Genome wide SNP identification in chickpea for use in development of a high density genetic map and improvement of chickpea reference genome assembly

BMC Genomics

Bunyamin Tar’an¹  Thomas D Warkentin¹  Kirstin Bett¹  Anoop Sindhu¹  Marwan Diapari¹  Andrew G Sharpe²  Larissa Ramsay¹  Amit A Deokar¹ 

[1] Crop Development Centre, Department of Plant Sciences, University of Saskatchewan, 51 Campus Dr, Saskatoon, SK S7N 5A8, Canada;National Research Council Canada, 110 Gymnasium Place, Saskatoon, SK S7N 0 W9, Canada

关键词: Illumina GoldenGate and Genetic mapping;    Restriction site associated DNA (RAD) markers;    Genotyping-by-sequencing;    Cicer arietinum;   

Others  :  1141331
DOI  :  10.1186/1471-2164-15-708

 received in 2014-03-25, accepted in 2014-07-31,  发布年份 2014

【 摘 要 】

Background

In the whole genome sequencing, genetic map provides an essential framework for accurate and efficient genome assembly and validation. The main objectives of this study were to develop a high-density genetic map using RAD-Seq (Restriction-site Associated DNA Sequencing) genotyping-by-sequencing (RAD-Seq GBS) and Illumina GoldenGate assays, and to examine the alignment of the current map with the kabuli chickpea genome assembly.

Results

Genic single nucleotide polymorphisms (SNPs) totaling 51,632 SNPs were identified by 454 transcriptome sequencing of Cicer arietinum and Cicer reticulatum genotypes. Subsequently, an Illumina GoldenGate assay for 1,536 SNPs was developed. A total of 1,519 SNPs were successfully assayed across 92 recombinant inbred lines (RILs), of which 761 SNPs were polymorphic between the two parents. In addition, the next generation sequencing (NGS)-based GBS was applied to the same population generating 29,464 high quality SNPs. These SNPs were clustered into 626 recombination bins based on common segregation patterns. Data from the two approaches were used for the construction of a genetic map using a population derived from an intraspecific cross. The map consisted of 1,336 SNPs including 604 RAD recombination bins and 732 SNPs from Illumina GoldenGate assay. The map covered 653 cM of the chickpea genome with an average distance between adjacent markers of 0.5 cM. To date, this is the most extensive genetic map of chickpea using an intraspecific population. The alignment of the map with the CDC Frontier genome assembly revealed an overall conserved marker order; however, a few local inconsistencies within the Cicer arietinum pseudochromosome 1 (Ca1), Ca5 and Ca8 were detected. The map enabled the alignment of 215 unplaced scaffolds from the CDC Frontier draft genome assembly. The alignment also revealed varying degrees of recombination rates and hotspots across the chickpea genome.

Conclusions

A high-density genetic map using RAD-Seq GBS and Illumina GoldenGate assay was developed and aligned with the existing kabuli chickpea draft genome sequence. The analysis revealed an overall conserved marker order, although some localized inversions between draft genome assembly and the genetic map were detected. The current analysis provides an insight of the recombination rates and hotspots across the chickpea genome.

【 授权许可】

   
2014 Deokar et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Figure 7.

Figure 8.

Figure 9.

