SSR analysis of genetic diversity and structure of the germplasm of faba bean (Vicia faba L.)

Assessing the diversity and genetic structure of faba bean (Vicia faba L.) germplasm is essential to improve the quality and yield of this economically important crop. In this study, simple sequence repeats (SSRs) were utilized to evaluate the diversity and structure of 35 faba bean genotypes originating from three different geographical regions (Northern Africa, Eastern Africa, and Near East). All 15 SSR loci generated a total of 100 alleles. The allele number per locus varied from 4 to 11, with a mean of 6.67. The expected heterozygosity (H_e) of SSR loci ranged between 0.51 and 0.81, with a mean of 0.63. The PIC value also varied from 0.44 to 0.78, with an average of 0.58. The expected heterozygosity of 22 faba bean genotypes was higher than the observed one. Interestingly, AMOVA analysis showed that much of variability resided within accessions (79.2%). A highly significant difference among regions was also evidenced, and represented 5.3% of the total variation. Moreover, cluster analysis divided the 35 faba bean genotypes into two main clusters. The first main cluster comprised all faba bean genotypes originating from the Near East region, whereas the second main cluster comprised all the genotypes originating from the Northern and Eastern Africa regions, indicating that the Northern and Eastern African faba bean genotypes were more closely related to each other than to the Near East genotypes. Structure analysis also revealed that the 35 faba bean genotypes might be assigned to two populations, in complete accordance with cluster analysis data. In conclusion, this study showed high levels of diversity in the analysed genotypes of faba bean, and could be utilized in future breeding programmes to develop new cultivars of high yield.     

Genetic diversity in recent elite faba bean lines using AFLP markers

Amplified fragment length polymorphism (AFLP) markers were used to study the genetic diversity among a large set (n = 79) of inbred lines of recent elite faba bean (Vicia faba L.) cultivars with Asian, European (Northern and Southern) and North African origin. The inbred lines were analyzed using eight selected AFLP primer combinations that produced 477 polymorphic fragments. Errors when scoring repeated lanes of one pre-amplification reaction on one gel were negligible, whereas errors when scoring lanes of two individuals of the same inbred line run on different gels were markedly higher. Scoring across gels should be backed by replicates and several appropriate check entries. Based on clustering with Jaccard's similarity coefficient and Principal Coordinate Analysis, only the Asian lines were distinct as a group, the other lines showed no marked further grouping. Nevertheless, several known pedigree relationships were verified. A priori grouping of inbred lines (geographic origin and seed size) and AFLP data corroborate available information on the history of spread and cultivation of faba bean in the studied regions. Based on the diversity observed, studies especially concerning the relationship between genetic similarity based on AFLP markers and hybrid performance within the European elite germplasm have been launched.Communicated by H.F. Linskens     

Assessment of genetic diversity in Azadirachta indica using AFLP markers

 Genetic diversity was estimated in 37 neem accessions from different eco-geographic regions of India and four exotic lines from Thailand using AFLP markers. Seven AFLP selective primer combinations generated a total of 422 amplification products. The average number of scorable fragments was 60 per experiment, and a high degree (69.8%) of polymorphism was obtained per assay with values ranging from 58% to 83.8%. Several rare and accession-specific bands were identified which could be effectively used to distinguish the different genotypes. Genetic relationships within the accessions were evaluated by generating a similarity matrix based on the Jaccard index. The phenetic dendrogram generated by UPGMA as well as principal correspondence analysis separated the 37 Indian genotypes from the four Thai lines. The cluster analysis indicated that neem germplasm within India constitutes a broad genetic base with the values of genetic similarity coefficient ranging from 0.74 to 0.93. Also, the Indian genotypes were more dispersed on the principal correspondence plot, indicating a wide genetic base. The four lines from Thailand, on the other hand, formed a narrow genetic base with similarity coefficients ranging from 0.88 to 0.92. The lowest genetic similarity coefficient value (0.47) was observed between an Indian and an exotic genotype. The level of genetic variation detected within the neem accessions with AFLP analysis suggests that it is an efficient marker technology for delineating genetic relationships amongst genotypes and estimating genetic diversity, thereby enabling the formulation of appropriate strategies for conservation and tree improvement programs. Received: 20 October 1998 / Accepted: 28 November 1998     

