Agronomy 2012, 2, 199-221; doi:10.3390/agronomy2030199 OPEN ACCESS agronomy ISSN 2073-4395 www.mdpi.com/journal/agronomy Review Impact of Genomic Technologies on Chickpea Breeding Strategies Pooran M. Gaur *, Aravind K. Jukanti and Rajeev K. Varshney Grain Legumes Research Program, International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru 502 324, Andhra Pradesh, India; E-Mails: [email protected] (A.K.J.); [email protected] (R.K.V.) * Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +91-40-3071-3356; Fax: +91-40-3071-3074. Received: 11 June 2012; in revised form: 10 August 2012 / Accepted: 13 August 2012 / Published: 23 August 2012 Abstract: The major abiotic and biotic stresses that adversely affect yield of chickpea (Cicer arietinum L.) include drought, heat, fusarium wilt, ascochyta blight and pod borer. Excellent progress has been made in developing short-duration varieties with high resistance to fusarium wilt. The early maturity helps in escaping terminal drought and heat stresses and the adaptation of chickpea to short-season environments. Ascochyta blight continues to be a major challenge to chickpea productivity in areas where chickpea is exposed to cool and wet conditions. Limited variability for pod borer resistance has been a major bottleneck in the development of pod borer resistant cultivars. The use of genomics technologies in chickpea breeding programs has been limited, since available genomic resources were not adequate and limited polymorphism was observed in the cultivated chickpea for the available molecular markers. Remarkable progress has been made in the development of genetic and genomic resources in recent years and integration of genomic technologies in chickpea breeding has now started. Marker-assisted breeding is currently being used for improving drought tolerance and combining resistance to diseases. The integration of genomic technologies is expected to improve the precision and efficiency of chickpea breeding in the development of improved cultivars with enhanced resistance to abiotic and biotic stresses, better adaptation to existing and evolving agro-ecologies and traits preferred by farmers, industries and consumers. Agronomy 2012, 2 200 Keywords: chickpea; genomics; molecular markers; marker assisted-selection; marker-assisted breeding 1. Introduction Chickpea (Cicer arietinum L.) is second only to the common bean (Phaseolus vulgaris L.) among food legumes for production worldwide. During 2010, the global chickpea area was 12.0 million ha, production was 10.9 million MT and yield was 913 kg ha−1 [1]. Chickpea is grown in over 50 countries with 90% of its area in developing countries. Southern and South-Eastern Asia accounts for 79% of the global chickpea production. India is the largest chickpea producing country, with 68% of world chickpea production. The other major chickpea producing countries include Australia, Pakistan, Turkey, Myanmar, Ethiopia, Iran, Mexico, Canada and USA. The number of chickpea importing countries has increased from 64 in 1990 to 142 in 2009 [1] suggesting increasing global demand of the chickpea. The genus Cicer belongs to the Leguminosae family and to the sub-family Papilionoideae. Most of the Cicer species are diploids with 2n = 2x = 16 chromosomes and a genome of about 740 Mb [2]. There are two distinct types of chickpea, “Kabuli” (also known as macrosperma) and “Desi” (also known as microsperma) differing in their geographic distribution and different plant type. The desi types are found in central Asia and in the Indian subcontinent while the kabuli types are mostly found in the Mediterranean region. Kabuli types are usually taller and have large beige or cream seed color and “ram’s head” seeds with white flowers. Desi types are generally shorter, possessing small leaflets, pods, seeds and predominantly pink-colored flowers. Kabuli chickpea seems to have evolved from the desi types [3]. Chickpea is a good and cheap source of protein for people in developing countries (especially in South Asia), who are largely vegetarian either by choice or because of economic reasons. Additionally, chickpea is rich in minerals (phosphorus, calcium, magnesium, iron and zinc), fiber, unsaturated fatty acids and β-carotene. Recently, Jukanti et al., [4] summarized the nutritional qualities and health benefits of chickpea. Chickpea also improves soil fertility by fixing atmospheric nitrogen, meeting up to 80% of its nitrogen (N) requirement from symbiotic nitrogen fixation [5]. Chickpea returns a significant