cit> .
for each linkage map used in this study , recombination rate was estimated at individual whole-chromosome scale as the ratio between genetic  and physical  distances.
one-way analysis of variance  was applied with tukey’s pairwise post-hoc test.
the levene’s test for homoscedasticity and the shapiro-wilk test for normal distribution were also applied to check the assumptions for the applicability of the anova.
in the case of violation, the non-parametric kruskal-wallis test was applied instead, with the mann-whitney pairwise post-hoc comparison and the bonferroni correction.
RESULTS
four linkage maps were used to improve the peach genome.
txe and cxa were already available  <cit>  and were enriched using a targeted approach.
another map was the result of a de-novo mapping of the pxf progeny  <cit>  obtained using the ipsc 9 k snp array  <cit>  and tripling the mapping progeny size.
finally, the mdxsd map, also obtained with the ipsc 9 k snp array, was used to specifically address some inconsistencies at the top of linkage group  <dig> .
targeted ssr identification and mapping in txe
a total of  <dig> ssrs  were identified in the peach v <dig>  assembly and primers were designed and then tested in the txe bin set .
twenty markers were developed on the major unmapped scaffolds,  <dig> were individuated within the randomly oriented scaffolds and  <dig> were identified within the  <dig> uncovered scaffold ends.
thirty-eight out of the total were monomorphic in txe and  <dig> were bin mapped .
out of  <dig> microsatellites falling within genic regions,  <dig>  were polymorphic.
three polymorphic markers  were mapped in txe, and found in successive analysis to have their primer pair on the same flanking side of the microsatellite region.
these three markers were retained and reclassified as indel markers.
these incidental length polymorphisms in the txe interspecific progeny reflect the different genomic structure of the closely related almond and peach species.
the same length variation was observed in six out of the seven knox genes whose fragment size indicated differences between the two parents ranging from 2 bp to 20 bp in length  <cit> .
a much higher level of polymorphism  with rflps in the txe progeny had been already observed  in comparison to that  of the intraspecific pxf progeny  <cit> .
thirty-two well-spaced ssrs, out of the  <dig> polymorphic ones, were mapped in the whole txe progeny to resolve orientation, misassembly, or ordering discrepancies.
in the preliminary steps of the peach genome assembly  <dig> ssrs  <cit>  targeting specific regions had been mapped in the whole progeny .
in addition, seven markers  targeting the peach knox genes  <cit>  had also been included.
the final map  is composed of  <dig> markers and covers  <dig>  cm.
three hundred and twenty-nine markers, having sequence information associated, were used for the peach v <dig>  map-sequence integration.
they cover  <dig>  cm  and 220 mb .
only four gaps larger than 10 cm are present.
in addition,  <dig> roscos bin mapped markers  <cit>  and four snps from the prunus-malus consensus sequence  <cit>  were included in this study and integrated with the wgs scaffolds.
furthermore,  <dig> snps from the cxa f <dig> parent and  <dig> ssrs isolated in this study  were also bin mapped.
in total  <dig>  txe markers were integrated within the assembly,  <dig> bin mapped , and  <dig> mapped in the whole progeny .table  <dig> anchoring statistics of the peach v <dig>  assembly
number of markers integrateda
<dig>
<dig>
225
total
554
374
483
570
288
407
392
355
<dig>
57
51
52
total
10
9
10
7
5
9
4
4
58
<dig>
<dig>
pxf f1
<dig>
<dig>
<dig>
<dig> , <dig>
<dig> , <dig>
pxf f1
<dig> , <dig>
<dig> , <dig>
<dig> , <dig>
total no of bases anchored
<dig> ,208
<dig> ,870
<dig> ,013
<dig> ,236
<dig> ,696
<dig> ,980
<dig> ,811
for each map and for each chromosome the number of markers, number of anchored scaffolds, genetic and physical distance covered with the integrated markers and the total number of anchored bases are reported
ain brackets the bin mapped markers in txe
targeted snp identification and mapping in cxa
from the resequencing of the cxa f <dig> parent,  <dig> snps have been developed and included in a total of ten iplex assays, with an average number of  <dig>  snp each iplex.
among the tested snps,  <dig> were useful for mapping while  <dig> were not: of these,  <dig> were monomorphic ,  <dig> showed only two over three expected genotypes and ten presented more than 25% of missing data.
the latter group also included five markers with no amplification in the whole progeny, possibly due to assay failure.
the fraction of scorable polymorphic snps ranged from  <dig> % to  <dig> % in different iplex assays, with an average success rate of  <dig> % .
the relatively negative result on iplex  <dig> and  <dig> is likely due to forcing the iplex design to develop markers in target regions.
the observed snp calling efficiency was evaluated by comparing our results with those obtained by verde et al.
<cit> , which used more stringent parameters for snp calling.
this a posteriori analysis showed that  <dig> % of the non-polymorphic snps were false positives  but at the same time five true snps over the  <dig> mapped ones  would have been lost using the more stringent conditions .
finally, with the more stringent parameters, the total efficiency of snp design would have increased from  <dig> % to  <dig> % .
the cxa map was first obtained with  <dig> ssrs on 169 f <dig> progeny  <cit> .
to improve the chromosome-scale assembly the number of progeny was increased to  <dig>  the additional individuals were genotyped with ssr markers, adding  <dig> targeted polymorphic snps to the map.
fifteen of these snps were already linked on lg <dig> to a candidate gene controlling maturity date in peach  <cit> .
another set of twelve snps on lg <dig> surrounding the nectarine g locus had been described  <cit> .
a total of  <dig> snps were identified on unmapped scaffolds, ten on the randomly oriented scaffolds and  <dig> in putative chimeric regions.
fifty-three of these snps were also bin mapped in txe, as already described in the previous paragraph.
the final cxa map  includes  <dig> markers  corresponding to  <dig> unique genetic positions, covering  <dig>  cm with only four gaps larger than 10 cm.
all the pseudomolecules are almost completely covered , with the exception of pp <dig> lacking the bottom portion  and pp <dig> missing the upper portion .
ipsc 9 k snp array mapping in pxf
the  <dig> trees of the pxf progeny were genotyped with the ipsc 9 k snp array  <cit> .
out of the  <dig>  placed on the array, a total of  <dig>  polymorphic snps  were identified;  <dig>  snps were informative for the f <dig> parent ,  <dig> were informative for both parents  and  <dig>  were informative for the recurrent parent .
of the three types of segregation, only the first two were used for the map-sequence integration because the unequal recombination frequencies between the two parents of the cross inhibited efficient joining of their genetic information.
in fact, the recurrent parent  displays a marked reduction in recombination frequency in comparison to that of the f <dig> parent .
as a consequence, the map-sequence integration was unreliable due to the heavily skewed order of markers in regions with strong differences in recombination frequencies between the two parents.
moreover, the distribution of the informative snps for the recurrent parent was uneven across the genome resulting in a fragmented linkage map with  <dig> groups.
in fact, five groups were split in two groups each , one was completely missing  resulting in a reduced genome coverage .
the inspection of the ipsc 9 k snp array markers segregating in the recurrent parent highlighted that they did not give additional information for integration except for two contiguous minor scaffolds  found in the upper part of the pp <dig>  seven markers located in these two scaffolds informative for the recurrent parent, were selected and integrated into the map.
the presence of bridge markers in that region  segregating in a 1:2: <dig> ratio and the low number of 1: <dig> markers informative for the f <dig> parent  enabled a reliable integration.
when a single snp was mapped in a linkage group that differed from the expected placement from the array information, and no further evidence of misassembly was observed in the same genetic region , this marker was deemed as putatively duplicated  and excluded.table  <dig> genetic/physical ratio  for each map and each chromosome
lg-ppa
a
lg linkage group, pp pseudomolecule
after filtering, we mapped  <dig>  snps in total  corresponding to  <dig> unique genetic loci.
the map  covers  <dig>  cm  with only one gap larger than 10 cm.
integration of unmapped scaffolds in peach v <dig>  pseudomolecules
one hundred and ninety-four scaffolds , had not been included in the eight peach v <dig>  pseudomolecules.
to anchor the larger unmapped scaffolds  in peach v <dig> , we first used the txe map.
twenty ssrs  were targeted in the unmapped portion of the peach genome and  <dig> polymorphic ones  enabled anchoring of the ten major scaffolds  and fixing the orientation for two of them .
the same was done using the cxa map .
this map , in addition to being from an intraspecific cross, was obtained with a large mapping progeny  providing a fine estimation of the recombination frequencies even at a small scale .
with this approach, we were able to confirm the anchoring of the ten major scaffolds  and anchoring of an extra minor scaffold .
the high resolution of the cxa map also enabled the ordering of two contiguous small scaffolds  on pseudomolecule  <dig>  that in txe were unordered and unoriented, and fixing the orientation of five scaffolds .
only  <dig> markers of the ipsc 9 k snp array were located in the unmapped portion of the genome and six of them were polymorphic in pxf.
the position of five of the ten major scaffolds previously mentioned  was confirmed using these markers.
together, these analyses positioned  <dig> unmapped scaffolds on the v <dig>  pseudomolecules .
they cover  <dig>  mb of sequence ; five of them were also orientable .
ordering and orientation of peach v <dig>  randomly oriented sequences
ten mapped scaffolds  of the peach v <dig>  genome were placed with random orientation due to the lack of recombination among markers or because they were anchored with only one marker.
to resolve the orientation of these scaffolds, previously bin mapped markers, located by the ends of the non-oriented scaffolds were mapped in the whole txe progeny.
for scaffolds where no bin mapped markers were available,  <dig> ssr primer pairs  were designed towards the end, tested on the txe bin set and the most physically distant polymorphic markers  were mapped in the whole progeny.
