Transferability of STS markers in studying genetic relationships of marvel grass (Dichanthium annulatum).

Transferability of sequence-tagged-sites (STS) markers was assessed for genetic relationships study among accessions of marvel grass (Dichanthium annulatum Forsk.). In total, 17 STS primers of Stylosanthes origin were tested for their reactivity with thirty accessions of Dichanthium annulatum. Of these, 14 (82.4%) reacted and a total 106 (84 polymorphic) bands were scored. The number of bands generated by individual primer pairs ranged from 4 to 11 with an average of 7.57 bands, whereas polymorphic bands ranged from 4 to 9 with an average of 6.0 bands accounts to an average polymorphism of 80.1%. Polymorphic information content (PIC) ranged from 0.222 to 0.499 and marker index (MI) from 1.33 to 4.49. Utilizing Dice coefficient of genetic similarity dendrogram was generated through un-weighted pair group method with arithmetic mean (UPGMA) algorithm. Further, clustering through sequential agglomerative hierarchical and nested (SAHN) method resulted three main clusters constituted all accessions except IGBANG-D-2. Though there was intermixing of few accessions of one agro-climatic region to another, largely groupings of accessions were with their regions of collections. Bootstrap analysis at 1000 scale also showed large number of nodes (11 to 17) having strong clustering (>50). Thus, results demonstrate the utility of STS markers of Stylosanthes in studying the genetic relationships among accessions of Dichanthium.

Assessment of genetic variation in lucerne (Medicago sativa L.) using protease inhibitor activities and RAPD markers.

The aim of the present study is to identify and characterize lucerne lines resistance to weevil infestation. After three years of field screening for resistance to weevil infestation, 13 lines of lucerne were selected to assess the genotypic variations for lucerne weevil (Hypera postica Gyll.) at biochemical and molecular levels. Total phenols varied from 0.15 to 0.91 mg g-1 (DM) in these genotypes. The highest trypsin (11.11 unit mg-1 protein) and chymotrypsin (93.0 unit mg-1 protein) inhibitors activities were recorded in G-1-02 and B-4-03 lines respectively, whereas highest a-amylases inhibitor activity (14.2 unit mg-1 protein) in C-6-01. Zymogram patterns for trypsin inhibitor activity showed quantitative variations among the lines. In total 262 DNA fragments were generated when 45 deca-mer random primers were employed. Genetic variation in terms of genetic distance ranged from 0.65 to 0.85. Sequential Agglomerative Hierarchical and Nested (SAHN) clustering using the Un-weighted Pair Group Method with Arithmetic mean (UPGMA) algorithm yielded two clusters (cluster I and II) which converged at 72% similarity level. Cluster I contained most of the lines having low level of weevil infestation. High bootstrap values (>40) indicated the significance of nodes embodied in these two clusters. However, SDSPAGE analysis of the leaf proteins of these 13 lines showed no major variations except minor difference in the protein bands of molecular weights between 14 to 20 kD.

Use of EST database markers from M. truncatula in the transferability to other forage legumes.

In general tropical forage legumes lack microsatellites or simple sequence repeat (SSR) markers. Development of genic SSR markers from expressed sequence tagged (EST) database is an alternate and efficient approach to generate the standard DNA markers for genome analysis of such crop species. In the present paper a total of 816 EST-SSRs containing perfect repeats of mono (33.5%), di (14.7%), tri (39.3%), tetra (2.7%), penta (0.7%) and hexa (0.4%) nucleotides were identified from 1,87,763 ESTs of Medicago truncatula. Along with, 70 (8.5%) SSRs of a compound type were also observed. Seven primer pairs of tri repeats were tested for cross transferability in 19 accessions of forage legumes comprising 11 genera. At two different annealing temperatures (55 and 60oC) all primer pairs except AJ410087 reacted with many accessions of forage legumes. A total of 51 alleles were detected with six M. truncatula EST-SSRs primer-pairs against DNA from 19 accessions representing 11 genera where number of alleles ranged from 2 to 13. The cross-transferability of these EST-SSRs was 40.6% at 55oC and 32.3% at 60oC annealing temperature. 24 alleles of the total 50 (48%) at 55oC and 27 of 51 (53%) at 60oC were polymorphic among the accessions. These 27 polymorphic amplicons identified could be used as DNA markers. This study demonstrates the developed SSR markers from M. truncatula ESTs as a valuable genetic markers and also proposes the possibility of transferring these markers between species of different genera of the legumes of forage importance. It was evident from the results obtained with a set of Desmanthus virgatus accessions where Sequential Agglomerative Hierarchical and Nested (SAHN) cluster analysis based on Dice similarity and Unweighted Pair Group Method with Arithmetic mean Algorithm (UPGMA) revealed significant variability (24 to 74%) among the accessions. High bootstrap values (>30) supported the nodes generated by dendrogram analysis of accessions.

