Rohtas Singh

Microsatellite markers: an overview of the recent progress in plants

In recent years, molecular markers have been utilized for a variety of applications including examination of genetic relationships between individuals, mapping of useful genes, construction of linkage maps, marker assisted selections and backcrosses, population genetics and phylogenetic studies. Among the available molecular markers, microsatellites or simple sequence repeats (SSRs) which are tandem repeats of one to six nucleotide long DNA motifs, have gained considerable importance in plant genetics and breeding owing to many desirable genetic attributes including hypervariability, multiallelic nature, codominant inheritance, reproducibility, relative abundance, extensive genome coverage including organellar genomes, chromosome specific location and amenability to automation and high throughput genotyping. High degree of allelic variation revealed by microsatellite markers results from variation in number of repeat-motifs at a locus caused by replication slippage and/or unequal crossing-over during meiosis. In spite of limited understanding of the functions of the SSR motifs within the plant genes, SSRs are being widely utilized in plant genome analysis. Microsatellites can be developed directly from genomic DNA libraries or from libraries enriched for specific microsatellites. Alternatively, microsatellites can also be found by searching public databases such as GenBank and EMBL or through cross-species transferability. At present, EST databases are an important source of candidate genes, as these can generate markers directly associated with a trait of interest and may be transferable in close relative genera. A large number of SSR based techniques have been developed and a quantum of literature has accumulated regarding the applicability of SSRs in plant genetics and genomics. In this review we discuss the recent developments (last 4–5 years) made in plant genetics using SSR markers.

Evaluation of genetic fidelity of in vitro raised plants of Dendrocalamus asper (Schult. & Schult. F.) Backer ex K. Heyne using DNA-based markers

Dendrocalamus asper, an edible bamboo is valued for its tender edible shoots in the food industry. However, overexploitation of natural stands of D. asper coupled with minimal conservation and reforestation efforts has led to its rapid depletion in nature. Therefore protocol for rapid multiplication of D. asper via direct regeneration using nodal segments from mature clumps was standardized and more than 25,000 plants were transferred to the field (Singh et al. 2012a). However, genetic fidelity of these in vitro raised plants needs to be authenticated for commercial scale application of the developed micropropagation protocol. PCR-based molecular markers have emerged as simple, fast, reliable and labor-effective tools for testing the genetic fidelity of in vitro raised plants. This study report the genetic fidelity analysis of in vitro raised plants of D. asper for the first time using arbitrary (Random Amplified Polymorphic DNA, RAPD), semi-arbitrary (Inter-Simple Sequence Repeat, ISSR; Amplified Fragment Length Polymorphism, AFLP), and sequence-based (Simple Sequence Repeat, SSR) markers. Bulked DNA samples of 20 in vitro raised shoots (collected after every three subculture cycles starting from 3rd to 30th passage) and field transferred plantlets were compared with the mother plant DNA using 90 primer combinations (25 each of RAPD, ISSR, SSR, and 15 AFLP) and scorable bands were produced by 78 (22 RAPD, 24 ISSR, 21 SSR, and 11 AFLP) primers. A total of 146 distinct and scorable bands were produced by 22 RAPD primers with an average of 6.6 bands per primer while the number of bands for ISSR primers varied from 3 (ISSR-4 and 9) to 13 (ISSR-17), with an average of 7.1 bands per primer. Similarly, SSR markers also showed wide variation in number of bands, ranging from 2 (RM 261) to 12 (RM 44, 140, and 224) with an average of 7.8 bands. AFLP primer combinations could generate 35–72 bands with an average of 48.7 bands per primer pair. Amplification of monomorphic bands with all primer combinations authenticated the true to type nature of the in vitro raised plants of D. asper which underwent up to 30 subculture passages over a period of approximately 2 years thereby supporting the commercial utilization of the developed micropropagation protocol.