in this way, five integrated scaffolds  representing  <dig>  mb of sequence were oriented along the pseudomolecules.
moreover, the attempt to orient a scaffold on pp <dig>  revealed that it was incorrectly placed along the pseudomolecule due to a mismapped marker  in txe.
this scaffold was correctly positioned at the top of the pp <dig>  the higher genetic resolution of cxa map  confirmed the position of super_ <dig> at the top of pp <dig> and the correct orientation of a major scaffold at the bottom of pp <dig> .
it also rectified the orientation of two wrongly oriented scaffolds due to mapping artifacts in txe: one on top of pp <dig>  and the other in the middle portion of pp <dig> .
the high density and resolution of the pxf map further confirmed the orientation of the above-mentioned scaffolds and enabled the correct orientation of three other scaffolds in peach v <dig>  .
in a region of pp <dig> , indicating high recombination frequency suppression, five scaffolds  were ordered with low probability in txe.
the higher resolution of cxa and pxf helped to resolve uncertainties .
however, the orientation of super_ <dig> and super_ <dig> was indeterminate in v <dig>  since the corresponding markers cosegregated in all maps.
moreover, it was not possible to have certainty of the order of super_ <dig> and super_ <dig> located in the same region .
in fact, markers on those scaffolds were ordered in txe with low probability .
in cxa and in pxf only one of these two scaffolds was anchored in each map  giving no additional information on their order.
for these scaffolds, the peach v <dig>  order, based on txe, was retained.
however, recently published maps  <cit>  obtained using the ipsc 9 k snp array  <cit>  have enabled verification of their order and orientation.
in particular in the mxr_ <dig> map  <cit>  two snps  mapping on super_ <dig> at  <dig>  and  <dig>  cm, respectively  and other four  mapping on super_ <dig> at  <dig> ,  <dig> ,  <dig>  and  <dig>  cm, respectively  revealed that the order established in peach v <dig>  is incorrect and will be inverted in a future release.
this is also confirmed by the dvss map  <cit>  in which two snps  were mapped at  <dig> and 32 cm, respectively.
the map obtained by sánchez et al.
<cit>  confirmed the orientation of super_ <dig> established only by pxf map and v <dig>  orientation of super_ <dig> was shown to be correct, though it was anchored by only one marker in txe and cxa.
together, all the ten scaffolds randomly oriented in peach v <dig>  and two wrongly placed  were correctly oriented in peach v <dig>  pseudomolecules.
currently, only six minor scaffolds  summing up  <dig>  mb are randomly oriented in this release.
moreover, only a known mis-order  and a random order  in the central part of chromosome  <dig> are still present in peach v <dig>  .
these will be rectified in a future release of the peach genome.
scaffold ends checking and correction of misassembled sequences
within the  <dig> scaffolds composing the peach v <dig>  pseudomolecules,  <dig> terminals were not covered with molecular markers for at least 500 kb of their length,  <dig> having an uncovered portion larger than 1 mb with the largest one being of  <dig>  mb.
these regions are potential sites of misassembled sequence.
to check scaffold consistency, ssr and snp markers were developed in the distal part of these uncovered regions.
markers mapped in txe and cxa maps  helped to reveal five out of these  <dig> uncovered scaffold ends as sites of misassembly: three in pseudomolecule  <dig>  one in pseudomolecule  <dig> and one in pseudomolecule  <dig>  on pseudomolecule  <dig>  there were two scaffolds bearing two different chimeric regions, resulting in six pieces in total that needed to be relocated on different chromosomes.
for this purpose, the two most distant polymorphic markers in each chimeric region were mapped in the whole txe progeny in order to locate and orient the new broken scaffolds within the peach pseudomolecules.
a particular case occurred in the v <dig>  integrated scaffolds scf_ <dig> and scf_ <dig> located on pseudomolecule  <dig> and pseudomolecule  <dig>  respectively.
they originated from a chimeric scaffold that had been broken in a wrong position in the peach v <dig>  assembly due to insufficient marker coverage within the putative chimeric region .
in v <dig> , with the help of the new mapping data, we refined the breakpoint, so that 385 kb from v <dig>  scf_ <dig> in pseudomolecule  <dig> were re-joined to the formerly broken scf_ <dig> to form the new super_ <dig> on v <dig>  pp <dig> .
the whole genome mapping approach of the pxf map confirmed all of the chimeric scaffolds and enabled identification of two other cases of misassembly on the top of v <dig>  pseudomolecule  <dig>  in this region two scaffolds  were chimeric and needed to be broken in one point each.
three of the broken portions,  <dig>  mb of sequence , had to be rearranged within the same chromosomal region .
to support the rearrangements at the top of v <dig>  pseudomolecule  <dig>  we used additional information from the mdxsd map.
in this map, lg <dig> is composed of  <dig> snp markers for a total of  <dig> single genetic positions covering a genetic distance of  <dig>  cm with an average of  <dig>  cm between markers and a major gap of  <dig>  cm .
the total physical distance covered amounts to  <dig>  mb .
twenty-one mdxsd markers mapped in the  <dig>  mb region  confirm the order of the three broken scaffolds highlighted by pxf and the orientation of two of them .
without high resolution and high-density linkage maps highlighting discrepancies in this  <dig>  mb region , this problem could not have been solved since the txe and cxa maps have low marker density in that region.
scaffold ends were also checked using additional information from recently published linkage maps .
in the current assembly, only  <dig> scaffold ends larger than 500 kb are still not covered with markers, all of them lying in highly repeated centromeric regions, with the largest being a 594 kb stretch of sequence .
in total,  <dig>  mb  of sequence from peach v <dig> , were relocated in their correct chromosomal positions with the correct orientation.
base accuracy and contiguity improvement through reference accession resequencing
the ‘lovell’ double haploid  was resequenced from  <dig> libraries , producing  <dig> , <dig> paired-end reads, corresponding to 43-fold paired end fragment sequence coverage  and 21-fold mate pair coverage .
this set of reads was assembled using abyss   <cit> , producing  <dig>  scaffolds greater than 500 bp for a total of  <dig>  mb of sequence.
scaffolds produced in the abyss assembly were broken into contigs, and a total of  <dig> contigs representing  <dig>  mb of sequence were used to close  <dig> peach v <dig>  gaps, with a gain of  <dig>  kb .
the overall contiguity was improved, with the total number of contigs in peach v <dig>  decreased by  <dig> % dropping from  <dig>  to  <dig>  and the contig l <dig> increased by  <dig> % .
finally,  <dig> homozygous snps and  <dig>  indels were corrected using ~43x paired end fragment illumina reads .table  <dig> summary of gap patching and indel and snp correction
totals:
206
212
<dig>
<dig> ,737
<dig> ,936
<dig>
<dig>
859
chromosome-scale assembly and comparison to other published genomes
in the new map-sequence integration, a total of  <dig>  markers  from three linkage maps were used in the integration of the original raw peach assembly obtained prior to the build of peach v <dig>  pseudomolecules:  <dig>  markers were integrated in txe ,  <dig> in pxf and  <dig> in cxa .
the improved txe map  enabled the anchoring of  <dig> scaffolds  and orienting of  <dig> scaffolds .
the cxa map  anchored  <dig> scaffolds  and oriented  <dig> .
the pxf map  anchored  <dig> scaffolds  and oriented  <dig> .
finally,  <dig> snps mapped in mdxsd on lg  <dig> were instrumental in confirming some faults at the top of pp <dig> .
important amendments were made in the v <dig>  assembly including the portion of mapped and oriented sequences and the identification and correction of discernible misassemblies.
a visual comparison between the two versions of the peach genome  is shown with the mareymap plot .
high-quality assemblies are those in which the plots are characterized by a monotonically increasing function.
both peach v <dig>  and v <dig>  show a general increasing tendency, however, in few v <dig>  regions the function decreases highlighting this release faults .
furthermore, differences in the physical length between the two assemblies, observed in all but pp <dig> pseudomolecule, reflect the inclusion of previously unmapped scaffolds and the correction of inter-pseudomolecule misassemblies .fig.
<dig> plots of genetic-by-physical distances .
comparison of v <dig>  and v <dig>  physical distances  and pxf genetic distances .
dots represent the mapped markers.
the vertical bars indicate the putative position of the centromere.
the solid line represents the recombination rate plotted along the  <dig> pseudomolecules
the total number of major wgs scaffolds  after filtering and scaffold breaking and prior to pseudomolecule construction was  <dig>  spanning  <dig> ,381 bp with a scaffold n50/l <dig> of 10/ <dig>  mb .
the final chromosome-scale assembly is arranged in  <dig> stretches of non-contiguous sequences  spanning  <dig> ,381 bp with a contig length of  <dig> ,928 bp and a scaffold contig coverage of  <dig> % .
fifty-eight scaffolds spanning  <dig>  mb of sequences were integrated into the  <dig> peach v <dig>  pseudomolecules corresponding to  <dig> % of the total sequence .
a comparison between the statistics of the two chromosome-scale assemblies, peach v <dig>  and peach v <dig>  is also shown in table  <dig>  fifty-two scaffolds, summing up  <dig>  mb , are correctly oriented in the new assembly .
the unmapped portion of the genome comprises  <dig> scaffolds with a length of  <dig>  mb  containing only  <dig> predicted genes  <cit> .
additional file 1: table s <dig> resumes the integration of peach v <dig>  assembly, reporting the scaffolds mapped in the  <dig> pseudomolecules, their position in peach v <dig>  and peach v <dig> , the orientation information in all the maps and the size of uncovered scaffold ends.table  <dig> summary statistics of the peach v <dig>  chromosome-scale assembly statistics and its comparison with the v <dig>
table  <dig> reports a comparison among some of the major plant genome assemblies released to date.
the table was adapted and updated from verde et al.
