dataesr/scientific-paragraphs-categorization huggingface dataset:
| Barnī is a true-to-type cultivar in al-'Ul a region We found that the 23 barnī from al-'Ul a are genetically identical, except barnī_00010. Indeed, those 22 accessions cluster together in both the PCA (Figure  4b ) and the tree (Figure  4c ; Figure  S3 ). Further, the King-robust kinship among them ranges from 0. 424 to 0. 491, nearing the theorical 0. 5 expectation and the 0. 481-0. 499 range observed among the known clones (Figure  4a ; Dataset S3). On a technical note, we hypothesized that lower kinship estimates among the 22 barnī, compared with the known clones, may be due to a lower coverage in the former (Dataset S3). We tested the relationship between the fraction of sites with information for two individuals (used to calculate pairwise kinship) and the King-robust kinship estimate, and indeed found that they are highly positively correlated (Notes S3; Figure  S4 ).
On a separate note, we found that the male accession used to calibrate the genetic identity analyses (ḏakar_00254) is closely related to barnī, with a King-robust kinship estimate ranging from 0. 194 to 0. 284 (Figure  4 ; Dataset S3). Since barnī is by far the most cultivated named type in al-'Ul a and that males mostly arise from accidental seedlings, this male is probably an offspring of barnī.
It is also at the crossroads of date palm diversity, between the distinct North African and West Asian genepools  (Flowers et al. , 2019; Hazzouri et al. , 2015). Figures  S5  and S6 ), reconstructing a phylogenetic tree (Figure  5b ) and applying a PCA (Figure  5c ; Dataset S4) using the SNP data. All three analyses (Figure  5 ) corroborated previous results on date palms, that is, that they can be split in two main clusters (North Africa and West Asia) with Egyptian accessions being a mix between the two (reviewed by  Gros-Balthazard & Flowers, 2021). Regarding the diversity in al-'Ul a, our results revealed that it may represent a unique mixing among West Asian date palms (Figure  5 ): The three al-'Ul a palms indeed cluster with West Asian date palms, but the male ḏakar shows influence from North African diversity.
, 2019; Gros-Balthazard & Flowers, 2021). The oasis of al-'Ul a has long been connected to the Levantine region and i1ts ports on the Mediterranean Sea. Whether the genetic make-up of its date palms also displays ancestry from P. theophrasti, reflecting this possible bond, remains to be determined. To evaluate whether the date palms from al-'Ul a showed evidence of introgression, both D-and f 4 -statistics were computed (Figure  6 ; Methods S2; Datasets S5-S6). This revealed that both barnī and 'asel a do not show any evidence of introgression (Figure  6 ). On the other hand, North African date palms, and the ancient Judean date palms, that were previously shown to be admixed, displayed significant positive D-and f 4 -statistics (Figure  6 ;  Flowers et al. , 2019; Gros-Balthazard et al. , 2021).
Interestingly, this is also the case for the male ḏakar_00254 from al-'Ul a (Figure  6 ). We further estimated the fraction of theophrasti ancestry in the date palms showing evidence of admixture according to D-and f 4statistics (Figure  6 ; Dataset S7), including the male ḏakar_00254. We found that the latter displays about 1. 1% of its genome from Phoenix theophrasti. This is in the lower range of what is observed in North African and Judean date palms (average 11. 23%, ranging from 0. 59 to 21. 58%; Dataset S7). Balthazard et al. , 2021). Indeed, a changeover from absence or low P. theophrasti ancestry to $10% of P. theophrasti ancestry coincided with a shift in imperial control of the region in favor of the Romans. The region of al-'Ul a, with the famous site of Hegra, was part of the Nabatean kingdom, and a stop on the trading routes connecting southern and eastern Arabia to Petra and the Mediterranean Sea.
theophrasti, respectively; and O is the outgroup, that is, P. reclinata. It was calculated only for samples showing evidence of an excess of shared alleles with P. theophrasti as evidenced by D-and f 4 -statistics, that is all samples except barnī and 'asel a. A negative D and f 4 in this context imply a greater degree of allele sharing between West Asian samples and theophrasti, whereas positive values would imply greater sharing between the test sample and theophrasti. More details on the methods may be found in Methods S2 while the data are in Datasets S5, S6, and S7. strictly vegetatively propagated, even by Bedouins in remote desert areas and in different palm grove farming systems. But geographic scale matters: barnī from al-'Ul a oasis is distinct from that of Oman, and we therefore confirmed the homonymy foreseen by  Popenoe and Bennett (1913).
