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SNP marker development in a QTL region associated with drought tolerance traits in chickpea (Cicer arietinum L.)
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SNP marker development in a QTL region associated with drought tolerance traits in chickpea (Cicer arietinum L.)

  1. Financial support from Generation Challenge program (GCP), Australia- India Strategic Research Fund (AISRF) is greatly appreciated. Sincere thanks to Mr. Vinay Kumar for the technical help. Inclusive Market-Oriented Development (IMOD) – our approach to bringing prosperity in the drylands. ICRISAT is a member of the CGIAR Consortium. Development of cleaved amplified polymorphic sequence (CAPS) markers Abstract Summary SNP marker development in a QTL region associated with drought tolerance traits in chickpea (Cicer arietinum L.) Deepa Jaganathan1*, Sarwar Azam1, Mahendar Thudi1, Pooran M Gaur1, Henry Nguyen2, Jacqueline Batley3, David Edwards3,4 , Tim Sutton5, Rajeev K Varshney1,6 1International Crops Research Institute for the Semi-Arid Tropics, Hyderabad, India, 2University of Missouri, Columbia, USA 3School of Agriculture and Food Sciences, University of Queensland, QLD, Australia 4Australian Centre for Plant Functional Genomics, University of Queensland, QLD, Australia 5Australian Centre for Plant Functional Genomics, University of Adelaide, SA, Australia 6CGIAR Generation Challenge Programme, c/o CIMMYT, Mexico DF, Mexico *Email deebiotech@gmail.com Drought is a major abiotic stress greatly limiting chickpea production, resulting in up to 50% yield loss. To understand the genetic complexity and mechanism of drought tolerance in chickpea, extensive phenotyping for drought tolerance traits and genotyping using SSR markers was peformed on the ICC 4958 ICC 1882 recombinant inbred line (RIL) population. As a result, a genomic hot spot region (~35 cM) harbouring several QTL for drought tolerance related traits was identified on linkage group 04 (CaLG04). This QTL region initially included four SSR markers (TAA170, ICCM0249, STMS11 and GA24). To enhance the marker density in this QTL region, a genotyping by sequencing (GBS) approach was adopted. In this context, GBS data (using the ApeKI enzyme) with coverage of 5x and 0.2x was generated for parental lines and 232 RILs, respectively. Using stringent measures, data was assembled to genotype 828 SNPs in the population. The extended genetic map comprises a total of 1,013 marker loci spanning a distance of 723.64 cM. Detailed analysis, based on the updated genetic map combined with additional phenotyping data identified 49 SNP markers in the QTL region and refined the interval containing the QTL to 14 cM. In order to utilize these SNP markers for molecular breeding, all 49 SNPs present in the QTL region were converted into cleaved amplified polymorphic sequence (CAPS) markers. Therefore these markers can serve as a foreground markers in marker assisted selection. In addition to that, with the aim of integrating more markers in the QTL region, the restriction site associated DNA (RAD) data of both the parents were aligned against the available chickpea genome (CDC frontier). A total of 118 SNPs were detected and a 96-plex Veracode assay was designed for SNP genotyping through Beadxpress. Hopefully the integration of the new markers and subsequent QTL analysis will result in refinement of the QTL hot spot region and provide tools to facilitate fine mapping. SNP ( C A ) at 11547141bp CAPS-CaLG040016NCPGR23654.4 TR2054.6 NCPGR23154.8 NCPGR3055.5 H4G1156.0 H1A1956.5 