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.     

Molecular marker-based genetic diversity assessment of Striga-resistant maize inbred lines

Striga-resistant maize inbred lines are of interest to maize breeding programs in the savannas of Africa where the parasitic weed is endemic and causes severe yield losses in tropical maize. Assessment of the genetic diversity of such inbred lines is useful for their systematic and efficient use in a breeding program. Diversity analysis of 41 Striga-resistant maize inbred lines was conducted using amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers to examine the genetic relationships among these lines and to determine the level of genetic diversity that exists within and between their source populations. The two marker systems generated 262 and 101 polymorphic fragments, respectively. Genetic similarity (GS) values among all possible pairs of inbred lines varied from 0.45 to 0.95, with a mean of 0.61±0.002 for AFLPs, and from 0.21 to 0.92, with a mean of 0.48±0.003, for SSRs. The inbred lines from each source population exhibited a broad range of GS values with the two types of markers. Both AFLPs and SSRs revealed similar levels of within population genetic variation for all source populations. Cluster and principal component analysis of GS estimates with the two markers revealed clear differentiation of the Striga-resistant inbred lines into groups according to their source populations. There was clear separation between early- and late-maturing Striga-resistant inbred lines. Considering the paucity of germplasm with good levels of resistance to Striga in maize, the broad genetic diversity detected within and among source populations demonstrates the genetic potential that exists to improve maize for resistance to Striga.     

Genetic diversity of cultivated flax (Linum usitatissimum L.) germplasm assessed by retrotransposon-based markers

Retrotransposon segments were characterized and inter-retrotransposon amplified polymorphism (IRAP) markers developed for cultivated flax (Linum usitatissimum L.) and the Linum genus. Over 75 distinct long terminal repeat retrotransposon segments were cloned, the first set for Linum, and specific primers designed for them. IRAP was then used to evaluate genetic diversity among 708 accessions of cultivated flax comprising 143 landraces, 387 varieties, and 178 breeding lines. These included both traditional and modern, oil (86), fiber (351), and combined-use (271) accessions, originating from 36 countries, and 10 wild Linum species. The set of 10 most polymorphic primers yielded 141 reproducible informative data points per accession, with 52% polymorphism and a 0.34 Shannon diversity index. The maximal genetic diversity was detected among wild Linum species (100% IRAP polymorphism and 0.57 Jaccard similarity), while diversity within cultivated germplasm decreased from landraces (58%, 0.63) to breeding lines (48%, 0.85) and cultivars (50%, 0.81). Application of Bayesian methods for clustering resulted in the robust identification of 20 clusters of accessions, which were unstratified according to origin or user type. This indicates an overlap in genetic diversity despite disruptive selection for fiber versus oil types. Nevertheless, eight clusters contained high proportions (70?C100%) of commercial cultivars, whereas two clusters were rich (60%) in landraces. These findings provide a basis for better flax germplasm management, core collection establishment, and exploration of diversity in breeding, as well as for exploration of the role of retrotransposons in flax genome dynamics.     

Comparative SNP and haplotype analysis reveals a higher genetic diversity and rapider LD decay in tropical than temperate germplasm in maize

Understanding of genetic diversity and linkage disequilibrium (LD) decay in diverse maize germplasm is fundamentally important for maize improvement. A total of 287 tropical and 160 temperate inbred lines were genotyped with 1943 single nucleotide polymorphism (SNP) markers of high quality and compared for genetic diversity and LD decay using the SNPs and their haplotypes developed from genic and intergenic regions. Intronic SNPs revealed a substantial higher variation than exonic SNPs. The big window size haplotypes (3-SNP slide-window covering 2160 kb on average) revealed much higher genetic diversity than the 10 kb-window and gene-window haplotypes. The polymorphic information content values revealed by the haplotypes (0.436-0.566) were generally much higher than individual SNPs (0.247-0.259). Cluster analysis classified the 447 maize lines into two major groups, corresponding to temperate and tropical types. The level of genetic diversity and subpopulation structure were associated with the germplasm origin and post-domestication selection. Compared to temperate lines, the tropical lines had a much higher level of genetic diversity with no significant subpopulation structure identified. Significant variation in LD decay distance (2-100 kb) was found across the genome, chromosomal regions and germplasm groups. The average of LD decay distance (10-100 kb) in the temperate germplasm was two to ten times larger than that in the tropical germplasm (5-10 kb). In conclusion, tropical maize not only host high genetic diversity that can be exploited for future plant breeding, but also show rapid LD decay that provides more opportunity for selection.     