Notes S3 On the relationship between coverage and the King-robust kinship estimator. Read processing and genome alignment. Reads were demultiplexed and those passing Illumina quality control filters were processed with Trimmomatic  (Bolger et al. , 2014)  v. 0. 39 to remove contaminating adapter sequences. For adapter removal, we used the adapter and Nextera transposase sequence database included with Trimmomatic (v. 0. 39) downloaded with the following setting ILLUMINACLIP:<adapter library>:2:30:10 and only reads pairs where both reads in a pair were 76 bp or longer following trimming were retained for subsequent steps. We aligned reads to the reference genome from  Hazzouri et al. , 2019. This genome is a PacBio/Illumina genome assembly from a male date palm from a fourth-generation backcross (BC4) with a female of the Barhee cultivar.
Its size is ~772 Mb and it contains 18 primary contigs (49. 9% of total length) along with 2,371 unplaced contigs. The nuclear and chloroplast genomes were combined to form a single modified reference sequence that was used for variant calling. Site filtering was nonetheless carried out separately. Processed reads were aligned to this unmasked genome using bwa mem  (Li, 2013)  v. 0. 7. 15-r1140. The bwa mem aligner was run with the -M option to mark supplementary reads (0x800 bitwise flag) as secondary (0x100). Sample alignments were processed with SamSort from Picard-tools (https://github. com/broadinstitute/picard) v. 2. 8. 2 to coordinate-sort the alignments. We used MarkDuplicates (Picard-tools) to flag duplicate read pairs. Processed alignments were summarized with samtools v. 1. 9  (Li et al.
Reconstruction of a chloroplast DNA tree We called Single Nucleotide Polymorphisms (SNPs) and genotypes for the chloroplast genome SNPs within 10 bp of an indel in the raw (unfiltered) indel call set were also removed. Finally, we excluded any site with missing data or with one or more heterozygote genotype calls as they may represent instances of heteroplasmy or genotyping artifacts. Remaining homozygous genotypes were converted to haploid to produce a sequence alignment with 52 SNPs. This alignment was used to construct a cpDNA tree with uncorrected distances using the Neighborjoining method with the phangorn (v. 2. 8. 1) package in R.
Methods S2 We tested for the presence and extent of admixture between date palms and Phoenix theophrasti using the R package admixr  (Petr et al. , 2019)  v. 0. 9. 1, which provides an implementation of ADMIXTOOLS  (Patterson et al. , 2012). We used a script available online to generate the input in EIGENSTRAT format from our VCF (https://github. com/joanam/scripts/blob/ master/convertVCFtoEigenstrat. sh). We ran the tool using the full Single Nucleotide Polymorphisms (SNPs) set. We focused on determining whether al-'Ulā date palms show evidence of gene flow with P. theophrasti as evidenced in North African cultivars, a few West Asian date palms, and most of the ancient Judean date palms  (Flowers et al. , 2019; Gros-Balthazard et al. , 2021). We use the same pattern of test, fitting the phylogenetic relationships among Phoenix, as in Gros- Balthazard et al.
Notes S3 On the relationship between coverage and the King-robust kinship estimator. We investigated why the King-robust kinship estimator is lower within the barnī accessions we believe to be clones compared to that found within each quartet/triplets of known clones. The latter being sequenced at a higher coverage, we hypothesized that it could affect the estimator, although we used genotype likelihoods rather than calls and a relatedness estimator presumably robust to Single Nucleotide Polymorphisms (SNPs) ascertainment biases linked to low coverage  (Waples et al. , 2019). Based on the estimated kinships (Fig. 4ab ; Dataset S3), we found three types of relationships among the barnī samples: 1) barnī_00010 is unrelated to other barnī, 2) barnī_Oman is unrelated to other barnī, and 3) all the other barnī are clones.
So far, no studies have focused on the origin and extent of the diversity of Northwestern Saudi Arabia date palms, including al-'Ul a oasis. A few studies focused on the genetic diversity of date palms varieties in Saudi Arabia(Aleid et al. , 2015; Al-Khalifah & Askari, 2003; Al-Qurainy et al. , 2011)  but how those varieties, some potentially cultivated in al-'Ul a, relate to cultivars from other regions remain to be elucidated. Here, we analyzed the genome of the barnī of al-'Ul a and of two other accessions from this oasis, one of the 'asel a variety ('asel a_000169A) and a male (ḏakar_00254), along with that of 112 Phoenix spp. (Dataset S1). Given the genetic identity of all barnī palms from al-'Ul a, we picked the accession with the highest coverage, namely barnī_000268, for downstream analyses.
