SSR and AFLP based genetic diversity of soybean germplasm differing in photoperiod sensitivity

Forty-four soybean genotypes with different photoperiod response were selected after screening of 1000 soybean accessions under artificial condition and were profiled using 40 SSR and 5 AFLP primer pairs. The average polymorphism information content (PIC) for SSR and AFLP marker systems was 0.507 and 0.120, respectively. Clustering of genotypes was done using UPGMA method for SSR and AFLP and correlation was 0.337 and 0.504, respectively. Mantel's correlation coefficients between Jaccard's similarity coefficient and the cophenetic values were fairly high in both the marker systems (SSR = 0.924; AFLP = 0.958) indicating very good fit for the clustering pattern. UPGMA based cluster analysis classified soybean genotypes into four major groups with fairly moderate bootstrap support. These major clusters corresponded with the photoperiod response and place of origin. The results indicate that the photoperiod insensitive genotypes, 11/2/1939 (EC 325097) and MACS 330 would be better choice for broadening the genetic base of soybean for this trait.     

Genetic diversity analysis of maintainer and restorer accessions in upland cotton (Gossypium hirsutum L.)

Genetic diversity amongst 91 upland cotton accessions (50 maintainer, ‘B’ and 41 restorer ‘R’ lines) and three wild species viz., G. aridum, G. thurberi and G. anomalum was analyzed using SSR and RAPD markers. A total of 53 primers (30 SSR and 23 RAPD) were sampled for screening 94 accessions, of which 26 SSR and 17 RAPD primers were polymorphic. Average polymorphism detected by SSR, RAPD and SSR + RAPD markers was 72.5, 62 and 66.66 per cent, respectively. A unique marker CIR-200₂₆₀ that distinguishes G. thurberi from all upland accessions has been identified. Similarity coefficient values within and between B and R lines ranged from 0.65–0.95, 0.61–0.98 and 0.53–0.93 for SSR and 0.72–0.98, 0.73–0.97 and 0.69–0.98 for RAPD markers. UPGMA cluster analysis was consistent with the pedigree and genotypic background of the accessions. RAPD and SSR matrices showed significant positive product moment correlation (r?=?0.93 and 0.92) with the RAPD + SSR combined data matrix, respectively. The result indicates a moderate level of genetic diversity in B and R accessions of upland cotton. Genetically diverse combinations were identified to further evaluate heterotic performance. The maintainer, AKH-108, AKH-118 and AKH-2173; and restorer AKH-31 and AKH 4943 accession were identified as most distinct and divergent, could be used as candidate parental genotypes in hybrid and varietal development programme and also development of mapping population for trait mapping in cotton.     

RAPD polymorphisms in spring wheat cultivars and lines with different level of Fusarium resistance

Random amplified polymorphic DNA (RAPD) markers have been used to characterize the genetic diversity among 35 spring wheat cultivars and lines with different levels of Fusarium resistance. The objectives of this study were to determine RAPD-based genetic similarity between accessions and to derive associations between Fusarium head blight (FHB) and RAPD markers. Two bulked DNA from either highly resistant lines or susceptible lines were used to screen polymorphic primers. Out of 160 screened primers, 17 primers generated reproducible and polymorphic fragments. Genetic similarity calculated from the RAPD data ranged from 0.64 to 0.98. A dendrogram was prepared on the basis of a similarity matrix using the UPGMA algorithm, which corresponded well with the results of principal component analysis and separated the 35 genotypes into two groups. Association analysis between RAPD markers and the FHB index detected three RAPD markers, H19(1000), F2(500) and B1(2400), significantly associated with FHB-resistant genotypes. These results suggest that a collection of unrelated genotypes can be used to identify markers linked to an agronomically important quantitative trait like FHB. These markers will be useful for marker-assistant breeding and can be used as candidate markers for further gene mapping and cloning.     