Inter retrotransposon based genetic diversity and phylogenetic analysis among the Musa germplasm accessions

In the present investigation, the insertional polymorphisms of retro-elements were studied in the Musa germplasm available at ICAR-NRCB field gene bank using IRAP markers. The maximum number of polymorphic bands were produced by the primer pair Nikita and LTR 6150 (48) followed by LTR 6149 and 3′LTR (47) and minimum of 35 bands were produced by the primer pair Sukkula and LTR 6150. The bands produced were scored as 0 (absent) and 1 (present) and the resultant binary data was subjected to diversity analysis. The dendrogram consisted of two major clusters with members of Eumusa and Rhodochlamys in one indicating their genetic closeness and members of the genus Ensete in another cluster. Results of principal coordinate analysis were congruent to those obtained in hierarchial cluster analysis. The molecular markers used in this study could reveal intra and inter-group diversity among the Musa germplasm accessions with similarity co-efficient ranging from 0.41 to 0.99. IRAP marker system has performed excellently clustering the accessions based on both genomic and subgroup levels. The entire germplasm was found to be robust with no duplications indicating the diverse group of accessions available at ICAR-NRCB field gene bank. It has also exhibited high polymorphism and hence could be effectively used to detect the genetic relatedness among diverse genome of Musa.     

中国木耳栽培种质生物学特性及遗传多样性分析

对中国32个木耳Auricularia auricula-judae主要栽培菌株的生物学特性进行了系统分析,在此基础上分别对27个生物学特性指标赋值,运用UPGMA聚类法及PCO主坐标分析法对木耳种质资源的遗传多样性进行研究。研究结果显示中国木耳栽培种质的遗传多样性丰富,32个供试菌株在生物学特性指标上均存在着一定差异;供试菌株聚为3个主要类群,分别包含大部分东北地区菌株,中部及东南部地区菌株,华北华南及部分中部地区菌株;表明生物学特性与地域来源呈一定相关性,同一地域来源的栽培菌株相似度更高,部分菌株间存在着同物异名现象。     

Microsatellite marker-based genetic diversity assessment among exotic and native maize inbred lines of Bangladesh

Hybrid development is basically dependent on the variability among available genetic resources. Polymorphism among the maize inbreds is essentially needed for maize hybridization. This study aimed at the assessment of diversity among 22 maize inbreds by 18 microsatellite markers. The study identified 187 alleles at 18 SSR loci. The amplified allele frequency per microsatellite locus was 10.4 and the highest allele per locus was 17 in SSR primer pair phi026. SSR primer set p-umc1292, phi074 and phi090 showed the lowest 6 alleles per genotype per locus. The locus phi026 showed the highest degree of gene diversity (0.92), and the locus p-umc1292 had the lowest of gene diversity (0.77) with a mean value of 0.862 among the microsatellites. At each site, the most prevalent allele varied between 0.14 (bnlg371) and 0.36. (p-umc1292). At any given locus, an average of 0.22 out of the 22 selected maize inbred lines had a common major allele. The average value of the polymorphic information content (PIC) was 0.85, within the range of 0.74 at the lowest to 0.92 at the highest. The higher PIC values of phi026 and nc013 established them to be the best markers for maize inbred lines. The UPGMA clustering generated seven distinct groups having 12.5% of similarity coefficient. The results revealed that inbred lines E10, E27, E19, E34, E35, E4, E43, E28, E11, E21, E17, E38, E25, E34, E14, E16, E39 and E3 were more diversified. These lines are promising to be used as parent materials for hybrid maize development in the future.     

An analysis of genetic diversity across the maize genome using microsatellites

How domestication bottlenecks and artificial selection shaped the amount and distribution of genetic variation in the genomes of modern crops is poorly understood. We analyzed diversity at 462 simple sequence repeats (SSRs) or microsatellites spread throughout the maize genome and compared the diversity observed at these SSRs in maize to that observed in its wild progenitor, teosinte. The results reveal a modest genome-wide deficit of diversity in maize relative to teosinte. The relative deficit of diversity is less for SSRs with dinucleotide repeat motifs than for SSRs with repeat motifs of more than two nucleotides, suggesting that the former with their higher mutation rate have partially recovered from the domestication bottleneck. We analyzed the relationship between SSR diversity and proximity to QTL for domestication traits and observed no relationship between these factors. However, we did observe a weak, although significant, spatial correlation for diversity statistics among SSRs within 2 cM of one another, suggesting that SSR diversity is weakly patterned across the genome. Twenty-four of 462 SSRs (5%) show some evidence of positive selection in maize under multiple tests. Overall, the pattern of genetic diversity at maize SSRs can be explained largely by a bottleneck effect with a smaller effect from selection.     