【 参考文献 】

• [1]FAOSTAT: FAO Statistical databases. Rome: Food and Agriculture Organization (FAO) of the United Nations; 2011. http://faostat.fao.org/site/339/default.aspx webcite
• [2]Clarke H, Siddique K: Response of chickpea genotypes to low temperature stress during reproductive development. Field Crop Res 2004, 90(2):323-334.
• [3]Pande S, Siddique K, Kishore G, Bayaa B, Gaur P, Gowda C, Bretag T, Crouch J: Ascochyta blight of chickpea (Cicer arietinum L.): a review of biology, pathogenicity, and disease management. Crop Pasture Sci 2005, 56(4):317-332.
• [4]Anbessa Y, Warkentin T, Vandenberg A, Ball R: Inheritance of time to flowering in chickpea in a short-season temperate environment. J Hered 2006, 97(1):55-61.
• [5]Millan T, Clarke HJ, Siddique KH, Buhariwalla HK, Gaur PM, Kumar J, Gil J, Kahl G, Winter P: Chickpea molecular breeding: new tools and concepts. Euphytica 2006, 147(1–2):81-103.
• [6]Varshney RK, Ribaut J-M, Buckler ES, Tuberosa R, Rafalski JA, Langridge P: Can genomics boost productivity of orphan crops? Nat Biotechnol 2012, 30(12):1172-1176.
• [7]Massman JM, Jung H-JG, Bernardo R: Genomewide selection versus marker-assisted recurrent selection to improve grain yield and stover-quality traits for cellulosic ethanol in maize. Crop Science 2013, 53(1):58-66.
• [8]Upadhyaya HD, Thudi M, Dronavalli N, Gujaria N, Singh S, Sharma S, Varshney RK: Genomic tools and germplasm diversity for chickpea improvement. Plant Genet Resour 2011, 9(1):45.
• [9]Tar'an B, Warkentin T, Tullu A, Vandenberg A: Genetic mapping of ascochyta blight resistance in chickpea (Cicer arietinum L.) using a simple sequence repeat linkage map. Genome 2007, 50(1):26-34.
• [10]Millan T, Winter P, Jüngling R, Gil J, Rubio J, Cho S, Cobos M, Iruela M, Rajesh P, Tekeoglu M: A consensus genetic map of chickpea (Cicer arietinum L.) based on 10 mapping populations. Euphytica 2010, 175(2):175-189.
• [11]Gaur R, Azam S, Jeena G, Khan AW, Choudhary S, Jain M, Yadav G, Tyagi AK, Chattopadhyay D, Bhatia S: High-throughput SNP discovery and genotyping for constructing a saturated linkage map of chickpea (Cicer arietinum L.). DNA Res 2012, 19(5):357-373.
• [12]Stephens A, Lombardi M, Cogan NO, Forster JW, Hobson K, Materne M, Kaur S: Genetic marker discovery, intraspecific linkage map construction and quantitative trait locus analysis of ascochyta blight resistance in chickpea (Cicer arietinum L.). Mol Breed 2014, 33(2):297-313.
• [13]Kudapa H, Azam S, Sharpe AG, Taran B, Li R, Deonovic B, Cameron C, Farmer AD, Cannon SB, Varshney RK: Comprehensive Transcriptome Assembly of Chickpea (Cicer arietinum L.) Using Sanger and Next Generation Sequencing Platforms: Development and Applications. PloS One 2014, 9(1):e86039.
• [14]Shendure J, Ji H: Next-generation DNA sequencing. Nat Biotechnol 2008, 26(10):1135-1145.
• [15]Kwok PY: Methods for genotyping single nucleotide polymorphisms. Annu Rev Genom Hum Genet 2001, 2(1):235-258.
• [16]Külheim C, Yeoh SH, Maintz J, Foley W, Moran G: Comparative SNP diversity among four Eucalyptus species for genes from secondary metabolite biosynthetic pathways. BMC Genom 2009, 10(1):452. BioMed Central Full Text
• [17]Labate JA, Baldo AM: Tomato SNP discovery by EST mining and resequencing. Mol Breed 2005, 16(4):343-349.
• [18]Gujaria N, Kumar A, Dauthal P, Dubey A, Hiremath P, Prakash AB, Farmer A, Bhide M, Shah T, Gaur PM: Development and use of genic molecular markers (GMMs) for construction of a transcript map of chickpea (Cicer arietinum L.). Theor Appl Genet 2011, 122(8):1577-1589.
