Identification and characterization of simple sequence repeat markers from Brassica napus expressed sequences

The availability of expressed sequence data derived from gene discovery programs enables mining for simple sequence repeats (SSR), providing useful genetic markers for crop improvement. These markers are inexpensive, require minimal labour to produce and can frequently be associated with functionally annotated genes. This study presents the development and characterization of 24 expressed sequence tags (EST)‐SSR markers from Brassica napus and their cross‐amplification across Brassica species. The markers show reliable amplification, genome specificity and considerable polymorphism, demonstrating the utility of EST‐SSRs for genetic analysis of wild Brassica populations and commercial Brassica germplasm.     

Sixteen new simple sequence repeat markers from Brassica juncea expressed sequences and their cross‐species amplification

The availability of expressed sequence data derived from gene discovery programs enables mining for simple sequence repeats (SSR), providing useful genetic markers for crop improvement. These markers are inexpensive, require minimal labour to produce and can frequently be associated with functionally annotated genes. This study presents the development and characterization of 16 expressed sequence tags (EST)‐SSR markers from Brassica juncea and their cross‐amplification across Brassica species. Sixteen primer pairs were assessed for polymorphism in all genomes of the diploid and amphidiploid Brassica species. The markers show reliable amplification, considerable polymorphism and high transferability across species, demonstrating the utility of EST‐SSRs for genetic analysis of brassicas.     

Development and use of simple sequence repeat SSR markers in Rubus species

The isolation of polymorphic codominant microsatellite markers in Rubus and in particular red raspberry will provide a tool to investigate gene flow between cultivated and wild raspberries. Microsatellite loci were isolated by screening a PstI size selected genomic library with AC₍₁₃₎ and AG₍₁₃₎. Positive clones were sequenced and primer pairs designed to the sequences flanking identified SSRs. One primer of each pair was fluorescently labelled to facilitate polymerase chain reaction (PCR) product identification on an automated DNA sequencer. We describe 10 polymorphic microsatellite loci developed and demonstrate their usefulness in different Rubus species.     

Characterization of simple sequence repeat markers derived in silico from Brassica rapa bacterial artificial chromosome sequences and their application in Brassica napus

A collaborative Brassica rapa genome sequencing project is currently in progress to aid the identification of agronomically important traits in Brassica species. As an initial stage, the ends of over 110 000 bacterial artificial chromosome clones were sequenced and mined for simple sequence repeats (SSRs). We present the characterization of 40 of these SSRs and their application in Brassica napus. The markers were screened against six Brassica species and Arabidopsis, and demonstrated reliable amplification, genome specificity, cross‐amplification and significant polymorphism. These SSRs will be useful for genetic analysis of Brassica germplasm.     

Identification of species-diagnostic inter simple sequence repeat markers for ten Phyllanthus species

Phyllanthus has been widely used in traditional medicine as an antipyretic, a diuretic, and to treat liver diseases and viral infections. Correct genotype identification of medicinal plant material remains important for the botanical drug industry. Limitations of chemical and morphological approaches for authentication have generated the need for newer methods in quality control of botanicals. In the present study, attempts were made to identify species-diagnostic markers for ten Phyllanthus species using the inter simple sequence repeat-polymerase chain reaction (ISSR-PCR) fingerprinting method. PCR amplification using seven ISSR primers resulted in significant polymorphism among the populations from different species. P. angustifolius and P. urinaria showed monomorphic frequency of maximum (63.88%) and minimum (20.64%), respectively. Seventeen species-diagnostic markers were identified for seven species (P. acidus, P. emblica, P. fraternus, P. urinaria, P. rotundifolius, P. amarus, and P. angustifolius) while no marker was detected for P. reticulatus, P. nivosus, and P. virgulatus. A maximum of six species-diagnostic markers were identified for P. acidus and a minimum of only one of 755 bp was available for P. amarus. Among the seventeen markers, nine were present in all individuals of particular species. The species-specific differences in fragment numbers and sizes could be used as diagnostic markers to distinguish the Phyllanthus species quickly.     

Development and characterization of non-coding RNA based simple sequence repeat markers in Capsicum species

Plant growth and development are largely regulated by non-coding RNAs (ncRNA); thus ncRNA based markers would be rewarding in molecular breeding. In the present study, for the first time we developed total 623 ncRNA based SSRs including 119 microRNASSRs (miRNASSRs) and 504 long non-coding RNASSRs (lncRNASSRs) distributed across 12 Capsicum chromosomes. Out of 623 ncRNASSRs, 120 (including 60 each miRNASSRs and lncRNASSRs) were used for genotyping of 96 Capsicum accessions belonging to C. annuum, C. chinense and C. frutescens; and 75% SSRs were polymorphic. Model-based and distance-based cluster analyses identified three species specific clusters, i.e. cluster-I (C. annuum), cluster-II (C. frutescens) and cluster-III (C. chinense); therefore, these SSRs may have a potential role to play in interspecific Capsicum breeding. Tissue specific expression of SSR containing ncRNAs and versatile functions of their targets suggested the usefulness of SSRs for mapping of genes/QTLs and breeding of wide range of traits in Capsicum.     