Assessing genetic diversity in a sugarcane germplasm collection using an automated AFLP analysis

An assessment of genetic diversity within and between Saccharum, Old World Erianthus sect. Ripidium, and North American E.giganteus (S.giganteum) was conducted using Amplified Fragment Length Polymorphism (AFLP^TM) markers. An automated gel scoring system (GelCompar^TM) was successfully used to analyse the complex AFLP patterns obtained in sugarcane and its relatives. Similarity coefficient calculations and clustering revealed a genetic structure for Saccharum and Erianthus sect. Ripidium that was identical to the one previously obtained using other molecular marker types, showing the appropriateness of AFLP markers and the associated automated analysis in assessing genetic diversity in sugarcane. A genetic structure that correlated with cytotype (2n=30, 60, 90) was revealed within the North American species, E. giganteus (S.giganteum). Complex relationships among Saccharum, Erianthus sect. Ripidium, and North American E.giganteus were revealed and are discussed in the light of a similar study which involved RAPD markers. This revised version was published online in July 2006 with corrections to the Cover Date.     

High throughput analysis of grape genetic diversity as a tool for germplasm collection management

Using 20 SSR markers well scattered across the 19 grape chromosomes, we analyzed 4,370 accessions of the INRA grape repository at Vassal, mostly cultivars of Vitis vinifera subsp. sativa (3,727), but also accessions of V. vinifera subsp. sylvestris (80), interspecific hybrids (364), and rootstocks (199). The analysis revealed 2,836 SSR single profiles: 2,323 sativa cultivars, 72 wild individuals (sylvestris), 306 interspecific hybrids, and 135 rootstocks, corresponding to 2,739 different cultivars in all. A total of 524 alleles were detected, with a mean of 26.20 alleles per locus. For the 2,323 cultivars of V. vinifera, 338 alleles were detected with a mean of 16.9 alleles per locus. The mean genetic diversity (GDI) was 0.797 and the level of heterozygosity was 0.76, with broad variation from 0.20 to 1. Interspecific hybrids and rootstocks were more heterozygous and more diverse (GDI?=?0.839 and 0.865, respectively) than V. vinifera cultivars (GDI?=?0.769), Vitis vinifera subsp. sylvestris being the least divergent with GDI?=?0.708. Principal coordinates analysis distinguished the four groups. Slight clonal polymorphism was detected. The limit between clonal variation and cultivar polymorphism was set at four allelic differences out of 40. SSR markers were useful as a complementary tool to traditional ampelography for cultivar identification. Finally, a set of nine SSR markers was defined that was sufficient to distinguish 99.8% of the analyzed accessions. This set is suitable for routine characterization and will be valuable for germplasm management.     

Analysis of plant diversity with retrotransposon-based molecular markers

Retrotransposons are both major generators of genetic diversity and tools for detecting the genomic changes associated with their activity because they create large and stable insertions in the genome. After the demonstration that retrotransposons are ubiquitous, active and abundant in plant genomes, various marker systems were developed to exploit polymorphisms in retrotransposon insertion patterns. These have found applications ranging from the mapping of genes responsible for particular traits and the management of backcrossing programs to analysis of population structure and diversity of wild species. This review provides an insight into the spectrum of retrotransposon-based marker systems developed for plant species and evaluates the contributions of retrotransposon markers to the analysis of population diversity in plants.     

香稻资源遗传多样性的比较

利用60个水稻SSR标记, 对来自国内外的370份香稻材料的遗传多样性进行了比较分析。结果共检测到361个等位基因, 每个位点的等位基因变幅为2~10个, 平均Nei基因多样性指数(He)为0.663, 变幅为0.104(RM308)~0.885(RM2634)。籼粳亚种间的遗传多样性具有明显差异, 籼稻的等位基因数和Nei基因多样性指数均高于粳稻。地方品种的遗传多样性高于选育品种, 选育品种等位基因数仅为地方品种的86.5%。分子方差分析表明, 香稻材料中总变异的43.08%是由于亚种间的遗传差异引起的。不同稻区的遗传分化程度总体介于1.69%~14.40%之间。其中, 华南与西南、华中与西南地方品种间遗传差异的分化程度达显著水平。聚类分析将参试材料明显分为籼粳两大类, 同时地域相同(稻区)、相邻省份的香稻材料基本归为同一类群。     

