1. Task Title : Completing genotyping of composite set of chickpea:

2. Involved institutions:

1. International Crops Research Institute for the semi-Arid Tropics (ICRISAT), Patancheru, India 502 324.

2. International Center for Agricultural Research in the Dry Areas (ICARDA), P.O. Box 5466, Aleppo, Syria.

3. Task Leader: HD Upadhyaya, ICRISAT

4. Scientists involved:

         - ICRISAT: Legume Genomics Scientist, PM Gaur, CLL Gowda, S Chandra

         - ICARDA: SM Udupa, BJ Furman, M Baum

5. Background information:

Chickpea ranks third among pulses, fifth among grain legumes, and 15^th among grain crops of the world. Chickpea accounts for 12% of the world pulses production, with a 1.9% annual growth rate of chickpea production during the last 20 years. There are two major types of chickpea: Desi types and kabuli types. Desi and Kabuli production ratio is 3:1. Chickpea is a rich and cheap source of protein (US $ 0.5 to 1.0 kg^-1) in comparison with animal protein (US $ 3 kg^-1). It is grown on 9.89 million ha in 43 countries of the world, with an annual productivity of 0.79 t ha^-1. Asia is the largest producer of chickpea (87.4% of the 7.8 M t) followed by North Central America (5.2%) and Africa (4.5%). chickpea productivity is rather low due to various biotic and abiotic stresses. Drought accounts for about half of the total yield loss caused by both biotic and abiotic stresses. Maximum yield losses due to drought across SAT and WANA region, where chickpea is an important crop in various production systems, range from 40-60%. Global yield loss due to drought in chickpea is estimated to be around 3.7 million tons and around 2.1 million tons of this can be recovered through crop improvement efforts.

An important goal of the Generation Challenge Program (GCP) is extensive genetic characterization, using molecular markers, of vast genetic resources (including wild relatives, landraces, breeding materials, cultivars and genetic stocks) held by the participating institutions. Through GCP commissioned funds in 2004, we developed a global composite collection of 3,000 accessions drawn from the core collections, trait donor parental lines, landraces, elite germplasm and cultivars, and wild Cicer species. Of these 3,000 accessions, 288 have been genotyped with 50 SSR loci during 2004. This commissioned grant is proposed to genotype the remaining 2712 (2714, because 2 accessions out of 288 have been replaced) accessions using the same 50 SSR loci, which have been used to characterize the earlier subset of 288 accessions.

Microsatellite have become extremely popular molecular markers for their application in phylogenetic analysis (Udupa et al. 1999)  and molecular mapping (Winter et al. 2000, Udupa and Baum 2003) in chickpea.  Most of the observed variations in microsatellite length are by the 1 repeat unit ( Udupa and Baum 2001).

Winter P, Pfaff T, Udupa SM, Huettel B, Sharma PC, Sahi S, Arreguin-Espinoza R, Weigand F, Muehlbauer FJ, Kahl G(1999) Characterization and mapping of sequence-tagged microsatellite sites in the chickpea (Cicer arietinum L.) genome. Mol Gen Genet 262:90–101

Udupa SM, Robertson LD, Weigand F, Baum M, Kahl G (1999) Allelic variation at (TAA)n microsatellite loci in a world collection of chickpea (Cicer arietinum L.) germplasm. MolGen Genet 261:354–363

Udupa SM, Baum M (2001) High mutation rate and mutational bias at (TAA)n microsatellite loci of chickpea (Cicer arietinum L.). Mol Genet Genomics 265:1097–1103

Udupa SM, Baum M (2003) Genetic dissection of pathotypespecific resistance to ascochyta blight disease in chickpea (Cicer arietinum L.) using microsatellite markers. Theor Appl Genet 106:1196–1202

6. Rationale and objective:

The application of anonymous markers to germplasm of a given crop has a very high added value. This allows structuring the collections, targeting new perspectives, rationalizing breeding strategies etc. It also allows determination of representative collections for phenotyping efforts and association studies using candidate genes orthologous to those that are proven on the most advanced crops.

The main objective of this commissioned grant is the marker analysis of a subset of 2714 chickpea accessions. This will result in defining the genetic structure of the global chickpea composite germplasm collection for functional and comparative genomics studies in the challenge program.

The analysis of genetic diversity will help to elucidate population structures that influence the analysis of the associations between markers and phenotypes, which will be performed later in the GCP. Based on the genotypic data set generated, a subset of about 300 accessions will be identified for detailed study of various traits of economic importance including intensive functional genomics analysis. Subsequently, additional SSR and orthologous markers will be screened across  this subset. Genetically broad-based mapping and breeding populations could be developed using information generated in this project.

7. Foreseen activities:

a) Growing plants and extracting DNA for 2714 accessions (2 out of the 288 accessions used earlier have been replaced by ICARDA) accessions. ICRISAT will grow and extract DNA from all 2714 accessions and share aliquots with ICARDA. This will result in uniform methodology and quality of DNA.

b) Genotyping for 50 SSR loci. ICRISAT will work on 35 SSR markers and ICARDA on 15 SSR markers on all the 2714 accessions. PCR primer pairs to be utilized for amplification of genomic microsatellite DNA sequences are the same which have been tested, optimized and used in year 1 on 288 accessions.

Data will be analyzed statistically using the appropriate software recommended by the SP4.

ICRISAT: Multifluorophore fragment analysis will be carried out on an ABI3700 and with GenMapper software.

ICARDA: Multifluorophore fragment analysis can be carried out on an ABI prism ^TM 377 DNA Sequencer and analyzed with GeneScan^TM analysis software version 2.0.2 and Genotyper ^TM analysis software version 2.0 (PE Applied Biosystems) and ABI3100 capillary sequencer with GenMapper software (partially bought with financial support of the Challenge Program).

8. In-kind contributions:

The in-kind contribution from ICRISAT and ICARDA will be in terms of salaries of the scientists and technicians, equivalent to budget for salary/benefits available from CP.

9. Budget: Total budget 137k US $

                   Budget by Activity

Activity 1 = Growing plants and extracting DNA for 2714 accessions.

Activity 2 = Screening with 50 SSR markers

Budget lines by Institution

(A)   ICRISAT

(B)   ICARDA