Yellow dots correspond to North African date palms. Full blue dots correspond to West Asian date palms, the empty ones are those having a fraction of their ancestry from the African cluster in (a). The variance explained by each principal component (PC) is given in parentheses. Coordinates on PC1 and 2, and additional PCs may be found in Dataset S4; (d) fraction of heterozygote sites across 88 date palm genomes calculated from 105,236,083 sites including 1,007,281 SNPs. Same legend as panel (c) The evidence of gene flow from P. theophrasti found in date palms of the Levant has been hypothesized to be related to the growing control of the Roman Empire in the region 2000 years ago, favoring exchange of goods, including dates, with North Africa (Gros-.
ACKNOWLEDGEMENTS We are grateful to the farmers, who opened their gardens and farms to us, shared their knowledge and know-how, and offered us unfailing hospitality. We thank  Claire Newton  for providing a sample;  Marc Arnoux ,  Nizar Drou ,  Michael Dhar , and the  New York University Abu Dhabi Bioinformatics Core  for assistance with DNA sequencing and bioinformatic analyses;  Luis Rivera Serna  from the  Center for Desert Agriculture at KAUST  for assistance with bioinformatic analyses. This work was supported in part through the  NYU IT High Performance Computing resources , services, and staff expertise. We are particularly grateful to  Shenglong Wang  for his unwavering support while using NYU cluster. This work was funded by the  French Agency for AlUla Development (AFALULA)  with his Saudi partner the  Royal Commission for AlUla (RCU)  through a grant awarded to  Vincent Battesti  and  Muriel Gros-Balthazard  (project al-'Ul a DPA: Ethnographic, genetic, and morphometric analyses of the date palm agrobiodiversity in al-'Ul a oasis).
Date palm sampling. (a) Location of al-'Ul a and Khaybar oases, in Saudi Arabia, are presented in the top-left map, while the main map shows the sampling in al-'Ul a, (b) a farmer, workforce from Qena (Egypt), is climbing a barnī date palm for harvest, in al-Lut at, an old palm grove of al-'Ul a. The same technique was used by us or the farmers to sample leaflets for analysis. F I G U R E 1 October 25, 2021. Picture: Vincent Battesti Overall, a total of 146 Phoenix spp. genomes were analyzed, of This includes 88 date palms from 13 countries of North Africa and which 34 were new to this study and 112 were retrieved from West Asia, 8 Phoenix sylvestris, 18 Phoenix. theophrasti, and 1 Phoenix GenBank SRA (Table 1; Figure 1; Dataset S1). reclinata.
A detailed account of the experiments is provided in the Materials and Methods section, together with schematics showing a step-by-step procedure for each experiment. The NR data were collected and analyzed systematically after each adsorption step to characterize (i) the bare silicon block, (ii) the planar SLBs formed on the surface of the silicon block, (iii) the DNA-decorated planar SLBs (for the relevant experiments), and (iv) the particle/vesicle layer. Bare silicon blocks, SLBs, and DNA-decorated SLBs NR data and fits for the bare silicon blocks in D2O and H2O (Figure  S13a ) revealed SiOx layers with thicknesses, 𝑡 SiO x ≈ 1 nm, for all blocks, with a low, but variable, interfacial roughness, 𝜎 SiO x = 3. 5-5. 5 Å (Table  S1 ). The experimental NR data and the fits for the planar SLBs formed on the top of the silicon blocks are shown in Figure  S13b.
The results demonstrate that the SLBs formed on all blocks were of high quality, with an interfacial roughness 𝜎 SLB ≤ 5. 2 Å and covering 96 to > 99% of the solid surface exposed to the neutron beam (Table  S1  and Figure  S13c ). For all SLBs, the modeling was performed using the lipid plugin from the NR fitting software, Aurore. 76  The plugin applies several molecular constraints to ensure that the fitted parameters represent a physically realistic SLB. NR measurements were also performed on SLBs after in situ binding of AB-DNA or XY-DNA; in all cases, there were no detectable differences in the NR data before and after the DNA binding step (Figure  S14 ), indicating that the presence of the DNA molecules did not perturb the SLB structure and that their volume fraction was below the detection limit.