• [19]Jhanwar S, Priya P, Garg R, Parida SK, Tyagi AK, Jain M: Transcriptome sequencing of wild chickpea as a rich resource for marker development. Plant Biotechnol J 2012, 10(6):690-702.
• [20]Thudi M, Bohra A, Nayak SN, Varghese N, Shah TM, Penmetsa RV, Thirunavukkarasu N, Gudipati S, Gaur PM, Kulwal PL: Novel SSR markers from BAC-end sequences, DArT arrays and a comprehensive genetic map with 1,291 marker loci for chickpea (Cicer arietinum L.). PloS One 2011, 6(11):e27275.
• [21]Hiremath PJ, Kumar A, Penmetsa RV, Farmer A, Schlueter JA, Chamarthi SK, Whaley AM, Carrasquilla‒Garcia N, Gaur PM, Upadhyaya HD: Large‒scale development of cost‒effective SNP marker assays for diversity assessment and genetic mapping in chickpea and comparative mapping in legumes. Plant Biotechnol J 2012, 10(6):716-732.
• [22]Hyten DL, Song Q, Choi IY, Yoon MS, Specht JE, Matukumalli LK, Nelson RL, Shoemaker RC, Young ND, Cregan PB: High-throughput genotyping with the GoldenGate assay in the complex genome of soybean. Theor Appl Genet 2008, 116(7):945-952.
• [23]Akhunov E, Nicolet C, Dvorak J: Single nucleotide polymorphism genotyping in polyploid wheat with the Illumina GoldenGate assay. Theor Appl Genet 2009, 119(3):507-517.
• [24]Ganal MW, Durstewitz G, Polley A, Berard A, Buckler ES, Charcosset A, Clarke JD, Graner E-M, Hansen M, Joets J: A large maize (Zea mays L.) SNP genotyping array: development and germplasm genotyping, and genetic mapping to compare with the B73 reference genome. PloS One 2011, 6(12):e28334.
• [25]Parida SK, Mukerji M, Singh AK, Singh NK, Mohapatra T: SNPs in stress-responsive rice genes: validation, genotyping, functional relevance and population structure. BMC Genomics 2012, 13(1):426. BioMed Central Full Text
• [26]Bachlava E, Taylor CA, Tang S, Bowers JE, Mandel JR, Burke JM, Knapp SJ: SNP discovery and development of a high-density genotyping array for sunflower. PloS One 2012, 7(1):e29814.
• [27]Sharpe AG, Ramsay L, Sanderson L-A, Fedoruk MJ, Clarke WE, Li R, Kagale S, Vijayan P, Vandenberg A, Bett KE: Ancient orphan crop joins modern era: gene-based SNP discovery and mapping in lentil. BMC Genomics 2013, 14(1):192. BioMed Central Full Text
• [28]Hyten DL, Cannon SB, Song Q, Weeks N, Fickus EW, Shoemaker RC, Specht JE, Farmer AD, May GD, Cregan PB: High-throughput SNP discovery through deep resequencing of a reduced representation library to anchor and orient scaffolds in the soybean whole genome sequence. BMC Genomics 2010, 11(1):38. BioMed Central Full Text
• [29]Antanaviciute L, Fernández-Fernández F, Jansen J, Banchi E, Evans KM, Viola R, Velasco R, Dunwell JM, Troggio M, Sargent DJ: Development of a dense SNP-based linkage map of an apple rootstock progeny using the Malus Infinium whole genome genotyping array. BMC Genomics 2012, 13(1):203. BioMed Central Full Text
• [30]Sim S-C, Van Deynze A, Stoffel K, Douches DS, Zarka D, Ganal MW, Chetelat RT, Hutton SF, Scott JW, Gardner RG: High-density SNP genotyping of tomato (Solanum lycopersicum L.) reveals patterns of genetic variation due to breeding. PloS One 2012, 7(9):e45520.
• [31]Elshire RJ, Glaubitz JC, Sun Q, Poland JA, Kawamoto K, Buckler ES, Mitchell SE: A robust, simple genotyping-by-sequencing (GBS) approach for high diversity species. PloS One 2011, 6(5):e19379.
• [32]van Orsouw NJ, Hogers RC, Janssen A, Yalcin F, Snoeijers S, Verstege E, Schneiders H, van der Poel H, van Oeveren J, Verstegen H: Complexity reduction of polymorphic sequences (CRoPS™): a novel approach for large-scale polymorphism discovery in complex genomes. PloS One 2007, 2(11):e1172.