Analysis of genetic diversity and relationships in East African banana germplasm

The genetic diversity and phylogenetic relationships of 29 East African highland banana (Musa spp.) cultivars and two outgroup taxa, M. acuminata Calcutta 4 and Agbagba were surveyed by RAPD analysis. A genetic similarity matrix was established based on the presence or absence of polymorphic amplified fragments. Phylogenetic relationships were determined by UPGMA cluster analysis. RAPDs showed that the highland bananas are closely related with a narrow genetic base. Nevertheless, there were sufficient RAPD polymorphisms that were collectively useful in distinguishing the cultivars. The dendrogram was divisible into a major cluster composed of all the AAA highland banana cultivars and Agbagba (AAB) and a minor cluster consisting of Kisubi (AB), Kamaramasenge (AB) and Calcutta 4 (AA). Several subgroups are recognized within the major cluster. RAPD data did not separate beer and cooking banana cultivars. Our study showed that RAPD markers can readily dissect genetic differences between the closely related highland bananas and provide a basis for the selection of parents for improvement of this germplasm. Received: 28 June 2000 / Accepted: 1 August 2000     

RAPD analysis of the genetic diversity of mango (Mangifera indica) germplasm in Brazil

We evaluated genetic variability of mango (Mangifera indica) accessions maintained in the Active Germplasm Bank of Embrapa Meio-Norte in Teresina, Piauí, Brazil, using RAPDs. Among these accessions, 35 originated from plantings in Brazil, six from the USA and one from India. Genomic DNA, extracted from leaf material using a commercial purification kit, was subjected to PCR with the primers A01, A09, G03, G10, N05, and M16. Fifty-five polymorphic loci were identified, with mean of 9.16 ± 3.31 bands per primer and 100% polymorphism. Application of unweighted pair group method using arithmetic average cluster analysis demonstrated five genotypic groups among the accessions examined. The genotypes Rosa 41, Rosa 48 and Rosa 49 were highly similar (94% similarity), whereas genotypes Sensation and Rosa 18 were the most divergent (only 7% similarity). The mango accessions were found to have considerable genetic variability, demonstrating the importance of analyzing each genotype in a collection in order to efficiently maintain the germplasm collection.     

Fine genetic characterization of elite maize germplasm using high-throughput SNP genotyping

To investigate the genetic structure of Chinese maize germplasm, the MaizeSNP50 BeadChip with 56,110 single nucleotide polymorphisms (SNPs) was used to genotype a collection of 367 inbred lines widely used in maize breeding of China. A total of 41,819 informative SNPs with minor allele number of more than 0.05 were used to estimate the genetic diversity, relatedness, and linkage disequilibrium (LD) decay. Totally 1,015 SNPs evenly distributed in the genome were selected randomly to evaluate the population structure of these accessions. Results showed that two main groups could be determined i.e., the introduced germplasm and the local germplasm. Further, five subgroups corresponding to different heterotic groups, that is, Reid Yellow Dent (Reid), Lancaster Sure Crop (Lancaster), P group (P), Tang Sipingtou (TSPT), and Tem-tropic I group (Tem-tropic I), were determined. The genetic diversity of within subgroups was highest in the Tem-Tropic I and lowest in the P. Most lines in this panel showed limited relatedness with each other. Comparisons of gene diversity showed that there existed some conservative genetic regions in specific subgroups across the ten chromosomes, i.e., seven in the Lancaster, seven in the Reid, six in the TSPT, five in the P, and two in the Tem-Tropical I. In addition, the results also revealed that there existed fifteen conservative regions transmitted from Huangzaosi, an important foundation parent, to its descendants. These are important for further studies since the outcomes may provide clues to understand why Huangzaosi could become a foundation parent in Chinese maize breeding. For the panel of 367 elite lines, average LD distance was 391 kb and varied among different chromosomes as well as in different genomic regions of one chromosome. This analysis uncovered a high natural genetic diversity in the elite maize inbred set, suggesting that the panel can be used in association study, esp. for temperate regions.     