<cit>  with newly published genomes and the updated genome releases  available on phytozome  <cit> .
among the wgs assemblies, peach v <dig>  displays one of the largest portions of sequences mapped on chromosomes .
the scaffold n50/l <dig> , prior to pseudomolecules build, shows the best values among those available.
chromosome-scale n50/l <dig>  is comparable with other chromosome-scale assemblies.
if we exclude the finished genomes  and those almost finished , the peach genome shows one of the best contig n50/l <dig> , including the recent wgs pineapple genome  <cit>   obtained combining the third generation single molecule long reads  with the ngs short reads .
the long read sequencing in pineapple was instrumental in increasing the scaffold l <dig> metric from  <dig> to 640 kb and the contig l <dig> from  <dig>  to  <dig>  kb  <cit> .
pacbio long reads were also useful to improve the assembly of the a. thaliana ler genome  <cit> ; the integration of 17x pacbio long-reads with the illumina short reads improved the scaffold l <dig> that increased from  <dig>  mb to  <dig>  mb.
the long read single molecule sequencing technologies are playing an increasingly important role in sequencing projects.
in fact, the bioinformatics efforts of the assembling procedure are assisted and empowered by the availability of sequence stretches entirely spanning the repetitive regions, which are a major contributor to gaps present in assemblies.table  <dig> comparison of the peach genome to other published plant genomes
arabidopsis thaliana
sorghum bicolor
physcomitrella patens
an50/l <dig> statistics of the wgs assembly prior to pseudomolecule build
bn50/l <dig> statistics of the chromosome-scale assembly
c
arabidopsis assembly, obtained using bac by bac approach, represents the golden standard for plant genome.
statistics were calculated from tair <dig> release.
drice assembly, obtained using bac by bac approach, represents the golden standard for plant genome.
statistics were calculated from irgsp releases build  <dig>
edata retrieved from schmutz et al.
<cit> ; they recalculated the original statistics to better match chromosome-scale assemblies
fdata from recent releases retrieved from phytozome
gdata were recalculated based on the original statistics reported in the paper
we compared the statistics of the current peach release with the standards established by chain et al.
<cit> .
according to these standards, the peach v <dig>  assembly can be classified as “improved high-quality draft” since actions have been taken in assigning almost the whole sequence to chromosomes, in solving discernible misassemblies, filling gaps to reduce the number of contigs and correcting base errors.
moreover, the annotation of the release  has greatly improved gene completeness using a large amount of rna-seq data, as well as the annotated repeats which include low copy repeats and a complete set of helitron transposons.
the number of gene models in v <dig> a decreased to  <dig>   resulting also in less fragmented gene models.
the average number of transcripts per gene model increased to  <dig>  from  <dig>  .
the annotation improvements, not described in this work, are briefly reported on phytozome  <cit> , gdr  <cit>  and iga  <cit>  websites.
for all these features the improved peach release  can be further classified according to chain et al.
<cit>  as an “annotation-directed improvement”, making it a useful tool for genome comparison and evolutionary studies, including gene studies such as alternative splicing analysis and metabolic pathway reconstruction.
physical vs genetic distance comparison and identification of centromeric regions
the availability of the txe, cxa and pxf linkage maps covering most of the peach genome enabled a chromosome-scale comparison of the recombination frequencies along the genome.
in fig.
<dig>  additional file 3: figure s <dig>  additional file 4: figure s <dig>  and additional file 5: figure s <dig>  mareymaps are plotted together with a function describing the genetic/physical ratio .
average physical/genetic distance ratios for each individual chromosome and cross were calculated  to be  <dig>  cm/mb in the interspecific cross txe and  <dig>  cm/mb and  <dig>  cm/mb in the intraspecific crosses cxa and pxf, respectively.
the mareymap plots , constructed for the three different mapping progenies, indicates the expected monotonic increase along each chromosome except for a flat region where a marked suppression of recombination can be observed in each pseudomolecule.
the survey of these regions on peach v <dig>  jbrowse  <cit>  revealed the almost complete absence of predicted genes and the abundance of repetitive elements.
this concurrent evidence suggest that these are likely the centromeric regions  which are known to be mainly composed of interspersed tandem repeats and retrotransposons.
despite the centromeres have highly conserved function during cell division, their dna sequences are not conserved  <cit> .
in particular, in plants, the centromeric satellite dna repeats are species-specific.
recent studies suggest that these sequences underwent a rapid evolution revealing no sequence similarity among species diverged more than  <dig> mya  <cit> .
other important components of the centromeric regions are represented by the retrotransposons directly involved in the centromere evolution and function  <cit> .
a blast analysis against the peach genome was performed using a set  <cit>  of  <dig> centromeric retrotransposon sequences belonging to  <dig> different plant species.
in particular, eight plant centromere retrotransposons indicated similarity with the peach putative centromeric regions: three from medicago truncatula , two from pinus taeda , one from picea glauca , one from beta vulgaris , one from vitis vinifera .
these results strongly support that these regions are the peach centromeres.
the accuracy of the peachv <dig>  assembly and the peach v <dig> a annotation enabled the positioning of the centromeric regions, which are generally very difficult to assemble due to their highly repetitive sequence composition.
linkage maps are of little use to assemble fragmented centromeric regions due to the suppression of recombination.
as an example, the putative peach centromeric region in pp <dig>  indicated no recombination in the nearly  <dig> meiosis analyzed in the three different mapping progenies, leaving the related scaffolds unoriented and unordered.
the identification of putative centromeric regions for all of the eight peach v <dig>  chromosomes attests the completeness of the current peach genome assembly.
to verify whether there were significant differences in the recombination frequencies among the three maps, an analysis of variance was performed.
since the pxf map was obtained by map data of the two parents of the cross , the individual maps of the two parents were also considered in the analysis.
both the anova and the non-parametric kruskal-wallis statistics were applied showing no significant difference among the five maps .
however, due to the low coverage  of some linkage groups  chromosomes with coverage lower than 50% of the total length  were excluded thus revealing significant differences with the anova test .
moreover, to enlarge the comparison panel, two recently published maps, obtained with the ipsc 9 k snp array, were included: the peach intraspecific ‘nj weeping’ x ‘bounty’ f <dig> map  <cit>   and the interspecific dvss bc <dig> map obtained by backcrossing a hybrid between peach and p. davidiana as donor with peach as recurrent  <cit> .
both maps indicate > 80% coverage for each individual chromosome, globally 93% and 95% of peach v <dig>  length, respectively.
the physical distances covered by these two latter maps were recalculated based on the peach v <dig>  assembly.
the anova comparison was then applied giving significant differences among the seven maps .
the lowest recombination frequency, displayed by the recurrent parent , was not significantly different  from that of the two interspecific hybrids .
the pxf f <dig> map, having the highest recombination frequency , was significantly different from the two interspecific maps   as well as from pxf recurrent map  but not significantly different from the other intraspecific maps .
the extreme reduction of recombination rate, observed in the pxf recurrent parent  is comparable to that of the interspecific crosses.
‘jh hale’ is an old cultivar, one of the founders of the so-called modern western germplasm, carrying the male sterility allele  while ‘bonanza’ is a dwarf accession.
the if <dig> recurrent parent is classified as an accession with intermediate tree vigor   <cit>  belonging to the western subgroup  <cit> .
no differences in the recombination frequency were observed among the other peach f <dig> individuals .
the other parents of the crosses analyzed in this study have different origin: ‘contender’, ‘ambra’ and ‘bounty’ all derive from western breeding programs while ferganensis belongs to the eastern subgroup  <cit>  and nj weeping is believed to have japanese origin  <cit> .
our results are in agreement with those reported in arabidopsis thaliana  <cit>  where recombination rates, calculated in 17 f <dig> populations derived from  <dig> accessions, do not correlate with genetic distances between intraspecific parental accessions.
an exception in this study is the pxf recurrent parent, which displays a reduction in recombination rate similar to that of the interspecific crosses.table  <dig> tuckey's pairwise comparison test among the different maps
<dig>
<dig>
<dig>
pxf f1
<dig>
<dig>
<dig>
<dig>
significant p-values  are reported in bold
CONCLUSIONS
in this paper an improved and refined version of the peach genome assembly based on high quality linkage maps and resequencing data is presented.
this new assembly release has been improved in terms of completeness and accuracy, including the increase of mapped and oriented sequences, repositioning of misassembled portions, enhancement of contiguity and correction of base errors.
high density  and high resolution  maps are important tools to assist wgs efforts.
in fact, even if unanchored wgs assemblies are able to catch the full gene complement, they defect in depicting the whole genome view thus being of little use for comparative genomics.
the late high-throughput genotyping technologies such as snp arrays or genotyping-by-sequencing platforms are essential for developing saturated and high-resolution maps in short times with minimal cost, even in species with a narrow genetic base like peach and other self-pollinating species.
third generation and ngs technologies can be efficiently used in newly or already available genome sequences to obtain a high quality assembly.
additional files
additional file 1: tables <dig>  ssr markers developed in this study from peach v <dig> .
for each primer, the locus name, primer sequences, position in peach v <dig>  and v <dig> , repeated motif, polymorphism in txe  and location in genic or intergenic regions are reported.
table s <dig>  snp markers developed in this study from the resequencing of the cxa f <dig> parent.
for each primer, the locus name, position in peach v <dig>  and v <dig> , primer sequences, bin position and location in genic or intergenic regions are reported.
table s <dig>  illumina short reads libraries.
all libraries were sequenced as 2x250s using illumina sequencing.
reads were filtered on quality, screening for reads that were predominantly simple sequence, and residual phix contamination.
table s <dig>  txe linkage map.
for each locus, the genetic position in linkage groups and physical position in peach v <dig>  are reported.
only integrated markers are reported.
table s <dig>  sequenom snp development and their efficiency with the snp calling parameters used in this study.
a posteriori efficiency was calculated using more stringent parameters as in verde et al.
