P. K. Ranjekar

Identification of inter simple sequence repeat (ISSR) markers associated with seed size in wheat

Abstract The feasibility of identifying inter-simple sequence repeat markers associated with seed weight in hexaploid wheat was tested using 113 recombinant inbred lines developed by the single-seed descent method, from a cross between Rye selection111, an Indian genetic stock obtained through the introgression of genes for bold seed size from rye, and Chinese Spring having small seed size. Three markers were associated with low seed size with gene effects of 14.8%, 9.5%, and 6%, while four markers with contributions of 8%, 4.66%, 2.92% and 2.61% were found to be linked to high seed size, together contributing 31% of the phenotypic variance in seed size. Nulli-tetrasomic and di-telosomic analysis revealed the presence of three low seed size QTL-associated markers on three chromosomes, 6BL, 2DL, and 1DS respectively. This study clearly demonstrates that ISSRs are highly useful for finding markers associated with major and minor genes controlling agronomically important traits in wheat.

Genetic relationships among annual and perennial wild species of Cicer using inter simple sequence repeat (ISSR) polymorphism

Wild Cicer germplasm is known to havegenes for disease resistance, stresstolerance and other important traits, andhence could be exploited for improvingcultivated genotypes. However, only fewCicer species are interfertile and itis essential to overcome crossabilitybarriers to utilize the germplasm moreeffectively. Genetic diversity analysis ofCicer species can give importantclues in understanding speciesrelationships and may assist in developingand planning breeding strategies. Weselected 6 annual and 7 perennial wildspecies, which were amplified using 15 ISSRprimers and UPGMA and AMOVA were used toevaluate the genetic diversity. On anaverage, 6.6 polymorphic bands per primerwere observed. Cluster analysis using theUPGMA algorithm indicated three majorgroups of species at the similarity valueof 0.60 with many subclusters. Theclustering pattern was in agreement withthe data based on crossability, seedstorage protein, isozyme, allozyme and RAPDmarker analysis. The among-populationcomponent of annual and perennial groupscalculated using AMOVA accounted for39.00%. Our results suggested that wildannuals of Cicer were notmonophyletic in nature.

Genetic analysis of kernel hardness in bread wheat using PCR-based markers

Abstract In wheat, kernel hardness is a complex genetic trait involving various directly and indirectly contributing components such as kernel hardness per se, protein content, hectolitre weight and 1,000-kernel weight. In an attempt to identify DNA markers associated with this trait, 100 recombinant inbred lines (RILs) derived from a cross between a hard grain land-race, NP4, and a soft grain variety, HB 208, were screened with 100 ISSR and 360 RAPD primers. Eighteen markers were assigned to seven linkage groups covering 223.6 cM whereas 11 markers remained unlinked. A multiple-marker model explained the percentage of phenotypic variation for kernel hardness as 20.6%, whereas that for protein content, hectolitre weight and 1,000-kernel weight was 18.8%, 13.5% and 12.1%, respectively. Our results indicate that phenotypic expression of kernel hardness is controlled by many QTLs and is interdependent on various related traits.

Arbitrarily primed-PCR based diversity assessment reflects hierarchical groupings of Indian tetraploid wheat genotypes

Abstract Genetic diversity analysis using PCR with arbitrary decamer primers (RAPD — random amplified polymorphic DNA) was carried out in a set of 63 tetraploid wheat genotypes which comprised 24 durum landraces, 18 durum cultivars, nine dicoccum cultivars, ten less commonly cultivated species and two wild tetraploid species. The durum and dicoccum wheat genotypes are a part of the germplasm used in Indian tetraploid wheat breeding programs. A total of 206 amplification products were obtained with 21 informative primers, of which 162 were polymorphic. The highest degree of polymorphism was seen in the wild and less commonly cultivated species (68.9%). Durum released cultivars showed greater polymorphism (50.6%) than landraces (44.8%), while dicoccum cultivars showed a considerably low level of polymorphism (23.6%). Cluster analysis led to the separation of wild and cultivated genotypes, and among cultivated emmer wheat distinct groups were formed by the durum cultivars, durum landraces and dicoccum cultivars. The subgroupings of landraces had no relation to their geographical distribution. The durum cultivars formed subgroups based on common parentage in their pedigree. Among species, wild timopheevi wheat (T. araraticum) and its cultivated form (T. timopheevi) formed a distinct group distant from all other genotypes. The present study is a first attempt at determining the genetic variation in Indian tetraploid wheats at the molecular level.

Molecular analysis of Ascochyta rabiei (Pass.) Labr., the pathogen of ascochyta blight in chickpea

Abstract Genetic diversity in Ascochyta rabiei (Pass.) Labr., the causative agent of ascochyta blight of chickpea, was determined using 37 Indian, five American (USA), three Syrian, and two Pakistani isolates. A total of 48 polymorphic RAPD markers were scored for each isolate and the data used for cluster analysis. Most of the isolates clustered in the dendrogram essentially according to geographic origin. Based on the two major clusters A and B, Indian isolates were grouped into two categories, type-A and type-B. Isolates of A. rabiei within the Punjab state were more diverse than isolates from other states in northwestern India. A DNA marker (ubc7561.6 kb), specific to Indian isolates was identified. This is the first report of a molecular diversity analysis of Indian isolates of A. rabiei. The information may assist Indian chickpea breeders in the proper deployment of blight-resistant cultivars and in disease management.

