A HapMap leads to a Capsicum annuum SNP infinium array: a new tool for pepper breeding

Abstract

The Capsicum genus (Pepper) is a part of the Solanacae family. It has been important in many cultures worldwide for its key nutritional components and uses as spices, medicines, ornamentals and vegetables. Worldwide population growth is associated with demand for more nutritionally valuable vegetables while contending with decreasing resources and available land. These conditions require increased efficiency in pepper breeding to deal with these imminent challenges. Through resequencing of inbred lines we have completed a valuable haplotype map (HapMap) for the pepper genome based on single-nucleotide polymorphisms (SNP). The identified SNPs were annotated and classified based on their gene annotation in the pepper draft genome sequence and phenotype of the sequenced inbred lines. A selection of one marker per gene model was utilized to create the PepperSNP16K array, which simultaneously genotyped 16 405 SNPs, of which 90.7% were found to be informative. A set of 84 inbred and hybrid lines and a mapping population of 90 interspecific F₂ individuals were utilized to validate the array. Diversity analysis of the inbred lines shows a distinct separation of bell versus chile/hot pepper types and separates them into five distinct germplasm groups. The interspecific population created between Tabasco (C. frutescens chile type) and P4 (C. annuum blocky type) produced a linkage map with 5546 markers separated into 1361 bins on twelve 12 linkage groups representing 1392.3 cM. This publically available genotyping platform can be used to rapidly assess a large number of markers in a reproducible high-throughput manner for pepper. As a standardized tool for genetic analyses, the PepperSNP16K can be used worldwide to share findings and analyze QTLs for important traits leading to continued improvement of pepper for consumers. Data and information on the array are available through the Solanaceae Genomics Network.

References

[1] USDA National Agriculture Statistics Service . 1994-2013; https://www.nass.usda.gov/.

[2] Kim S, Park M, Yeom SI, Kim YM, Lee JM, Lee HA et al. Genome sequence of the hot pepper provides insights into the evolution of pungency in Capsicum species. Nat Genet 2014; 46: 270-278.

[3] Moscone EA, Baranyi M, Ebert I, Greilhuber J, Ehrendorfer F, Hunziker AT . Analysis of nuclear DNA content in Capsicum (Solanaceae) by flow cytometry and Feulgen densitometry. Ann Bot 2003; 92: 21-29.

[4] Zonneveld BJM, Leitch IJ, Bennett MD . First nuclear DNA amounts in more than 300 angiosperms. Annals of Botany 2005; 96: 229-244.

[5] Sato S, Tabata S, Hirakawa H, Asamizu E, Shirasawa K, Isobe S et al. The tomato genome sequence provides insights into fleshy fruit evolution. Nature 2012; 485: 635-641.

[6] Xu X, Pan SK, Cheng SF, Zhang B, Mu DS, Ni PX et al. Genome sequence and analysis of the tuber crop potato. Nature 2011; 475: 189-U194.

[7] Lu FH, Cho MC, Park YJ . Transcriptome profiling and molecular marker discovery in red pepper, Capsicum annuum L. TF68. Mol Biol Rep 2012; 39: 3327-3335.

[8] Lu F-H, Yoon M-Y, Cho Y-I, Chung J-W, Kim K-T, Cho M-C et al. Transcriptome analysis and SNP/SSR marker information of red pepper variety YCM334 and Taean. Scientia Horticulturae 2011; 129: 38-45.

[9] Ashrafi H, Hill T, Stoffel K, Kozik A, Yao J, Chin-Wo SR et al. De novo assembly of the pepper transcriptome (Capsicum annuum): a benchmark for in silico discovery of SNPs, SSRs and candidate genes. BMC Genomics 2012; 13: 571.

[10] Hill TA, Ashrafi H, Reyes-Chin-Wo S, Yao J, Stoffel K, Truco MJ et al. Characterization of Capsicum annuum genetic diversity and population structure based on parallel polymorphism discovery with a 30K unigene Pepper GeneChip. PLoS One 2013; 8: e56200.

[11] Qin C, Yu C, Shen Y, Fang X, Chen L, Min J et al. Whole-genome sequencing of cultivated and wild peppers provides insights into Capsicum domestication and specialization. Proc Natl Acad Sci USA 2014; 111: 5135-5140.