<dig>  table s <dig>  cxa linkage map.
for each locus, the genetic position in linkage groups and physical position in peach v <dig>  are reported.
table s <dig>  pxf linkage map.
for each locus, the genetic position in linkage groups and physical position in peach v <dig>  are reported.
table s <dig>  linkage map of pxf f <dig> parent.
for each locus, the genetic position in linkage groups and physical position in peach v <dig>  are reported.
table s <dig>  linkage map of pxf recurrent parent .
for each locus, the genetic position in linkage groups and physical position in peach v <dig>  are reported.
table s <dig>  composition of the  <dig> peach v <dig>  pseudomolecules.
start and end of each scaffold, total scaffold length, scaffold denomination in v <dig>  and in v <dig> , v <dig>  position of each v <dig>  scaffold, length of the scaffold ends uncovered with markers and mapping and orientation information achieved with the linkage maps used in this study, are reported.
table s <dig>  linkage map of lg <dig> of mdxsd.
for each locus, the genetic position  in linkage groups and physical position in peach v <dig>  are reported.
table s <dig>  peach v <dig>  assembly statistics  prior to the pseudomolecules build.
table s <dig>  peach v <dig>  assembly statistics post chromosome-scale build.
additional file 2: figure s <dig>  anchoring of the peach scaffolds to the three genetic maps.
colored bars represent the  <dig> linkage groups: pink for pxf, purple for txe and blue for cxa.
wgs scaffolds were positioned in each pseudomolecule  with the corresponding genetic markers and are depicted in three different colors ; genetic markers are in the same colors of the corresponding wgs scaffolds.
the zero  denotes the six scaffolds placed with random orientation along the pseudomolecules.
the asterisk  indicates the two scaffolds with random order.
the crosshatch  indicates the two scaffolds with the wrong order in peach v <dig>  that need to be inverted in a future release.
additional file 3: figure s <dig>  mareymap plot of pxf linkage maps .
vertical bars indicate the putative position of the centromere.
the solid line represents the recombination rate plotted along the  <dig> pseudomolecules calculated using the cubic spline method.
additional file 4: figure s <dig>  mareymap plot of txe linkage map.
vertical bars indicate the putative position of the centromere.
the solid line represents the recombination rate plotted along the  <dig> pseudomolecules calculated using the cubic spline method.
additional file 5: figure s <dig>  mareymap plot of cxa linkage map.
vertical bars indicate the putative position of the centromere.
the solid line represents the recombination rate plotted along the  <dig> pseudomolecules calculated using the cubic spline method.
the authors thank the centre for applied biomedical research  of bologna for valuable contributions to the sequenom analyses and elisa banchi from fem for performing the snp genotyping on iscan platform.
the authors also thank fondazione cassa di risparmio in bologna for supporting crba.
funding
the work conducted in italy was funded by the ministero delle politiche agricole alimentari e forestali -italy  through the project “drupomics: “sequenziamento del genoma del pesco ed utilizzo della sequenza in programmi di miglioramento della qualità del frutto del pesco e della resistenza alle malattie"  and the european union-funded project “fruitbreedomics: “integrated approach for increasing breeding efficiency in fruit tree crops" .
the work conducted by the us department of energy joint genome institute, was supported by the office of science of the us department of energy under contract no.
de-ac02-05ch <dig>
availability of data and materials
the dataset supporting the conclusions of this article are partly included within the article and its additional files  <dig>   <dig>   <dig>   <dig> and  <dig>
further datasets supporting the conclusions of this article are available at the following repositories.
the peach genome sequence can be accessed at following databases urls:
phytozome ;
genome database for rosaceae ;
applied genomic institute  : .
this whole genome shotgun project had been deposited at ddbj/embl/genbank under the accession akxu <dig>  the version described in this paper is accessible at the accession akxu02000000
illumina short reads are deposited into ncbi short read archive under accession numbers srx <dig>  srx <dig>  srx <dig>  srx <dig>  srx2392650
ssr primer sequences were deposited into ncbi probe database under accession numbers from pr <dig> to pr <dig>
authors’ contributions
iv, st and js conceived and designed the experiments; jj, ld, sm, gp ev, rp, va lg, lr, ss and jg performed the experiments; iv, jj, ld, sm, ev, ss, jg, st, mtd and js analyzed the data; iv, lr, db, mt, ss, jg, st and js contributed reagents/materials/analysis tools; iv, jj and st wrote the manuscript.
ld, sm, gp, ev, rp, db, mt, st, mtd and js critically revised the manuscript.
all authors read and approved the final manuscript.
competing interests
the authors declare that they have no competing interests.
publisher’s note
springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

 the wgs approach for sequencing complex eukaryotic genomes has contributed to the assembling many genomes of non-model and crop species. poplar and grape were the first plant genomes sequenced with this approach. the advantages of wgs sequencing, as compared to the bac by bac approach are the speed of sequencing and the reduced cost. however, a weakness of the wgs sequencing approach is it tends to produce a more fragmented assembly with reduced contiguity, also coupled with the risk of large-scale misassemblies. this is especially true for complex eukaryotic genomes and the assembly process can be confounded with recent duplication events, large regions of expanded repeats, and residual heterozygosity. the result is the production of a fragmented sequence with poor contiguity metrics such as the n and the l. the potential issues are greater if a highly heterozygous individual is chosen as reference. however, in the bac by bac approach, the use of local sequence information mitigates the risk of large-scale misassembly. the advent of next generation sequencing technologies has exacerbated wgs assembly drawbacks, typically producing a more fragmented assembly. ngs produces shorter sequence reads compared to the sanger method, making genome assembly more difficult and requiring the development of a range of dedicated bioinformatics tools and novel alignment algorithms. a de novo short-read ngs assembly needs high genome coverage, mainly to overcome the reduced overlap length and improve the contiguity of the resulting assembly. henson et al. calculated that by increasing the reads length from 50 bp to 1000 bp, the contig l value of the human genome can theoretically increase from 3 kb to about  kb. moreover, the lack of a chromosome-scale assembly, combined with putative misassemblies precludes extensive use in evolutionary and comparative genomics studies, as well as genome wide association studies. the availability of a chromosome-scale assembly is, therefore, crucial to maximally leverage the advantages of the wgs sequences. the coupling of wgs genome assembly with highly saturated and high resolution molecular genetic maps has been proposed to cope with the lack of chromosome-scale wgs genomes. genetic maps enable the reconstruction of a chromosome-scale sequence by positioning wgs scaffolds in their correct order and orientation to arrange them in long stretches of dna, representing the individual chromosomes, called “pseudomolecules” or “pseudochromosomes”. inconsistencies between the position of markers on the map and in the assembly can highlight putative misassembled sequences that can be further resolved by breaking the chimeric scaffolds and rearranging the broken pieces in their correct order and orientation. fragmented genome assemblies, such as those obtained with short reads ngs data, need dense genetic maps for a large fraction of the assembled sequence to be anchored on chromosomes. the availability of wgs genome assemblies in many species combined with ngs platforms has fueled variant discovery through alignment of resequenced reads of different accessions to the reference genome. millions of single nucleotide polymorphisms and small insertions/deletions dispersed throughout the genome have been discovered in different species. this discovery, coupled with high-throughput genotyping technologies, such as snp arrays and genotyping by sequencing, has accelerated the construction of high-resolution genetic maps, enabling map-sequence integration of wgs scaffolds in highly fragmented de novo ngs assemblies. medium and high-throughput genotyping tools have been developed in prunus for peach and cherry and in other rosaceae species such as apple and strawberry. to overcome the limitations of short read sequencing technologies third generation methodologies, based on single molecule sequencing, have been recently released such as the one of pacific bioscience and moleculo. these methodologies, are capable of obtaining much longer reads, up to 50 kb, with the trade-off of an increase in error rates of 13-15% in comparison to – % in illumina short reads. to solve this problem, several strategies have been proposed such as the integration of long reads with more accurate ngs reads. this hybrid sequencing strategy has been recently used in pineapple apple and arabidopsis thaliana ler. the peach genome sequence was obtained by the international peach genome initiative and is an -fold wgs high quality draft sequence with long contiguity, high base accuracy, and a large portion of sequences mapped on chromosomes. sequences were obtained using the sanger methodology and a complete homozygous reference accession, the ‘lovell’ double haploid plov2-2n. the prunus reference map was used to anchor the first release of the peach genome obtaining eight pseudomolecules representing the eight prunus chromosomes. approximately half of the markers were placed by genotyping only six seedlings using the bin mapping strategy. thus, even if the bin mapped markers were useful to assign scaffolds to chromosomes and check scaffold integrity, in most cases they did not provide sufficient information for anchoring the scaffolds on chromosomes. moreover, the txe map is an interspecific map and was obtained with a limited number of individuals resulting in a reduced recombination frequency in some regions, providing only a rough estimation of the recombination frequency at a short physical distance. the anchoring markers are unevenly distributed along the linkage groups leaving portions of the genome uncovered. later analyses of the peach genome revealed that 4% of the sequence of the peach v was not included in the pseudomolecules; with ten scaffolds anchored with unknown orientation and a large number of mapped scaffolds had no markers on their ends, making it difficult to detect putative chimerism. in this work, we describe the efforts aimed at improving the peach chromosome-scale build using a set of linkage maps and resequencing the reference accession. two mapping strategies were used: i) a targeted approach where markers were targeted in specific regions of the peach genome and mapped in two already available linkage maps; ii) a whole genome approach that made use of the ipsc 9 k snp array v to genotype a large set of progeny. moreover, illumina ngs resequencing of the reference accession at high coverage was performed to correct sequencing errors and to close a number of gaps in the peach v assembly increasing the contiguity of the final peach genome. methods plant material, dna extraction and quality test four biparental mapping populations were used to refine the peach genome:i) seedlings of the ‘texas’ x ‘earligold’ f population, an interspecific cross between almond and peach, maintained at the experimental station of crea-fru in rome, italy; ii) seedlings of the peach selection if x ferganensis bc population maintained at the experimental station of crea-fru; iii) seedlings of the ‘contender’ x ‘ambra’ f population maintained in a farm belonging to the municipality of castel san pietro leased to astra; iv) seedlings of the maria dolce x sd f cross maintained at the experimental station of crea-fru. young leaves were collected from each seedling and lyophilized. dna was extracted with the dneasy plant mini kit, quantified with the nanodrop spectrophotometer and with the picogreen® assay for samples genotyped on the ipsc 9 k snp array. for sequenom analysis, dna was extracted from the seedlings of the cxa progeny after mercado et al.. target ssrs identification and mapping in txe progeny target ssrs were selected from among the identified within the peach v genome sequence to increase marker density in order to leverage in large unanchored scaffolds, and correct order/orientation of anchored scaffolds. sequences of ~600 bp flanking both sides of the repeated motif were first blasted against peach v, using the blast facility available on phytozome, and only non-repetitive regions were selected to design specific primer pairs by the primer software. only single locus ssr markers were used for further analysis. the selected ssr primers were first tested on the bin set and parents of the txe progeny. only the most physically distant polymorphic markers in each target region were genotyped on the whole txe progeny. to check for scaffold integrity, some ssrs were developed in the distal region of uncovered scaffold ends. in the presence of suspected chimeric regions, further markers spaced approximately every 100 kb were identified, developed, and mapped to restrict the size of misassembly containing regions. primer sequences and features are listed in additional file 1: table s all pcr reactions were carried out in a 10 μl volume with a final concentration of 1x pcr buffer,  mm mgcl 200 μm of each dntp and  μm of each primer, 10 ng genomic dna, and u of platinum®taq dna polymerase. the amplification profile was: one cycle at 94 °c for 5 min, followed by touchdown cycles, with a decrement of the annealing step temperature of  °c/cycle, starting with a denaturation at 94 °c for 30 s, an annealing step five degrees above the primer-specific annealing temperature for 30 s, and an elongation at 72 °c for 30 s, followed by 25 cycles at 94 °c for 30 s, ta °c for 30 s, 72 °c for 30 s, and a final elongation of 30 min at 72 °c. pcr products were then separated on a 3% high-resolution agarose gel in tbe 1x with a voltage of ∼ 5 v/cm and stained with the gelred™. markers that could not be easily scored on agarose gel were separated by capillary electrophoresis on a ceq genetic analyzer. ssrs genotyped in the whole txe progeny were integrated with the previous txe dataset and mapped using the software mapmaker, grouping them at a lod score higher than they were located by using the try and ripple commands. after mapping, the error detection command of mapmaker was used and putative double recombinants were manually checked. snp identification and mapping in the cxa progeny the f parent of the cxa progeny was resequenced with an illumina platform. the clc genomics workbench was used for read alignment and snp calling. only reads aligning to a single location with at least 92% identity over at least 90% of their length were considered. for snp calling, variants were retained when: i) the coverage ranged between and x of the average coverage; ii) minor allele frequency > 30%; iii) the polymorphic nucleotide phred-scaled quality score ≥  and the average quality ≥  for the 11 bp surrounding the putative snp. snps were manually selected based on their distribution on the peach genome. snps surrounded by repetitive sequences and/or located within a stretch of bases identical to that of the snp itself were avoided. the surrounding sequences were obtained from the peach gbrowse available on the iga website and blasted against the v peach genome at gdr to verify their uniqueness. only unique snp-surrounding sequences were used to design the assays, and combine them in multiplex reactions by the software mass array design. all locus-specific pcr primers and probes were blasted against the peach genome to further verify their specificity. only the snps that passed all these quality checks were retained for further analyses. a total of ten iplex were designed to attain the optimal genome coverage, including two iplex with snps selected in specific uncovered regions, or where snps previously tested were not useful. information about the primers used for the genotyping is listed in additional file 1: table s genotyping in the cxa progeny was performed using iplex gold technology and mass array high-throughput dna analysis mass spectrometry at the centre for applied biomedical research of bologna. the sequenom data for each snp were first verified by checking the heterozygosity of the cxa f parent and its consistency with the two grandparents ‘contender’ and ‘ambra’. all the snp data were integrated with the dataset of ssr markers genotyped following eduardo et al., and then analyzed by joinmap software with the default parameters and the kosambi mapping function. linkage groups were established at lod value higher than all these snps were also tested on the bin set of the txe mapping progeny. ipsc 9 k snp array genotyping and mapping the ipsc 9 k snp array was used to genotype individuals of the pxf and of the mdxsd progenies, using the illumina infinium ii design probes, and the dual color channel assay. snp genotypes were scored with the genotyping module of the genome studio data analysis software. snps with a genetrain score ≥ and less than 10% missing data were retained. allele segregation was also checked and snps showing unexpected segregations or unexpected genotype classes, were inspected using genome studio. if possible they were re-clustered using the “define cluster” function. those still showing missing or unexpected classes were discarded. linkage analysis and map construction were performed with joinmap using the cp and bc population types for pxf and mdxsd, respectively. linkage groups were established at lod value higher than as described above. the multipoint maximum likelihood mapping algorithm was used with the default parameters. kosambi units were used; for the cp population type, haldane map distances were manually converted in kosambi units using the formula provided in joinmap manual. map-sequence integration the original raw version of the peach v assembly, post filtering organelle, repetitive, and small scaffolds sequences, was used for the new map-sequence integration. markers were placed on the wgs scaffolds using two methods as reported in verde et al.. ssr and snp markers having primer sequences were placed using three successive rounds of electronic pcr with n =  n =  and n =  markers with a known sequence, including rflp and snp markers or ssrs whose primers had not been found with the three rounds of epcr, were placed with blastn. the additional breaks were made in regions of low bac/fosmid coverage and the broken sequences reordered according to the new maps. the mapped wgs scaffolds were joined as described in verde et al to form pseudomolecules. each map join is denoted by  n bps. in this work, to avoid confusion between v and v releases, scaffolds composing the peach v assembly were named and are hereafter referred to as “scaffold_##”. the wgs scaffolds included in the v pseudomolecules were named as “scf_##”. the wgs scaffolds composing the v pseudomolecules were named as “super_##”. lovell dh resequencing resequencing of the ‘lovell’ double haploid was performed using the miseq illumina platform. paired-end reads were assembled with the abyss software after quality checking and filtering. the resulting contigs were used to patch gaps in the peach v assembly after the new breaks and joins described above were applied. contigs were aligned to the repeat masked modified v assembly using blat. contigs whose ends aligned to either side of a gap, with at least 1/ of the contig length anchoring to the edges of the gap at ≥95% identity, were used to patch the gap. sequence and quality scores were then integrated into the v modified assembly. finally, homozygous snps and indels were corrected using ~43x illumina reads. reads were aligned using bwa and variants called using the standard gatk pipeline including base quality score recalibration, indel realignment, and duplicate removal. physical vs genetic distance comparison and identification of centromeric regions mareymaps were obtained, for each mapping progeny, by plotting the genetic positions of molecular markers against their physical position on the peach v. cumulative recombination curves for each chromosome were estimated using the cubic spline interpolation method with default parameters and the cross-validation type present in the mareymap package. the recombination value per position was obtained calculating the slope per markers and their curves were plotted for each chromosome. in order to identify the putative centromeric region of each chromosome, regions displaying the lowest recombination rate, as highlighted by the mareymaps, were manually checked on the peach v using the jbrowse available on phytozome for the absence of transcripts and the abundance of repeated elements associated with centromeric and pericentromeric regions. sequences retrieved from neumann et al., representing a catalog of plant repeated elements associated with centromeric regions, were aligned with blastn to the peach genome assembly. sequences aligning within the putative peach centromeric regions, with at least 63% of identity and an e-value greater than x 10− were retained. recombination frequency was compared among all progenies by multiple comparison statistics implemented in past. for each linkage map used in this study, recombination rate was estimated at individual whole-chromosome scale as the ratio between genetic and physical distances. one-way analysis of variance was applied with tukey’s pairwise post-hoc test. the levene’s test for homoscedasticity and the shapiro-wilk test for normal distribution were also applied to check the assumptions for the applicability of the anova. in the case of violation, the non-parametric kruskal-wallis test was applied instead, with the mann-whitney pairwise post-hoc comparison and the bonferroni correction.  