Ca4_1592593658.8 CaM190359.0 Ca4_1594238859.3 Ca4_1565180459.4 Ca4_1627860059.7 Ca4_1593490159.8 TR860.2 Ca4_1627867160.3 Ca4_1593460760.7 Ca4_1592616061.1 Ca4_1595707061.2 Ca4_16149998 Ca4_1604692861.3 ICCM024964.5 ICCM006566.3 Ca4_1255854167.7 Ca4_1372466668.5 Ca4_13588956 Ca4_1369966368.7 Ca4_13840484 Ca4_1384025168.8 Ca4_1127622569.5 Ca4_1139888969.6 NCPGR12769.8 Ca4_11276413 Ca4_1123413070.0 Ca4_11441735 Ca4_11435380 Ca4_1143565170.1 Ca4_11343257 Ca4_11343230 Ca4_11547141 Ca4_11246164 Ca4_11398699 Ca4_11334350 Ca4_11246093 Ca4_11273405 70.2 Ca4_11398682 Ca4_11517582 Ca4_11246173 Ca4_11517517 Ca4_11277574 Ca4_11491864 Ca4_11273328 Ca4_11490100 Ca4_11334343 Ca4_11490496 Ca4_11274281 Ca4_11244334 Ca4_11244395 Ca4_11276484 Ca4_11275171 Ca4_11465075 70.3 Ca4_11465057 Ca4_11441604 Ca4_1144167370.4 Ca4_1298242072.4 Ca4_13787649 Ca4_1378744872.8 Ca4_1384134072.9 Ca4_1384022773.0 Ca4_1383879673.1 Ca4_13839288 Ca4_13839294 Ca4_1368745673.2 TAA17077.4 NCPGR2178.3 STMS1178.8 cpPb-68055279.6 CaM061582.6 CaM084583.8 CaM085684.0 Tp68496486.3 SNP ( A G ) at 13839294bp CAPS-CaLG040048 NCPGR23654.4 TR2054.6 NCPGR23154.8 NCPGR3055.5 H4G1156.0 H1A1956.5 Ca4_1592593658.8 CaM190359.0 Ca4_1594238859.3 Ca4_1565180459.4 Ca4_1627860059.7 Ca4_1593490159.8 TR860.2 Ca4_1627867160.3 Ca4_1593460760.7 Ca4_1592616061.1 Ca4_1595707061.2 Ca4_16149998 Ca4_1604692861.3 ICCM024964.5 ICCM006566.3 Ca4_1255854167.7 Ca4_1372466668.5 Ca4_13588956 Ca4_1369966368.7 Ca4_13840484 Ca4_1384025168.8 Ca4_1127622569.5 Ca4_1139888969.6 NCPGR12769.8 Ca4_11276413 Ca4_1123413070.0 Ca4_11441735 Ca4_11435380 Ca4_1143565170.1 Ca4_11343257 Ca4_11343230 Ca4_11547141 Ca4_11246164 Ca4_11398699 Ca4_11334350 Ca4_11246093 Ca4_11273405 70.2 Ca4_11398682 Ca4_11517582 Ca4_11246173 Ca4_11517517 Ca4_11277574 Ca4_11491864 Ca4_11273328 Ca4_11490100 Ca4_11334343 Ca4_11490496 Ca4_11274281 Ca4_11244334 Ca4_11244395 Ca4_11276484 Ca4_11275171 Ca4_11465075 70.3 Ca4_11465057 Ca4_11441604 Ca4_1144167370.4 Ca4_1298242072.4 Ca4_13787649 Ca4_1378744872.8 Ca4_1384134072.9 Ca4_1384022773.0 Ca4_1383879673.1 Ca4_13839288 Ca4_13839294 Ca4_1368745673.2 TAA17077.4 NCPGR2178.3 STMS1178.8 cpPb-68055279.6 CaM061582.6 CaM084583.8 CaM085684.0 Tp68496486.3 SNP ( C T ) at 11517582bp CAPS-CaLG040023 HphI 1-ICC 283 2-ICC 4958 3-ICC 1882 4-ICC 8261 5-PI 489777 L-100bp Ladder Restriction Enzyme : Marker : CaLG040016 CaLG040023 CaLG040048 PCR amplification After enzyme digestion HaeIII MseI L LL L LL CaLG04 Pseudomolecule 4 ICCM0249 STMS11 10077164bp 13840227bp 3.76Mb SNP Discovery CaLG04 Conversion of SNP into CAPS • By using GBS approach 49 SNP markers were integrated into the genetic map in the QTL region • A 400bp flanking sequence of each SNP was extracted and primers were designed • The priducts were amplified and digested with respective enzymes • All 49 SNPs were converted into CAPS markers SNP Discovery in the QTL region • QTL hot spot region (STMS11 – ICCM0249) was mapped to the reference genome (CDC frontier) of chickpea • The 7Mb (9102731bp – 16180797bp) region of the pseudomolecule 4 of physical map was identified as the QTL region • SNPs between parents (ICC 4958 and ICC 1882) were identified in the QTL hot spot region using RAD data • 118 novel SNPs were selected • Finally 96 plex veracode assay were designed for Illumina's Beadxpress  Integrating these SNP markers into the existing genetic map and further QTL analysis will refine the hot spot region and facilitate fine mapping Acknowledgements QTL hotspot Colour codes: Blue – SSR markers Red - SNP markers
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