Molecular mapping and characterization of an RGA locus RGAPtokin1-2171 in chickpea

Resistance gene analog polymorphism (RGAP)is a targeted homology based method, which has been used in different crops to identify tightly linked markers for disease resistance genes and also to enrich the map with a different class of markers. In chickpea, using the RGA primers, which are designed based on the conserved motifs present in characterized R-genes, Bulk Segregant Analysis (BSA) was performed on a resistant bulk and a susceptible bulk along with parents for ascochyta blight resistance. Of all available RGAs and their48 different combinations, only one RGA showed polymorphism during BSA. This marker was evaluated in an F7:8 population of142 RILs from an interspecific cross ofC. arietinum (FLIP 84-92C) × C. reticulatum (PI 599072) and was mapped toCicer linkage map. The genomic location of chickpea RGA was compared with the locations of mapped chickpea R-genes. This is the first RGA marker mapped to chickpea linkage map.

Molecular Marker Analysis of Protein Content Using PCR-Based Markers in Wheat

Grain protein concentration (GPC) of hexaploid wheat is one of the important factors that determines the end-product quality, as well as playing a pivotal role in human nutrition. In an attempt to identify PCR-based DNA markers linked to GPC, 106 recombinant inbred lines (RILs) were developed from a cross between two wheat cultivars PH132 and WL711, which differ significantly in GPC, by the single seed descent method. The RILs were phenotyped for GPC at two diverse agroclimatic locations, namely Pune and Ludhiana, to study the influence of genotype and environment interactions on this trait. The parents were screened with 85 inter simple sequence repeat (ISSR) primers and 350 random primers. The selective genotyping and whole population analysis revealed nine DNA markers associated with the trait. Three markers (UBC8441100, UBC8801100, and OPA4800) were observed to be associated with the trait in both locations, whereas two markers (OPH41400 and UBC873750) were found to be specific to Pune, and four markers (OPM5870, OPO10870, OPV141200, and UBC8251000) were specific to Ludhiana. Together five markers at the Pune location representing five QTLs and seven markers at Ludhiana representing four QTLs accounted for 13.4 and 13.5% of total phenotypic variation, respectively. This study clearly demonstrates that GPC is highly influenced by the environment, and the applicability of ISSR and RAPD markers in finding regions on chromosomes associated with quantitative characters in wheat such as GPC.

Inheritance and identification of DNA markers associated with yellow berry tolerance in wheat (Triticum aestivum L.)

Yellow berry (YB) is a serious seed disorder in durum wheat, bread wheatand triticale, which arises due to deficiency in nitrogen concentration in thesoil. YB seriously affects the grain protein content (GPC) thereby affectingbread making quality in bread wheat and pasta making quality in durumwheat. In order to study the inheritance and to identify DNA markersassociated with YB tolerance, a recombinant inbred line (RIL) populationof 113 individuals was developed by making a cross between RyeSelection111 (RS111), highly resistant to YB and Chinese Spring (CS), asusceptible parent. Phenotyping of this population to YB incidenceindicated that, at least one major gene/QTL and few minor genes governthe tolerance to YB. DNA marker analysis revealed linkage of twomicrosatellite markers Xgwm174 and Xgwm190 from chromosome 5Dwith YB tolerance while one ISSR marker UBC842600 and oneRAPD marker OPR81000 from chromosome 6B were found to beassociated with YB tolerance in repulsion phase. Association of YBtolerance with that of GPC was analyzed using the markers associated withYB tolerance. It was found to be reciprocal in this population in accordancewith the previous reports.

Intraspecific genetic variability analysis of Neovossia indica causing Karnal bunt of wheat using repetitive elements

Abstract Neovossia indica (Tilletia indica), causing Karnal bunt of wheat, affects major wheat growing regions all over the world. Karnal bunt ranks as one of the major diseases of wheat causing quality losses and monetary losses due to international quarantine regulations. The present work is the first report of a genetic diversity analysis of Indian isolates of N. indica. A library of N. indica isolate Ni7 was constructed in a λZAPII system, and three repetitive elements were identified for molecular analysis. These repetitive elements generated complex hybridization profiles producing fingerprint patterns of all seven isolates. Copy-number estimation of these three elements, pNiR9, pNiR12 and pNiR16, indicated the presence of 32, 61 and 64 copies, respectively. Cluster analysis based on hybridization patterns grouped together moderately virulent isolates Ni1, Ni7 and Ni8, thus suggesting a positive correlation between virulence typing and cluster analysis based on molecular data. Variability analysis of N. indica isolates will aid in checking new resistant sources in host germplasm.

Note: Diversity Analysis of Indian Tetraploid Wheat Using Intersimple Sequence Repeat Markers Reveals Their Superiority over Random Amplified Polymorphic DNA Markers

Intersimple sequence repeat polymorphism (ISSR, Zietkiewicz et al., 1994) has emerged as a relatively new, reliable, and speedy marker system for germplasm evaluation and has been used in wheat for detection of polymorphism (Nagaoka and Ogihara, 1997), genetic mapping (Kojima et al., 1998) and tagging of quantitative traits (Ammirajuet al., in press) and in various other plants such as conifers (Tsumuraet al., 1996), citrus (Fanget al., 1997), grapevine (Moreno et al., 1998), and rice (Joshi et al., 2000) for assessment of variability in germplasm. In the present work, we have employed ISSR markers to characterize the variability in a set of Indian tetraploid wheat germplasm, which had been analyzed using RAPD markers in an earlier work by us (Pujar et al., 1999), and have compared the two marker systems for their suitability in the evaluation of tetraploid wheats.