Use of degenerate primers in rapid generation of microsatellite markers in Panicum maximum.

Guineagrass (Panicum maximum Jacq.) is an important forage grass of tropical and semi-tropical regions, largely apomictic and predominantly exist in tetraploid form. For molecular breeding work, it is prerequisite to develop and design molecular markers for characterization of genotypes, development of linkage map and marker assisted selection. Hence, it is an important researchable issue to develop molecular markers in those crops where such information is scanty. Among many molecular markers, microsatellites or simple sequence repeat (SSR) markers are preferred markers in plant breeding. Degenerate primers bearing simple sequence repeat as anchor motifs can be utilized in rapid development of SSR markers; however selection of suitable degenerate primers is a prerequisite for such procedure so that SSR enriched genomic library can be made rapidly. In the present study seven degenerated primers namely KKVRVRV(AG)10, KKVRVRV(GGT)5, KKVRVRV(CT)10, KKVRVRV(AAT)6, KKVRVRV(GTG)6, KKVRVRV(GACA)5 and KKVRVRV(CAA)6 were used in amplification of Panicum maximum genomic DNA. Primers with repeat motifs (GGT)5 and (AAT)6 have not reacted whereas (AG)10 , (GACA)5 and (CAA)6 highly informative as they have generated many DNA fragments ranging from 250 to 1600 bps as revealed from the results obtained with restriction digestion of recombinant plasmids. Primer with (CT)10 anchor repeat, amplified fragments of high molecular weight where as (GTG)6 primer generated only six bands with low concentration indicating less suitability of these primer in SSR markers development in P. maximum.

Isozyme, ISSR and RAPD profiling of genotypes in marvel grass (Dichanthium annulatum).

Genetic analysis of 30 accessions of marvel grass (Dichanthium annulatum Forsk.), a tropical range grass collected from grasslands and open fields of drier regions, was carried out with the objectives of identifying unique materials that could be used in developing the core germplasm for such regions as well as to explore gene (s) for drought tolerance. Five inter-simple sequence repeat (ISSR) primers [(CA)4, (AGAC)4, (GACA)4 ]; 27 random amplified polymorphic DNA (RAPD) and four enzyme systems were employed in the present study. In total, ISSR yielded 61 (52 polymorphic), RAPD 269 (253 polymorphic) and enzyme 55 isozymes (44 polymorphic) bands. The average polymorphic information content (PIC) and marker index (MI) across all polymorphic bands of 3 markers systems ranged from 0.419 to 0.480 and 4.34 to 5.25 respectively. Dendrogram analysis revealed three main clusters with all three markers. Four enzymes namely esterase (EST), polyphenol oxidase (PPO), peroxidase (PRX) and superoxide dismutase (SOD) revealed 55 alleles from a total of 16 enzyme-coding loci. Of these, 14 loci and 44 alleles were polymorphic. The mean number of alleles per locus was 3.43. Mean heterozygosity observed among the polymorphic loci ranged from 0.406 (SOD) to 0.836 (EST) and accession wise from 0.679 (IG3108) to 0.743 (IGKMD-10). Though there was intermixing of few accessions of one agro-climatic region to another, largely groupings of accessions were with their regions of collections. Bootstrap analysis at 1000 iterations also showed large numbers of nodes (11 to 17) having strong clustering (>50 bootstrap values) in all three marker systems. The accessions of the arid and drier regions forming one cluster are assigned as distinct core collection of Dichanthium and can be targeted for isolation of gene (s) for drought tolerance. Variations in isozyme allele numbers and high PIC (0.48) and MI (4.98) as observed with ISSR markers indicated their usefulness for germplasm characterization.

Studies on morphological and genetical similarities of Medicago murex and M. doliata to M. scutellata.

Lucerne (M. sativa L., 2n = 4x = 32) is susceptible to weevil (Hypera postica Gyll) insect, hence incorporation of desirable gene (s) from M. scutellata (2n = 30) is an important researchable issue. Incompatibility due to incongruous chromosomal arrangements in these two species necessitated the identification of closer species to M. scutellata (possibly progenitors). After screening 197 accessions comprising 50 Medicago species, M. murex (2n = 2x = 14) and M. doliata (2n = 2x = 16) have been identified as morphologically similar having compatible ploidy and genetically closer to M. scutellata as observed with 17 simple sequence repeats (SSR) and 8 enzymes based isozyme markers. The identified accessions namely IL-04-223 and IL-04-151 of M. doliata and M. murex respectively showing low levels (< 5%) of weevil infestation can be contemplated with diploid M. sativa (2n = 2x = 16) to generate weevil resistant lines.

Identification of species-specific RAPD markers in genus Cenchrus.