High-throughput linkage analysis of Mutator insertion sites in maize

Insertional mutagenesis is a cornerstone of functional genomics. High-copy transposable element systems such as Mutator ( Mu ) in maize ( Zea mays ) afford the advantage of high forward mutation rates but pose a challenge for identifying the particular element responsible for a given mutation. Several large mutant collections have been generated in Mu -active genetic stocks, but current methods limit the ability to rapidly identify the causal Mu insertions. Here we present a method to rapidly assay Mu insertions that are genetically linked to a mutation of interest. The method combines elements of MuTAIL (thermal asymmetrically interlaced) and amplification of insertion mutagenized sites (AIMS) protocols and is applicable to the analysis of single mutants or to high-throughput analyses of mutant collections. Briefly, genomic DNA is digested with a restriction enzyme and adapters are ligated. Polymerase chain reaction is performed with TAIL cycling parameters, using a fluorescently labeled Mu primer, which results in the preferential amplification and labeling of Mu -containing genomic fragments. Products from a segregating line are analyzed on a capillary sequencer. To recover a fragment of interest, PCR products are cloned and sequenced. Sequences with lengths matching the size of a band that co-segregates with the mutant phenotype represent candidate linked insertion sites, which are then confirmed by PCR. We demonstrate the utility of the method by identifying Mu insertion sites linked to seed-lethal mutations with a preliminary success rate of nearly 50%.     

Evaluation of maize microsatellite markers for genetic diversity analysis and fingerprinting in sugarcane.

The use of maize microsatellite markers as a potential cost-effective method for molecular analysis of sugarcane was evaluated. Of the 34 primer pairs obtained from maize genomic libraries, 14 showed repeatable amplifications in Saccharum species clones, commercial hybrids, and the related genera Erianthus, accounting for 41.17% cross transferability. Complex banding patterns were encountered in sugarcane with the number of amplified fragments ranging from 7 to 14 with an average of 10 per primer, indicating the high polyploidy and heterozygosity existing in sugarcane. Phenetic analysis of the SSR polymorphisms produced by nine primers could clearly differentiate the different species of Saccharum and Erianthus and revealed the relationships that existed between them. Genetic similarity co-efficient indicated low diversity existing among the S. officinarum clones (82%) and a relatively higher level of diversity in the S. spontaneum clones (69.7%). Higher level of divergence of Erianthus from Saccharum was also clearly estabilished. Five primers produced genus- and species-specific fragments for Erianthus, S. spontaneum, S. officinarum, and S. barberi. The polymorphic primers, when tested on a panel of 30 commercial sugarcane cultivars, revealed a broad range (32.4-83.3%) of pair-wise similarity values, indicating their ability to detect high levels of polymorphism. A combination of two primers could differentiate all the varieties, further emphasizing their potential in fingerprinting and varietal identification.     

Molecular characterization and assessment of genetic diversity of inbred lines showing variability for drought tolerance in maize

A set of 24 genotypes bred at different centres in India as well as in CIMMYT showing variability for drought tolerance were selected for molecular and morpho-physiological characterization. A set of 35 SSR markers, having genome-wide coverage, was chosen for genotyping the inbreds. These markers generated a total of 111 polymorphic alleles with an average of 3.17 alleles per locus. The minimum and maximum PIC value was 0.27 and 0.77 with a mean of 0.5. A total of 13 unique alleles were found in the 24 inbred lines. The coefficient of genetic dissimilarity ranged from 0.192 to 0.803. NJ-based tree suggested the presence of three major clusters of which, two of them had subgroups. Phenotyping of inbreds by morpho-physiological traits revealed that there was a positive relationship among root length, chlorophyll content, relative water content while anthesis-silking interval was negative relationship with all these traits. Genotyping data complemented by morpho-physiological parameters were used to identify a number of pair-wise combinations for the development of mapping population segregating for drought tolerance and potential heterotic pairs for the development of drought tolerant hybrids.     

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.     

Development of Ty1-copia retrotransposon-based SSAP molecular markers for the study of genetic diversity in peach

Sequence-specific amplified polymorphism (SSAP) technology is a novel, anchored PCR approach derived from AFLP, which amplifies the region between a transposon insertion and an adjacent restriction site and have higher levels of polymorphism. In the current study, we developed 16 SSAP markers based on the long terminal repeat (LTR) sequences of Ty1-copia retrotransposons in the peach and used them for DNA profiling of 52 individual peaches: 44 peach cultivars and 8 ornamental peaches. These primer combinations produced a total of 1,553 fragments and 1,517 polymorphic bands with a polymorphism percentage of 97.7%. Furthermore, the Shannon's information index of each primer combination ranged from 0.1593 to 0.4456. Neighbor-joining analyses revealed two main genetic clusters, corresponding to the fruit flesh types: (A-1) MF (melting flesh) with clingstone and ornamental peaches; (A-2) MF with freestone and NMF (non-melting flesh) with clingstone. Finally, cluster analyses revealed that all ornamental peaches are closely related to the MF with clingstone peach cultivars. The application of these primer combinations identified using SSAP will facilitate future cultivar identification and germplasm management in peaches.