High-volume mapping of maize mutants with simple sequence repeat markers

Many genes in maize (Zea mays L.) are revealed by mutations that cause phenotypic variation from normal. These mutants are valuable resources of genetic information. From among the huge collection of maize mutants, it is ultimately necessary to establish which alleles are of the same genes and which are novel genes. Although any given mutant can be subjected to complementation tests or can be mapped by using conventional techniques, the number of mutants at this time makes these approaches prohibitive to encompass the whole collection. Here we describe procedures to efficiently map large numbers of mutants. Included are methods for generating polymorphic mapping progenies, for simply and rapidly preparing samples to use in polymerase chain reaction (PCR), for tissue pooling and application of simple sequence repeat (SSR), markers, and for stepwise determination of linkage followed by mapping to chromosomal region.     

Identification of inter simple sequence repeat (ISSR) markers associated with seed size in wheat

The feasibility of identifying inter-simple sequence repeat markers associated with seed weight in hexaploid wheat was tested using 113 recombinant inbred lines developed by the single-seed descent method, from a cross between Rye selection111, an Indian genetic stock obtained through the introgression of genes for bold seed size from rye, and Chinese Spring having small seed size. Three markers were associated with low seed size with gene effects of 14.8%, 9.5%, and 6%, while four markers with contributions of 8%, 4.66%, 2.92% and 2.61% were found to be linked to high seed size, together contributing 31% of the phenotypic variance in seed size. Nulli-tetrasomic and di-telosomic analysis revealed the presence of three low seed size QTL-associated markers on three chromosomes, 6BL, 2DL, and 1DS respectively. This study clearly demonstrates that ISSRs are highly useful for finding markers associated with major and minor genes controlling agronomically important traits in wheat. Received: 24 February 2000 / Accepted: 31 March 2000     

Development of genomic simple sequence repeat markers for linseed using next-generation sequencing technology

Linseed (Linum usitatissimum L.) is regarded as a cash crop of tomorrow because of the presence of nutraceutically important ??-linolenic acid (ALA) and lignan. However, only limited breeding progress has been made in this crop, mainly due to the lack of sufficient genetic and genomic resources. Among these, simple sequence repeats (SSR) are useful DNA markers for diversity analysis, genetic mapping and tagging traits because of their co-dominant and highly polymorphic nature. In order to develop SSR markers for linseed, we used three microsatellite isolation methods, viz., PCR Isolation of Microsatellite Arrays (PIMA), 5??-anchored PCR method, and Fast Isolation by AFLP of Sequences COntaining repeats (FIASCO). The amplified products from these methods were pooled and sequenced using the 454 GS-FLX platform. A total of 36,332 reads were obtained, which assembled into 2,183 contigs and 2,509 singlets. The contigs and the singlets contained 1,842 microsatellite motifs, with dinucleotide motifs as the most abundant repeat type (54%) followed by trinucleotide motifs (44%). Based on this, 290 SSR markers were designed, 52 of which were evaluated using a panel of 27 diverse linseed genotypes. Among the three enrichment methods, the 5??-anchored PCR method was most efficient for isolation of microsatellites, while FIASCO was most efficient for developing SSR markers. We show the utility of next-generation sequencing technology for efficiently discovering a large number of microsatellite markers in non-model plants.     

Detection of haplotypic variation and natural hybridization in halepensis-complex pine species using chloroplast simple sequence repeat (SSR) markers

In this investigation, nine chloroplast, paternally inherited simple-sequence repeat (cpSSR) markers were used to describe genetic variation of three closely related species belonging to the halepensis complex ( Pinus halepensis Ait., P. brutia Mill. and P. eldarica Medwed.). Both the infinite allele model (IAM) and stepwise-mutation model (SMM) have been applied to the analysis of the genetic structure of natural populations and the geographical distribution of haplotypic variation. SMM-based estimators performed better than IAM-based estimators for large values of within-population diversity and divergence between population pairs. Overall, large haplotypic variation and high genetic divergence were detected for both P. halepensis and P. brutia . The genetic structures of the three species are discussed with consideration to the evolutionary and ecological characteristics of these species. Three highly informative markers showing size variants distinguishing P. halepensis from the other two species were used to provide more information on the occurrence of natural hybridization in a Turkish sympatric population of P. halepensis and P. brutia . Strong evidence of introgression of ' halepensis ' chloroplast haplotypes into P. brutia seeds (but not vice versa) was detected. According to previous evidence from controlled crossings, matings between the above species seem to be successful only when P. halepensis is the pollen donor and P. brutia is the female parent (but not reciprocally). The existence of unidirectional gene flow in sympatric populations confirms previous evidence about partial reproductive barriers between P. halepensis and P. brutia . Implications of the above evidence for the evolutionary history of these species are discussed.     