amount of residual nitrogen to the soil and adds organic matter, improving soil health and fertility. Growing interest in chickpea consumption, coupled with increased preference for vegetable-based protein has led to an increase in global demand for chickpea. Additionally, awareness of both economic (premium prices for large seeded kabuli) and other benefits of chickpea including human health, crop diversification and sustainable agriculture have increased interest among farmers in cultivating chickpea. Conventional breeding approaches have given over 350 improved cultivars, which have contributed to improved productivity, reduced fluctuations in yield, and enhanced adaption of chickpea to new niches [6]. Remarkable progress has been made in recent years in developing novel genetic tools, such as molecular markers, genetic maps, and genome profiling techniques to identify genomic regions, quantitative trait loci (QTL) and genes governing traits of interest [7,8]. These new advances in genomics provide exciting opportunities to researchers and breeders to utilize these new technologies Agronomy 2012, 2 201 for improving and stabilizing chickpea yield for the benefit of farmers and consumers. The aim of this review is to provide an update on progress in the development and application of molecular breeding approaches to the improvement of chickpea. 2. Breeding Methods in Chickpea During the early phases of chickpea improvement, selections from native or introduced landraces were used to develop varieties. Most recent varieties have been developed through hybridization. Single, three-way or multiple crosses are used, depending on the objective of the breeding program. Backcross breeding is often used to incorporate one or few traits, from a germplasm line or a wild species, into a well-adapted variety. Pedigree, bulk or different modifications of these methods are used in handling segregating generations. The pedigree method is not widely used, due to the cumbersome data collection and the fact that this approach limits the breeding program to only a few crosses [6]. A combination of bulk and pedigree methods is widely used among many chickpea breeding programs. In early segregating generations (F –F ), selection is done for simple traits (disease 2 3 resistance and seed traits), since selection for yield at this stage is not very effective, due to high environment effects. Single plant selection for yield generally starts from F or F . A method that uses 4 5 early generation selection for yield for eliminating inferior crosses and inferior F -derived families [9] 2 is also used in some breeding programs. There is a need to enhance precision and efficiency of selections in the segregating generations for higher and rapid genetic gains. Precision in selection for resistance/tolerance to stresses can be improved by screening under controlled environmental conditions or at hot spot locations. Genomics-assisted breeding (GAB) approaches, such as marker-assisted selection (MAS), can greatly improve precision and efficiency of selection in crop breeding [10]. Several success stories of GAB to develop superior varieties are available in temperate cereals [11] and soybean [12]. Integration of genomics tools in chickpea breeding programs has a great potential for crop improvement [13]. For instance, molecular markers can facilitate indirect selection for traits that are difficult or inconvenient to score directly (e.g., root traits, resistance to root knot nematodes), pyramiding genes from different sources (e.g., bringing together ascochyta blight resistance genes from different donors) and combining resistance to multiple stresses (e.g., resistance to fusarium wilt and ascochyta blight). Single seed descent (SSD) and rapid generation advancement [14] methods are being used at present to reduce the time required to reach the desired level of homozygosity. Development of double-haploids is another desired approach for saving time in reaching homozygosity. There is a recent report on success in regenerating double-haploids through anther culture in chickpea [15], which opens opportunities for exploiting haploid technology in chickpea breeding programs. The wild Cicer species are valuable gene pools, particularly for resistance to biotic and abiotic stresses [16,17]. These have largely remained under-utilized due to crossability barriers, but there are some examples of successful introgression of genes into the cultivated species from two closely related species, Cicer reticulatum and C. echinospermum [17]. Mutation breeding is another approach to improve a well-adapted variety for a deficient trait or creating a novel variability. Several chickpea varieties have been developed through mutation breeding [6]. Agronomy 2012, 2 202 Efficient transformation and regeneration protocols are now available for chickpea [18], which have made it possible to introduce any desired gene from any source into chickpea. Transgenic plants are being developed to improve traits which have no or limited genetic variability in the cultivated and cross-compatible wild species (primary and secondary gene pools), e.g., resistance to pod borer. 