Acknowledgments The authors acknowledge the support of the  Knut and Alice Wallenberg Foundation  (# 2019-0577 ), the  Swedish Research Council  ((# 2018-04900 ), and  Röntgen Ångstrom Cluster  ( 2015-06139 ). The authors thank  Emanuel Schneck  ( TU Darmstadt ) for support with preliminary xray reflectivity experiments and valuable discussions. Experiments at ISIS Neutron & Muon Source were supported by a beamtime allocation  RB1710273  from the  Science and Technology Facilities Council. Data is available under: https://doi. org/10. 5286/ISIS. E. RB1710273.
org/10. 5281/zenodo. 5727015). To exclude the possibility that detected mutations in the analysed regions are natural polymorphism (SNPs), we determined allelic variation in analysed regions in potato cv. Rywal and cv. Désirée (see Figure  3a, b, c ; "polymorphism in NT plants" sheet in Data S2, Data S3 and Figure  S6 ). We obtained several different types of mutations detected in different transgenic lines but also in different alleles of the same plant. For all MIR loci, the most abundant types of mutations were short deletions (up to 6 nt), but we also identified 1-nt insertions (T or G) and longer deletions (Figure  3a, b, c ; Data S2). Mutations were frequently, but not exclusively introduced in the expected (a few nucleotides upstream of PAM motif) cutting site (Figure  3a, b, c ). Only in one transgenic line, cr-MIR160a_line3_A.
Therefore, to investigate the effects of the introduced mutations, we predicted secondary structures for all altered pre-miRNAs of each transgenic line and assessed their stability and potential for efficient processing of miRNAs from these precursors (Data S2, Data S3, https:// doi. org/10. 5281/zenodo. 5727015). Minimal folding free energy (MFE) as a measure of folding stability has been one of the main criteria for accurate miRNA annotation. Plant miRNA precursors however exhibit AU bias, that is absent in other non-coding RNAs  [61]. Therefore,  To link the type and frequency of mutations with miRNA abundance, we followed relative miRNA abundance in the selected transgenic lines. Relative miRNA abundance in transgenic lines normalised to the averaged expression in non-transgenics (NT) is given.
NA: not applicable. miRNA targets abundance: relative expression of CathB and ARF10 in transformed plants determined by RT-qPCR. Impaired pre-miRNA processing: pre-miRNAs processing efficiency has been predicted as impaired if the MFEI values of pre-miRNAs were below 0. 85 (see methods) or internal loop (> 5 nt) has been detected in miRNA/miRNA * duplex region. Indicated are the number of pre-miRNAs with impaired processing compared to the number of all PCR products analysed in each line. b) Examples of perturbed secondary structures of pre-miRNA sequences. One example is given per each selected potato MIR loci, secondary structure of non-mutated pre-miRNA is shown for comparison, see https://doi. org/10. 5281/zenodo. 5727015 for all. Sequences of miRNAs coding regions are highlighted in colours (miR-5p -orange, miR-3p -blue).
Zhang et al. proposed the minimal folding free energy index (MFEI>0. 85) of pre-miRNAs, that considers this, as a better criterion for distinguishing pre-miRNAs from other RNAs. By calculating MFEI values we observed that 27 out of 52 mutated pre-miRNAs did not satisfy the criterion of MFEI values above 0. 85, but instead showed values more similar to tRNA, rRNAs and mRNAs (MFEI values ∼0. 6)  [61]. The vast majority  (24)  of these 27 altered pre-miRNAs harbour 4-nt or longer deletions and therefore exhibit highly altered secondary structures (Data S2, Data S3, https://doi. org/10. 5281/zenodo. 5727015) with reduced stability, which may affect DCL1 recognition and processing efficiency. Additionally, changes in the local structural features, such as the position of bulges and internal loops and their shape have been shown to reduce the processing efficiency and accuracy  [49, 65].
miRNA variants produced from mutated MIR loci are not functionally relevant To evaluate the effect of mutations in the 5p coding region of mature miRNAs in terms of conservation of biological function, all detected novel miRNA variants originating from altered precursors were subjected to target prediction screening using the psRNATarget tool to assess binding to their corresponding wild-type miRNA targets. In potato, miR160a-5p and miR160b-5p guide the cleavage of ARF10 and ARF17, whereas miR390a-5p directs cleavage of non-coding TAS3 transcripts  [27, 40, 59] , leading to the production of TAS3-tasiRNAs that negatively regulate ARF2, ARF3 and ARF4 mRNAs  [1]. Target analysis revealed that 5 out of 9 variants of cr-MIR160a lines retained the target binding to ARF10 and ARF17 (Data S6), but all were detected in minute quantities (Data S5).