four linkage maps were used to improve the peach genome. txe and cxa were already available and were enriched using a targeted approach. another map was the result of a de-novo mapping of the pxf progeny obtained using the ipsc 9 k snp array and tripling the mapping progeny size. finally, the mdxsd map, also obtained with the ipsc 9 k snp array, was used to specifically address some inconsistencies at the top of linkage group. targeted ssr identification and mapping in txe a total of ssrs were identified in the peach v assembly and primers were designed and then tested in the txe bin set. twenty markers were developed on the major unmapped scaffolds, were individuated within the randomly oriented scaffolds and were identified within the uncovered scaffold ends. thirty-eight out of the total were monomorphic in txe and were bin mapped. out of microsatellites falling within genic regions, were polymorphic. three polymorphic markers were mapped in txe, and found in successive analysis to have their primer pair on the same flanking side of the microsatellite region. these three markers were retained and reclassified as indel markers. these incidental length polymorphisms in the txe interspecific progeny reflect the different genomic structure of the closely related almond and peach species. the same length variation was observed in six out of the seven knox genes whose fragment size indicated differences between the two parents ranging from 2 bp to 20 bp in length. a much higher level of polymorphism with rflps in the txe progeny had been already observed in comparison to that of the intraspecific pxf progeny. thirty-two well-spaced ssrs, out of the polymorphic ones, were mapped in the whole txe progeny to resolve orientation, misassembly, or ordering discrepancies. in the preliminary steps of the peach genome assembly ssrs targeting specific regions had been mapped in the whole progeny. in addition, seven markers targeting the peach knox genes had also been included. the final map is composed of markers and covers  cm. three hundred and twenty-nine markers, having sequence information associated, were used for the peach v map-sequence integration. they cover  cm and 220 mb. only four gaps larger than 10 cm are present. in addition, roscos bin mapped markers and four snps from the prunus-malus consensus sequence were included in this study and integrated with the wgs scaffolds. furthermore, snps from the cxa f parent and ssrs isolated in this study were also bin mapped. in total txe markers were integrated within the assembly, bin mapped, and mapped in the whole progeny.table anchoring statistics of the peach v assembly number of markers integrateda 225 total 554 374 483 570 288 407 392 355 57 51 52 total 10 9 10 7 5 9 4 4 58 pxf f1,, pxf f1,,, total no of bases anchored,208,870,013,236,696,980,811 for each map and for each chromosome the number of markers, number of anchored scaffolds, genetic and physical distance covered with the integrated markers and the total number of anchored bases are reported ain brackets the bin mapped markers in txe targeted snp identification and mapping in cxa from the resequencing of the cxa f parent, snps have been developed and included in a total of ten iplex assays, with an average number of snp each iplex. among the tested snps, were useful for mapping while were not: of these, were monomorphic, showed only two over three expected genotypes and ten presented more than 25% of missing data. the latter group also included five markers with no amplification in the whole progeny, possibly due to assay failure. the fraction of scorable polymorphic snps ranged from % to % in different iplex assays, with an average success rate of %. the relatively negative result on iplex and is likely due to forcing the iplex design to develop markers in target regions. the observed snp calling efficiency was evaluated by comparing our results with those obtained by verde et al., which used more stringent parameters for snp calling. this a posteriori analysis showed that % of the non-polymorphic snps were false positives but at the same time five true snps over the mapped ones would have been lost using the more stringent conditions. finally, with the more stringent parameters, the total efficiency of snp design would have increased from % to %. the cxa map was first obtained with ssrs on 169 f progeny. to improve the chromosome-scale assembly the number of progeny was increased to the additional individuals were genotyped with ssr markers, adding targeted polymorphic snps to the map. fifteen of these snps were already linked on lg to a candidate gene controlling maturity date in peach. another set of twelve snps on lg surrounding the nectarine g locus had been described. a total of snps were identified on unmapped scaffolds, ten on the randomly oriented scaffolds and in putative chimeric regions. fifty-three of these snps were also bin mapped in txe, as already described in the previous paragraph. the final cxa map includes markers corresponding to unique genetic positions, covering  cm with only four gaps larger than 10 cm. all the pseudomolecules are almost completely covered, with the exception of pp lacking the bottom portion and pp missing the upper portion. ipsc 9 k snp array mapping in pxf the trees of the pxf progeny were genotyped with the ipsc 9 k snp array. out of the placed on the array, a total of polymorphic snps were identified; snps were informative for the f parent, were informative for both parents and were informative for the recurrent parent. of the three types of segregation, only the first two were used for the map-sequence integration because the unequal recombination frequencies between the two parents of the cross inhibited efficient joining of their genetic information. in fact, the recurrent parent displays a marked reduction in recombination frequency in comparison to that of the f parent. as a consequence, the map-sequence integration was unreliable due to the heavily skewed order of markers in regions with strong differences in recombination frequencies between the two parents. moreover, the distribution of the informative snps for the recurrent parent was uneven across the genome resulting in a fragmented linkage map with groups. in fact, five groups were split in two groups each, one was completely missing resulting in a reduced genome coverage. the inspection of the ipsc 9 k snp array markers segregating in the recurrent parent highlighted that they did not give additional information for integration except for two contiguous minor scaffolds found in the upper part of the pp seven markers located in these two scaffolds informative for the recurrent parent, were selected and integrated into the map. the presence of bridge markers in that region segregating in a 1:2: ratio and the low number of 1: markers informative for the f parent enabled a reliable integration. when a single snp was mapped in a linkage group that differed from the expected placement from the array information, and no further evidence of misassembly was observed in the same genetic region, this marker was deemed as putatively duplicated and excluded.table genetic/physical ratio for each map and each chromosome lg-ppa a lg linkage group, pp pseudomolecule after filtering, we mapped snps in total corresponding to unique genetic loci. the map covers  cm with only one gap larger than 10 cm. integration of unmapped scaffolds in peach v pseudomolecules one hundred and ninety-four scaffolds, had not been included in the eight peach v pseudomolecules. to anchor the larger unmapped scaffolds in peach v, we first used the txe map. twenty ssrs were targeted in the unmapped portion of the peach genome and polymorphic ones enabled anchoring of the ten major scaffolds and fixing the orientation for two of them. the same was done using the cxa map. this map, in addition to being from an intraspecific cross, was obtained with a large mapping progeny providing a fine estimation of the recombination frequencies even at a small scale. with this approach, we were able to confirm the anchoring of the ten major scaffolds and anchoring of an extra minor scaffold. the high resolution of the cxa map also enabled the ordering of two contiguous small scaffolds on pseudomolecule that in txe were unordered and unoriented, and fixing the orientation of five scaffolds. only markers of the ipsc 9 k snp array were located in the unmapped portion of the genome and six of them were polymorphic in pxf. the position of five of the ten major scaffolds previously mentioned was confirmed using these markers. together, these analyses positioned unmapped scaffolds on the v pseudomolecules. they cover  mb of sequence; five of them were also orientable. ordering and orientation of peach v randomly oriented sequences ten mapped scaffolds of the peach v genome were placed with random orientation due to the lack of recombination among markers or because they were anchored with only one marker. to resolve the orientation of these scaffolds, previously bin mapped markers, located by the ends of the non-oriented scaffolds were mapped in the whole txe progeny. for scaffolds where no bin mapped markers were available, ssr primer pairs were designed towards the end, tested on the txe bin set and the most physically distant polymorphic markers were mapped in the whole progeny. in this way, five integrated scaffolds representing  mb of sequence were oriented along the pseudomolecules. moreover, the attempt to orient a scaffold on pp revealed that it was incorrectly placed along the pseudomolecule due to a mismapped marker in txe. this scaffold was correctly positioned at the top of the pp the higher genetic resolution of cxa map confirmed the position of super_ at the top of pp and the correct orientation of a major scaffold at the bottom of pp. it also rectified the orientation of two wrongly oriented scaffolds due to mapping artifacts in txe: one on top of pp and the other in the middle portion of pp. the high density and resolution of the pxf map further confirmed the orientation of the above-mentioned scaffolds and enabled the correct orientation of three other scaffolds in peach v. in a region of pp, indicating high recombination frequency suppression, five scaffolds were ordered with low probability in txe. the higher resolution of cxa and pxf helped to resolve uncertainties. however, the orientation of super_ and super_ was indeterminate in v since the corresponding markers cosegregated in all maps. moreover, it was not possible to have certainty of the order of super_ and super_ located in the same region. in fact, markers on those scaffolds were ordered in txe with low probability. in cxa and in pxf only one of these two scaffolds was anchored in each map giving no additional information on their order. for these scaffolds, the peach v order, based on txe, was retained. however, recently published maps obtained using the ipsc 9 k snp array have enabled verification of their order and orientation. in particular in the mxr_ map two snps mapping on super_ at and  cm, respectively and other four mapping on super_ at,, and  cm, respectively revealed that the order established in peach v is incorrect and will be inverted in a future release. this is also confirmed by the dvss map in which two snps were mapped at and 32 cm, respectively. the map obtained by sánchez et al. confirmed the orientation of super_ established only by pxf map and v orientation of super_ was shown to be correct, though it was anchored by only one marker in txe and cxa. together, all the ten scaffolds randomly oriented in peach v and two wrongly placed were correctly oriented in peach v pseudomolecules. currently, only six minor scaffolds summing up  mb are randomly oriented in this release. moreover, only a known mis-order and a random order in the central part of chromosome are still present in peach v. these will be rectified in a future release of the peach genome. scaffold ends checking and correction of misassembled sequences within the scaffolds composing the peach v pseudomolecules, terminals were not covered with molecular markers for at least 500 kb of their length, having an uncovered portion larger than 1 mb with the largest one being of  mb. these regions are potential sites of misassembled sequence. to check scaffold consistency, ssr and snp markers were developed in the distal part of these uncovered regions. markers mapped in txe and cxa maps helped to reveal five out of these uncovered scaffold ends as sites of misassembly: three in pseudomolecule one in pseudomolecule and one in pseudomolecule on pseudomolecule there were two scaffolds bearing two different chimeric regions, resulting in six pieces in total that needed to be relocated on different chromosomes. for this purpose, the two most distant polymorphic markers in each chimeric region were mapped in the whole txe progeny in order to locate and orient the new broken scaffolds within the peach pseudomolecules. a particular case occurred in the v integrated scaffolds scf_ and scf_ located on pseudomolecule and pseudomolecule respectively. they originated from a chimeric scaffold that had been broken in a wrong position in the peach v assembly due to insufficient marker coverage within the putative chimeric region. in v, with the help of the new mapping data, we refined the breakpoint, so that 385 kb from v scf_ in pseudomolecule were re-joined to the formerly broken scf_ to form the new super_ on v pp. the whole genome mapping approach of the pxf map confirmed all of the chimeric scaffolds and enabled identification of two other cases of misassembly on the top of v pseudomolecule in this region two scaffolds were chimeric and needed to be broken in one point each. three of the broken portions,  mb of sequence, had to be rearranged within the same chromosomal region. to support the rearrangements at the top of v pseudomolecule we used additional information from the