Assessment of genetic diversity among okra genotypes using SSR markers

Okra (Abelmoschus esculentus) is an important nutritious vegetable. Despite its high economic and industrial value, very little attention has been paid to assess genetic diversity of okra at molecular level. For effective conservation and proper deployment of germplasm, a study on diversity analysis of okra germplasm was conducted with DNA markers. Microsatellite/Simple sequence repeat (SSR) markers were utilized to evaluate the genetic diversity among 96 accessions of Abelmoschus, of which 92 accessions were of A. esculentus and one accession each of A. tuberculatus, A. moschatus, A. moschatus subspecies tuberosus and A. manihot. A set of 40 SSR primers were tested, of which 30 primers gave reproducible amplification which were used further for diversity analysis. With a mean of 7.1 bands per SSR, DNA amplification with 30 SSRs generated a total 213 bands, of which 60.66 % were recorded polymorphic. Polymorphic information content ranged between 0.11 and 0.80 with an average of 0.52, indicating that the majority of primers were informative. The Jaccard’s coefficient ranged from 0.107 to 0.969. The UPGMA analysis grouped Abelmoschus genotypes into three main clusters at a cut-off of 0.20. Results of present study revealed that sufficient variation exists among the studied accessions and GAO-5 which was found highly diverse can be exploited for okra improvement. The outcome of present research would assist to make use of Ablemoschus germplasm for okra breeding.     

Morphological and physiological characterization of different genotypes of faba bean under heat stress

Heat stress (HS) is the major constraint to crop productivity worldwide. The objective of the present experiment was to select the tolerant and sensitive genotype(s) on the basis of morpho-physiological and biochemical characteristics of ten Vicia faba genotypes. These genotypes were as follows: Zafar 1, Zafar 2, Shebam 1, Makamora, Espan, Giza Blanka, Giza 3, C4, C5 and G853. The experimental work was undertaken to study the effects of different levels of temperature (control, mild, and modest) on plant height (PH) plant⁻¹, fresh weight (FW) and dry weight (DW) plant⁻¹, area leaf⁻¹, content of leaf relative water (RWC), proline content (Pro) and total chlorophyll (Total Chl), electrolyte leakage (EL), malondialdehyde level (MDA), hydrogen peroxide (H₂O₂), and activities of catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD) enzymes. HS significantly affected growth performance of all genotypes. However, the magnitude of reduction in genotypes ‘C5’ was relatively low, possibly due to its better antioxidant activities (CAT, POD and SOD), and accumulation of Pro and Total Chl, and leaf RWC. In the study, ‘C5’ was noted to be the most HS tolerant and ‘Espan’ most HS sensitive genotypes. It was concluded that the heat-tolerant genotypes may have better osmotic adjustment and protection from free radicals by increasing the accumulation of Pro content with increased activities of antioxidant enzyme.     

Determination of genetic diversity among Saccharina germplasm using ISSR and RAPD markers