3. Major Constraints to Chickpea Production The major constraints limiting chickpea production globally include various abiotic and biotic stresses. Drought is the most important constraint to yield in chickpea, accounting for about 50% yield reduction globally. It generally occurs at the terminal stage as the crop is mostly raised on conserved soil moisture under rain-fed conditions. In addition to terminal drought, heat stress at the reproductive stage has become a major constraint to chickpea production because of (1) a large shift in chickpea area from cooler long-season environments to warmer short-season environments, (2) increased area under late sown conditions, due to increased cropping intensity, and (3) an expected overall increase in temperatures due to climate change. Soil salinity is another important abiotic constraint in some chickpea growing areas of the world. Fusarium wilt (FW), caused by Fusarium oxysporum f. sp. ciceri, dry root rot caused by Rhizoctonia bataticola, and collar rot, caused by Sclerotium rolfsi, are the important root diseases of chickpea in areas where the chickpea growing season is dry and warm, e.g., southern and eastern Asia and eastern Africa. All these diseases cause plant mortality. In general, collar rot and early- stage fusarium wilt causes mortality at the seedling stage, whereas dry root rot and late- fusarium wilt kills plants during flowering and the pod-filling stages. Ascochyta blight (AB), caused by Ascochyta rabiei (Pass.) Labr., and botrytis grey mold (BGM) caused by Botrytis cineria Pres., are the important foliar diseases of chickpea in the areas where the chickpea growing season is cool and humid. AB is important in west and central Asia, North Africa, North America, Australia, northern India and Pakistan, while BGM is important in Nepal, Bangladesh, northern India and Australia. Sources with absolute resistance (score 1 on a scale of 1–9, where 1 = resistant and 9 = susceptible) to these diseases are not available in the cultivated and the wild species accessions examined so far. Pod borer (Helicoverpa armigera Hubner) is the most important pest of chickpea worldwide. It is a highly polyphagous pest and can feed on various plant parts, such as leaves, tender shoots, flower buds, and immature seeds. The extent of global losses to chickpea by this pest is estimated at over US$ 500 million [19]. The viral diseases, rust (Uromyces ciceris-arietini), root nematodes (Meloidogyne sp.), Phytophthora root rot (Phytophthora medicaginis), cutworm (Agrotis sp.) and leaf miner (Liriomyza cicerina) are also important in some chickpea growing areas. 4. Breeding Achievements Over 350 improved varieties of chickpea have been released globally, and about half of these have been released in India, which has the largest national chickpea breeding program in the world [6]. Excellent progress has been made in the development of early maturing cultivars, which can escape terminal drought and heat stresses [20]. The first landmark variety was ICCV 2, which matures in about 85 days, and it is perhaps the world’s earliest maturing variety of kabuli chickpea. It has been instrumental in extending kabuli chickpea cultivation to short-season environments of India and its Agronomy 2012, 2 203 neighboring country Myanmar. This short-duration variety covers over 50% of the chickpea area in Myanmar [21]. Several short-duration high yielding varieties of chickpea, both in desi and kabuli types, have been developed [6,20,22]. Further advancements have been made in breeding for super-earliness in chickpea. Two super early desi chickpea lines, ICCV 96029 and ICCV 96030, were developed which mature in 75 to 80 days in southern India [23]. High root biomass has been found to be associated with drought tolerance [24–26], and a positive relationship has been established between root biomass and seed yield under drought conditions [27]. Efforts are being made to develop varieties with a vigorous and deeper root system to improve drought tolerance [28]. A large genetic variation for heat tolerance has been identified in chickpea [8,29]. A heat tolerant breeding line ICCV 92944 has been released for cultivation in Myanmar (Yezin 6) and India (JG 14). Few varieties with tolerance to moderate levels of salinity (ECe ranging from 4 to 6 dS/m) have been developed, e.g., Karnal Chana 1 (CSG 8962) in India and Genesis 836 (ICCV 96836) in Australia [30]. Recent screening for salinity tolerance at ICRISAT identified several lines that gave a higher yield than the salinity tolerant cultivar Karnal Chana 1 under saline conditions [29,31]. Excellent progress has been made in the development of cultivars with high levels of resistance to FW, both in desi and kabuli types, because of the availability of highly resistant sources and simple and effective field screening techniques. Similar success has not been possible in breeding for resistance to ascochyta blight [32], BGM [33] and any other disease. Nevertheless, there has been a significant impact of incorporating current levels of resistance to ascochyta blight into adapted backgrounds in countries such as Australia. Development of cultivars resistant to pod borer also remained a challenge due to non-availability of sources with high levels of resistance. Higher levels of resistance were observed in some wild species [34]. Efforts are also being made to combine the non-preference (antixenosis) mechanism of resistance identified in the cultigen (e.g., ICC 506 EB) and antibiosis mechanism of resistance identified in C. reticulatum [17]. 5. Genetic and Genomic Resources in Chickpea 5.1. Germplasm The chickpea germplasm is maintained by different institutes across the world, including the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), India, and the International Centre for Agricultural Research in Dryland Areas (ICARDA), Syria (Table 1). Although a large number of cultivated and wild chickpea accessions are available, there is a large void in utilizing these accessions in breeding programs. The major limitation preventing their utilization has been lack of information on major economic traits. Additionally, screening a large number of germplasm lines in multi-location trials is both expensive and time-consuming. After evaluating about 16,990 chickpea accessions for 13 traits, a core collection of 1956 accessions has been developed at ICRISAT [35]. Further, a mini-core collection of 211 chickpea accessions has also been developed [36]. A reference set of 300 lines has been developed jointly by ICRISAT and ICARDA under the Generation Challenge Program (GCP) of the CGIAR to represent genetic variability present in the germplasm available at the two institutions [37]. The core, mini-core and reference sets of germplasm provide cost-effective and manageable entry points to initiate screening for different Agronomy 2012, 2 204 traits [38]. With the objective of trait mapping using genome-wide association studies, the reference set of chickpea is being genotyped with a large number of DArT and SNP markers and is being phenotyped for many traits both in the field and under controlled environmental conditions. Table 1. Chickpea germplasm held at global research institutes *. Institute Number of Accessions 1 International Crops Research Institute for the Semi-Arid 20,267 Tropics (ICRISAT), India 2 National Bureau of Plant Genetics Resource (NBPGR), 14,704 India 3 International Centre for Agricultural Research in 13,462 Dryland Areas (ICARDA), Syrian Arab Republic 4 Australian Temperate Field Crops Collection, Australia 8,655 5 Western Regional Plant Introduction Station, 6,763 USDA-ARS, Washington State University, USA 6 National Plant Gene Bank of Iran, Iran 5,700 7 Plant Genetic Resources Institute, Pakistan 2,146 8 N.I. Vavilov All-Russian Scientific Research Institute 2,091 of Plant Industry, Russian Federation 9 Plant Genetic Resources Department, Turkey 2,076 10 Institute of Biodiversity Conservation, Ethiopia 1,173 * adapted from Upadhyaya et al., [8]. 