Comparative Evaluation of RAPD and AFLP Based Genetic Diversity in Brinjal (Solanum melongena)

The present investigation was carried out with an objective of evaluating genetic diversity in brinjal (Solanum melongena) using DNA markers. A total of 38 brinjal accessions including one wild-species, Solanum sisymbrifolium were characterized using random amplified polymorphic DNA (RAP D) and amplified fragment length polymorphism (AFLP) techniques. Out of 45 primers employed to generate RAPD profiles, reproducible patterns were obtained with 32 primers and 30 (93.7%) of these detected polymorphism. A total of 149 bands were obtained, out of which 108 (72.4%) were polymorphic. AFLP analysis was carried out using four primer combinations. Each of these primers was highly polymorphic. Out of 253 fragments amplified from these four primer combinations, 237 (93.6%) were polymorphic. The extent of pair-wise similarity ranged from 0.264 to 0.946 with a mean of 0.787 in RAPD, in contrast to a range of 0.103 to 0.847 with a mean of 0.434 in AFLP. The wild species clustered separately from the brinjal genotypes. In the dendrogram constructed separately using RAPD and AFLP markers, the brinjal genotypes were grouped into clusters and sub-clusters, and the varieties released by IARI remained together on both the dendrograms. All the 30 RAPD primers in combination and each of the four primer pairs in AFLP could distinguish the brinjal accessions from each other. AFLP was thus found to be more efficient than RAPD in estimation of genetic diversity and differentiation of varieties in brinjal.     

Genetic variation in a wild population of the 'sleep' passion fruit (Passiflora setacea) based on molecular markers

Little is known about the molecular genetic diversity of most Passiflora species. We used RAPD markers to evaluate the genetic diversity of 24 genotypes of the 'sleep' passion fruit (Passiflora setacea). Twelve primers generated 95 markers, 88% of which were polymorphic. The genetic distance estimated by the complement of the Dice index ranged from 0.29 (among accessions Ps-G1 and Ps-G13) to 0.69 (among accessions Ps-G21 and Ps-G23). Genotype grouping based on the UPGMA algorithm showed considerable variability among genotypes. We conclude that P. setacea has a broad genetic base that could be exploited in breeding programs.     

Analysis of Genetic Diversity in Cicer arietinum L Using Random Amplified Polymorphic DNA Markers

PCR-based random amplified polymorphic DNA (RAPD) markers were employed to assess genetic diversity in 23 chickpea genotypes. Forty of the 100 random primers screened revealed polymorphism among the genotypes. Most of the primers revealed single polymorphic band, and only 14.1 2% of the products were polymorphic. Estimates of genetic similarity based on Jaccard’s coefficient ranged from 0.92 to 0.99, indicating narrow genetic variability among the genotypes based on RAPD markers.The 23 chickpea genotypes formed two major clusters in the dendrogram.The low RAPD polymorphism among chickpea genotypes suggests that more number of polymorphic primers need to be analysed to determine genetic relationships. It was observed that RAPD analysis employing 30 polymorphic primers could provide better estimates of genetic relationships in chickpea.     

山东省无花果种质资源多样性的RAPD分析

利用随机扩增多态性DNA(RAPD)技术,对58份山东省无花果(Ficus caricaL.)资源的多样性进行了评价,经15个随机引物的初步分析表明,其中至少有23个不同的基因型。继而从36个随机引物中筛选出28个多态性好的引物,进一步对这23个不同基因型材料进行RAPD扩增分析,共得到309个清晰稳定的扩增位点,其中多态性位点236个,占76.4%。利用UPGMA法分析发现,这些基因型间的遗传相似系数在0.592~0.960之间,在此基础上,构建了各基因型间的遗传关系树状图。结果表明,山东省内的无花果资源具有较为丰富的遗传多样性。     

Genetic characterization of Iranian safflower (Carthamus tinctorius) using inter simple sequence repeats (ISSR) markers

Safflower (Carthamus tinctorious L.) is valued as a source of high quality vegetable oil. 20 ISSR primers were used to assess the genetic diversity of 18 accessions of safflower collected from different geographical regions of Iran. The ISSR primers combinations revealed 57.6 % polymorphism, among 338 genetic loci amplified from the accessions. The sum of effective number of alleles and observed number of alleles were 29.76 and 36.77, respectively. To understand genetic relationships among these cultivars, Jacquards’ similarity coefficient and UPGMA clustering algorithm were applied to the ISSR marker data set. ISSR markers grouped accessions into two main clusters and four sub clusters. Also, the principal coordinate analysis (PCoA) supported the cluster analysis results. The results showed these genotypes have high genetic diversity, and can be used for alternative safflower breeding program.     

