Sequence-tagged Microsatellite Research Articles

Saturation and mapping of a majorFusarium head blight resistance QTL on chromosome 3BS of Sumai 3 wheat

Fusarium head blight (FHB) is a destructive disease in wheat. The major quantitative trait locus (QTL) on 3BS from Sumai 3 and its derivatives has been used as a major source of the resistance to FHB worldwide, but the discrepancy in reported location of the major QTL could block its using in map based cloning and marker assisted selection. In this study, Chinese Spring-Sumai 3 chromosome 3B substitution line was used as resistant parent of the mapping population to reduce the confounded effect of genetic background in Sumai 3. The major QTL region was saturated with the Sequence Tagged Microsatellite (STM) and Sequence Tagged Site (STS) markers. A linkage map of chromosome 3B with 36 markers covering a genetic distance of 112.4 cM was constructed. Twelve new markers were inserted into the chromosome region where the major QTL was located. The average interval distance between markers was 1.5 cM. Multiple QTL Models (MQM) mapping indicated that the major QTL was located in the interval of Xgwm533-Xsts9-1, and explained 45.6% of phenotypic variation of the resistance to FHB. The SSR (simple sequence repeat) marker Xgwm533 and STM marker Xstm748tcac are closely linked to the major QTL.

Genetic and pathogenic diversity inPyrenophora teresf.maculatain barley crops of Victoria, Australia

Forty-four Pyrenophora teres f. maculata isolates collected from barley crops in Victoria, Australia, were used in an analysis of genetic and pathogenic diversity. Genetic analysis with molecular markers revealed high levels of diversity. Fifteen sequence-tagged microsatellite primers revealed polymorphism among the 44 isolates, with two to five alleles (average 2.87 ± 0.42) amplified at each locus. Each isolate exhibited a unique genotype, which was likely to be the result of sexual recombination by random mating in the Victorian pathogen population. The two mating types of P. teres f. maculata were found at almost a 1: 1 ratio in the 44 isolate set indicating that sexual recombination is possible. Consistent with international studies of this pathogen, the high genetic diversity detected did not correlate with pathogenic variation. Adifferential set developed in Australia that consisted of 21 barley varieties was evaluated for reaction response towards the 44 P. teres f. maculata isolates as seedlings. None of the isolates tested were virulent on barley varieties known to possess the Rpt4 gene for seedling resistance, or the Ha4 allele associated with adult plant resistance. This indicates that use of the Rpt4 gene, Ha4 allele and other sources of resistance will provide effective control of spot form of net blotch (caused by P. teres f. maculata) in Victoria. However, the high level of genetic diversity indicates significant potential for the pathogen population to evolve rapidly to adapt to changes in selective pressures such as the deployment of host resistance.

GENE SPECIFIC MOLECULAR MARKERS FOR HOP (HUMULUS LUPULUS L.)

The development of DNA technology has provided a number of methods, which eliminate any influence of environmental factors, to detect differences at the DNA sequence level for evaluation of hop genotypes. Most of these methods in hops used unknown DNA sequences (e.g., AFLP) or specific DNA sequences (e.g., SSR) in non-coding regions of the genome. A new trend in molecular methods is to develop new molecular markers in specific sequences, near or within structural genes. Recently, markers in intron, 5' and 3'UTR were identified in several hop genes. Increasing sequence information of hop genes in Genebank provides additional opportunities to develop new gene specific molecular markers. In this study, we report new efficient and reliable expressed sequence tag microsatellite repeat (EST-SSR) molecular markers. We used forty primer pairs for twenty-nine gene specific sequences. We evaluated them for identification and clustering in eight hops cultivars. Gene-specific based clustering was compared to clustering based on SSR, AFLP markers and on a combination of molecular markers. These molecular markers are ideal for DNA fingerprinting, for control of authenticity and purity of varieties, for evaluation of genetic diversity in genetic resources, etc. They are readily applicable in breeding, and for genetic linkage mapping and quantitative trait loci (QTL) analysis.

Mutagenic induction of double-podding trait in different genotypes of chickpea and their characterization by STMS marker

With 1 figure and 2 tables Abstract A gene that confers double-podding in chickpea is considered to be important for breeding higher yielding cultivars. Double-podded mutants were produced from five desi- and four kabuli-type chickpea genotypes through induced mutations and stabilty was checked up to M13 generation. Desi-type produced higher number of mutants as compared with kabuli-type. The inheritance studies in induced mutants of six genotypes showed that the double-podded trait was governed by single recessive gene. Different genotypes and their double-podded mutants were also characterized through sequence-tagged microsatellite site marker, TA-80. Allelic variations were found in single-podded genotypes and eight different alleles were identified, while for double-poddedness no allelic variants were found in all the analysed mutants. Addition of bases in the double-podded mutants showed that there might be involvement of transposable elements in the production of double-podded mutants through mutagens.

