Integrated physical, genetic and genome map of chickpea (Cicer arietinum L.)

Abstract

Physical map of chickpea was developed for the reference chickpea genotype (ICC 4958) using bacterial artificial chromosome (BAC) libraries targeting 71,094 clones (~12× coverage). High information content fingerprinting (HICF) of these clones gave high-quality fingerprinting data for 67,483 clones, and 1,174 contigs comprising 46,112 clones and 3,256 singletons were defined. In brief, 574 Mb genome size was assembled in 1,174 contigs with an average of 0.49 Mb per contig and 3,256 singletons represent 407 Mb genome. The physical map was linked with two genetic maps with the help of 245 BAC-end sequence (BES)-derived simple sequence repeat (SSR) markers. This allowed locating some of the BACs in the vicinity of some important quantitative trait loci (QTLs) for drought tolerance and reistance to Fusarium wilt and Ascochyta blight. In addition, fingerprinted contig (FPC) assembly was also integrated with the draft genome sequence of chickpea. As a result, ~965 BACs including 163 minimum tilling path (MTP) clones could be mapped on eight pseudo-molecules of chickpea forming 491 hypothetical contigs representing 54,013,992 bp (~54 Mb) of the draft genome. Comprehensive analysis of markers in abiotic and biotic stress tolerance QTL regions led to identification of 654, 306 and 23 genes in drought tolerance "QTL-hotspot" region, Ascochyta blight resistance QTL region and Fusarium wilt resistance QTL region, respectively. Integrated physical, genetic and genome map should provide a foundation for cloning and isolation of QTLs/genes for molecular dissection of traits as well as markers for molecular breeding for chickpea improvement.

Fig. 1

Insert size estimation of chickpea BAC clones by pulsed field gel electrophoresis (PFGE). A set of 28 clones can be visualized from a CAH0000 library (constructed using HindIII) and b CAE0000 library (constructed using EcoRI). PFG marker can be seen in two lanes on either side of the 28 clones from each library. The insert size of BAC library is ~120 and ~124 kb for CAH0000 and CAE0000 libraries, respectively

Fig. 2

Distribution of number of clones in 1,174 contigs in chickpea FPC assembly. The range of clones in each contig varied from 2 to 3,007 with an average of 39.27 clones/contig

Fig. 3

Anchoring physical map with two chickpea genetic maps. The number of marker hits on BAC contigs varied from 1 to 5; unique hits were obtained for 131 markers

Fig. 4

A snapshot of anchoring BAC clones to the “QTL-hotspot” region harbouring QTLs for several drought tolerance-related traits in chickpea. Two BAC contigs namely ctg198 and ctg1769 were assigned to “QTL-hotspot” region as SSR markers namely CaM0232 and CaM1328 derived from end sequences of BACs from the above mentioned contigs were mapped in the same region on inter-specific (Thudi et al. 2011) and intra-specific (Varshney et al. 2014) genetic maps, respectively

Fig. 5

A snapshot showing coverage of Ascochyta blight resistance QTL regions with BAC clones in chickpea. The BAC ctg1390 on LG2 of inter-specific genetic map developed by Thudi et al. (2011) based on RIL population ICC 4958 × PI 489777 is closer to the SSR marker GA16 that flanks the ar2a QTL identified for Ascochyta blight resistance by Udupa and Baum (2003). Similarly, the BAC contig 198 on LG04 of inter-specific genetic map developed by Thudi et al. (2011) is closer to SSR marker TA130 that flanks ar2b QTL identified for Ascochyta blight resistance by Udupa and Baum (2003)

Fig. 6

Summary of steps involved in BES mapping analysis for integrating chickpea FPC contig assembly with the high-density genetic map and the reference chickpea genome sequence. A set of 53,316 BESs from 28,147 clones were used for anchoring contig physical map with the draft genome sequence of chickpea. BLASTN analysis of 53,316 BESs provided 16,086 unique (non-repeat) BES. The unique BESs (16,086) were tried for their mapping onto the chickpea genome sequence covering eight pseudo-molecules (Ca1 to Ca8) and un-anchored scaffolds (Ca0). As a result, a total of 965 BAC clones were successfully mapped onto draft genome of Kabuli chickpea, while 13,868 BES had multiple hits, and 1,252 had either low similarity or no hits