Genetic and geographic polymorphism of cultivated tobaccos (Nicotiana tabacum) in Turkey

Nicotiana tabacum (2n = 48) is a natural amphidiploid and shows a distribution over a geographical area in eastern Anatolia. Random amplified polymorphic DNA (RAPD) technique was used to evaluate both genetic diversity among 21 primitive tobacco accessions comparing flue cure virginia genotype (FCV) and their geographical polymorphism as a source of genetic variations for breeding programs. Only 13 of all the 60 random primers used in RAPD showed polymorphism acceptable for characterization of these accessions. Totally 118 RAPD fragments were generated from 13 decamer primer and 64 of them were found polymorphic (54.2%). Mus and FCV showed the smallest genetic distance among accessions cultivated in the eastern Anatolia. These results shows that the RAPD assay is a powerful approach for identifying genetic and geographic polymorphism. This article was submitted by the authors in English.     

Estimation of genetic distance based on RAPDs between 11 cotton accessions varying in heat tolerance

The genetic distance of 11 cotton genotypes varying in heat tolerance was studied using RAPD markers. Fifty-three random decamer primers were used for the estimation of genetic distance. Among the 53 RAPD primers, which were custom synthesized by GeneLink Inc., UK, 32 were polymorphic and 21 were monomorphic. The 32 polymorphic primers produced 273 fragments, with a mean of 8.3 fragments per primer. The number of polymorphic bands produced in the 11 cotton accessions ranged from 1 to 31. Primer GLC-20 produced 31 polymorphic bands, while two primers, GLB-5 and GLC-12, produced one polymorphic band each. A range of 88.89 to 42.48% genetic similarity was observed among the 11 cotton accessions. The highest genetic similarity was observed between FH-945 and BH-160 (88.89%), whereas the lowest value was found between NIAB-801/2 and FH-945 (42.48%). Unique amplification profiles were produced by most of the cultivars; the differences were sufficient to distinguish them from other genotypes. This confirms the efficacy of RAPD markers for the identification of plant genotypes. An accumulative analysis of amplified products generated by RAPDs was sufficient to assess the genetic diversity among the genotypes. This information should be helpful for formulating breeding and genome mapping programs.     

Assessment of genetic diversity in Colletotrichum falcatum Went accessions based on RAPD and ISSR markers

Sugarcane is susceptible to red rot disease caused by phytopathogenic fungus Colletotrichum falcatum Went which ultimately affect the economy of farmers as well as sugar based industry. One of the various ways to control this devastating disease is to develop disease resistance sugarcane cultivar and this requires the complete understanding of genetic makeup of pathogen. Although South Gujarat is well known sugarcane cultivating area, less published data can be found about PCR-based genetic diversity in prevalent C. falcatum accessions. So, present investigation aims at finding molecular variation among the ten accessions of C. falcatum using RAPD and ISSR molecular markers. A total of 35 RAPD and 39 ISSR primers were screened across 10 C. falcatum accessions, of which 15 RAPD and 21 ISSR primers have showed consistent amplification. Statistics related to genetic variation were estimated using NTSYS-PC by means of Dice’s coefficient. The results revealed 80.6% and 68.07% polymorphism and similarity coefficient ranged from 0.43 to 0.91 and 0.73 to 0.93 in RPAD and ISSR analysis respectively. The dendrogram generated using RAPD, ISSR and combined RAPD-ISSR grouped accessions into different clusters which reveal considerable level molecular variation among the C. falcatum accessions. It is also evident from PCA plots that accessions are rather dispersed with tested marker systems indicating good genetic base. So, in nut shell, we found considerable genetic variation and relatedness within C. falcatum accessions collected from different areas of south Gujarat, India using RAPD and ISSR markers.     