5.2. Mapping Populations The mapping populations generally used in linkage mapping include F , F -derived F progeny, 2 2 3 backcrosses, doubled haploid, recombinant inbred lines (RILs) and near-isogenic lines (NILs). Some of the studies have used F populations and F progenies in linkage mapping of chickpea [39–41], but 2 3 most of the studies have used RILs. The RIL mapping populations are of immense value in linkage mapping, as they are immortal and phenotyping can be replicated over locations and years. Several interspecific and intraspecific RIL populations are available in chickpea. The traits being targeted for mapping include resistance to AB, FW and BGM; root traits; salinity tolerance and protein content. Development of a multi-parent advanced generation inter-cross (MAGIC) population is in progress at ICRISAT. This population is being developed from eight parents and includes cultivars and elite breeding lines from India and Africa. Twenty-eight two-way, 14 four-way and 7 eight-way crosses were made to develop this MAGIC population. The large number of accumulated recombination events in MAGIC populations increase novel rearrangements of alleles and bring about greater genetic diversity. MAGIC populations provide a platform for a community-based approach to gene discovery, characterization and deployment of genes for understanding complex traits [38]. In addition to trait mapping, the highly recombined MAGIC population may be used directly as source material for the development of improved cultivars. Agronomy 2012, 2 205 5.3. Development of Molecular Markers Important pre-requisites for undertaking molecular breeding are molecular markers, genetic maps and markers associated with traits [42]. Isozyme markers were used for map development in chickpea during the early days of genomic studies. But, expression of these markers was influenced by the environment and their number was small, coupled with a low level of polymorphism in cultivated genotypes of chickpea. In some earlier studies, RFLP and RAPD markers were also used for genetic mapping and diversity studies [43]. However, the extensive use of molecular markers in chickpea genetics and breeding started only after development of simple sequence repeat (SSR) or microsatellite markers. SSR markers have become the marker of choice in plant breeding due to their multi-allelic and co-dominant nature [44]. Both genomic and transcript datasets have been utilized to develop SSR markers. Several hundred SSR markers have been developed from genomic DNA libraries [45–48]. Recently, Nayak et al., [49] have developed a set of 311 novel SSR markers, designated as “ICRISAT Chickpea Microsatellite” (ICCM) markers, obtained from a SSR-enriched genomic library of chickpea accession, ICC 4958. SSR markers have also been mined from expressed sequence tags (ESTs) [50,51]. Varshney et al., [52] have identified a total of 3728 SSR and designed primer pairs for 177 new EST-SSR markers. In another study, generating and mining of 435,018 454/FLX sequences and 21,491 Sanger ESTs produced a total of 103,215 tentative unique sequences (TUSs) [53]. The TUSs were used to identify 26,252 SSRs and primer pairs were designed for 3172 SSRs. This resulted in 728 non-redundant SSR primer pairs. Another source for developing SSR markers is BAC libraries. Lichtenzveig et al., [54] developed and characterized 233 SSRs from BAC libraries. In another study, Thudi et al., [55] obtained 6845 SSRs by mining 46,270 BESs and primer pairs that were designed for only 1344 SSRs. In summary, primer pairs are available for about 2000 SSR markers. Single nucleotide polymorphism (SNP) markers have drawn greater attention in recent years due to their higher abundance and amenability to high-throughput approaches. About 21,405 high confidence SNPs were identified from 742 EST contigs along with several SSR and EST-SSR markers [52]. In another study by Gujaria et al., [51] a total of 1893 SNPs were identified by scanning 220 candidate genic regions in chickpea. Chickpea transcriptomic analysis identified a set of 495 SNPs from 103,215 TUSs [53]. The average frequency of SNPs (1/35.83 bp) discovered in this study was higher than previously reported by Rajesh and Muehlbauer [56], 1/61 bp in coding regions and 1/71 bp genomic regions. Diversity arrays technology (DArT) is a high throughput genome analysis method enabling a rapid and economical approach for screening a large number of marker loci in parallel [57]. DArT technology utilizes the microarray platform to analyze DNA polymorphism. DArT markers have been used for different purposes: (i) developing high density genetic maps and (ii) studying genetic diversity in crops like barley [58], wheat [59] and pearl millet [60]. In the case of chickpea, DArT arrays comprising 15,360 DArT clones generated from 94 diverse genotypes have been developed [55]. This study showed polymorphism with a total of 5397 markers in the