Base pairing here is critical for miRNA-mediated target repression so miRNA may not be functionally relevant  [30]. To assess the impact of reduced levels of mature miR-NAs due to CRISPR-Cas9 induced mutations, along with the fact that also some miRNA variants can also maintain target binding, we measured the expression levels of miRNA targets in the selected transgenic lines. We found that the expression of ARF10, target of miR160a-5p and miR160b-5p and expression of CathB, target of miR160a-3p, are negatively correlated with miRNA abundance. This shows that CRISPR-Cas9 induced mutations efficiently reduce mature miRNAs levels and that five miRNA variants which were predicted to retain target binding, do not contribute to the regulation of the mature miRNAs targets, which is also consistent with their low abundance (Figure  4a , Data S6, Data S5).
Following protoplasts transfection, functionalities of sgRNAs were determined by HRM and Sanger sequencing. We were able to confirm mutated alleles in MIR160a and MIR160b by both methods (Data S1). The presence of mutations could in MIR390a-edited protoplasts only be confirmed by HRM. This could be explained by low editing efficiency of designed sgRNAs and relatively low number of analysed individual PCR products (Data S1) as following stable transformation we did obtain mutated lines with the same set of sgRNAs. Determined by sequencing, low editing efficiency in potato protoplasts transfection set-up was observed in our experiment (20% and 10% in case of MIR160a and MIR160b, respectively), but was also shown by others  [2, 52]. Therefore, we conclude that HRM is fast and reliable screening method for the selection of functional sgRNAs and additional sequencing is not required at this step of the procedure.
Similarly, 1 bp-indels in mature miR528 region did not abolish miRNA function in rice  [64]. The quantity of these newly generated miRNA variants was however much lower compared to the wild-type miRNAs (Data S5), indicating lower efficiency of precursor processing also in case of short indels. By analysing the phenotype of transgenic plants, we observed that MIR160a-edited plants with lower miR160a abundance have sickle-shaped leaves and exhibit lamina growth arrest (Figure  3 , Figure  4 ). This is in accordance with the results published by Damodharan et al. [15] , who observed abnormal leaf phenotypes in miR160a-edited tomato plants. The phenotype of MIR160b-edited plants did not change due to the dominant role of MIR160a loci over MIR160b. MIR160a loci is not affected in cr-MIR160bedited plants and since the identical miRNA (miR160a-5p) is produced from MIR160a loci this does not affect the final phenotype.
Editing efficiency to mutate all four alleles in miRNA coding region is target-dependent. In our study, all sequences were mutated in 20% of analysed transgenic lines of cv. Désirée for MIR160a, 50% for MIR160b and 67% for MIR390a. Our results are in agreement with the results reported by others who also showed that editing efficiency in potato is not consistent when different guide sequences are used, however, their results have not been obtained for miRNA coding regions  [2, 10]. The complete pipeline for CRISPR-Cas9-mediated modulation of miRNA expression that we established considers pros and cons of different approaches. As Agrobacterium-mediated stable transformation is time consuming, transient transfection of protoplasts following screening by HRM for the selection of optimal sgRNAs in a transient set-up prior stable transformation in combination with ICE software-dependent selection of lines with desired mutations is a significant improvement.
Genotypisation of transgenic lines 800 bp-long regions surrounding coding sequence for each miRNA were amplified from genomic DNA and purified as explained above. For the screening of transgenic lines with desired mutations, PCR products were Sanger sequenced (Eurofins Genomics) with primers specific for amplicons (Figure  S11 ). To determine types of mutations and polymorphism, PCR products were for the selected transgenic lines and NT plants (Data S2) cloned into pJET and transformed into E. coli as explained above. Next, the constructs were isolated, sequenced and aligned as explained above. Next, the constructs were isolated from 9 or 10 colonies per PCR product using GenElute™ Plasmid Miniprep Kit (Sigma) and used for Sanger sequencing (Eurofins Genomics) with primers specific for amplicons (Figure  S11 ).
Inference of CRISPR edits analysis Inference of CRISPR Edits (ICE) analysis was performed using ICE v2 CRISPR Analysis Tool (  [23] ; https://ice. synthego. com) according to the provider's instructions. The used parameters were the following: GTCGTGTA-CACGTATATGCC and GAGCTTTGCAAATGGCATAC as guide sequences and DZW775 (Désirée) and CCP522 (Rywal) as control ab1. files for MIR160a, AGGAG-TAAGAATGATGTGCC and GGTGAAAGTGTGTGGCATAC as guide sequences and DZW857 as a control ab1. file for MIR160b and ATGGAGAATCTGTAAAGCTC and AAGATCAATTGAGTCATCCA as guide sequences and EBE422 as a control ab1. file for MIR390a. Note that the obtained scores are slightly higher than expected due to a polymorphism as the program predicted a mutation on the position of the natural polymorphism.