Genetic diversity and association mapping in a collection of selected Chinese soybean accessions based on SSR marker analysis

For broadening the narrow genetic base of modern soybean cultivars, 159 accessions were selected from the Chinese soybean collection which contained at least one of seven important agronomic traits: resistance to soybean cyst nematode (SCN) or soybean mosaic virus (SMV), tolerance to salt, cold, or drought, high seed oil content or high protein content. Genetic diversity evaluation using 55 microsatellite loci distributed across the genome indicated that a large amount of genetic diversity (0.806) and allelic variation (781) were conserved in this selected set, which captured 65.6% of the alleles present in Chinese soybean collection (1,863 accessions). On average, 9.4 rare alleles (frequency <5%) per locus were present, which were highly informative. Using model-based Bayesian clustering in STRUCTURE we distinguished four main clusters and a set of accessions with admixed ancestry. The four clusters reflected different geographic regions of origin of the accessions. Since the clusters were also clearly different with respect to the seven agronomic traits, the inferred population structure was introduced when association analysis was conducted. A total of 21 SSR markers on 16 chromosomes were identified as significantly (P < 0.01) associated with high oil content (6), high protein content (1), drought tolerance (5), SCN resistance (6) and SMV resistance (3). Twelve of these markers were located in or near previously identified quantitative trait loci (QTL). The results for both genetic relationship and trait-related markers will be useful for effective conservation and utilization of soybean germplasm.     

Genetic and geographic polymorphism of cultivated tobaccos (Nicotiana tabacum) in Turkey

Nicotiana tabacum (2n = 48) is a natural amphidiploid and shows a distribution over a geographical area in eastern anatolia. Random amplified polymorphic DNA (RAPD) technique was used to evaluate both genetic diversity among 21 primitive tobacco accessions comparing flue cure virginia genotype (FCV) and their geographical polymorphism as a source of genetic variations for breeding programs. Only 13 of all the 60 random primers used in RAPD showed polymorphism acceptable for characterization of these accessions. Totally 118 RAPD fragments were generated from thirteen decamer primer and sixtyfour of them were found polymorphic (54.2%). Mus and FCV showed the smallest genetic distance among accessions cultivated in the eastern anatolia. These results shows that the RAPD assay is a powerful approach for identifying genetic and geographic polymorphism.     

Identification of RAPD and ISSR markers for drought tolerance in wheat (Triticum aestivum L.)

Drought tolerance is the essential trait that needs to be incorporated in cereal crops, particularly those grown under the rainfed cultivation. Drought tolerance being contributed by several regions of the genome requires identification of these regions, using suitable molecular markers. Therefore, present investigation was aimed at analyzing the genetic diversity present among the cultivars of rainfed and the irrigated areas with respect to the drought tolerant trait. In all, 14 RAPD and 90 ISSR markers were used to identify these genomic regions. Out of 14 RAPD markers, one RAPD primer exhibited polymorphic banding pattern with 18.6 % polymorphism, clearly separating drought tolerant and drought susceptible genotypes. Out of 90 ISSR primers, only 3 ISSR primers revealed polymorphism in relation to the drought tolerance trait exhibiting 21.38 % polymorphism.     

Genetic relationships among Native American maize accessions of the Great Plains assessed by RAPDs

Genetic variation among 15 accessions of Native American maize from the Great Plains was investigated using random amplified polymorphic DNA (RAPD). RAPDs revealed very high levels of polymorphism among accessions. Banding patterns ranged in percentage polymorphism from 46.7% to 86.2% with an overall mean of 70.7% for the primers analyzed. The construction of genetic relationships using cluster analysis and principal coordinates analysis revealed that RAPDs are successful in confirming hypothesized relationships and in identifying misclassified specimens. Furthermore, the phenogram fails to reveal a strong correspondence between genetic relationships and the geographical position of Native Americans prior to contact. This provides support for the hypothesis that multiple introductions of maize into the Great Plains via trade may have resulted in the great morphological variation found among accessions in the region. Based on these data, it is unlikely that a separate Great Plains race of maize can be distinguished. In general, we conclude that RAPDs are potentially very useful in organizing seed collections and understanding intraspecific genetic differentiation. Received: 10 March 1999 / Accepted: 25 March 1999     

