Genome-wide clonal variability in European pear “Rocha” using high-throughput sequencing

Abstract

Pears (Pyrus) are one of the most economically important fruits worldwide. The Pyrus genus is characterized by a high degree of genetic variability between species and interspecific hybrids, and several studies have been performed to assess this variability for both cultivated and wild accessions. These studies have mostly been limited by the resolving power of traditional molecular markers, although in the recent past the availability of reference genome sequences or SNP arrays for pear have enhanced the capability of high-resolution genomics studies. These tools can also be applied to better understand the intra-varietal (or clonal) variability in pear. Here we report the first high resolution genomics analysis of a pear clonal population using whole genome sequencing (WGS). Results showed unique signatures for the accumulation of mutations and transposable element insertions in each clone, which are likely related to their history of propagation and cultivation. The nucleotide diversity remained low in the clonal collection with the exception of few genomic windows, suggesting that balancing selection may be occurring. These windows included mainly genes related to plant fertility. Regions with higher mutational load were partially associated with transcription factors, probably reflecting the distinctive phenotypes in the collection. The annotation of variants also revealed the theoretical disruption of relevant genes in pear. Taken together, the results from this study show that pear clones accumulate mutations differently, and that those mutations can play a role on pear phenotypes, meaning that the study of pear clonal populations can be relevant in genetic studies, mainly when comparing with traditional association studies.

References

[1] FAOSTAT. http://www.fao.org/faostat/en/#data/QCL 2021.

[2] Asanidze Z, Akhalkatsi M, Gvritishvili M . Comparative morphometric study and relationships between the Caucasian species of wild pear (Pyrus spp.) and local cultivars in Georgia . Flora-Morphol Distrib Funct Ecol Plants. 2011; 206: 974-86.

[3] Silva GJ, Souza TM, Barbieri RL et al. Origin, domestication, and dispersing of pear (Pyrus spp.) . Adv Agric. 2014; 2014: e541097.

[4] Quinet M, Wesel J-P . Botany and Taxonomy of Pear. In: Korban SS, ed. The Pear Genome.Compendium of Plant Genomes. Springer, Cham, 2019, 1-33.

[5] Jiang S, Zheng X, Yu P et al. Primitive genepools of Asian pears and their complex hybrid origins inferred from fluorescent sequence-specific amplification polymorphism (SSAP) markers based on LTR retrotransposons. PLoS One. 2016; 11: e0149192.

[6] Katayama H, Amo H, Wuyun T et al. Genetic structure and diversity of the wild Ussurian pear in East Asia. Breed Sci. 2016; 66: 90-9.

[7] Xue L, Liu Q, Qin M et al. Genetic variation and population structure of “Zangli” pear landraces in Tibet revealed by SSR markers. Tree Genet Genomes. 2017; 13: 26.

[8] Zong Y, Sun P, Yue X et al. Variation in microsatellite loci reveals a natural boundary of genetic differentiation among Pyrus betulaefolia populations in northern China. J Am Soc Hortic Sci. 2017; 142: 319-29.

[9] Volk GM, Richards CM, Henk AD et al. Diversity of wild Pyrus communis based on microsatellite analyses. J Am Soc Hortic Sci. 2006; 131: 408-17.

[10] Wolko Ł, Antkowiak W, Lenartowicz E et al. Genetic diversity of European pear cultivars (Pyrus communis L.) and wild pear (Pyrus pyraster (L.) Burgsd.) inferred from microsatellite markers analysis . Genet Resour Crop Evol. 2010; 57: 801-6.

[11] Wolko Ł, Bocianowski J, Antkowiak W et al. Genetic diversity and population structure of wild pear (Pyrus pyraster (L.) Burgsd.) in Poland . Open Life Sci. 2015; 10: 19-29.

[12] Zheng X, Cai D, Potter D et al. Phylogeny and evolutionary histories of Pyrus L. revealed by phylogenetic trees and networks based on data from multiple DNA sequences. Mol Phylogenet Evol. 2014; 80: 54-65.

[13] Wu J, Wang Z, Shi Z et al. The genome of the pear (Pyrus bretschneideri Rehd.). Genome Res. 2013; 23: 396-408.

[14] Chagné D, Crowhurst RN, Pindo M et al. The draft genome sequence of European pear (Pyrus communis L. ‘Bartlett’). PLoS One. 2014; 9: e92644.

[15] Linsmith G, Rombauts S, Montanari S et al. Pseudo-chromosome-length genome assembly of a double haploid ‘Bartlett’ pear (Pyrus communis L.). GigaScience. 2019; 8: giz138.

[16] Xue H, Wang S, Yao J-L et al. Chromosome level high-density integrated genetic maps improve the Pyrus bretschneideri ‘DangshanSuli’ v1.0 genome. BMC Genomics. 2018; 19: 833.

