黄瓜DAMD反应体系的建立及种质遗传资源研究

通过L16(45)正交试验,研究Mg2+、dNTPs、Taq酶、引物和模板DNA对黄瓜DAMD(direct amplificationof minisatellite region DNA)反应的影响.结果表明,适宜的DAMD反应体系包括1×Taq酶缓冲液、1.5 mmol/LMg2+、0.25 mmol/L dNTPs、1.2 UTaq酶、2.0μmol/L引物和10~80 ng模板DNA,总体积20μL.采用该体系并结合温度梯度PCR,确定了15种小卫星核心DNA引物的适宜退火温度.利用这些引物对20份不同类型的黄瓜材料进行扩增,引物平均检测到10.2个位点,平均多态性位点7.3个,平均多态性百分率71.4%.PCA分析表明,20份材料明显可分为3个区域,第I区包含绝大部分华南型黄瓜,第II区包含绝大部分华北型黄瓜,第III区为野生型黄瓜.此结果与SSR标记的研究结果基本一致,证明本实验构建的DAMD反应体系的稳定性及DAMD标记的可行性.     

Genetic variability and population structure of Bergenia ciliata (Saxifragaceae) in the Western Himalaya inferred from DAMD and ISSR markers

Genetic variability and population structure of Bergenia ciliata (Haw.) Sternb., commonly known as “Pashanbheda” (Stone-breaker), collected from the Western Himalayan region of India were estimated using two DNA fingerprinting methods viz., directed amplification of minisatellite DNA (DAMD) and inter simple sequence repeats (ISSR). The cumulative data analysis of DAMD and ISSR markers for 74 accessions from eight populations showed 86.1% polymorphism. Analysis of molecular variance (AMOVA) showed highest percentage of variation within individuals of populations (73.6%) and 21.7% among populations. STRUCTURE and PCoA analyses on the hierarchical partitioning of genetic diversity showed strong admixture of individuals among the eight assumed geographical populations of B. ciliata. The data suggests that high genetic flow is one of the major factors responsible for low genetic differentiation. Preservation of genetic diversity of B. ciliata is important, both to promote adaptability of the populations to changing environment as well as to preserve a large gene pool for future prospection. The present study using DAMD and ISSR markers, therefore, provide the means of rapid characterization of accessions within the populations, and thus enable the selection of appropriate accessions for further utilization in conservation and prospection programmes.     

Genetic diversity and gene flow estimation in Prosopis cineraria (L.) Druce: A key stone tree species of Indian Thar Desert

Selected amplicon data obtained through our earlier study using ISSR and DAMD markers were utilized for determination of diversity within and among the populations of Prosopis cineraria (L.) accessions collected from different districts of Rajasthan (India). A total of 83 bands were generated from eight ISSR and five DAMD primers of which 79 were found to be polymorphic (95.18%). Nei’s gene diversity (h) ranged between 0.185 and 0.301 with overall diversity of 0.316 while Shannon’s information index (I) values recorded between 0.253 and 0.438 with an average value of 0.243. The gene flow value (1.713) and the diversity among populations (0.226) demonstrated higher genetic variation within the population. It is concluded that P. cineraria is accompanied by high genetic diversity within the population and elevated gene flow showing indications of adaptation to callous and fragile dry conditions of arid environment.     

Detecting DNA polymorphism and genetic diversity in Lentil (Lens culinaris Medik.) germplasm: comparison of ISSR and DAMD marker

Genetic diversity and interrelationships among 31 lentil genotypes were evaluated using 10 Inter-Simple Sequence Repeat (ISSR) and 10 directed amplification of minisatellite DNA region (DAMD) primers. A total of 43 and 48 polymorphic bands were amplified by ISSR and DAMD markers, respectively. Average polymorphism information content (PIC) for ISSR and DAMD markers were 0.37 and 0.41, respectively. All 31 lentil genotypes could be distinguished by ISSR markers into three groups and by DAMD markers into two groups. Various molecular markers show a different efficiency for evaluating DNA polymorphism in lentil and indicate that the patterns of variation are clearly influenced by the genetic marker used. Comparatively, the genetic diversity of examined lentil genotypes by two different marker techniques (ISSR and DAMD) was high and indicated that ISSR and DAMD are effective and promising marker systems for fingerprinting in lentil and give useful information on its genetic relationships.     