用RAPD和ISSR检测的橡胶树野生种质和栽培品种的遗传多样性

用19个RAPD引物和12个ISSR引物对14份野牛橡胶树种质和我国的37份栽培品种进行了遗传多样性分析。RAPD引物共产生132条带,多态性带占88．6％,相似系数变化范围在0．432—0．947。ISSR引物其产生101条带,多态性带占87．1％,相似系数为0．505—0．941。平均基因杂合度分析表明野生种质比栽培品种具有较高的遗传多样性。根据UPGMA法对51份材料进行聚类分析,结果表明,ISSR分析中所有材料可分为2类：第一类为野生种质,第二类为栽培品种：而RAPD分析中野牛种质和栽培品种不能被分为明显的两人类。虽然ISSR和RAPD的聚类分析结果存在差异,但对两种方法进行的相关分析表明,他们之间仍存在极显著相关性,相关系数为0．574。品种PR107、热研217等一些栽培品种可以通过特异带在51份供试材料中被区分开。这些结果可以对橡胶树的育种上作起到一定的指导作用,同时RAPD和ISSR技术也是进行橡胶树品种鉴定和遗传多样性研究的有效手段。     

Analysis of genetic diversity among medicinal therapist <Emphasis Type="Italic">Trigonella foenum</Emphasis>-<Emphasis Type="Italic">graecum</Emphasis> L. genotypes through RAPD and SSR markers

Trigonella is recognized as a medicinal therapist throughout the globe due to its multifaceted rare medicinal properties. It is indigenous from Iran to Northern India but has gained global acceptance towards cultivation and consumption for its yellow-to-amber colored seed which substantially contributes to food, pharmaceutical, nutraceutical and cosmetic industry. Genetic diversity serves as an excellent tool for developing improved crop varieties with breeder preferred traits. Unfortunately, very little information available on variability existing in commercial Trigonella genotypes considerably impedes the crop improvement. In this study, ninety Trigonella genotypes belonging to most productive North Indian states were subjected to multilocus genotyping using RAPD (49) and SSR (13) primers and detected an average of 55.60 and 50.16% polymorphism, respectively. The percentage polymorphism range (RAPD, 16.7–90.90; SSR, 33.30–66.66) average band informativeness (RAPD, 0.182–0.85; SSR, 0.21–0.91) and resolving power (RAPD, 0.95–9.984; SSR, 1.68–7.28) obtained revealed the wide range of diversity prevailing among these genotypes. Hierarchical clustering of genotypes in nine different clusters showed Trigonella’s genetic variability has wide genetic distribution across different agro-climatic zones. No consistency was observed while grouping Trigonella varieties based on eco-geographical region. Eventually, knowledge of these genetic differences significantly contributes in designing intra-specific crosses with potential interest to spice breeding programs. To the best of our knowledge, this is the first report of genetic diversity using SSR molecular markers in Trigonella foenum-graecum L.     

Comparative evaluation of genetic diversity using RAPD,SSR and cytochrome P450 gene based markers with respect to calcium content in finger millet (<Emphasis Type="Italic">Eleusine coracana</Emphasis> L. Gaertn.)

