Integrative Physical and Genetic Mapping of the Chickpea Genome for Fine Mapping and Analysis of Agronomic Traits

Abstract

Chickpea is the third most important pulse crop in the world and ranks first in the Middle East; however, it has been subjected to only limited research in modern genomics. In the first period of this project (US-3034-98R) we constructed two large-insert BAC and BIBAC libraries, developed 325 SSR markers and mapped QTLs controlling ascochyta blight resistance (ABR) and days to first flower (DTF). Nevertheless, the utilities of these tools and results in gene discovery and marker-assisted breeding are limited due to the absence of an essential platform. The goals of this period of the project were to use the resources and tools developed in the first period of the project to develop a BAC/BIBAC physical map for chickpea and using it to identify BAC/BIBACcontigs containing agronomic genes of interest, with an emphasis on ABR and DTF, and develop DNA markers suitable for marker-assisted breeding. Toward these goals, we proposed: 1) Fingerprint ~50,000 (10x) BACs from the BAC and BIBAC libraries, assemble the clones into a genome-wide BAC/BIBAC physical map, and integrate the BAC/BIBAC map with the existing chickpea genetic maps (Zhang, USA); 2) fine-map ABR and DTFQTLs and enhance molecular tools for chickpea genetics and breeding (Shahal, Sherman and DaniShtienberg, Israel; Chen and Muehlbauer; USA); and 3) integrate the BAC/BIBAC map with the existing chickpea genetic maps (Sherman, Israel; Zhang and Chen, USA). For these objectives, a total of $460,000 was requested originally, but a total of $300,000 was awarded to the project. We first developed two new BAC and BIBAC libraries, Chickpea-CME and Chickpea- CHV. The chickpea-CMEBAC library contains 22,272 clones, with an average insert size of 130 kb and equivalent to 4.0 fold of the chickpea genome. The chickpea-CHVBIBAC library contains 38,400 clones, with an average insert size of 140 kb and equivalent to 7.5 fold of the chickpea genome. The two new libraries (11.5 x), along with the two BAC (Chickpea-CHI) and BIBAC (Chickpea-CBV) libraries (7.1 x) constructed in the first period of the project, provide libraries essential for chickpea genome physical mapping and many other genomics researches. Using these four libraries we then developed the proposed BAC/BIBAC physical map of chickpea. A total of 67,584 clones were fingerprinted, and 64,211 (~11.6 x) of the fingerprints validated and used in the physical map assembly. The physical map consists of 1,945 BAC/BIBACcontigs, with each containing an average of 39.2 clones and having an average physical length of 559 kb. The contigs collectively span ~1,088 Mb, being 1.49 fold of the 740- Mb chickpea genome. Third, we integrated the physical map with the two existing chickpea genetic maps using a total of 172 (124 + 48) SSR markers. Fourth, we identified tightly linked markers for ABR-QTL1, increased marker density at ABR-QTL2 and studied the genetic basis of resistance to pod abortion, a major problem in the east Mediterranean, caused by heat stress. Finally, we, using the integrated map, isolated the BAC/BIBACcontigs containing or closely linked to QTL4.1, QTL4.2 and QTL8 for ABR and QTL8 for DTF. The integrated BAC/BIBAC map resulted from the project will provide a powerful platform and tools essential for many aspects of advanced genomics and genetics research of this crop and related species. These includes, but are not limited to, targeted development of SNP, InDel and SSR markers, high-resolution mapping of the chickpea genome and its agronomic genes and QTLs, sequencing and decoding of all genes of the genome using the next-generation sequencing technology, and comparative genome analysis of chickpea versus other legumes. The DNA markers and BAC/BIBACcontigs containing or closely linked to ABR and DTF provide essential tools to develop SSR and SNP markers well-suited for marker-assisted breeding of the traits and clone their corresponding genes. The development of the tools and knowledge will thus promote enhanced and substantial genetic improvement of the crop and related legumes.