Various species of genus Saccharina are economically important brown macroalgae cultivated in China. The genetic background of the conserved Saccharina germplasm was not clear. In this report, DNA-based molecular markers such as inter simple sequence repeats (ISSR) and random amplified polymorphic DNA (RAPD) were used to assess the genetic diversity and phylogenetic relationships among 48 Saccharina germplasms. A total of 50 ISSR and 50 RAPD primers were tested, of which only 33 polymorphic primers (17 ISSR and 16 RAPD) had an amplified clear and reproducible profile, and could be used. Seventeen ISSR primers yielded a total of 262 bands, of which 256 were polymorphic, and 15.06 polymorphic bands per primer were amplified from 48 kelp gametophytes. Sixteen RAPD primers produced 355 bands, of which 352 were polymorphic, and 22 polymorphic bands per primer were observed across 48 individuals. The simple matching coefficient of ISSR, RAPD and pooled ISSR and RAPD dendrograms ranged from 0.568 to 0.885, 0.670 to 0.873, and 0.667 to 0.862, revealing high genetic diversity. Based on the unweighted pair group method with the arithmetic averaging algorithm (UPGMA) cluster analysis and the principal components analysis (PCA) of ISSR data, the 48 gametophytes were divided into three main groups. The Mantel test revealed a similar polymorphism distribution pattern between ISSR and RAPD markers, the correlation coefficient r was 0.62, and the results indicated that both ISSR and RAPD markers were effective to assess the selected gametophytes, while matrix correlation of the ISSR marker system (r = 0.78) was better than that of the RAPD marker system (r = 0.64). Genetic analysis data from this study were helpful in understanding the genetic relationships among the selected 17 kelp varieties (or lines) and provided guidance for molecular-assisted selection for parental gametophytes of hybrid kelp breeding.     

Nutritional and antinutritional attributes of faba bean (Vicia faba L.) germplasms growing in Bihar,India

Eleven germplasms of faba bean seeds from four agroclimatic regions of Bihar, India, have been investigated to estimate their nutritional (soluble protein, free amino acids, starch, reducing and non reducing sugar, total soluble sugar) and antinutritional (total extractable phenol and condensed tannin/proanthocyanidin) parameters. These parameters were found in varying concentration in all genotypes studied. The highest concentration of total extractable phenol and proanthocyanidin (condensed tannin) (2.56 and 1.59 % leucocyanidin equivalents respectively on dry matter basis) were found in Samastipur while the lowest from Patna (0.95 and 0.426 % leucocyanidin equivalent on dry matter basis). The different nutritional parameters were also found to be in variable concentration among different germplasms viz. total soluble protein ≈ 20–32 %, free amino acids ≈ 188–348 mg/100 g, starch ≈ 27–33 %, reducing sugars ≈ 85–188 mg/100 g, non reducing sugars ≈ 0.7–1.7 % and total soluble sugars ≈ 0.8–1.9 %.     

Development of SCAR markers linked to a gene controlling absence of tannins in faba bean

Faba beans are inexpensive, nutrient-dense sources of plant protein, but anti-nutritional factors such as condensed tannins reduce the biological value of their protein. Two recessive genes, zt-1 and zt-2, control the absence of tannins in faba bean seeds and also determine a white flower character on the plant. However, crosses between them produce coloured F₁ plants with tannins that contaminate the crop. Therefore, it is important to identify the gene present in all tannin-free cultivars and gene bank accessions to enable breeders to choose appropriate genitors for their crosses. The aim of this study was the identification of markers linked to zt-1, one of the genes governing free tannin content in faba bean. A segregating F₂ population derived from the cross between the coloured flower and high tannin content genotype Vf6 and a zt-1 line was developed and characterized phenotypically. Bulked Segregant Analysis (BSA) was used to identify Random Amplified Polymorphic DNA (RAPD) markers linked to the zt-1 gene. Four RAPD loci (OPC5₅₅₁, OPG15₆₀₀, OPG11₁₁₇₁ and OPAF20₇₇₆) showed polymorphism between the contrasting bulks. The markers were sequenced to develop specific Sequence Characterised Amplified Regions (SCARs). Amplification of SCC5₅₅₁ produced a single product which was only observed in the white flowered and zero tannin content genotypes, whereas SCAR SCG11₁₁₇₁only produced a band in F₂ plants with coloured flower and high tannin content. SCARs SCC5₅₅₁ and SCG11₁₁₇₁ were tested for their applicability for routine screening in 37 faba bean genotypes differing in flower colour and tannin content. SCC5₅₅₁, allowed the prediction of the zt-1 genotypes with a 95% of accuracy, underscoring the potential of this SCAR marker as a cost-effective tool for MAS in large faba bean breeding populations.     