Agronomic characterization, genetic diversity and association analysis of cotton cultivars using simple sequence repeat molecular markers

Cotton is the most important textile plant in the world and is one of the most important crops for the production of oilseed. Because of its worldwide economic importance, new cultivars are constantly being released in the world and consequently in the Greek market, as Greece is the largest producer in Europe. We used simple sequence repeat (SSR) markers for the identification and the phylogenetic analysis of the most widely cultivated cotton cultivars in Greece. Initially, we used 12 pairs of SSR molecular markers for the analysis of 29 cultivars of Gossypium hirsutum and an interspecific hybrid (G. hirsutum x G. barbadense). Of the 12 pairs of SSR primers, 11 amplified polymorphic products, while one pair did not amplify any product. Globally, 17 polymorphic marker loci were identified. Two to four different alleles were amplified at each genomic locus, with a mean of 2.53 alleles per locus. Among the 30 genotypes that we analyzed, the polymorphism information content ranged from 0 to 0.548, with a mean of 0.293. Three main groups were formed among the 30 genotypes when a phylogenetic analysis was performed using UPGMA. Computational analysis of each molecular marker separately showed an association of SSR markers with agronomic traits such as fiber quality. To our knowledge, this is the first in-depth molecular analysis of cotton cultivars grown in Greece using SSR markers. An analysis of association of SSR markers with fiber quality traits of 29 cotton cultivars is reported for the first time.     

Characterization of 215 simple sequence repeat markers in creeping bentgrass (Agrostis stolonifera L.)

Creeping bentgrass (Agrostis stolonifera L.) is a versatile, cross-pollinated, temperate and perennial turfgrass species. It occurs naturally in a wide variety of habitats and is also cultivated on golf courses, bowling greens and tennis courts worldwide. Isozymes and amplified fragment length polymorphisms (AFLPs) have been used to determine genetic diversity, and restriction fragment length polymorphisms (RFLPs) and random amplified polymorphic DNA (RAPDs) were used to construct a genetic linkage map of this species. In the current report, we developed and characterized 215 unique genomic simple sequence repeat (SSR) markers in creeping bentgrass. The SSRs reported here are the first available markers in creeping bentgrass to date. Eight hundred and eighteen alleles were amplified by 215 SSR loci, an average of 3.72 alleles per locus. Fifty-nine per cent of those alleles segregated in a 1:1 Mendelian fashion (P > 0.05). Twenty-two per cent had a distorted segregation ratio (P ≤ 0.05). These SSR markers will be useful for assessing genetic diversity in creeping bentgrass and will be important for the development of genetic linkage maps and identifying quantitative trait loci. These markers could enhance breeding programmes by improving the efficiency of selection techniques.     

Strain-typing of Lentinula edodes in China with inter simple sequence repeat markers

To validate strain typing by inter simple sequence repeat (ISSR) analysis in Lentinula edodes cultivars, 17 Chinese L. edodes strains including 15 cultivated strains cultivated on a large scale and two wild strains were analyzed with the ISSR technique. With the use of two ISSR primers, a total of 32 DNA products were detected, of which, 31 DNA products (96.9% of the detected products) were polymorphic between two or more strains. The profiles of those two primers could be employed to differentiate all of the tested strains. A cluster analysis based on ISSR data revealed that the 17 strains could be classified into two distinct groups. One group consisted of eight strains in which the cultivated strains were H (high-temperature)-type or B (broad-temperature)-type, and the other group comprised cultivated strains that were of the L (low-temperature)-type or M (medium-temperature)-type. In contrast to the two wild strains, the genetic diversity of 15 cultivated strains was very rich based on a similarity coefficient analysis.     

