Novel SSR markers from BAC-end sequences, DArT arrays and a comprehensive genetic map with 1,291 marker loci for chickpea (Cicer arietinum L.)

Abstract

Chickpea (Cicer arietinum L.) is the third most important cool season food legume, cultivated in arid and semi-arid regions of the world. The goal of this study was to develop novel molecular markers such as microsatellite or simple sequence repeat (SSR) markers from bacterial artificial chromosome (BAC)-end sequences (BESs) and diversity arrays technology (DArT) markers, and to construct a high-density genetic map based on recombinant inbred line (RIL) population ICC 4958 (C. arietinum)×PI 489777 (C. reticulatum). A BAC-library comprising 55,680 clones was constructed and 46,270 BESs were generated. Mining of these BESs provided 6,845 SSRs, and primer pairs were designed for 1,344 SSRs. In parallel, DArT arrays with ca. 15,000 clones were developed, and 5,397 clones were found polymorphic among 94 genotypes tested. Screening of newly developed BES-SSR markers and DArT arrays on the parental genotypes of the RIL mapping population showed polymorphism with 253 BES-SSR markers and 675 DArT markers. Segregation data obtained for these polymorphic markers and 494 markers data compiled from published reports or collaborators were used for constructing the genetic map. As a result, a comprehensive genetic map comprising 1,291 markers on eight linkage groups (LGs) spanning a total of 845.56 cM distance was developed (http://cmap.icrisat.ac.in/cmap/sm/cp/thudi/). The number of markers per linkage group ranged from 68 (LG 8) to 218 (LG 3) with an average inter-marker distance of 0.65 cM. While the developed resource of molecular markers will be useful for genetic diversity, genetic mapping and molecular breeding applications, the comprehensive genetic map with integrated BES-SSR markers will facilitate its anchoring to the physical map (under construction) to accelerate map-based cloning of genes in chickpea and comparative genome evolution studies in legumes.

Figure 1. Distribution of Class I and Class II repeats in newly isolated chickpea microsatellites.

Class I microsatellites contain >20 nucleotides, Class II repeats perfect SSRs with >12 but <20 nucleotides. Among Class I repeats, tri-nucleotide repeats were most abundant, followed by di-nucleotide repeats, while in Class II repeats, penta-nucleotide repeats were most prevalent, followed by hexa-repeats. N, mono-nucleotide repeats; NN, di-nucleotide repeats; NNN, tri-nucleotide repeats; NNNN, tetra-nucleotide repeats; NNNNN, penta-nucleotide repeats, NNNNNN, hexa-nucleotide repeats.

Figure 2. Distribution of microsatellites with varying repeat units in BAC-end sequences.

N: mono-nucleotide repeats; NN: di-nucleotide repeats; NNN: tri-nucleotide repeats; NNNN: tetra-nucleotide repeats; NNNNN: penta-nucleotide repeats and NNNNNN: hexa-nucleotide repeats.

Figure 3. Number of BES-SSR markers in different PIC value classes.

The number of di-, tri-, tetra-, penta-, hexa- nucleotide repeats and compound SSRs in different PIC value classes are in blue, light blue, yellow, purple, dark red and green respectively.

Figure 4. Number of DArT markers in different PIC value classes.

Polymorphic markers have been grouped into five classes of PIC values namely 0.01– 0.10, 0.11–0.20, 0.21– 0.30, 0.31– 0.40 and 0.41–0.50.

Figure 5. Interspecific reference genetic map with 1,291 loci, spanning 845.56 cM.

The map distance is indicated on the left and the marker names on the right side of each linkage group. Each linkage group is divided into 10 cM bins. Marker series are colour coded: CaM (red), DArT (brown), ICCeM (green), CISR (light green), COS-SNP (pink), CAPS (blue) and legacy markers (black).