Assessment of genetic diversity in a Morus germplasm collection using fluorescence-based AFLP markers

To meet various breeding objectives and to conserve the existing genetic resources of mulberry for future use, the present study was undertaken to investigate the amount of genetic diversity and to establish the relationships between mulberry genotypes using fluorescence-based AFLP markers. Genetic diversity was estimated in 45 mulberry accessions from different eco-geographic regions of Japan and other parts of the world. Five primer combinations amplified an average of 110 AFLP markers per primer combination, ranging in size from 35 to 500 bp. A high degree of polymorphism was revealed by these combinations that ranged from 69.7 to 82.3% across all the genotypes studied. Several rare genotype-specific bands were also identified which could be effectively utilized to distinguish different genotypes. The wide range in genetic similarity coefficients (0.58–0.99) indicated that the mulberry germplasm collection represents a genetically diverse popu-lation. The phenetic dendrogram generated by the UPGMA method grouped 45 accessions into four major clusters, which was in agreement with the results from conventional methods. Clustering of some genotypes into strictly separate groups was not readily apparent and no clear interrelationships could be depicted, in spite of their different geographic origin. In addition, AFLP analysis provided sufficient polymorphism for DNA typing and contributed additional insights into the genetic structure of the mulberry germplasm. These results will help in the formulation of appropriate strategies for conservation and variety improvement in mulberry, for which little or no knowledge of genetic diversity is currently available. Received: 30 December 1999 / Accepted: 14 March 2000     

Description and analysis of genetic diversity between commercial bean lines (Phaseolus vulgaris L.)

The effectiveness of RFLP, DAMD-PCR, ISSR and RAPD markers in assessing polymorphism and relationships between 24 commercial lines of Phaseolus vulgaris L.was evaluated. We have used a Phaseolus-specific minisatellite sequence as a probe, which enabled 23 of the bean lines tested to be fingerprinted. Based on the sequence information obtained, primers corresponding to the bean-specific minisatellite core sequence were used in subsequent PCR amplifications. Our observations indicated that while the DAMD-PCR was sensitive in detecting genetic variation between bean species and between accessions of P. vulgaris, when used alone it may be limited in its ability to detect genetic variation among cultivated bean lines due to the low number of loci amplified. Only one out of the five ISSR primers tested was efficient in generating multiple band profiles, which was insufficient to distinguish all the different bean lines. Reproducible RAPD profiles were obtained, and these allowed us to differentiate all the genotypes tested with seven primers. We ultimately used only results from RFLP and RAPD markers to explore the genetic diversity among commercial bean lines. Both analyses led to the same clustering of the bean lines according to their geographical origins (United States or Europe). With respect to the European lines, the results obtained from RAPD data also enable the lines to be clustered according to their creators. Received: 15 January 2000 / Accepted: 21 March 2000     

Assessment of genetic diversity among 16 promising cultivars of ginger using cytological and molecular markers

Ginger (Zingiber officinale Roscoe) is an economically important plant, valued all over the world. The existing variation among 16 promising cultivars as observed through differential rhizome yield (181.9 to 477.3 g) was proved to have a genetic basis using different genetic markers such as karyotype, 4C nuclear DNA content and random amplified polymorphic DNA (RAPD). The karyotypic analysis revealed a differential distribution of A, B, C, D and E type of chromosomes among different cultivars as represented by different karyotype formulas. A significant variation of 4C DNA content was recorded in ginger at an intraspecific level with values ranging from 17.1 to 24.3 pg. RAPD analysis revealed a differential polymorphism of DNA showing a number of polymorphic bands ranging from 26 to 70 among 16 cultivars. The RAPD primers OPC02, OPA02, OPD20 and OPN06 showing strong resolving power were able to distinguish all 16 cultivars. The extent of genetic diversity among these cultivars was computed through parameters of gene diversity, sum of allele numbers per locus and Shannon's information indices. Cluster analysis, Nei's genetic similarity and genetic distances, distribution of cultivars into special distance classes and principal coordinate analysis and the analysis of molecular variance suggested a conspicuous genetic diversity among different cultivars studied. The genetic variation thus detected among promising cultivars of ginger has significance for ginger improvement programs.     