germplasm examined in the study. The number of polymorphic markers in intra- and inter-specific chickpea populations ranged from 35–496 and 210–906, respectively. Polymorphism information content (PIC) values obtained for chickpea were comparable to other crops, such as sorghum and cassava [61,62]. Agronomy 2012, 2 206 5.4. Linkage Mapping The limited polymorphism exhibited by cultivated chickpea for the molecular markers developed in the early phase forced researchers to use interspecific crosses in linkage mapping of chickpea. The first linkage map of chickpea was developed by Gaur and Slinkard [39,40] using isozyme markers and inter-specific crosses of C. arietinum with C. reticulatum and C. echinospermum. Later, DNA-based molecular markers, such as randomly amplified polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP) were integrated into the chickpea linkage map by Simon and Muehlbauer [43]. The RIL population of C. arietinum (ICC 4958) × C. reticulatum (PI 489777) has been considered as the reference mapping population and extensively used for genome mapping [51,55,63]. The first integrated genetic map based on this population comprised 37 inter simple sequence repeats (ISSRs), 70 amplified fragment length polymorphisms (AFLPs), 118 sequence-tagged microsatellite sites (STMSs), 96 DNA amplification finger printings (DAFs), 17 RAPDs, 3 cDNAs, 8 isozymes and 2 sequence characterized amplified regions (SCARs), covering a total distance of 2077.9 centimorgans (cM) [63]. An advanced genetic map with 521 markers, including simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs), with an inter marker distance of 4.99 cM spanning 2602.1 cM, was developed from the above population [49]. In this map, Nayak et al., [49] integrated 71 SNP loci, based on gene-specific primers developed by Choi et al., [64]. This effort demonstrated the power of a comparative genomics approach (Medicago truncatula/sativa and Cicer arietinum) to identify molecular tools. The genetic maps developed based on genes are also referred to as transcript maps [65]. In the case of chickpea, Gujaria et al., [51] has developed a transcript map that comprises of 300 loci (including 126 genic molecular markers [GMMs]) and spans a genomic region of 766.56 cM. Cho et al., [66] developed the first intra-specific map of cultivated chickpea from ICCV 2 × JG 62 RILs. The map was developed using 3 ISSRs, 20 RAPDs, 55 STMSs along with two phenotypic markers comprising 14 linkage groups and spanning 297.5 cM. The map was used to map genes for important morphological traits along with the double podded character. An intraspecific chickpea genome map consisting of 51 STMS, 3 inter-simple sequence repeats (ISSRs) and 12 resistance gene analogs (RGA) mapped to eight linkage groups was developed using an F population of chickpea [67]. 2 Another map developed based on a “Kabuli × Desi” cross included a total of 134 molecular markers (3 ISSR, 13 STMSs AND 118 RAPDs) mapped to 10 linkage groups [68]. This map spanned a genomic region of 534.5 cM with an average marker interval of 8.1 cM. Radhika et al., [69] developed an integrated intraspecific map spanning a region of 739.6 cM, including 230 markers at an average distance of 3.2 cM between markers. Another map developed recently included 144 markers assigned to 11 linkage groups, spanning 442.8 cM, with an average marker interval of 3.3 cM [70]. Consensus genetic maps using both interspecific and intraspecific populations were also developed. A consensus map based on five interspecific (C. arietinum × C. reticulatum) and five intraspecific (Desi × Kabuli types) populations was developed [71]. It integrated 555 marker loci including, RAPDs (251), STMSs (149), AFLPs (47), 33 cross-genome markers, 28 gene-specific markers, 10 isozyme markers, 10 inter-simple sequence repeats (ISSRs) and 7 RGA loci. Several other linkage maps were developed using different mapping populations with different morphological and molecular Agronomy 2012, 2 207 markers [66,67,72–74]. More recently, a comprehensive genetic map spanning 845.56 cM was developed using RILs from C. arietinum (ICC 4958) × C. reticulatum (PI 489777) [55]. In total, it included 1291 markers on eight linkage groups (LGs). The highest number of markers per LG was on LG 3 (218) and the lowest was on LG 8 (68), with an average inter-marker distance of 0.65 cM. In several cases, the mapping populations used for developing the maps were also phenotyped for the segregating traits. Analysis of phenotyping data together with