Identification of Quantitative Trait Loci Controlling Root and Shoot Traits Associated with Drought Tolerance in a Lentil (Lens culinaris Medik.) Recombinant Inbred Line Population.

Drought is one of the major abiotic stresses limiting lentil productivity in rainfed production systems. Specific rooting patterns can be associated with drought avoidance mechanisms that can be used in lentil breeding programs. In all, 252 co-dominant and dominant markers were used for Quantitative Trait Loci (QTL) analysis on 132 lentil recombinant inbred lines based on greenhouse experiments for root and shoot traits during two seasons under progressive drought-stressed conditions. Eighteen QTLs controlling a total of 14 root and shoot traits were identified. A QTL-hotspot genomic region related to a number of root and shoot characteristics associated with drought tolerance such as dry root biomass, root surface area, lateral root number, dry shoot biomass and shoot length was identified. Interestingly, a QTL (QRSratioIX-2.30) related to root-shoot ratio, an important trait for drought avoidance, explaining the highest phenotypic variance of 27.6 and 28.9% for the two consecutive seasons, respectively, was detected. This QTL was closed to the co-dominant SNP marker TP6337 and also flanked by the two SNP TP518 and TP1280. An important QTL (QLRNIII-98.64) related to lateral root number was found close to TP3371 and flanked by TP5093 and TP6072 SNP markers. Also, a QTL (QSRLIV-61.63) associated with specific root length was identified close to TP1873 and flanked by F7XEM6b SRAP marker and TP1035 SNP marker. These two QTLs were detected in both seasons. Our results could be used for marker-assisted selection in lentil breeding programs targeting root and shoot characteristics conferring drought avoidance as an efficient alternative to slow and labor-intensive conventional breeding methods.

Chickpea hybridization using in vitro techniques.

Tissue culture techniques play an important role in the utilization of wild Cicer species for the improvement- of cultivated chickpea. Utilization of wild Cicer species has become essential as a series of evolutionary bottlenecks have narrowed the genetic base of chickpea, thus making it susceptible to a range of diseases and pests. Crosses with wild Cicer can broaden its genetic base and introduce useful traits. Except for two wild species, none of the other Cicer species are cross-compatible. To use a range of Cicer species for the improvement of chickpea, embryo rescue and tissue culture techniques are necessary. The success of the cross with incompatible species depended on a range of techniques including the application of growth regulators to pollinated pistils and saving aborting embryos in vitro. Further, the chances of successful transfer of hybrid shoots to soil are greater if the hybrid shoots are grafted to chickpea stocks.

A BAC/BIBAC-based physical map of chickpea, Cicer arietinum L.

BACKGROUND: Chickpea (Cicer arietinum L.) is the third most important pulse crop worldwide. Despite its importance, relatively little is known about its genome. The availability of a genome-wide physical map allows rapid fine mapping of QTL, development of high-density genome maps, and sequencing of the entire genome. However, no such a physical map has been developed in chickpea. RESULTS: We present a genome-wide, BAC/BIBAC-based physical map of chickpea developed by fingerprint analysis. Four chickpea BAC and BIBAC libraries, two of which were constructed in this study, were used. A total of 67,584 clones were fingerprinted, and 64,211 (~11.7 x) of the fingerprints validated and used in the physical map assembly. The physical map consists of 1,945 BAC/BIBAC contigs, with each containing an average of 28.3 clones and having an average physical length of 559 kb. The contigs collectively span approximately 1,088 Mb. By using the physical map, we identified the BAC/BIBAC contigs containing or closely linked to QTL4.1 for resistance to Didymella rabiei (RDR) and QTL8 for days to first flower (DTF), thus further verifying the physical map and confirming its utility in fine mapping and cloning of QTL. CONCLUSION: The physical map represents the first genome-wide, BAC/BIBAC-based physical map of chickpea. This map, along with other genomic resources previously developed in the species and the genome sequences of related species (soybean, Medicago and Lotus), will provide a foundation necessary for many areas of advanced genomics research in chickpea and other legume species. The inclusion of transformation-ready BIBACs in the map greatly facilitates its utility in functional analysis of the legume genomes.

Analysis of genome organization, composition and microsynteny using 500 kb BAC sequences in chickpea.

The small genome size (740 Mb), short life cycle (3 months) and high economic importance as a food crop legume make chickpea (Cicer arietinum L.) an important system for genomics research. Although several genetic linkage maps using various markers and genomic tools have become available, sequencing efforts and their use are limited in chickpea genomic research. In this study, we explored the genome organization of chickpea by sequencing approximately 500 kb from 11 BAC clones (three representing ascochyta blight resistance QTL1 (ABR-QTL1) and eight randomly selected BAC clones). Our analysis revealed that these sequenced chickpea genomic regions have a gene density of one per 9.2 kb, an average gene length of 2,500 bp, an average of 4.7 exons per gene, with an average exon and intron size of 401 and 316 bp, respectively, and approximately 8.6% repetitive elements. Other features analyzed included exon and intron length, number of exons per gene, protein length and %GC content. Although there are reports on high synteny among legume genomes, the microsynteny between the 500 kb chickpea and available Medicago truncatula genomic sequences varied depending on the region analyzed. The GBrowse-based annotation of these BACs is available at http://www.genome.ou.edu/plants_totals.html . We believe that our work provides significant information that supports a chickpea genome sequencing effort in the future.

Stem and Crown Rot of Chickpea in California Caused by Sclerotinia trifoliorum.