Development and optimization of sequence-tagged microsatellite site markers to detect genetic diversity withinColletotrichum capsici,a causal agent of chilli pepper anthracnose disease

Genomic libraries enriched for microsatellites from Colletotrichum capsici, one of the major causal agents of anthracnose disease in chilli pepper (Capsicum spp.), were developed using a modified hybridization procedure. Twenty-seven robust primer pairs were designed from microsatellite flanking sequences and were characterized using 52 isolates from three countries India, Sri Lanka and Thailand. Highest gene diversity of 0.857 was observed at the CCSSR1 with up to 18 alleles among all the isolates whereas the differentiation ranged from 0.05 to 0.45. The sequence-tagged microsatellite site markers developed in this study will be useful for genetic analyses of C. capsici populations.

Genetic Diversity Among Chinese Pea ( Pisum sativum L.) Landraces as Revealed by SSR Markers

To reveal the genetic diversity of China cultivated pea ( Pisum sativum L.) and the genetic relationships among germplasm from different sowing area and provinces, a total of 1,221 landraces were sampled from 19 provinces in China consisting of 784 spring-sowing and 437 winter-sowing landrace. Using 21 sequence tagged microsatellites (STMS) markers, 104 polymorphic bands were detected with an average of total 4.95 alleles per locus and 3.10 (62.52%) effective alleles per locus for diversity analysis. Among the different provinces, the alleles of landrace group distributed evenly, whereas the number of effective alleles and the Shannon's information index ( I) varied markedly. The largest genetic diversity appeared in landraces from Inner Mongolia, followed by Gansu, Sichuan, Yunnan, and Tibet, and Liaoning had the lowest genetic diversity. According to 3-dimension principal component analysis (PCA), 3 gene pools were detected in Chinese pea landraces, which were typically represented by the landraces from Inner Mongolia and Shaanxi (Gene Pool I), Henan (Gene Pool II), and other provinces (Gene Pool III). The pairwise Nei's (1978) genetic distance ranged from 5.159 to 27.586 in provinces. The provincial accessions were clustered into 2 groups and 8 subgroups with interactions to the gene pools. The cluster result suggested that the genetic diversity of Chinese pea landraces was associated with their ecological and geographical distribution.

High-resolution mapping and gene prediction of Xanthomonas Oryzae pv. Oryzae resistance gene Xa7

Bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo) is a devastating disease in rice worldwide. The resistance gene Xa7, which provides dominant resistance against the pathogen with avirulence (Avr) gene AvrXa7, has proved to be durably resistant to BB. A set of SSR markers were selected from the “gramene” database based on the Xa7 gene initial mapping region on chromosome 6. These markers were used to construct a high-resolution genetic map of the chromosomal region surrounding the Xa7 gene. An F2 mapping population with 721 highly susceptible individuals derived from a cross between the near isogenic lines (NILs) IRBB7 and IR24 were constructed to localize the Xa7 gene. In a primary analysis with eleven polymorphic SSR markers, Xa7 was located in approximately the 0.28-cM region. To walk closer to the target gene, recombinant F2 individuals were tested using newly developed STMS (sequence tagged microsatellite) markers. Finally, the Xa7 gene was mapped to a 0.21-cM interval between the markers GDSSR02 and RM20593. The Xa7-linked markers were landed on the reference sequence of cv. Nipponbare through bioinformatics analysis. A contig map corresponding to the Xa7 gene was constructed. The target gene was assumed to span an interval of approximately 118.5-kb which contained a total of fourteen genes released by the TIGR Genome Annotation Version 5.0. Candidate-gene analysis of Xa7 revealed that the fourteen genes encode novel domains that have no amino acid sequence similar to other cloned Xa(xa) genes.

Locus-specific microsatellite markers in common bean (Phaseolus vulgaris L.): isolation and characterization

This study aimed at the development of microsatellite markers for Phaseolus vulgaris L. by two techniques: hybridization-based detection, and transfer of microsatellite markers developed for chickpea to the common bean. Small-insert genomic libraries of common bean were screened for simple sequence repeat (SSR)-containing fragments with a set of microsatellite-specific oligonucleotide probes. Twenty-five (GA)10 positive clones detected by a dinucleotide repeat probe were selected for sequencing. Sixteen of 18 primer pairs, complementary to the SSR-flanking regions and tested in eight P. vulgaris L. accessions, are polymorphic at an intra-specific level. Mendelian inheritance of the sequence-tagged microsatellite site (STMS) markers was demonstrated using a set of recombinant inbred lines (RILs) and their parents. A total of 46 chickpea STMS markers of 447 different primer combinations amplified loci in the genome of common bean. Sequencing of amplified products from Phaseolus with these primer combinations demonstrated that the sequence of microsatellite marker TA 176s proved to be significantly similar to transcription factor SCOF1 of soy bean, controlling the response to cold stress, and also to transcription factor zinc finger protein (a Kruppel-like zinc finger protein, or TFIIIA), coordinating the reaction to osmotic stress in Medicago truncatula. Sequence analysis of this site in the two common bean parental lines BAT 477 and DOR 364 revealed that the sequence of BAT 477 was 5 bp longer than the corresponding sequence of DOR 364. Amplification of this fragment in six RILs of common bean detected the existence of sequence polymorphisms between the different lines.