Cenchrus is an important component of major grass cover of world. Similar to the other major tropical grasses most of the species in genus Cenchrus are also apomictic in nature hence correct and precise identification of accessions and species are problematic and dubious. In the present study 187 decamer oligonucleotide primers were tested for PCR-based DNA amplification of six prominent species of genus Cenchrus. Of these, 32 potential repetitive and polymorphic primers were tested for identification of species-specific markers for C. ciliaris, C. setigerus, C. pennisetiformis, C. prieurri, C. biflorus and C. myosuroides. These primers yielded 51 unique RAPD markers either specific to a species (37) or shared by two or more species (14). Maximum markers were shared between C. ciliaris and C. setigerus confirming their more closeness to each other. Primers like OPF09, OPF11, OPR15, OPAJ11, OPQ10 and OPAK20 generated strong intense bands can be used on priority in identifying the species from their natural habitat for the development of species-specific core germplasm. Due to apomictic nature this is the prime method of developing cultivars, as morphological characters are largely unable to distinguish them. The level of variation observed clearly suggest RAPD as an appropriate marker for genetic studies and in identifying the lines with species-specific markers for Cenchrus germplasm management activity and also maintaining identity and purity for proprietary reasons.

Bacillus thuringiensis (Bt) transgenic crop: an environment friendly insect-pest management strategy.

Introduction of DDT (dichloro-diphenyl-trichloroethane) and following move towards indiscriminate use of synthetic chemical insecticides led to the contamination of water and food sources, poisoning of non-target beneficial insects and development of insect-pests resistant to the chemical insecticides. Increased public concems about the adverse environmental effects of indiscriminate use of chemical insecticides prompted search of altemative methods for insect-pest control. One of the promising alternatives has been the use of biological control agents. There is well-documented history of safe application of Bt (B. thuringiensis, a gram positive soil bacterium) as effective biopesticides and a number of reports of expression of delta-endotoxin gene(s) in crop plants are available. Only a few insecticidal sprays are required on Bt transgenic crops, which not only save cost and time, but also reduce health risks. Insects exhibit remarkable ability to develop resistance to different insecticidal compounds, which raises concern about the unsystematic use of Bt transgenic technology also. Though resistance to Bt products among insect species under field conditions has been rare, laboratory studies show that insects are capable of developing high levels of resistance to one ormore Cry proteins. Now it is generally agreed that 'high-dose/refuge strategy' is the most promising and practical approach to prolong the effectiveness of Bt toxins. Although manybiosafety concerns, ethical and moral issues exist, area under Bt transgenic crops is rapidly increasing and they are cultivated on more than 32 million hectares world over Even after reservation of European Union (EU) for acceptance of geneticaly modified (GM) crops, 6 out of 25 countries have already adopted Bt crops and many otherindustrial countries will adopt Bt transgenic crops in near future. While the modem biotechnology has been recognized to have a great potential for the promotion of human well-being, adoption of biosafety protocol is necessary to protect human health and environment from the possible adverse effects of the products of genetic engineering. The debate between proponents and opponents of GM technology has created major obstacles in hamessing benefits of the technology It has now become clear that transgenics willbe accepted by the public only when doubts related with general risks and environmental safety are adequately dispelled. Thus, there is need to organize public awareness and present the benefits of Bt transgenic crops to improve social attitude for their rational deployment. In this review, an attempt has been made to discuss social and environmental safety issues of Bt transgenic crops.

Effect of water stress on carbon isotope discrimination and its relationship with transpiration efficiency and specific leaf area in Cenchrus species.

Carbon isotope discrimination (CID) has been proposed in estimating transpiration efficiency (TE) in plants indirectly To identify variations for TE and specific leaf area (SLA) and their association with CID, a glasshouse experiment was conducted using six prominent species of Cenchrus. A significant increase in TE (3.50 to 3.87 g kg(-1)) and decrease in SLA (219.50 to 207.99 cm2 g(-1)) and CID (13.72 to 13.23% per hundred) was observed from well watered to stress condition. Results indicated a direct relationship of SLA with CID (r = 0.511* and 0.544*) and inverse relationship between TE and CID (r = -0.229 and -0.270) However the relationship of TE with CID was insignificant. A positive and significant relationship was visualized between TE and dry matter production in both control (r = 0.917**) and stress (0.718**) treatments. Relationships of total dry matter with SLA and CID were monitored insignificant and negative in control and positive in stress treatment indicated difference in dry matter production under two treatments. It seems that, in Cenchrus species, CID was influenced more by the photosynthetic capacity than by stomatal conductance, as indicated by its positive relationship with SLAin both control (r = 0.511) and stress (r = 0.544) conditions and negative relationship with root dry matter production under control (r = -0.921**) and stress (r = -0.919***) condition. Results showed good correspondence between CID and SLA, indicating that lines having high TE and biomass production can be exploited for their genetic improvement for drought.