Single primer amplification reaction methods reveal exotic and indigenous mulberry varieties are similarly diverse

Mulberry is the sole food source for mulberry silkworm and a number of indigenous and exotic varieties are used in sericulture. Studies on assessment of genetic diversity have been done amongst a few mulberry varieties using one or at the most two methods. However, no comprehensive study on a large number of varieties has been carried out. In present study, single primer amplification reaction (SPAR) methods have been used for determination of diversity in 27 mulberry varieties (exotic as well as indigenous), using four minisatellite core sequence primers for directed amplification of minisatellite DNA (DAMD), three simple sequence repeat (SSR) motifs as primers for inter simple sequence repeat (ISSR) and 20 arbitrary sequence decamer primers for random amplified polymorphic DNA (RAPD) reactions. The Jaccard coefficients were determined for the DAMD, ISSR and RAPD band data (total of 58, 39 and 235 bands respectively). All three methods revealed wide range of distances supporting a wide range of mulberry genetic diversity. A cumulative analysis of the data generated by three methods resulted in a neighbour-joining (NJ) tree that gave a better reflection of the relatedness and affinities of the varieties to each other. Comparison of the three methods by marker indices and the Mantel test of correlation indicated that though all methods were useful for the assessment of diversity in mulberry, the DAMD method was better. When considered as two groups (10 exotic and 17 indigenous varieties), the mulberry varieties in the exotic group were found to have slightly greater diversity than the indigenous ones. These results support the concept of naturalization of mulberry varieties at locales distant from their origins. NBRI communication No. 542.     

Assessment of genetic diversity in indigenous turmeric (Curcuma longa) germplasm from India using molecular markers

Curcuma longa L., commonly known as turmeric, is one of the economically and medicinally important plant species. It is predominantly cultivated in the tropical and sub tropical countries. India is the largest producer, and exporter of turmeric in the world, followed by China, Indonesia, Bangladesh and Thailand. In the present study, Directed Amplification of Minisatellite DNA (DAMD) and Inter Simple Sequence Repeats (ISSR), methods were used to estimate the genetic variability in indigenous turmeric germplasm. Cumulative data analysis for DAMD (15) and ISSR (13) markers resulted into 478 fragments, out of which 392 fragments were polymorphic, revealing 82 % polymorphism across the turmeric genotypes. Wide range of pairwise genetic distances (0.03–0.59) across the genotypes revealed that these genotypes are genetically quite diverse. The UPGMA dendrogram generated using cumulative data showed significant relationships amongst the genotypes. All 29 genotypes studied grouped into two clusters irrespective of their geographical affiliations with 100 % bootstrap value except few genotypes, suggesting considerable diversity amongst the genotypes. These results suggested that the current collection of turmeric genotypes preserve the vast majority of natural variations. The results further demonstrate the efficiency and reliability of DAMD and ISSR markers in determining the genetic diversity and relationships among the indigenous turmeric germplasm. DAMD and ISSR profiling have identified diverse turmeric genotypes, which could be further utilized in various genetic improvement programmes including conventional as well as marker assisted breeding towards development of new and desirable turmeric genotypes.     

Genetic variability and population structure in Sapindus emarginatus Vahl from India

Sapindus emarginatus is an economically important tropical tree species sparsely distributed in different geographical provinces like Gangetic Plains, Western Ghats, and Deccan Plateau in India. In the present paper estimation of genetic variability within and among 41 accessions representing five populations was carried out using 3 single primer amplification reaction (SPAR) methods viz. RAPD, DAMD and ISSR. The cumulative data analysis was carried out for all three SPAR methods, and showed 82.32% polymorphism across all the accessions of S. emarginatus. Jaccard's similarity values among 41 accessions ranged from 0.15 to 0.49 with an average value of 0.37. The intra-population genetic diversity revealed highest values of Nei's genetic diversity (0.19,) Shannon information index (0.29) and polymorphic loci (55.18%), among the accessions of Gujarat (GJ) population, while the corresponding lowest values were (0.10), (0.15) and (26.40%) respectively among the accessions of Rajasthan (RJ) population. The maximum inter-population average genetic distance (0.20) was between Karnataka (KA) and RJ, while the corresponding least genetic distance (0.06) was between Allahabad (AL) and Varanasi (VS) populations. The analysis of molecular variance (AMOVA) revealed maximum percentage of variation among individuals of populations (72%) followed by 16% among regions and 12% among populations. Principal coordinate analysis (PCA) of cumulative data also supported the clustering pattern in the UPGMA dendrogram. These results suggest that genetic diversity is corroborating with the geographical diversity. Mantel's test was performed which revealed a highly significant correlation between cumulative vs RAPD, and showed the maximum (0.93) correlation coefficient, followed by cumulative vs ISSR (0.78) and cumulative vs DAMD (0.91) respectively, and this clearly indicates that the SPAR methods (RAPD, DAMD and ISSR) are sufficiently informative and are suitable to analyze the genetic variability within and among the populations of S. emarginatus.     