Genetic relationships among 52 Eleusine coracana (finger millet) genotypes collected from different districts of Uttarakhand were investigated by using randomly amplified polymorphic DNA (RAPD), simple sequence repeat (SSR) and cytochrome P450 gene based markers. A total of 18 RAPD primers, 10 SSR primers, and 10 pairs of cytochrome P450 gene based markers, respectively, revealed 49.4%, 50.2% and 58.7% polymorphism in 52 genotypes of E. coracana. Mean polymorphic information content (PIC) for each of these marker systems (0.351 for RAPD, 0.505 for SSR and 0.406 for cyt P450 gene based markers) suggested that all the marker systems were effective in determining polymorphisms. Pair-wise similarity index values ranged from 0.011 to 0.999 (RAPD), 0.010 to 0.999 (SSR) and 0.001 to 0.998 (cyt P450 gene based markers) and mean similarity index value of 0.505, 0.504 and 0.499, respectively. The dendrogram developed by RAPD, SSR and cytochrome P450 gene based primers analyses revealed that the genotypes are grouped in different clusters according to high calcium (300–450 mg/100 g), medium calcium (200–300 mg/100 g) and low calcium (100–200 mg/100 g). Mantel test employed for detection of goodness of fit established cophenetic correlation values above 0.95 for all the three marker systems. The dendrograms and principal coordinate analysis (PCA) plots derived from the binary data matrices of the three marker systems are highly concordant. High bootstrap values were obtained at major nodes of phenograms through WINBOOT software. Comparison of RAPD, SSR and cytochrome P450 gene based markers, in terms of the quality of data output, indicated that SSRs and cyt P450 gene based markers are particularly promising for the analysis of plant genome diversity. The genotypes of finger millet collected from different districts of Uttarakhand constitute a wide genetic base and clustered according to calcium contents. The identified genotypes could be used in breeding programmes and amajor input into conservation biology of cereal crops.     

Genetic Relationships Among Wild and Cultivated Accessions of Curry Leaf Plant (Murraya koenigii (L.) Spreng.), as Revealed by DNA Fingerprinting Methods

Murraya koenigii (L.) Spreng. (Rutaceae), is an aromatic plant and much valued for its flavor, nutritive and medicinal properties. In this study, three DNA fingerprinting methods viz., random amplification of polymorphic DNA (RAPD), directed amplification of minisatellite DNA (DAMD), and inter-simple sequence repeat (ISSR), were used to unravel the genetic variability and relationships across 92 wild and cultivated M. koenigii accessions. A total of 310, 102, and 184, DNA fragments were amplified using 20 RAPD, 5 DAMD, and 13 ISSR primers, revealing 95.80, 96.07, and 96.73% polymorphism, respectively, across all accessions. The average polymorphic information content value obtained with RAPD, DAMD, and ISSR markers was 0.244, 0.250, and 0.281, respectively. The UPGMA tree, based on Jaccard’s similarity coefficient generated from the cumulative (RAPD, DAMD, and ISSR) band data showed two distinct clusters, clearly separating wild and cultivated accessions in the dendrogram. Percentage polymorphism, gene diversity (H), and Shannon information index (I) estimates were higher in cultivated accessions compared to wild accessions. The overall high level of polymorphism and varied range of genetic distances revealed a wide genetic base in M. koenigii accessions. The study suggests that RAPD, DAMD, and ISSR markers are highly useful to unravel the genetic variability in wild and cultivated accessions of M. koenigii.     

Population structure and genetic diversity in bottle gourd [Lagenaria siceraria (Mol.) Standl.] germplasm from India assessed by ISSR markers

Genetic diversity analysis was undertaken in 42 geographically distant genotypes accessions of bottle gourd (Lagenaria siceraria) from India northeastern (14) and northern region (28) using inter-simple sequence repeat (ISSR) markers. A total of 209 amplified bands were obtained from 20 ISSR primers used in this study, of which 186 were polymorphic with 89.00 % band polymorphism. Various parameters namely, observed number of alleles, effective number of alleles, Nei’s gene diversity/heterozygosity, resolving power, Shannon’s information index and gene flow were estimated under experiment. Jaccard’s similarity coefficient matrix was generated for pairwise comparisons between individual ISSR profiles and UPGMA cluster analysis based on this matrix showed clustering into six groups. Jaccard’s coefficient of similarity values ranged from 0.409 to 0.847, with a mean of 0.628 revealing a moderate level of genetic diversity. The Bayesian model-based approach to infer hidden genetic population structures using the multilocus ISSR markers revealed two populations among the 42 genotypes. This is the first report on the assessment of genetic variation using ISSR markers in this medicinal vegetable plant, and this study of diversity analysis will be helpful in analyzing future hybrid breeding strategy and devising effective germplasm exploration and conservation strategy.