Biotechnological interventions in sea buckthorn ( Hippophae L.): current status and future prospects

Sea buckthorn (Hippophae L., Elaeagnaceae) is an economically and ecologically important medicinal plant comprising of species which are winter hardy, dioecious, wind-pollinated multipurpose shrubs bearing yellow or orange berries with nitrogen-fixing ability. It grows widely in cold regions of Indian Himalayas, China, Russia, Europe and many other countries. It is commonly known as ‘cold desert gold’ due to its high potential as a bio-resource for land reclamation, reducing soil erosion and its multifarious uses. The wild populations are being used for harvesting economic benefits with negligible plantation efforts. Although this plant has many excellent traits, it is still in an early phase of domestication. This woody plant is prone to many pests and diseases which destroy the plants and halt its commercial production. Limited progress has been made for improvement of sea buckthorn through breeding programs due to long juvenile period and lack of QTL linkage map, which makes screening of mapping populations a time-consuming and labor-intensive task. Conventional propagation methods, i.e. seeds, softwood and hardwood cuttings, and suckers are in place but are cumbersome and season dependent. Therefore, application of modern tools of biotechnology needs to be standardized for harnessing maximum benefits from this nutraceutical plant. Improvement of this genus through genetic transformation requires an efficient regeneration system, which is yet to be standardized. Taxonomic status of the genus is controversial and requires more inputs. Taxonomic delineation of species and subspecies and also the breeding programs can be more robustly addressed using molecular markers. This review summarizes the progress made and suggests some future directions of research for this important fruit species.

Characterization of seed storage proteins in high protein genotypes of cowpea [Vigna unguiculata (L.) Walp.]

Twenty one genotypes and two check varieties viz. CS-88 and V-240 of cowpea [Vigna unguiculata (L.) Walp. ] were screened for total proteins. The total protein content ranged from 22.4 (HC-3) to 27.9 % (HC-98-64) in 21 genotypes whereas in check varieties it was 25.6 (V-240) and 26.0 % (CS-88). Seven genotypes viz. HC-6, HC-5, CP-21, LST-II-C-12, CP-16, COVU-702 and HC-98-64 having high protein content (26.7 to 27.9 %) were selected for further characterization of their seed storage proteins. Globulins were the major protein fraction ranging from 55.6 (LST-II-C-12) to 58.8 % (CP-16 and HC-6) of total protein. Glutelins was the second major fraction ranging from 14.4 to 15.6 % followed by albumins (8.2 to 11.9 %) and prolamins (2.3 to 5.0 %). Content of free amino acids also showed variations amongst genotypes with COVU-702 having maximum and LST-II-C-12 having minimum content. Essential amino acid analysis revealed that S-amino acids (cysteine and methionine) were the first limiting amino acids followed by tryptophan. From the results presented here it could be suggested that two genotypes viz. LST-II-C-12 and HC-5 be used in breeding programmes aimed at developing high protein moth bean varieties with good quality.

Grain filling duration and temperature pattern influence on the performance of wheat genotypes under late planting

Terminal heat referred to as increase in temperature during grain filling, is one of the important stress factors for wheat production and is responsible for decline in wheat production in many environments worldwide. In order to meet the challenges of high temperature ahead of global warming, concerted efforts are needed to evaluate wheat genotypes for heat tolerance and develop genotypes suitable for such stressed environments. Twenty-seven advanced wheat genotypes developed for stress and normal environments by different research centres were evaluated during 2009–10 and 2010–11 under timely sown (normal) and late sown (heat stress) environments. Analysis of variance revealed that the genotypes differed significantly in grain filling duration (GFD), grain growth rate (GGR) and thousand-grain weight (TGW). Out of 27 genotypes, 16 were found to be tolerant for thousand-grain weight under late planting (heat stress) during 2009-10 but only 12 were tolerant during 2010–11. Many of the genotypes registered more reduction in thousand-grain weight during 2010–11 as compared to 2009–10; the temperatures during 2009–10 were higher. The differences in grain filling duration under two conditions during both seasons as well as difference in temperatures during first half of grain filling explain the reduction pattern in the genotypes. GFD had significant negative correlation with temperatures during post heading period and the difference in GFD under two environments had positive correlation with these temperatures. The reduction in GFD had regression of 33.3% on reduction in GGR and reduction in GGR had regression of 41.6% on reduction in TGW genotypes AKW 1071, DBW 17, HS 277, K 7903, K 9107, NW 1014 and RAJ 3765 had less sensitivity to stress environments during both years.