mdxsd map. in this map, lg is composed of snp markers for a total of single genetic positions covering a genetic distance of  cm with an average of  cm between markers and a major gap of  cm. the total physical distance covered amounts to  mb. twenty-one mdxsd markers mapped in the  mb region confirm the order of the three broken scaffolds highlighted by pxf and the orientation of two of them. without high resolution and high-density linkage maps highlighting discrepancies in this  mb region, this problem could not have been solved since the txe and cxa maps have low marker density in that region. scaffold ends were also checked using additional information from recently published linkage maps. in the current assembly, only scaffold ends larger than 500 kb are still not covered with markers, all of them lying in highly repeated centromeric regions, with the largest being a 594 kb stretch of sequence. in total,  mb of sequence from peach v, were relocated in their correct chromosomal positions with the correct orientation. base accuracy and contiguity improvement through reference accession resequencing the ‘lovell’ double haploid was resequenced from libraries, producing, paired-end reads, corresponding to 43-fold paired end fragment sequence coverage and 21-fold mate pair coverage. this set of reads was assembled using abyss, producing scaffolds greater than 500 bp for a total of  mb of sequence. scaffolds produced in the abyss assembly were broken into contigs, and a total of contigs representing  mb of sequence were used to close peach v gaps, with a gain of  kb. the overall contiguity was improved, with the total number of contigs in peach v decreased by % dropping from to and the contig l increased by %. finally, homozygous snps and indels were corrected using ~43x paired end fragment illumina reads.table summary of gap patching and indel and snp correction totals: 206 212,737,936 859 chromosome-scale assembly and comparison to other published genomes in the new map-sequence integration, a total of markers from three linkage maps were used in the integration of the original raw peach assembly obtained prior to the build of peach v pseudomolecules: markers were integrated in txe, in pxf and in cxa. the improved txe map enabled the anchoring of scaffolds and orienting of scaffolds. the cxa map anchored scaffolds and oriented. the pxf map anchored scaffolds and oriented. finally, snps mapped in mdxsd on lg were instrumental in confirming some faults at the top of pp. important amendments were made in the v assembly including the portion of mapped and oriented sequences and the identification and correction of discernible misassemblies. a visual comparison between the two versions of the peach genome is shown with the mareymap plot. high-quality assemblies are those in which the plots are characterized by a monotonically increasing function. both peach v and v show a general increasing tendency, however, in few v regions the function decreases highlighting this release faults. furthermore, differences in the physical length between the two assemblies, observed in all but pp pseudomolecule, reflect the inclusion of previously unmapped scaffolds and the correction of inter-pseudomolecule misassemblies.fig. plots of genetic-by-physical distances. comparison of v and v physical distances and pxf genetic distances. dots represent the mapped markers. the vertical bars indicate the putative position of the centromere. the solid line represents the recombination rate plotted along the pseudomolecules the total number of major wgs scaffolds after filtering and scaffold breaking and prior to pseudomolecule construction was spanning,381 bp with a scaffold n50/l of 10/  mb. the final chromosome-scale assembly is arranged in stretches of non-contiguous sequences spanning,381 bp with a contig length of,928 bp and a scaffold contig coverage of %. fifty-eight scaffolds spanning  mb of sequences were integrated into the peach v pseudomolecules corresponding to % of the total sequence. a comparison between the statistics of the two chromosome-scale assemblies, peach v and peach v is also shown in table  fifty-two scaffolds, summing up  mb, are correctly oriented in the new assembly. the unmapped portion of the genome comprises scaffolds with a length of  mb containing only predicted genes. additional file 1: table s resumes the integration of peach v assembly, reporting the scaffolds mapped in the pseudomolecules, their position in peach v and peach v, the orientation information in all the maps and the size of uncovered scaffold ends.table summary statistics of the peach v chromosome-scale assembly statistics and its comparison with the v table  reports a comparison among some of the major plant genome assemblies released to date. the table was adapted and updated from verde et al. with newly published genomes and the updated genome releases available on phytozome. among the wgs assemblies, peach v displays one of the largest portions of sequences mapped on chromosomes. the scaffold n50/l, prior to pseudomolecules build, shows the best values among those available. chromosome-scale n50/l is comparable with other chromosome-scale assemblies. if we exclude the finished genomes and those almost finished, the peach genome shows one of the best contig n50/l, including the recent wgs pineapple genome obtained combining the third generation single molecule long reads with the ngs short reads. the long read sequencing in pineapple was instrumental in increasing the scaffold l metric from to 640 kb and the contig l from to  kb. pacbio long reads were also useful to improve the assembly of the a. thaliana ler genome; the integration of 17x pacbio long-reads with the illumina short reads improved the scaffold l that increased from  mb to  mb. the long read single molecule sequencing technologies are playing an increasingly important role in sequencing projects. in fact, the bioinformatics efforts of the assembling procedure are assisted and empowered by the availability of sequence stretches entirely spanning the repetitive regions, which are a major contributor to gaps present in assemblies.table comparison of the peach genome to other published plant genomes arabidopsis thaliana sorghum bicolor physcomitrella patens an50/l statistics of the wgs assembly prior to pseudomolecule build bn50/l statistics of the chromosome-scale assembly c arabidopsis assembly, obtained using bac by bac approach, represents the golden standard for plant genome. statistics were calculated from tair release. drice assembly, obtained using bac by bac approach, represents the golden standard for plant genome. statistics were calculated from irgsp releases build edata retrieved from schmutz et al.; they recalculated the original statistics to better match chromosome-scale assemblies fdata from recent releases retrieved from phytozome gdata were recalculated based on the original statistics reported in the paper we compared the statistics of the current peach release with the standards established by chain et al.. according to these standards, the peach v assembly can be classified as “improved high-quality draft” since actions have been taken in assigning almost the whole sequence to chromosomes, in solving discernible misassemblies, filling gaps to reduce the number of contigs and correcting base errors. moreover, the annotation of the release has greatly improved gene completeness using a large amount of rna-seq data, as well as the annotated repeats which include low copy repeats and a complete set of helitron transposons. the number of gene models in v a decreased to resulting also in less fragmented gene models. the average number of transcripts per gene model increased to from. the annotation improvements, not described in this work, are briefly reported on phytozome, gdr and iga websites. for all these features the improved peach release can be further classified according to chain et al. as an “annotation-directed improvement”, making it a useful tool for genome comparison and evolutionary studies, including gene studies such as alternative splicing analysis and metabolic pathway reconstruction. physical vs genetic distance comparison and identification of centromeric regions the availability of the txe, cxa and pxf linkage maps covering most of the peach genome enabled a chromosome-scale comparison of the recombination frequencies along the genome. in fig.  additional file 3: figure s additional file 4: figure s and additional file 5: figure s mareymaps are plotted together with a function describing the genetic/physical ratio. average physical/genetic distance ratios for each individual chromosome and cross were calculated to be  cm/mb in the interspecific cross txe and  cm/mb and  cm/mb in the intraspecific crosses cxa and pxf, respectively. the mareymap plots, constructed for the three different mapping progenies, indicates the expected monotonic increase along each chromosome except for a flat region where a marked suppression of recombination can be observed in each pseudomolecule. the survey of these regions on peach v jbrowse revealed the almost complete absence of predicted genes and the abundance of repetitive elements. this concurrent evidence suggest that these are likely the centromeric regions which are known to be mainly composed of interspersed tandem repeats and retrotransposons. despite the centromeres have highly conserved function during cell division, their dna sequences are not conserved. in particular, in plants, the centromeric satellite dna repeats are species-specific. recent studies suggest that these sequences underwent a rapid evolution revealing no sequence similarity among species diverged more than mya. other important components of the centromeric regions are represented by the retrotransposons directly involved in the centromere evolution and function. a blast analysis against the peach genome was performed using a set of centromeric retrotransposon sequences belonging to different plant species. in particular, eight plant centromere retrotransposons indicated similarity with the peach putative centromeric regions: three from medicago truncatula, two from pinus taeda, one from picea glauca, one from beta vulgaris, one from vitis vinifera. these results strongly support that these regions are the peach centromeres. the accuracy of the peachv assembly and the peach v a annotation enabled the positioning of the centromeric regions, which are generally very difficult to assemble due to their highly repetitive sequence composition. linkage maps are of little use to assemble fragmented centromeric regions due to the suppression of recombination. as an example, the putative peach centromeric region in pp indicated no recombination in the nearly meiosis analyzed in the three different mapping progenies, leaving the related scaffolds unoriented and unordered. the identification of putative centromeric regions for all of the eight peach v chromosomes attests the completeness of the current peach genome assembly. to verify whether there were significant differences in the recombination frequencies among the three maps, an analysis of variance was performed. since the pxf map was obtained by map data of the two parents of the cross, the individual maps of the two parents were also considered in the analysis. both the anova and the non-parametric kruskal-wallis statistics were applied showing no significant difference among the five maps. however, due to the low coverage of some linkage groups chromosomes with coverage lower than 50% of the total length were excluded thus revealing significant differences with the anova test. moreover, to enlarge the comparison panel, two recently published maps, obtained with the ipsc 9 k snp array, were included: the peach intraspecific ‘nj weeping’ x ‘bounty’ f map and the interspecific dvss bc map obtained by backcrossing a hybrid between peach and p. davidiana as donor with peach as recurrent. both maps indicate > 80% coverage for each individual chromosome, globally 93% and 95% of peach v length, respectively. the physical distances covered by these two latter maps were recalculated