The identities of Sclerotinia isolates obtained from chickpea plants showing stem and crown rot were determined using morphological characteristics, variations in group I introns, and internal transcribed spacer (ITS) sequences. Isolates could be separated into two groups based on growth rates at 22°C, fast growing (about 40 mm per day) versus slow growing (about 20 mm per day). All fast-growing isolates induced stronger color change of a pH-indicating medium than did slow-growing isolates at 22°C. The slow-growing isolates contained at least one group I intron in the nuclear small subunit rDNA, whereas all fast-growing isolates lacked group I introns in the same DNA region. ITS sequences of the slow-growing isolates were identical to sequences of Sclerotinia trifoliorum. Those of the fast-growing isolates were identical to sequences of S. sclerotiorum. Finally, the slow-growing isolates showed ascospore dimorphism, a definitive character of S. trifoliorum, whereas the fast-growing isolates showed no ascospore dimorphism. Isolates of both species were pathogenic on chickpea and caused symptoms similar to those observed in the field. This study not only associated the differences between S. sclerotiorum and S. trifoliorum in growth rates, group I introns, ITS sequences, and ascospore morphology, but also represented the first report that S. trifoliorum causes stem and crown rot of chickpea in North America.

Genetics of Chickpea Resistance to Five Races of Fusarium Wilt and a Concise Set of Race Differentials for Fusarium oxysporum f. sp. ciceris.

Genetics of resistance in chickpea accession WR-315 to Fusarium wilt was investigated, and a concise set of differentials was developed to identify races of Fusarium oxysporum f. sp. ciceris. A population of 100 F7 recombinant inbred lines (RILs) from a cross of WR-315 (resistant) and C-104 (susceptible) was used to study genetics of resistance to races 1A, 2, 3, 4, and 5 of F. oxysporum f. sp. ciceris, and a population of 26 F2 plants from a cross between the same two parents was used to study inheritance of resistance to race 2. Segregations of the RILs for resistance to each of the five races suggest that single genes in WR-315 govern resistance to each of the five races. A 1:3 resistant to susceptible ratio in the F2 population indicated that resistance in WR-315 to race 2 was governed by a single recessive gene. A race-specific slow disease progress reaction was observed in chickpea line FLIP84-92C(3) to infection by race 2, a phenomenon termed as slow wilting, that is different from previously reported late wilting with respect to latent period, disease progress rate, and final disease rating. Twenty-nine germ plasm lines (27 Cicer arietinum and two C. reticulatum) including previously used differentials were evaluated for their reactions to infection by the five races. Only eight of the 29 germ plasm lines differentiated at least one of the five races based on either resistant or susceptible reactions, whereas the remaining germ plasm lines were either susceptible or resistant to all five races or differentiated them by intermediate reactions. A concise set of eight chickpea lines comprised of four genotypes and four F7 RILs with vertical resistance was developed as differentials for race identification in F. oxysporum f. sp. ciceris. These differential lines were characterized by early appearance of wilt symptoms, and clear and consistent disease phenotypes based on no wilt or 100% wilt incidence, which offers important improvements over previously available differential sets and provides more precise and unambiguous identification of the races.

Pathotype-specific genetic factors in chickpea (Cicer arietinum L.) for quantitative resistance to ascochyta blight.

Ascochyta blight in chickpea ( Cicer arietinum L.) is a devastating fungal disease caused by the necrotrophic pathogen, Ascochyta rabiei (Pass.) Lab. To elucidate the genetic mechanism of pathotype-dependent blight resistance in chickpea, F(7)-derived recombinant inbred lines (RILs) from the intraspecific cross of PI 359075(1) (blight susceptible) x FLIP84-92C(2) (blight resistant) were inoculated with pathotypes I and II of A. rabiei. The pattern of blight resistance in the RIL population varied depending on the pathotype of A. rabiei. Using the same RIL population, an intraspecific genetic linkage map comprising 53 sequence-tagged microsatellite site markers was constructed. A quantitative trait locus (QTL) for resistance to pathotype II of A. rabiei and two QTLs for resistance to pathotype I were identified on linkage group (LG)4A and LG2+6, respectively. A putative single gene designated as Ar19 (or Ar21d) could explain the majority of quantitative resistance to pathotype I. Ar19 (or Ar21d) appeared to be required for resistance to both pathotypes of A. rabiei, and the additional QTL on LG4A conferred resistance to pathotype II of A. rabiei. Further molecular genetic approach is needed to identify individual qualitative blight resistance genes and their interaction for pathotype-dependent blight resistance in chickpea.

Molecular mapping of Fusarium oxysporum f. sp. ciceris race 3 resistance gene in chickpea.

Sequence-tagged microsatellite site (STMS) and sequence-tagged site (STS) markers linked closely to Fusarium oxysporum f. sp. ciceris race 3 resistance gene in chickpea were identified, and linkage between three wilt resistance genes was elucidated. The resistance to race 3 in chickpea germplasm accession WR-315 was inherited as a single gene, designated foc-3, in 100 F(7) recombinant inbred lines derived from the cross of WR-315 (resistant) x C-104 (susceptible). The foc-3 gene was mapped 0.6 cM from STMS markers TA96 and TA27 and STS marker CS27A. Another STMS marker, TA194, at 14.3 cM, flanked the gene on the other side. Linkage between foc-3 and two other chickpea wilt resistance genes, foc-1 (syn. h(1)) and foc-4, was established. foc-3 was mapped 9.8 cM from foc-1 and 8.7 cM from foc-4, whereas foc-1 and foc-4 are closely linked at 1.1 cM. The identification of closely linked markers to resistance genes will facilitate marker-assisted selection for introgression of the race 3 resistance gene to susceptible chickpea lines.