genotyping data in some cases identified molecular markers associated with the genes/quantitative trait loci (QTLs), controlling resistance to key diseases (ascochyta blight, fusarium wilt, botrytis grey mold, rust), morphological traits (single pod vs. double pod, flowering time and flower color), seed yield and yield components, etc. (Table 2). Table 2. List of some QTLs/genes identified for different traits in chickpea. Trait Number & Name Marker References of Gene/QTL Ascochyta blight AR2, ar1, ar1a, SSRs, RAPDs, DAF [73–77] ar1b, ar2a, ar2b, Ar19 QTL , QTL SCARs, SSRs, [78,79] AR1 AR2 RAPDs 13 QTL SSRs, RAPDs [41,63,67,75,80,81] 5 QTL (1-5) SSRs [82] 3 QTL SSR [83] Fusarium wilt foc-0, foc-1, foc-2, SSRs, STSs, RAPDs, [63,68,75,78,84] foc-3, foc-4, foc-5 Botrytis grey mold 3 QTL (1, 2, 3) SSRs [70] Rust 1 QTL SSR [85] Seed size traits 2 QTL SSRs [86–88] Seed yield 1 QTL SSRs [89] Seed yield components 1 QTL SSRs [89] Single or double pod s SSRs [66,68,90] Flowering time 2 QTL SSRs [87,91] 2 QTL SSRs [83] Beta carotene, leutin, 4 QTL for carotene; SSRs [92] seed weight 3 QTL for seed weight Flower color B/b SSR [68] Agronomy 2012, 2 208 5.5. Physical Mapping Several genetic linkage maps have been developed and markers linked to different traits have been identified in chickpea. Though these markers can be used in marker-assisted selection (MAS) for improving the trait, the molecular basis of traits remains unknown. Isolation and validation of genes underlying the QTL/genes for the traits of interest is an essential step to determine gene function. Development of a genome-wide physical map or local physical map around the QTL region and then sequencing those region(s) are the next steps in this direction. Large-insert arrayed DNA libraries, like BAC and plant transformation competent binary BAC libraries, are essential resources for developing the physical map. Large-insert BAC libraries representing several-fold coverage of the genome have been developed in several crops. In the case of chickpea, a BIBAC library consisting of 23,780 clones, with an average insert size of 100 kb and covering about 3.8X genomes of chickpea was developed [93]. However, multi-enzyme BAC libraries with higher genome coverage are required for comprehensive genome research [94–96]. Subsequently, Lichtenzveig et al., [54] developed a BAC and BIBAC library in chickpea from the Hadas genotype; digested with HindIII and BamHI enzymes, respectively. These BAC and BIBAC libraries consist of 14,796 and 23,040 clones, respectively, with an average insert size of 121 kb (BAC) and 145 kb (BIBAC). These libraries jointly represent about 7.0X genome coverage of chickpea. Furthermore, Zhang et al., [97] constructed BAC and BIBAC libraries consisting of 22,272 and 38,400 clones, respectively. Analysis of random clones showed combined genome coverage of 11.5X. Very recently, a BAC-library was developed from ICC 4958 that comprise 55,680 clones digested with HindIII [55]. Sequencing of 25,000 clones has provided 46,270 BAC-end sequences (BESs) that were used to develop SSR markers. SSR markers (157), derived from BESs, have been integrated into the genetic map based on the ICC 4958 × PI 489777 population. In terms of physical mapping, Zhang et al., [97] developed a BAC/BIBAC-based physical map of chickpea. It consists of 1945 contigs and each contig contains an average of 28.3 clones and has an average physical length of 559 kb. In total, the contigs span about 1088 Mb. Using this map, they were able to identify BAC/BIBAC contigs containing or close to QTL, governing resistance to Didymella rabiei and QTL—responsible for days to first flower. A pioneer work towards integration of genetic- and chromosome-based physical maps has been done recently by Zatloukalová et al., [98]. They have been able to assign linkage groups (LG) in chickpea to different chromosomes using flow cytometry and PCR-based primers that amplify sequence-tagged microsatellite site markers. Using this approach, they were able to assign LGs: LG8 to chromosome H, LG5 to chromosome A, LG4 to chromosome E and LG3 to chromosome B. The two chromosomes (C & D) could not be sorted out; therefore, they were jointly assigned to LG6 and LG7. Similarly, LG1 and LG2 were assigned to chromosomes F and G. This ability to isolate individual chromosomes will be useful in high-throughput physical mapping. This could also be used to discover genes and determine the order of low-copy genic regions on a chromosome as was done in wheat [99,100] and barley [101,102].