Understanding the genetic structure of Symplocos laurina Wall. populations using nuclear gene markers.

To characterize the genetic diversity of present populations of Symplocos laurina, which grow in the montane forests in India, we analyzed the DNA sequences of a nuclear gene. Using the 881 bp sequence of cytosolic Glyceraldehyde-3-phosphate dehydrogenase gene, we detected 24 haplotypes among 195 individuals sampled from 14 populations. Two dominant haplotypes were distributed over the entire range of this species in India and several private haplotypes were found. Low genetic diversity within population, high differentiation, number of population specific haplotypes and deviation from neutral evolution characterized the present populations of S. laurina. An analysis of molecular variance indicated the presence of geographic structure within the haplotype distribution. The occurrence of S. laurina preglaciation in India is the most parsimonious explanation for the current geographic structure observed. The populations are presumably ancient and might have spread across its extant distribution range in India through a recent range expansion event.

Molecular mapping of stem and leaf rust resistance in wheat.

Stem rust caused by Puccinia graminis f. sp. tritici Eriks and Henn and leaf rust caused by Puccinia triticina Rob. ex Desm. are major constraints to wheat production worldwide. In the present study, F(4)-derived SSD population, developed from a cross between Australian cultivars 'Schomburgk' and 'Yarralinka', was used to identify molecular markers linked to rust resistance genes Lr 3 a and Sr 22. A total of 1,330 RAPD and 100 ISSR primers and 33 SSR primer pairs selected ob the basis of chromosomal locations of these genes were used. The ISSR marker UBC 840(540) was found to be linked with Lr 3 a in repulsion at a distance of 6.0 cM. Markers cfa 2019 and cfa 2123 flanked Sr 22 at a distance of 5.9 cM (distal) and 6.0 cM (proximal), respectively. The use of these markers in combination would predict the presence or absence of Sr 22 in breeding populations. A previously identified PCR-based diagnostic marker STS 638 linked to Lr 20 was validated in this population. This marker showed a recombination value of 7.1 cM with Lr 20.

Genetic diversity across natural populations of three montane plant species from the Western Ghats, India revealed by intersimple sequence repeats.

We analysed genetic diversity across the natural populations of three montane plant species in the Western Ghats, India; Symplocos laurina, Gaultheria fragrantissima and Eurya nitida using intersimple sequence repeat (ISSR) markers. These markers revealed genetic diversity within the populations of these plants from Nilgiri and also between two populations of S. laurina from Nilgiri and Amboli. Genetic variation within and between populations was analysed using various parameters such as total heterozygosity (HT), heterozygosity within population (HS), diversity between populations (DST), coefficient of population differentiation (GST), genetic distance (D) and gene flow (Nm). Total heterozygosity (HT) was higher for S. laurina (0.238) than for G. fragrantissima (0.172) and E. nitida (0.182). Two populations of S. laurina, separated by > 1000 km, showed a high within-population variation (53.7%) and a low gene flow (Nm = 0.447). upgma phenograms depicted a tendency of accessions to group according to their geographical locations in all the three plant species. The insight gained into the genetic structure of these plant populations might have implications in developing in situ and ex situ conservation strategies.

Marker assisted selection of bacterial blight resistance genes in rice.

Bacterial leaf blight caused by Xanthomonas oryzae pv. oryzae is one of the most important diseases affecting rice production in Asia. We were interested in surveying rice genotypes that are popularly used in the Indian breeding program for conferring resistance to bacterial blight, using 11 STMS and 6 STS markers. The basis of selection of these DNA markers was their close linkage to xa5, xa13, and Xa21 genes and their positions on the rice genetic map relative to bacterial blight resistance genes. Eight lines were found to contain the xa5 gene while two lines contained Xa21 gene and none of the lines contained the xa13 gene with the exception of its near-isogenic line. Using the polymorphic markers obtained in the initial survey, marker-assisted selection was performed in the F3 population of a cross between IR-64 and IET-14444 to detect lines containing multiple resistance genes. Of the 59 progeny lines analyzed, eight lines contained both the resistance genes, xa5 and Xa4.

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, UBC8801000, and OPA4800) were observed to be associated with the trait in both locations, whereas two markers (OPH41400) and UBC873750) werefound 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.

DNA fingerprinting to detect genetic variation in rice using hypervariable DNA sequences.

The suitability of miniand microsatellite related DNA sequences capable of detecting multiple loci was investigated for their ability to generate DNA fingerprints in rice. These included R18.1, a cattle-derived probe, the M13 repeat probe, pV47, a human minisatellite probe; and repeats in the Per gene, telomere, chi sequence and 3' hypervariable region of apolipoprotein B. With the R18.1, pV47 and M13 repeat probes, the level of polymorphism was high enough to identify all of the cultivars and wild rice species used in this study. R18.1, which showed the highest level of polymorphism, was estimated to identify up to 2.5×10(20) genotypes of rice. In a F2 population of a 'Basmati-370' and 'Taichung-65' cross, loci detected by R18.1 segregated in a Mendelian fashion. DNA fingerprints were somatically stable and the hybridization patterns were identical among different plants of the same cultivar. Application of the above molecular genetic markers for identification of rice genotypes is reported here for the first time.

DNA fingerprinting in rice using oligonucleotide probes specific for simple repetitive DNA sequences.

In this report we describe the use of five oligonucleotide probes, namely (GATA)4, (GACA)4, (GGAT)4, (GAA)6 and (CAC)5, to reveal highly polymorphic DNA regions in rice. With each of the oligonucleotide probes, the level of polymorphism was high enough to distinguish several rice genotypes. Moreover, individual plants of one cultivar showed the same cultivar-specific DNA fingerprint. The multilocus fingerprint patterns were somatically stable. Our study demonstrates that microsatellite-derived DNA fingerprints are ideally suited for the identification of rice genotypes. As the majority of the probes detected a high level of polymorphism, they can be very useful in monitoring and aiding gene introgression from wild rice into cultivars.

Identification of a dispersed MboI repeat family in five higher plant genomes.

Digestion of nuclear DNAs of five plants, namely Cucurbita maxima (red gourd), Trichosanthes anguina (snake gourd), Cucumis sativus (cucumber), Cajanus cajan (pigeon pea) and Phaseolus vulgaris (french bean) with the restriction endonuclease MboI yielded discrete size classes with molecular weights in the range of 0.5 to 5 kbp. The MboI digestion pattern of Cot 0.1 DNA in french bean is comparable with that of total DNA, indicating that these bands represented highly repeated DNA sequences. Cleavage of the DNAs with varying amounts of MboI indicated the dispersed nature of the repeat families. Southern hybridization studies using french bean highly repetitive DNA as a probe indicated more homology with repeats of pigeon pea and less homology with red gourd, snake gourd and cucumber repeats.