[12] Xu X, Chao J, Cheng X, Wang R, Sun B, Wang H et al. Mapping of a novel race specific resistance gene to phytophthora root rot of pepper (Capsicum annuum) using bulked segregant analysis combined with specific length amplified fragment sequencing strategy. PLoS One 2016; 11: e0151401.

[13] Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 2009; 25: 2078-2079.

[14] Nagy I, Stagel A, Sasvari Z, Roder M, Ganal M . Development, characterization, and transferability to other Solanaceae of microsatellite markers in pepper (Capsicum annuum L.). Genome 2007; 50: 668-688.

[15] Van Ooijen J JoinMap 4. Software for the calculation of genetic linkage maps in experimental populations Kyazma BV, Wageningen, Netherlands 2006.

[16] Manly KF, Cudmore RH Jr, Meer JM . Map Manager QTX, cross-platform software for genetic mapping. Mamm Genome 2001; 12: 930-932.

[17] Voorrips RE . MapChart: software for the graphical presentation of linkage maps and QTLs. J Hered 2002; 93: 77-78.

[18] Hill T, Ashrafi H, Chin-Wo SR, Stoffel K, Truco MJ, Kozik A et al. Ultra-high density, transcript-based genetic maps of pepper define recombination in the genome and synteny among related species. G3 (Bethesda) 2015; 5: 2341-2355.

[19] Pritchard JK, Stephens M, Donnelly P . Inference of population structure using multilocus genotype data. Genetics 2000; 155: 945-959.

[20] Evanno G, Regnaut S, Goudet J . Detecting the number of clusters of individuals using the software STRUCTURE: a simulation study. Mol Ecol 2005; 14: 2611-2620.

[21] Wu F, Eannetta NT, Xu Y, Durrett R, Mazourek M, Jahn MM et al. A COSII genetic map of the pepper genome provides a detailed picture of synteny with tomato and new insights into recent chromosome evolution in the genus Capsicum. Theor Appl Genet 2009; 118: 1279-1293.

[22] Poland JA, Brown PJ, Sorrells ME, Jannink JL . Development of high-density genetic maps for barley and wheat using a novel two-enzyme genotyping-by-sequencing approach. PLoS One 2012; 7: e32253.

[23] Mascher M, Muehlbauer GJ, Rokhsar DS, Chapman J, Schmutz J, Barry K et al. Anchoring and ordering NGS contig assemblies by population sequencing (POPSEQ). Plant J 2013; 76: 718-727.

[24] Boutet G, Alves Carvalho S, Falque M, Peterlongo P, Lhuillier E, Bouchez O et al. SNP discovery and genetic mapping using genotyping by sequencing of whole genome genomic DNA from a pea RIL population. BMC Genomics 2016; 17: 121.

[25] Xavier A, Muir WM, Rainey KM . Impact of imputation methods on the amount of genetic variation captured by a single-nucleotide polymorphism panel in soybeans. BMC Bioinformatics 2016; 17: 55.

[26] Hulse-Kemp AM, Lemm J, Plieske J, Ashrafi H, Buyyarapu R, Fang DD et al. Development of a 63K SNP array for cotton and high-density mapping of intraspecific and interspecific populations of Gossypium spp. G3 (Bethesda) 2015; 5: 1187-1209.

[27] Lee YG, Jeong N, Kim JH, Lee K, Kim KH, Pirani A et al. Development, validation and genetic analysis of a large soybean SNP genotyping array. Plant J 2015; 81: 625-636.

[28] Singh N, Jayaswal PK, Panda K, Mandal P, Kumar V, Singh B et al. Single-copy gene based 50K SNP chip for genetic studies and molecular breeding in rice. Sci Rep 2015; 5: 11600.

[29] Wahyuni Y, Ballester AR, Sudarmonowati E, Bino RJ, Bovy AG . Metabolite biodiversity in pepper (Capsicum) fruits of thirty-two diverse accessions: variation in health-related compounds and implications for breeding. Phytochemistry 2011; 72: 1358-1370.

[30] Stewart C Jr, Kang BC, Liu K, Mazourek M, Moore SL, Yoo EY et al. The Pun1 gene for pungency in pepper encodes a putative acyltransferase. Plant J 2005; 42: 675-688.