[17] Shirasawa K, Itai A, Isobe S . Chromosome-scale genome assembly of Japanese pear (Pyrus pyrifolia) variety ‘Nijisseiki’. DNA Res. 2021; 28: dsab001.

[18] Ou C, Wang F, Wang J et al. A de novo genome assembly of the dwarfing pear rootstock Zhongai 1. Sci Data. 2019; 6: 281.

[19] Dong X, Wang Z, Tian L et al. De novo assembly of a wild pear (Pyrus betuleafolia) genome. Plant Biotechnol J. 2020; 18: 581-95.

[20] Wu J, Wang Y, Xu J et al. Diversification and independent domestication of Asian and European pears. Genome Biol. 2018; 19: 77.

[21] Gabay G, Dahan Y, Izhaki Y et al. High-resolution genetic linkage map of European pear (Pyrus communis) and QTL fine-mapping of vegetative budbreak time. BMC Plant Biol. 2018; 18: 175.

[22] Oh S, Oh Y, Kim K et al. Construction of high-resolution genetic linkage map in pear pseudo-BC1 ((Pyrus pyrifolia × P. communis) × P. pyrifolia) using GBS-SNPs and SSRs. Hortic Environ Biotechnol. 2020; 61: 745-53.

[23] Kumar S, Kirk C, Deng C et al. Genotyping-by-sequencing of pear (Pyrus spp.) accessions unravels novel patterns of genetic diversity and selection footprints. Hortic Res. 2017; 4: 1-10.

[24] Nishio S, Hayashi T, Shirasawa K et al. Genome-wide association study of individual sugar content in fruit of Japanese pear (Pyrus spp.). BMC Plant Biol. 2021; 21: 378.

[25] Zhang M-Y, Xue C, Hu H et al. Genome-wide association studies provide insights into the genetic determination of fruit traits of pear. Nat Commun. 2021; 12: 1144.

[26] Kumar S, Kirk C, Deng CH et al. Marker-trait associations and genomic predictions of interspecific pear (Pyrus) fruit characteristics. Sci Rep. 2019; 9: 9072.

[27] Lee HS, Kim GH, Kwon SI et al. Analysis of ‘Fuji’ apple somatic variants from next-generation sequencing. Genet Mol Res. 2016; 15: gmr.15038185.

[28] Zhao J, Shen F, Gao Y et al. Parallel bud mutation sequencing reveals that fruit sugar and acid metabolism potentially influence stress in malus. Int J Mol Sci. 2019; 20: 5988.

[29] Calderón L, Mauri N, Muñoz C et al. Whole genome resequencing and custom genotyping unveil clonal lineages in ‘Malbec’ grapevines (Vitis vinifera L.). Sci Rep. 2021; 11: 7775.

[30] Roach MJ, Johnson DL, Bohlmann J et al. Population sequencing reveals clonal diversity and ancestral inbreeding in the grapevine cultivar chardonnay. PLoS Genet. 2018; 14: e1007807.

[31] Gambino G, Molin AD, Boccacci P et al. Whole-genome sequencing and SNV genotyping of ‘Nebbiolo’ (Vitis vinifera L.) clones. Sci Rep. 2017; 7: 17294.

[32] Vondras AM, Minio A, Blanco-Ulate B et al. The genomic diversification of grapevine clones. BMC Genomics. 2019; 20: 972.

[33] Queiroz Á, Guimarães JB, Sánchez C et al. Genetic diversity and structure of the Portuguese pear (Pyrus communis L.) germplasm. Sustainability. 2019; 11: 5340.

[34] Silva JM, Barba NG, Barros MT et al. ‘Rocha’, the pear from Portugal. Acta Hortic. 2005; 671: 219-22.

[35] Martin M . Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet j. 2011; 17: 10-2.

[36] Andrew, S. FastQC A Quality Control tool for High Throughput Sequence Data. (2010).

[37] Li H. Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM. arXiv:1303.3997v2 [q-bio.GN].

[38] Li H, Handsaker B, Wysoker A et al. The sequence alignment/map format and SAMtools. Bioinformatics. 2009; 25: 2078-9.

[39] Picard toolkit. Broad Institute, GitHub repository (2019).

[40] Okonechnikov K, Conesa A, García-Alcalde F . Qualimap 2: advanced multi-sample quality control for high-throughput sequencing data. Bioinformatics. 2016; 32: 292-4.

[41] Poplin R, Ruano-Rubio V, DePristo MA et al. Scaling accurate genetic variant discovery to tens of thousands of samples. bioRxiv 201178. 2018.

[42] Li H. A statistical framework for SNP calling, mutation discovery, association mapping and population genetical parameter estimation from sequencing data. Bioinformatics. 2011; 27: 2987-93.

[43] Danecek P, Auton A, Abecasis G et al. The variant call format and VCFtools. Bioinformatics. 2011;27:2156-8.