Assessment and comparison of AFLP and SSR based molecular genetic diversity in Indian isolates of Ascochyta rabiei, a causal agent of Ascochyta blight in chickpea (Cicer arietinum L.)

Ascochyta blight (AB), caused by Ascochyta rabiei (Pass.) Labr. (anamorph), is the most damaging disease of chickpea (Cicer arietinum L.) and is a serious biotic stress constraint for chickpea production. To understand the molecular diversity in A. rabiei populations of India, a total of 64 isolates collected from AB-infected chickpea plants from different agroclimatic regions in the North Western Plain Zone (NWPZ) of India were analyzed with 11 AFLP (amplified fragment length polymorphism) and 20 SSR (simple sequence repeat) markers. A total of 9 polymorphic AFLP primer pairs provided a total of 317 fragments, of which 130 were polymorphic and showed an average PIC value 0.28. Of the SSR markers, 12 showed polymorphism and provided a total of 29 alleles with an average PIC value 0.35. To the best of our knowledge, this is the first report on a comparison of AFLP and SSR diversity estimates in A. rabiei populations. The dendrogram developed based on AFLP and SSR data separately, as well as on the combined marker dataset, grouped the majority of AB isolates as per geographic regions. Model based population structure analysis revealed four distinct populations with varying levels of ancestral admixtures among 64 isolates studied. Interestingly, several AFLP primer combinations and SSR markers showed the locus/allele specific to AB isolates of certain regions, e.g., Hisar, Sriganganagar, Gurdaspur, and Sundarnagar. Genetic variability present in AB isolates of the NWPZ of India suggests the continuous monitoring of changes in A. rabiei population to anticipate the breakdown of AB resistance in chickpea cultivars grown in India.     

Assessment of genetic diversity within and among germplasm accessions in cultivated sorghum using microsatellite markers

Microsatellite markers are increasingly being used in crop plants to discriminate among genotypes and as tools in marker-assisted selection. Here we evaluated the use of microsatellite markers to quantify the genetic diversity within as well as among accessions sampled from the world germplasm collection of sorghum. Considerable variation was found at the five microsatellite loci analysed, with an average number of alleles per locus equal to 2.4 within accessions and 19.2 in the overall sample of 25 accessions. The collection of sorghum appeared highly structured genetically with about 70% of the total genetic diversity occurring among accessions. However, differentiation among morphologically defined races of sorghum, or among geographic origins, accounted for less than 15% of the total genetic diversity. Our results are in global agreement with those obtained previously with allozyme markers. We were also able to show that microsatellite data are useful in identifying individual accessions with a high relative contribution to the overall allelic diversity of the collection. Received: 10 August 1999 / Accepted: 27 August 1999     

AFLP assessment of genetic diversity among Indian Mucuna accessions

Amplified fragment length polymorphism (AFLP) marker was used to assess diversity in germplasm collection of Mucuna species which has gained tremendous attention in the recent past due to its promising nutritional, agronomic and medicinal attributes. Twenty five accessions comprising five species, collected from seven states of India were evaluated with twelve AFLP primer combinations that generated a total of 1,612 fragments with an average of 134 fragments per primer combination. The values of polymorphic information content (PIC), marker index (MI) and the resolving power (Rp) demonstrated the utility of the primer combinations used in the present study for discriminating the Mucuna accessions. UPGMA and Principal coordinate analysis (PCoA) of the genotypic data revealed clustering of accessions as per phenetic and genetic relationships. The Jaccard’s similarity coefficient values suggested good variability among the M. pruriens accessions indicating their utility in breeding programs. Molecular diversity presented in this study combined with the datasets on other morphological/agronomic traits will be highly useful for selecting appropriate accessions for plant improvement through conventional as well as molecular breeding approaches and for evolving suitable conservation strategies.     