based on the peach v assembly. the anova comparison was then applied giving significant differences among the seven maps. the lowest recombination frequency, displayed by the recurrent parent, was not significantly different from that of the two interspecific hybrids. the pxf f map, having the highest recombination frequency, was significantly different from the two interspecific maps as well as from pxf recurrent map but not significantly different from the other intraspecific maps. the extreme reduction of recombination rate, observed in the pxf recurrent parent is comparable to that of the interspecific crosses. ‘jh hale’ is an old cultivar, one of the founders of the so-called modern western germplasm, carrying the male sterility allele while ‘bonanza’ is a dwarf accession. the if recurrent parent is classified as an accession with intermediate tree vigor belonging to the western subgroup. no differences in the recombination frequency were observed among the other peach f individuals. the other parents of the crosses analyzed in this study have different origin: ‘contender’, ‘ambra’ and ‘bounty’ all derive from western breeding programs while ferganensis belongs to the eastern subgroup and nj weeping is believed to have japanese origin. our results are in agreement with those reported in arabidopsis thaliana where recombination rates, calculated in 17 f populations derived from accessions, do not correlate with genetic distances between intraspecific parental accessions. an exception in this study is the pxf recurrent parent, which displays a reduction in recombination rate similar to that of the interspecific crosses.table tuckey's pairwise comparison test among the different maps pxf f1 significant p-values are reported in bold  in this paper an improved and refined version of the peach genome assembly based on high quality linkage maps and resequencing data is presented. this new assembly release has been improved in terms of completeness and accuracy, including the increase of mapped and oriented sequences, repositioning of misassembled portions, enhancement of contiguity and correction of base errors. high density and high resolution maps are important tools to assist wgs efforts. in fact, even if unanchored wgs assemblies are able to catch the full gene complement, they defect in depicting the whole genome view thus being of little use for comparative genomics. the late high-throughput genotyping technologies such as snp arrays or genotyping-by-sequencing platforms are essential for developing saturated and high-resolution maps in short times with minimal cost, even in species with a narrow genetic base like peach and other self-pollinating species. third generation and ngs technologies can be efficiently used in newly or already available genome sequences to obtain a high quality assembly. additional files additional file 1: tables ssr markers developed in this study from peach v. for each primer, the locus name, primer sequences, position in peach v and v, repeated motif, polymorphism in txe and location in genic or intergenic regions are reported. table s snp markers developed in this study from the resequencing of the cxa f parent. for each primer, the locus name, position in peach v and v, primer sequences, bin position and location in genic or intergenic regions are reported. table s illumina short reads libraries. all libraries were sequenced as 2x250s using illumina sequencing. reads were filtered on quality, screening for reads that were predominantly simple sequence, and residual phix contamination. table s txe linkage map. for each locus, the genetic position in linkage groups and physical position in peach v are reported. only integrated markers are reported. table s sequenom snp development and their efficiency with the snp calling parameters used in this study. a posteriori efficiency was calculated using more stringent parameters as in verde et al. table s cxa linkage map. for each locus, the genetic position in linkage groups and physical position in peach v are reported. table s pxf linkage map. for each locus, the genetic position in linkage groups and physical position in peach v are reported. table s linkage map of pxf f parent. for each locus, the genetic position in linkage groups and physical position in peach v are reported. table s linkage map of pxf recurrent parent. for each locus, the genetic position in linkage groups and physical position in peach v are reported. table s composition of the peach v pseudomolecules. start and end of each scaffold, total scaffold length, scaffold denomination in v and in v, v position of each v scaffold, length of the scaffold ends uncovered with markers and mapping and orientation information achieved with the linkage maps used in this study, are reported. table s linkage map of lg of mdxsd. for each locus, the genetic position in linkage groups and physical position in peach v are reported. table s peach v assembly statistics prior to the pseudomolecules build. table s peach v assembly statistics post chromosome-scale build. additional file 2: figure s anchoring of the peach scaffolds to the three genetic maps. colored bars represent the linkage groups: pink for pxf, purple for txe and blue for cxa. wgs scaffolds were positioned in each pseudomolecule with the corresponding genetic markers and are depicted in three different colors; genetic markers are in the same colors of the corresponding wgs scaffolds. the zero denotes the six scaffolds placed with random orientation along the pseudomolecules. the asterisk indicates the two scaffolds with random order. the crosshatch indicates the two scaffolds with the wrong order in peach v that need to be inverted in a future release. additional file 3: figure s mareymap plot of pxf linkage maps. vertical bars indicate the putative position of the centromere. the solid line represents the recombination rate plotted along the pseudomolecules calculated using the cubic spline method. additional file 4: figure s mareymap plot of txe linkage map. vertical bars indicate the putative position of the centromere. the solid line represents the recombination rate plotted along the pseudomolecules calculated using the cubic spline method. additional file 5: figure s mareymap plot of cxa linkage map. vertical bars indicate the putative position of the centromere. the solid line represents the recombination rate plotted along the pseudomolecules calculated using the cubic spline method. the authors thank the centre for applied biomedical research of bologna for valuable contributions to the sequenom analyses and elisa banchi from fem for performing the snp genotyping on iscan platform. the authors also thank fondazione cassa di risparmio in bologna for supporting crba. funding the work conducted in italy was funded by the ministero delle politiche agricole alimentari e forestali -italy through the project “drupomics: “sequenziamento del genoma del pesco ed utilizzo della sequenza in programmi di miglioramento della qualità del frutto del pesco e della resistenza alle malattie" and the european union-funded project “fruitbreedomics: “integrated approach for increasing breeding efficiency in fruit tree crops". the work conducted by the us department of energy joint genome institute, was supported by the office of science of the us department of energy under contract no. de-ac02-05ch availability of data and materials the dataset supporting the conclusions of this article are partly included within the article and its additional files and further datasets supporting the conclusions of this article are available at the following repositories. the peach genome sequence can be accessed at following databases urls: phytozome; genome database for rosaceae; applied genomic institute:. this whole genome shotgun project had been deposited at ddbj/embl/genbank under the accession akxu the version described in this paper is accessible at the accession akxu02000000 illumina short reads are deposited into ncbi short read archive under accession numbers srx srx srx srx srx2392650 ssr primer sequences were deposited into ncbi probe database under accession numbers from pr to pr authors’ contributions iv, st and js conceived and designed the experiments; jj, ld, sm, gp ev, rp, va lg, lr, ss and jg performed the experiments; iv, jj, ld, sm, ev, ss, jg, st, mtd and js analyzed the data; iv, lr, db, mt, ss, jg, st and js contributed reagents/materials/analysis tools; iv, jj and st wrote the manuscript. ld, sm, gp, ev, rp, db, mt, st, mtd and js critically revised the manuscript. all authors read and approved the final manuscript. competing interests the authors declare that they have no competing interests. publisher’s note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
four linkage maps with molecular markers were used to improve the portion of mapped and oriented sequences and enabled a more detailed identification of discernible misassemblies. the availability of the peach genome sequence has fostered relevant research in peach and related prunus species enabling the identification of genes underlying important horticultural traits as well as the development of advanced tools for genetic and genomic analyses. the first release of the peach genome represented a high-quality wgs chromosome-scale assembly with high contiguity, large portions of mapped sequences and high base accuracy. the improved high quality peach genome assembly represents a valuable tool for the analysis of the genetic diversity, domestication, and as a vehicle for genetic improvement of peach and related prunus species. the aim of this work was to improve the quality of the first assembly by increasing the portion of mapped and oriented sequences, correcting misassemblies and improving the contiguity and base accuracy using high-throughput linkage mapping and deep resequencing approaches. moreover, the important phylogenetic position of peach and the absence of recent whole genome duplication events make peach a pivotal species for comparative genomics studies aiming at elucidating plant speciation and diversification processes.
 the availability of the peach genome sequence has fostered relevant research in peach and related prunus species enabling the identification of genes underlying important horticultural traits as well as the development of advanced tools for genetic and genomic analyses. the first release of the peach genome represented a high-quality wgs chromosome-scale assembly with high contiguity, large portions of mapped sequences and high base accuracy. the aim of this work was to improve the quality of the first assembly by increasing the portion of mapped and oriented sequences, correcting misassemblies and improving the contiguity and base accuracy using high-throughput linkage mapping and deep resequencing approaches.  four linkage maps with molecular markers were used to improve the portion of mapped and oriented sequences and enabled a more detailed identification of discernible misassemblies. the deep resequencing approach fixed homozygous snps and homozygous indels. moreover, the assembled ngs contigs enabled the closing of gaps with an improvement in the contig l of %.  the improved high quality peach genome assembly represents a valuable tool for the analysis of the genetic diversity, domestication, and as a vehicle for genetic improvement of peach and related prunus species. moreover, the important phylogenetic position of peach and the absence of recent whole genome duplication events make peach a pivotal species for comparative genomics studies aiming at elucidating plant speciation and diversification processes. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords prunus persicawgs assemblysnpsssrslinkage mappingngs resequencinggap patchingrecombination ratescentromeric regionshttp://dx.doi.org/ /501100005401ministero delle politiche agricole alimentari e forestalidm14999/7303/08verde ignazio http://dx.doi.org/ /100000015u.s. department of energyde-ac02-05ch11231schmutz jeremy http://dx.doi.org/ /501100004963seventh framework programmefp7-265582verde ignazio issue-copyright-statement© the author 2017 