EST-derived genic molecular markers: development and utilization for generating an advanced transcript map of chickpea

Abstract

Well-saturated linkage maps especially those based on expressed sequence tag (EST)-derived genic molecular markers (GMMs) are a pre-requisite for molecular breeding. This is especially true in important legumes such as chickpea where few simple sequence repeats (SSR) and even fewer GMM-based maps have been developed. Therefore, in this study, 2,496 ESTs were generated from chickpea seeds and utilized for the development of 487 novel EST-derived functional markers which included 125 EST-SSRs, 151 intron targeted primers (ITPs), 109 expressed sequence tag polymorphisms (ESTPs), and 102 single nucleotide polymorphisms (SNPs). Whereas ESTSSRs, ITPs, and ESTPs were developed by in silico analysis of the developed EST sequences, SNPs were identified by allele resequencing and their genotyping was performedusing the Illumina GoldenGate Assay. Parental polymorphism was analyzed between C. arietinum ICC4958 and C. reticulatum PI489777, parents of the reference chickpea mapping population, using a total of 872 markers: 487 new gene-based markers developed in this study along with 385 previously published markers, of which 318 (36.5%) were found to be polymorphic and were used for genotyping. The genotypic data were integrated with the previously published data of 108 markers and an advanced linkage map was generated that contained 406 loci distributed on eight linkage groups that spanned 1,497.7 cM. The average marker density was 3.68 cM and the average number of markers per LG was 50.8. Among the mapped markers, 303 new genomic locations were defined that included 177 gene-based and 126 gSSRs (genomic SSRs) thereby producing the most advanced gene-rich map of chickpea solely based on co-dominant markers.

GENETIC RELATIONSHIPS BETWEEN CULTIVARS OF Cicer arietinum AND ITS PROGENITOR GROWN IN TURKEY DETERMINED BY USING THE SSR MARKERS

“…Some researchers have previously demonstrated genetic variation in chickpea using SSR markers. For instance, Choudhary et al (2012) used 48 chickpea cultivars and detected 2 to 11 alleles and an average of 4.8 alleles per locus using 100 SSR markers. Saeed et al (2011) obtained 2 to 13 alleles among 44 chickpea genotypes using 16 SSR markers while Singh et al (2008) analyzed 21 chickpea cultivars using 18 SSR markers and detected 2 to 5 alleles.…”

Advances in Chickpea Genomics

“…Further, this map was integrated with BES-SSRs, DArT and genebased markers by Thudi et al ( 2011 ), which comprised of 1,291 loci. An advanced gene-rich map of chickpea comprising of 406 loci (including 177 gene-based markers) spanning 1,497.7 cM genetic distance has been developed for this reference population (Choudhary et al 2012b ). Recently, Hiremath et al ( 2012 ) developed large-scale KASPar assays for SNP genotyping and developed a genetic map comprising 1,328 marker loci including novel 625 CKAMs (Chickpea KAspar Assay Markers), 314 TOG-SNPs and 389 published marker loci for this reference population.…”

Development of gene-based markers for use in construction of the chickpea (Cicer arietinum L.) genetic linkage map and identification of QTLs associated with seed weight and plant height

“…The parameters set for identifying di-, tri-, tetra-and pentanucleotide motifs were a minimum of 5, 4, 3 and 3 repeats, respectively. Primer pairs flanking the SSRs were designed using the program Primer 3.0 (http://frodo.wi.mit.edu/primer3/) [31] according to the criteria mentioned by Choudhary et al [12]. The markers were designated as 'CESSR' in continuation with the series of CESSR markers reported by Choudhary et al [12].…”

“…The PIP markers were designated as 'PIP' in continuation with the series of PIP markers reported by Choudhary et al [12].…”

“…Studies related to linkage analysis and molecular mapping have been carried out in a large percentage of known plant species using various GMMs [5,[7][8][9]. Fewer reports are available on EST-based markers in chickpea [10][11][12][13][14]. Although chickpea genome has been sequenced [1] and around 11,000 gene-based microsatellite markers have been developed in the recent years from the chickpea ESTs [10][11][12] and next-generation transcript sequences [6,13,14], only a small percentage (*5-10 %) of these have been validated experimentally and used for various applications in chickpea such as genotyping and QTL mapping.…”