Assessment of genetic diversity among and within Achillea species using amplified fragment length polymorphism (AFLP)

Amplified fragment length polymorphism (AFLP) markers were used to assess the genetic diversity of 57 Achillea accessions belonging to five species, A. millefolium, A. filipendulina, A. tenuifolia, A. santolina and A. biebersteinii. Nine AFLP primer combinations were used, which produced 301 polymorphic bands. In most species, a high level of genetic variation was detected among the genotypes. The Jaccard's similarity indices (J), based on AFLP profiles, were subjected to UPGMA cluster analysis. Application of Mantel's test for cophenetic correlation to the cluster analysis indicated the high fitness of the accessions to a group (r = 0.918). The dendrogram generated revealed five major groups corresponding to five species. The principle coordinate analysis (PCoA) data confirmed the results of the clustering. Among the species, A. teunifolia and A. santolina showed the greatest and the least genetic diversity, respectively. A. filipendulina accessions were acquired primarily from the same ecological regions of western Iran. Accessions belonging to A. biebersteinii originated from the Isfahan province and were separated from other species at the root of the dendrogram. The results of the clustering method, based on AFLP markers, corresponded closely with the geographical origins of the genotypes. The results of the present study could contribute to a better understanding and management of conservation and exploitation of the Achillea germplasm.     

Analysis of genetic diversity in Aconitum kongboense L. revealed by AFLP markers

The systematic evaluation of the molecular diversity encompassed in Aconitum kongboense L. inbreds or parental lines offers an efficient means of exploiting the heterosis in A. kongboense as well as for management of biodiversity. An excellent and novel DNA-based molecular, amplified fragment length polymorphisms (AFLPs) markers, was firstly used to analysis the genetic diversity in A. kongboense genotypes. Out of 256 primers screened, a total of ten combinations successfully produced scorable, clear, reproducible and relatively high polymorphism bands, 64.12% of which were polymorphic. The values of number of polymorphic loci (NPL), percentage of polymorphic loci (PPB), observed number of alleles (Na) and effective number of alleles (Ne) were highest in population 3 (NPL = 77, PPB = 68.75%, Na = 1.688 ± 0.466, Ne = 1.412 ± 0.397) and lowest in population 2 (NPL = 57, PPB = 50.89%, Na = 1.509 ± 0.502, Ne = 1.273 ± 0.340). In addition, Jaccard's similarity coefficients among different populations varied from 0.45 to 1.00 with an average of 0.55. The data collected will contribute to identification, rational exploitation and conservation of germplasms of A. kongboense, and potentially useful to aid its breeding.     

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.     

Molecular assessment of genetic diversity in mung bean germplasm

RAPD profiles were used to identify the extent of diversity among 54 accessions of mung bean that included both improved and local land races. Out of the 40 primers screened, seven primers generated 174 amplification products with an average of 24.85 bands per primer. The RAPD profiles were analysed for Jaccard's similarity coefficients that was found to be in the range from 0 to 0.48, indicating the presence of wide range of genetic diversity at molecular level. Cluster analysis was carried out based on distances (1-similarity coefficient) using neighbour-joining method in Free Tree package. The dendrogram resolved all the accessions into two major clusters, I (with 11 accessions) and II (with 43 accessions). However, the cluster was further divided into four subclusters (II A with six, II B with nine, II C with 15 and II D with 13 accessions). The distribution of the accessions in different clusters and subclusters appears to be related to their performance in field conditions for 10 morphological traits that were scored. This study indicated that the RAPD profiles provide an easy and simple technique for preliminary genetic diversity assessment of mung bean accessions that may reflect morphological trait differences among them.     

中国甘蓝型油菜遗传多样性的RAPD分子标记

本文利用ＲＡＰＤ方法和统计学分析,对我国７省市和国外引进的总计４０份甘蓝型油菜品种的遗传多样性进行了研究。结果表明,４０个品种的甘蓝型油菜存在着广泛的遗传变异,根据ＲＡＰＤ指纹图谱,通过在ＤＮＡ分子水平上的聚类分析可以将它们分为３大类群,反映出这些品种之间的亲缘关系,并对如何引进甘蓝型油菜资源进行了初浅的讨论。