• [33]Baird NA, Etter PD, Atwood TS, Currey MC, Shiver AL, Lewis ZA, Selker EU, Cresko WA, Johnson EA: Rapid SNP discovery and genetic mapping using sequenced RAD markers. PloS One 2008, 3(10):e3376.
• [34]Chutimanitsakun Y, Nipper RW, Cuesta-Marcos A, Cistué L, Corey A, Filichkina T, Johnson EA, Hayes PM: Construction and application for QTL analysis of a Restriction Site Associated DNA (RAD) linkage map in barley. BMC Genomics 2011, 12(1):4. BioMed Central Full Text
• [35]Morris GP, Ramu P, Deshpande SP, Hash CT, Shah T, Upadhyaya HD, Riera-Lizarazu O, Brown PJ, Acharya CB, Mitchell SE: Population genomic and genome-wide association studies of agroclimatic traits in sorghum. Proc Natl Acad Sci 2013, 110(2):453-458.
• [36]Varshney RK, Song C, Saxena RK, Azam S, Yu S, Sharpe AG, Cannon S, Baek J, Rosen BD, Tar'an B: Draft genome sequence of chickpea (Cicer arietinum) provides a resource for trait improvement. Nat Biotechnol 2013, 31(3):240-246.
• [37]Jain M, Misra G, Patel RK, Priya P, Jhanwar S, Khan AW, Shah N, Singh VK, Garg R, Jeena G, Yadav M, Kant C, Sharma P, Yadav G, Bhatia S, Tyagi AK, Chattopadhyay D: A draft genome sequence of the pulse crop chickpea (Cicer arietinum L.). Plant J 2013, 74(5):715-729.
• [38]Thudi M, Li Y, Jackson SA, May GD, Varshney RK: Current state-of-art of sequencing technologies for plant genomics research. Brief Funct Genomics 2012, 11(1):3-11.
• [39]Warkentin T, Banniza S, Vandenberg A: CDC Frontier kabuli chickpea. Can J Plant Sci 2005, 85(4):909-910.
• [40]Eveland AL, McCarty DR, Koch KE: Transcript profiling by 3′-untranslated region sequencing resolves expression of gene families. Plant Physiol 2008, 146(1):32-44.
• [41]Parkin IA, Clarke WE, Sidebottom C, Zhang W, Robinson SJ, Links MG, Karcz S, Higgins EE, Fobert P, Sharpe AG: Towards unambiguous transcript mapping in the allotetraploid Brassica napus. Genome 2010, 53(11):929-938.
• [42]Margulies M, Egholm M, Altman WE, Attiya S, Bader JS, Bemben LA, Berka J, Braverman MS, Chen Y-J, Chen Z: Genome sequencing in microfabricated high-density picolitre reactors. Nature 2005, 437(7057):376-380.
• [43]Wu TD, Watanabe CK: GMAP: a genomic mapping and alignment program for mRNA and EST sequences. Bioinformatics 2005, 21(9):1859-1875.
• [44]Conesa A, Gotz S, Garcia-Gomez JM, Terol J, Talon M, Robles M: Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 2005, 21(18):3674-3676.
• [45]Moriya Y, Itoh M, Okuda S, Yoshizawa AC, Kanehisa M: KAAS: an automatic genome annotation and pathway reconstruction server. Nucleic Acids Res 2007, 35(suppl 2):W182-W185.
• [46]Cingolani P, Platts A, Coon M, Nguyen T, Wang L, Land SJ, Lu X, Ruden DM: 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 2012, 6(2):80-92.
• [47]Meglecz E, Costedoat C, Dubut V, Gilles A, Malausa T, Pech N, Martin JF: QDD: a user-friendly program to select microsatellite markers and design primers from large sequencing projects. Bioinformatics 2010, 26(3):403-404.
• [48]Wong MM, Cannon CH, Wickneswari R: Development of high-throughput SNP-based genotyping in Acacia auriculiformis x A. mangium hybrids using short-read transcriptome data. BMC Genomics 2012, 13:726. BioMed Central Full Text
• [49]Saghai-Maroof M, Soliman K, Jorgensen RA, Allard R: Ribosomal DNA spacer-length polymorphisms in barley: Mendelian inheritance, chromosomal location, and population dynamics. Proc Natl Acad Sci 1984, 81(24):8014-8018.
• [50]Langmead B, Trapnell C, Pop M, Salzberg SL: Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 2009, 10(3):R25. BioMed Central Full Text
• [51]Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R: The sequence alignment/map format and SAMtools. Bioinformatics 2009, 25(16):2078-2079.
