Genetic Relationship in Sainfoin (Onobrychis viciifolia) Landraces Cultivated East Anatolia by using RAPD and ISSR Markers

Background: Sainfoin (Onobrychis viciifolia) is a forage crop that yields high in arid and calcareous soils and is cultivated in large areas. There aren’t many genetic diversity studies on the varieties of cultured sainfoin. This study was conducted to determine the genetic diversity and the degree of relationship between 23 cultivated landraces and one registered variety. Methods: To take samples from the populations, seeds were sown in the field in 2014. Samples were taken from the young leaves of the plants and preserved at -80oC in same year. RAPD and ISSR primers were used in the study. The bands obtained as a result of PCR were recorded and the data of both methods were also evaluated by combining them. Result: In the study, 5 RAPD and 4 ISSR primers were used and a total of 49 bands were obtained. Of 29 bands obtained using RAPD primers, 20 were found to be polymorphic and of 20 bands obtained using ISSR primers, 15 were found to be polymorphic. It was found that there was a very low correlation between the two methods. Using RAPD and ISSR markers and RAPD + ISSR combination, the similarity index among populations was found to be between 0.25-0.95, 0.5-1.00 and 0.45-0.91, respectively. The Nei’s genetic diversity index was found to be between 0.3365, 0.2656 and 0.3018 with RAPD, ISSR primers and RAPD + ISSR combination, respectively. Based on the dendrograms obtained using RAPD, ISSR primers and RAPD + ISSR combination, the populations under analysis were classified into 3, 3 and 5 groups, respectively. With this study, the closest populations were identified and a significantly high genetic diversity was detected.

Morphological And Molecular Identification of Double Flowered Stock (Matthiola Incana R. Br) Cultivars With High Fertility

Garden stock (Matthiola incana R. Br.) is a commercially important horticultural crop owing to its ornamental effect. There are different stock cultivars varied in color and shape, especially flowered phenotype is an essential index evaluating its commercial value, because double flowered cultivars have more brilliant flowers compared to single flowered one. The present work aimed: (1) to make superior cultivars with different colors, high fertility, being capable of early selecting only double flowered seedlings by leaf color and to investigate morphological characteristics and (2) to select RAPD and ISSR primers for the cultivar certification and identification to culture and produce good commercial stock cultivars. Here we obtained new double flowered stock cultivars with different colors including pink, pale pink and white, through outcrossing between “white” cultivar (high fertile but unable to select double flower phenotype) and “pink” cultivar (vice. versa). Among newly obtained stock cultivars, single and double flower seedlings are distinguishable from each other by leaf color, having about 70% of fertility. Moreover RAPD and ISSR markers selected in this study can be applied to identify different stock cultivars in seed production, culture and to establish cultivar certification system.

Efficient in vitro plant regeneration from leaf derived callus and genetic fidelity assessment of an endemic medicinal plant of Ranunculus wallichianus Wight & Arnn using RAPD and ISSR markers

Ranunculus wallichianus is a medicinally important plant and an endemic species to Western Ghats of South India. An efficient and reliable indirect regeneration protocol system for R. wallichianus was developed from leaf explants in the present investigation. Leaf explants were cultured on both full-strength and half-strength MS (Murashige & Skoog) medium supplemented with different concentrations (1.0 mg L− 1 to 3.0 mg L− 1) of 2,4-D and NAA. Among the different concentrations tested, the highest percentage of yellowish green compact nodular callus formation was observed on half-strength MS medium with 2.0 mg L− 1 of 2, 4-D. Then, the in vitro raised organogenic callus was cultured on half strength MS medium containing various concentrations (1.0 mg L− 1 to 3.0 mg L− 1) of BA, KIN and TDZ with 0.5 mg L− 1 NAA and 10% CW for in vitro shoot regeneration. The highest percentage of regeneration response (97%) and maximum number of shoots formation (11.1 ± 0.13 shoots/culture with 9.2 ± 0.35 cm mean shoot length) were obtained from MS medium containing 2.5 mg L− 1 BA with 0.5 mg L− 1 NAA and 10% CW. The well elongated in vitro raised shoots were rooted in half strength MS medium with 2.5 mg L− 1 IBA + 250 mg L− 1 activated charcoal shows high frequency of root formation. The well rooted plantlets were successfully hardened and acclimatized with the survival rate of 94%. Clonal fidelity of in vitro raised plantlets was assessed by using DNA based RAPD and ISSR molecular markers. The total of 56 and 47 monomorphic bands were obtained from RAPD and ISSR markers respectively. This present in vitro propagation protocol system could be an effective for the conservation of R. wallichianus with their genetic purity and its further investigations.

Trichovariability in rhizosphere soil samples and their biocontrol potential against downy mildew pathogen in pearl millet

The present work is aimed to examine the genetic variability and the distribution pattern of beneficial Trichoderma spp. isolated from rhizosphere samples and their mode of action in improving the plant health. A total of 131 suspected fungi were isolated from the rhizospheric soil and 91 isolates were confirmed as Trichoderma spp. T. asperellum and T. harzianum were found high in the frequency of occurrence. Genetic diversity analysis using RAPD and ISSR revealed the diverse distribution pattern of Trichoderma spp. indicating their capability to adapt to broad agroclimatic conditions. Analysis of genetic diversity using molecular markers revealed intra-species diversity of isolated Trichoderma spp. The frequency of pearl millet (PM) root colonization by Trichoderma spp. was found to be 100%. However, they showed varied results for indole acetic acid, siderophore, phosphate solubilization, β-1,3-glucanase, chitinase, cellulase, lipase, and protease activity. Downy mildew disease protection studies revealed a strong involvement of Trichoderma spp. in direct suppression of the pathogen (mean 37.41) in the rhizosphere followed by inducing systemic resistance. Our findings highlights the probable distribution and diversity profile of Trichoderma spp. as well as narrate the possible utilization of Trichoderma spp. as microbial fungicides in PM cultivation across different agroclimatic zones of India.

Genetic diversity of the endangered endemic species Hedysarum sangilense Krasnoborov et Timokhina (Fabaceae)

Hedysarum sangilense Krasnoborov et Timokhina (Fabaceae) is а rare subendemic species of the Fabaceae family. It has been found in a restricted area, only in isolated habitats in Tyva Republic and Northern Mongolia. Two PCR techniques, using RAPD and inter-simple sequence repeat (ISSR) markers, were used to perform a comparative analysis of genetic diversity in this species. When amplifying DNA with three RAPD primers, we produced 51 bands, of which 21 (40.6 %) were polymorphic. Amplification of genomic DNA using ISSR analysis yielded 96 fragments, of which 35 (36.6 %) were polymorphic. Nei,s gene diversity (H) was estimated to be 0.105 within populations (range 0.086–0.150) and 0.191 at the species level. Genetic differentiation among populations (GST) was 0.383. The results indicate that both of the marker systems RAPD and ISSR, individually or in combination, can be effectively used in the determination of the genetic relationship among and within populations of H. sangilense.