Taxonomic resolution of the genus Bythotrephes Leydig using molecular markers and re-evaluation of its global distribution

Bythotrephes Leydig is a predatory, onychopod cladoceran native to Eurasia that typically inhabits oligo‐ and mesotrophic water bodies of the Palaearctic region. It recently invaded 70 North American lakes, prompting a re‐evaluation of the taxonomic status, global distribution, and determinants of local occurrence and abundance. European studies have reported two distinct species, B. longimanus, which lacks a kink on the caudal process, and B. cederstroemi, which possesses one. We employed sequencing of the mitochondrial gene cytochrome c oxidase subunit I (COI) in addition to Directed Amplification of Minisatellite‐region DNA (DAMD) using polymerase chain reactions (PCRs) to assay the genetic nature of these ‘species’. These analyses revealed that the two taxa are consistent with a single, common species, Bythotrephes longimanus Leydig 1860, thus resolving the nomenclatural issue. Furthermore, a common haplotype was identified between Lake Ontario and Lake Puruvesi, Finland, suggesting a potential invasion corridor via the nearby Baltic Sea. Statistical analysis revealed that the two forms also occur in similar habitats in Eurasia and North America. Habitat characteristics of water bodies in Northern Europe, where both forms are found and occasionally co‐occur, do not differ statistically. Similarly, no significant differences were detected between characteristics of Eurasian habitats that support longimanus‘forms’ and those of inland lakes in North America that support cederstroemi‘forms’. Human activities have had a strong effect on the distribution of Bythotrephes in both Europe and North America. Global and local distributions have been affected by ballast water transfer and by boating and fishing activities, respectively. Cultural eutrophication, oligotrophication and acidification alter the suitability of habitats for fish predators, and may indirectly influence Bythotrephes’ local occurrence and abundance.     

Single primer amplification reaction (SPAR) methods reveal subsequent increase in genetic variations in micropropagated plants of Nepenthes khasiana Hook. f. maintained for three consecutive regenerations

The genetic fidelity of in vitro-raised plants of three successive regenerations of Nepenthes khasiana Hook. f. was assessed using three different single primer amplification reaction (SPAR) methods, viz., random amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR) and direct amplification of minisatellite DNA region (DAMD) markers. Out of 80 RAPD primers screened, 14 primers reflected a genetic variation of 4.1% in the first regeneration which was increased to 9.4% in the third regeneration. In the case of ISSR, out of 36 primers screened for assessment of genetic homogeneity of the regenerated plantlets, 12 primers showed an increase of genetic variation from 4.3% to 10% from the first to the third regenerations. In DAMD profiling, 15 primers were used for the evaluation of genetic fidelity where 8.47% of polymorphism was observed in the first regeneration which was increased to 13.33% in the third regeneration. The cumulative analysis reflected a genetic variation of 5.65% in the first regeneration which increased subsequently to 7.77% in the second regeneration and 10.87% in the third regeneration. The present study demonstrates SPAR technique to be an efficient tool for the assessment of clonal fidelity of in vitro-raised plants.     

Assessment of genetic diversity and population structure of Bergenia stracheyi (Saxifragaceae) in the Western Himalaya (India)

Genetic diversity and population structure in Bergenia stracheyi, a threatened medicinal herb in the Western Himalaya of India was analysed using directed amplification of minisatellite DNA (DAMD) and inter simple sequence repeats (ISSR) markers. A total of 41 accessions of B. stracheyi representing three populations (Khillenmarg –KLM, Jalori Pass-JLP and Rohtang-RTG) were considered in the present study. The cumulative data analysis for 26 (10 DAMD + 16 ISSR) markers revealed 87.1% polymorphism. The maximum inter-population genetic distance was found between KLM and JLP, whereas the minimum genetic distance was found between RTG and JLP populations. The analysis of molecular variance (AMOVA) revealed maximum percentage of variation among individuals within populations (75%) than among the populations (25%). Clustering pattern of the three sample populations in STRUCTURE and PCoA analyses showed high genetic variation at population level. The present study revealed that distribution patterns, high altitudinal ranges, high habitat specificity, relatively high gene flow, small and isolated population size have shaped the current population structure of B. stracheyi in the Western Himalayan region. DAMD and ISSR markers have provided significant insights into characterization of B. stracheyi populations, and facilitate selection of appropriate accessions for further utilization in conservation and bioprospecting programmes.