• [52]Kozik A: MadMapper. Available athttp://cgpdb.ucdavis.edu/XLinkage/MadMapper/ webcite 2006:Accessed 30 Nov. 2013
• [53]Van Os H, Stam P, Visser RG, Van Eck HJ: RECORD: a novel method for ordering loci on a genetic linkage map. Theor Appl Genet 2005, 112(1):30-40.
• [54]Lorieux M: MapDisto: fast and efficient computation of genetic linkage maps. Mol Breed 2012, 30(2):1231-1235.
• [55]Chakravarti A, Lasher LK, Reefer JE: A maximum likelihood method for estimating genome length using genetic linkage data. Genetics 1991, 128(1):175-182.
• [56]Sekino M, Hara M: Linkage maps for the Pacific abalone (Genus Haliotis) based on microsatellite DNA markers. Genetics 2007, 175(2):945-958.
• [57]Kumar S, Banks TW, Cloutier S: SNP discovery through next-generation sequencing and its applications. Int J Plant Prod 2012, 831460:15.
• [58]Wang C, Chen S, Yu S: Functional markers developed from multiple loci in GS3 for fine marker-assisted selection of grain length in rice. Theor Appl Genet 2011, 122(5):905-913.
• [59]Lagudah ES, Krattinger SG, Herrera-Foessel S, Singh RP, Huerta-Espino J, Spielmeyer W, Brown-Guedira G, Selter LL, Keller B: Gene-specific markers for the wheat gene Lr34/Yr18/Pm38 which confers resistance to multiple fungal pathogens. Theor Appl Genet 2009, 119(5):889-898.
• [60]Thudi M, Upadhyaya HD, Rathore A, Gaur PM, Krishnamurthy L, Roorkiwal M, Nayak SN, Chaturvedi SK, Basu PS, Gangarao NV, Fikre A, Kimurto P, Sharma PC, Sheshashayee MC, Tobita S, Kashiwagi J, Ito O, Killian A, Varshney RK: Genetic Dissection of Drought and Heat Tolerance in Chickpea through Genome-Wide and Candidate Gene-Based Association Mapping Approaches. PloS One 2014, 9(5):e96758.
• [61]Thompson C, Tar'an B: Genetic characterization of the acetohydroxyacid synthase (AHAS) gene responsible for resistance to imidazolinone in chickpea (Cicer arietinum L.). Theor Appl Genet 2014. doi:10.1007/s00122-014-2320-0
• [62]Upadhyaya HD, Dwivedi SL, Baum M, Varshney RK, Udupa SM, Gowda CL, Hoisington D, Singh S: Genetic structure, diversity, and allelic richness in composite collection and reference set in chickpea (Cicer arietinum L.). BMC Plant Biology 2008, 8(1):106. BioMed Central Full Text
• [63]Choudhary P, Khanna S, Jain P: Genetic structure and diversity analysis of the primary gene pool of chickpea using SSR markers. Genet Mol Res 2012, 11(2):891-905.
• [64]Leonforte A, Sudheesh S, Cogan NO, Salisbury PA, Nicolas ME, Materne M, Forster JW, Kaur S: SNP marker discovery, linkage map construction and identification of QTLs for enhanced salinity tolerance in field pea (Pisum sativum L.). BMC Plant Biology 2013, 13(1):161. BioMed Central Full Text
• [65]Parkin IA, Koh C, Tang H, Robinson SJ, Kagale S, Clarke WE, Town CD, Nixon J, Krishnakumar V, Bidwell SL, Denoeud F, Belcram H, Links MJ, Just J, Clarke C, Bender T, Huebert T, Mason AS, Pires JC, Barker G, Moore J, Walley PG, Manoli S, Batley J, Edwards D, Nelson MN, Wang X, Paterson AH, King G, Bancroft I, et al.: Transcriptome and methylome profiling reveals relics of genome dominance in the mesopolyploid Brassica oleracea. Genome Biology 2014, 15(6):R77. BioMed Central Full Text
• [66]Radhika P, Gowda S, Kadoo N, Mhase L, Jamadagni B, Sainani M, Chandra S, Gupta V: Development of an integrated intraspecific map of chickpea (Cicer arietinum L.) using two recombinant inbred line populations. Theor Appl Genet 2007, 115(2):209-216.
• [67]Lu H, Romero-Severson J, Bernardo R: Chromosomal regions associated with segregation distortion in maize. Theor Appl Genet 2002, 105(4):622-628.