Genetic diversity analysis of Bt cotton genotypes in Pakistan using simple sequence repeat markers

The popularity of genetically modified insect resistant (Bt) cotton has promoted large scale monocultures, which is thought to worsen the problem of crop genetic homogeneity. Information on genetic diversity among Bt cotton varieties is lacking. We evaluated genetic divergence among 19 Bt cotton genotypes using simple sequence repeat (SSR) markers. Thirty-seven of 104 surveyed primers were found informative. Fifty-two primers selected on the basis of reported intra-hirsutum polymorphism in a cotton marker database showed a high degree of polymorphism, 56% compared to 13% for randomly selected primers. A total of 177 loci were amplified, with an average of 1.57 loci per primer, generating 38 markers. The amplicons ranged in size from 98 to 256 bp. The genetic similarities among the 19 genotypes ranged from 0.902 to 0.982, with an average of 0.947, revealing a lack of diversity. Similarities among genotypes from public sector organizations were higher than genotypes developed by private companies. Hybrids were found to be more distant compared to commercial cultivars and advanced breeding lines. Cluster analysis grouped the 19 Bt cotton genotypes into three major clusters and two independent entries. Cultivars IR-3701, Ali Akbar-802 and advanced breeding line VH-259 grouped in subcluster B2, with very narrow genetic distances despite dissimilar parentage. We found a very high level of similarity among Pakistani-bred Bt cotton varieties, which means that genetically diverse recurrent parents should be included to enhance genetic diversity. The intra-hirsutum polymorphic SSRs were found to be highly informative for molecular genetic diversity studies in these cotton varieties.     

Characterization and compilation of polymorphic simple sequence repeat (SSR) markers of peanut from public database

ABSTRACT: BACKGROUND: There are several reports describing thousands of SSR markers in the peanut (Arachis hypogaea L.) genome. There is a need to integrate various research reports of peanut DNA polymorphism into a single platform. Further, because of lack of uniformity in the labeling of these markers across the publications, there is some confusion on the identities of many markers. We describe below an effort to develop a central comprehensive database of polymorphic SSR markers in peanut. FINDINGS: We compiled 1,343 SSR markers as detecting polymorphism (14.5%) within a total of 9,274 markers. Amongst all polymorphic SSRs examined, we found that AG motif (36.5%) was the most abundant followed by AAG (12.1%), AAT (10.9%), and AT (10.3%).The mean length of SSR repeats in dinucleotide SSRs was significantly longer than that in trinucleotide SSRs. Dinucleotide SSRs showed higher polymorphism frequency for genomic SSRs when compared to trinucleotide SSRs, while for EST-SSRs, the frequency of polymorphic SSRs was higher in trinucleotide SSRs than in dinucleotide SSRs. The correlation of the length of SSR and the frequency of polymorphism revealed that the frequency of polymorphism was decreased as motif repeat number increased. CONCLUSIONS: The assembled polymorphic SSRs would enhance the density of the existing genetic maps of peanut, which could also be a useful source of DNA markers suitable for high-throughput QTL mapping and marker-assisted selection in peanut improvement and thus would be of value to breeders. KEYWORDS: SSR, motif, polymorphism, cultivated peanut.     

Analysis of genetic diversity of Guizotia abyssinica from Ethiopia using inter simple sequence repeat markers

Within and among population genetic diversity of 37 Guizotia abyssinica populations from Ethiopia were analyzed using inter simple sequence repeats (ISSRs). Five primers amplified a total of 118 genomic DNA fragments across a total of 370 individuals of which 106 were polymorphic (89.83%). The average number of polymorphic bands per primer was 21.2. More bands were generated by primer UBC 888 (BDB(CA)(7.) The total genetic diversity (Ht) and the coefficient of genetic differentiation (Gst) were 0.4115 and 0.0918 respectively, while the within population genetic diversity (Hs) and the among population genetic diversity(Dst) were 0.3738 and 0.03776 respectively suggesting more variability within the populations than among them. The standard genetic distances between the G. abyssinica populations of the eight regions ranged from 0.0281 (between Wollo and Gojam) to 0.1148 (between Jimma and Hararghe). Generally, the standard genetic distances are smaller between populations of neighboring regions and highest between those of Jimma and the other regions, ranging from 0.0696 (between Jimma and Shewa) to 0.1148 (between Jimma and Hararghe). The ISSR based UPGMA clustering using the standardized genetic distances matrix also placed populations from neighboring regions closer than those from farther apart areas, while the UPGMA clustering by regions based on the standard genetic distances produced three clusters following the proximity and the contiguity of the regions. The mean Shannon Weaver diversity indices for the populations of the eight regions ranged from 0.8197 (Jimma) to 0.9176 (Hararghe), with a mean of 0.8841 for the whole material.     

Development of simple sequence repeat markers for Botryosphaeria spp. with Fusicoccum anamorphs

We report the development of eight sets of microsatellite markers for the ascomycete fungus and tree pathogen, Botryosphaeria parva. The primers were identified after cloning and sequencing of fragments amplified using simple sequence repeat (SSR) primers. Genome walking was used to determine unknown sequences on either side of new SSRs. The primers were tested and proved useful in nine other Botryosphaeria species that all have Fusicoccum anamorphs, similar to B. parva.