[44] Untergasser A, Nijveen H, Rao X et al. Primer3Plus, an enhanced web interface to Primer3. Nucleic Acids Res. 2007; 35: W71-4.

[45] R Core Team R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, 2021.

[46] Pfeifer B, Wittelsbürger U, Ramos-Onsins SE et al. PopGenome: an efficient Swiss army knife for population genomic analyses in R. Mol Biol Evol. 2014; 31: 1929-36.

[47] Manichaikul A, Mychaleckyj JC, Rich SS et al. Robust relationship inference in genome-wide association studies. Bioinformatics. 2010; 26: 2867-73.

[48] Gogarten SM, Sofer T, Chen H et al. Genetic association testing using the GENESIS R/Bioconductor package. Bioinformatics. 2019; 35: 5346-8.

[49] Purcell S, Neale B, Todd-Brown K et al. PLINK: a tool set for whole-genome association and population-based linkage analyses. Am J Hum Genet. 2007; 81: 559-75.

[50] Wickham H. ggplot2: Elegant Graphics for Data Analysis . Springer-Verlag, New York, 2009.

[51] Bailey TL, Boden M, Buske FA et al. MEME SUITE: tools for motif discovery and searching. Nucleic Acids Res. 2009; 37: W202-8.

[52] Cingolani P, Platts A, Wang LL et al. A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3. Fly (Austin). 2012; 6: 80-92.

[53] Kofler R, Gómez-Sánchez D, Schlötterer C . PoPoolationTE2: comparative population genomics of transposable elements using Pool-Seq. Mol Biol Evol. 2016; 33: 2759-64.

[54] Keane TM, Wong K, Adams DJ . RetroSeq: transposable element discovery from next-generation sequencing data. Bioinformatics. 2013; 29: 389-90.

[55] Nelson MG, Linheiro RS, Bergman CM . McClintock: an integrated pipeline for detecting transposable element insertions in whole-genome shotgun sequencing data. G3 (Bethesda). 2017; 7: 2763-78.

[56] Montanari S, Bianco L, Allen BJ et al. Development of a highly efficient AxiomTM 70 K SNP array for Pyrus and evaluation for high-density mapping and germplasm characterization. BMC Genomics. 2019; 20: 331.

[57] Montanari S, Postman J, Bassil NV et al. Reconstruction of the largest pedigree network for pear cultivars and evaluation of the genetic diversity of the USDA-ARS National Pyrus Collection. G3 (Bethesda). 2020; 10: 3285-97.

[58] Dong X, Hong Z, Sivaramakrishnan M et al. Callose synthase (CalS5) is required for exine formation during microgametogenesis and for pollen viability in Arabidopsis. Plant J. 2005; 42: 315-28.

[59] Nishikawa S, Zinkl GM, Swanson RJ et al. Callose (beta-1,3 glucan) is essential for Arabidopsis pollen wall patterning, but not tube growth. BMC Plant Biol. 2005; 5: 22.

[60] Rajaraman J, Douchkov D, Hensel G et al. An LRR/Malectin receptor-like kinase mediates resistance to non-adapted and adapted powdery mildew fungi in barley and wheat. Front Plant Sci. 2016; 7: 1836.

[61] Wang D, Liang X, Bao Y et al. A malectin-like receptor kinase regulates cell death and pattern-triggered immunity in soybean. EMBO Rep. 2020; 21: e50442.

[62] Huck N, Moore JM, Federer M et al. The Arabidopsis mutant feronia disrupts the female gametophytic control of pollen tube reception. Development. 2003; 130: 2149-59.

[63] Rotman N, Gourgues M, Guitton A-E et al. A dialogue between the Sirène pathway in Synergids and the fertilization independent seed pathway in the central cell controls male gamete release during double fertilization in Arabidopsis. Mol Plant. 2008; 1: 659-66.

[64] Kou X, Qi K, Qiao X et al. Evolution, expression analysis, and functional verification of Catharanthus roseus RLK1-like kinase (CrRLK1L) family proteins in pear (Pyrus bretchneideri). Genomics. 2017; 109: 290-301.

[65] Jia M, Du P, Ding N et al. Two FERONIA-like receptor kinases regulate apple fruit ripening by modulating ethylene production. Front Plant Sci. 2017; 8: 1406.

[66] Bieluszewski T, Galganski L, Sura W et al. AtEAF1 is a potential platform protein for Arabidopsis NuA4 acetyltransferase complex. BMC Plant Biol. 2015; 15: 75.

[67] Malenica N, Maletic E, Simon S et al. Grapevine variety determination from herbarium and archeological specimens. Acta Hortic. 2014; 1046: 603-8.

[68] Franco-Zorrilla JM, López-Vidriero I, Carrasco JL et al. DNA-binding specificities of plant transcription factors and their potential to define target genes. Proc Natl Acad Sci U S A. 2014; 111: 2367-72.