• [68]Winter P, Benko-Iseppon A-M, Hüttel B, Ratnaparkhe M, Tullu A, Sonnante G, Pfaff T, Tekeoglu M, Santra D, Sant V: A linkage map of the chickpea (Cicer arietinum L.) genome based on recombinant inbred lines from a C. arietinum × C. reticulatum cross: localization of resistance genes for fusarium wilt races 4 and 5. Theor Appl Genet 2000, 101(7):1155-1163.
• [69]Abbo S, Molina C, Jungmann R, Grusak M, Berkovitch Z, Reifen R, Kahl G, Winter P, Reifen R: Quantitative trait loci governing carotenoid concentration and weight in seeds of chickpea (Cicer arietinum L.). Theor Appl Genet 2005, 111(2):185-195.
• [70]Rehman A, Malhotra R, Bett K, Tar'an B, Bueckert R, Warkentin T: Mapping QTL associated with traits affecting grain yield in Chickpea (Cicer arietinum L.) under terminal drought stress. Crop Science 2011, 51(2):450-463.
• [71]Castro P, Rubio J, Cabrera A, Millán T, Gil J: A segregation distortion locus located on linkage group 4 of the chickpea genetic map. Euphytica 2011, 179(3):515-523.
• [72]Taleei A, Kanouni H, Baum M: QTL analysis of ascochyta blight resistance in Chickpea. In Advances in Communication and Networking. Heidelberg: Springer; 2009:25-40.
• [73]Hamwieh A, Imtiaz M, Malhotra R: Multi-environment QTL analyses for drought-related traits in a recombinant inbred population of chickpea (Cicer arietinum L.). Theor Appl Genet 2013, 126:1025-1038.
• [74]Jensen-Seaman MI, Furey TS, Payseur BA, Lu Y, Roskin KM, Chen C-F, Thomas MA, Haussler D, Jacob HJ: Comparative recombination rates in the rat, mouse, and human genomes. Genome Research 2004, 14(4):528-538.
• [75]Henderson IR: Control of meiotic recombination frequency in plant genomes. Curr Opin Plant Biol 2012, 15(5):556-561.
• [76]Schnable PS, Hsia A-P, Nikolau BJ: Genetic recombination in plants. Curr Opin Plant Biol 1998, 1(2):123-129.
• [77]Drouaud J, Camilleri C, Bourguignon P-Y, Canaguier A, Bérard A, Vezon D, Giancola S, Brunel D, Colot V, Prum B: Variation in crossing-over rates across chromosome 4 of Arabidopsis thaliana reveals the presence of meiotic recombination “hot spots”. Genom Res 2006, 16(1):106-114.
• [78]Tian Z, Rizzon C, Du J, Zhu L, Bennetzen JL, Jackson SA, Gaut BS, Ma J: Do genetic recombination and gene density shape the pattern of DNA elimination in rice long terminal repeat retrotransposons? Genom Res 2009, 19(12):2221-2230.
• [79]Saintenac C, Faure S, Remay A, Choulet F, Ravel C, Paux E, Balfourier F, Feuillet C, Sourdille P: Variation in crossover rates across a 3-Mb contig of bread wheat (Triticum aestivum) reveals the presence of a meiotic recombination hotspot. Chromosoma 2011, 120(2):185-198.
• [80]Wright SI, Agrawal N, Bureau TE: Effects of recombination rate and gene density on transposable element distributions in Arabidopsis thaliana. Genom Res 2003, 13(8):1897-1903.
• [81]Ladizinsky G: Plant evolution under domestication. Dordrecht: Kluwer Academic Press; 1998:262.
• [82]Flandez-Galvez H, Ades PK, Ford R, Pang EC, Taylor PW: QTL analysis for ascochyta blight resistance in an intraspecific population of chickpea (Cicer arietinum L.). Theor Appl Genet 2003, 107(7):1257-1265.
• [83]Michael TP, Jackson S: The first 50 plant genomes. Plant Genome 2013, 6(2):1-7.
• [84]Alkan C, Sajjadian S, Eichler EE: Limitations of next-generation genome sequence assembly. Nat Methods 2010, 8(1):61-65.
• [85]Birney E: Assemblies: the good, the bad, the ugly. Nat Methods 2010, 8(1):59-60.
• [86]Yu X, Wang H, Zhong W, Bai J, Liu P, He Y: QTL mapping of leafy heads by genome resequencing in the RIL population of Brassica rapa. PloS One 2013, 8(10):e76059.