Evaluation and Validation of Four Molecular Markers Associated with Pre-harvest Sprouting Tolerance in Chinese Wheat

Four preharvest sprouting (PHS) tolerance-associated markers MST101, wmc104, Xgwm155, and Vp1B3, were used to screen 95 Chinese landraces and historical varieties of wheat ( Triticum aestivum L.), and aimed at assessing the efficiency of these markers in selecting genotypes with higher PHS tolerance. Among the 95 genotypes tested, 57, 19, and 19 accessions had germination indexes (GI) of less than 30%, 30–60%, and over 60%, respectively. The relationships between the variation of the PCR products of the 4 markers and germination indexes of the tested materials were analyzed. Variations at Vp1B3 and Xgwm155 were associated with PHS response, but the sequence-tagged site (STS) marker MST101 and sequence-tagged microsatellite site (STMS) marker wmc104 were not. Vp1B3 was more effective than Xgwm155 for selecting PHS resistant varieties. The selection efficiency will be improved if both markers are used.

Origin and frequency of 2 n gametes in Citrus sinensis × Poncirus trifoliata and their reciprocal crosses

A total of 293 putative citrange F 1 seedlings, derived from 13 crosses and visually identified by their trifoliate leaf morphology, were investigated using six characterized expressed sequence tag (EST) microsatellite primers. Their origins and ploidy levels were simultaneously and unambiguously determined by these markers. Besides a majority of diploid zygotic hybrids, as high as 24% triploid hybrids were produced, only by Citrus sinensis (sweet orange) × Poncirus trifoliata (trifoliate orange), but not the reciprocal crosses. The results indicated that 2 n eggs originated only from the maternal sweet orange parent, contributing the extra haploid genome in the triploids. It was further inferred that the 2 n eggs in sweet orange result from first division restitution (FDR), not second division restitution (SDR), according to the marker genotypes of these triploids. High percentages of self-cross and nucellar origin seedlings were found only from P. trifoliata × C. sinensis, but not from the reciprocals, simply due to their different reliabilities in the visual leaf identification of the seedlings. Survey of leaves indicated that adult triploid seedlings had predominantly monofoliate leaves, compared to diploid hybrids with mostly trifoliate leaves. The possible genetic mechanism and potential applications were discussed.

Comparison of genetic variation among accessions of Aegilops tauschii using AFLP and SSR markers

Genetic diversity of 54 accessions of Aegilops tauschii from five countries was assessed using sequence-tagged microsatellites (or simple sequence repeats, SSRs) and amplified fragment length polymorphisms (AFLPs). In the case of AFLP analysis, a total of 256 amplification products obtained, 234 of them were polymorphic across all the 54 accessions. A total of 224 fragments were obtained from the 24 SSR primers and 219 of fragments were polymorphic across all the genotypes screened. Based on both AFLP and SSR markers, the highest percentage of polymorphisms were obtained in Iranian and accessions of unknown origin. The highest polymorphic information content (PIC) value was observed for SSRs (0.82) while the highest marker index (MI) value was for AFLPs (8.5) reflecting the hyper-variability of the first and the distinctive nature of the second system. Principal co-ordinate analysis (PCO) revealed congruent patterns of genetic relationships for both data sets, but did not group accessions strictly according to their geographical origins. Poor correlation was found between AFLP and SSR marker loci. This low association may be due to low number of AFLP and SSR markers. These results show that molecular markers can help to organize the genetic variability and expose useful diversity for breeding purposes.

PRIMER NOTE: Isolation and characterization of polymorphic microsatellite markers in Anthyllis vulneraria

To study the reproductive system of the putative autogamous species Anthyllis vulneraria in Belgian calcareous grasslands, eight polymorphic microsatellite loci were developed using the sequence-tagged microsatellite profiling technique and 35 plants were typed for all loci. No linkage disequilibrium was found between any pair of loci. Over the loci, the number of alleles ranged from two to five. Observed and expected heterozygosities ranged from 0.053 to 0.688 and from 0.053 to 0.631, respectively. The estimated FIS values (FIS = 0.031 and 0.122) are not in accordance with a predominantly selfing reproductive system.

Diversity arrays technology (DArT) for high-throughput profiling of the hexaploid wheat genome

Despite a substantial investment in the development of panels of single nucleotide polymorphism (SNP) markers, the simple sequence repeat (SSR) technology with a limited multiplexing capability remains a standard, even for applications requiring whole-genome information. Diversity arrays technology (DArT) types hundreds to thousands of genomic loci in parallel, as previously demonstrated in a number diploid plant species. Here we show that DArT performs similarly well for the hexaploid genome of bread wheat (Triticum aestivum L.). The methodology previously used to generate DArT fingerprints of barley also generated a large number of high-quality markers in wheat (99.8% allele-calling concordance and approximately 95% call rate). The genetic relationships among bread wheat cultivars revealed by DArT coincided with knowledge generated with other methods, and even closely related cultivars could be distinguished. To verify the Mendelian behaviour of DArT markers, we typed a set of 90 Cranbrook x Halberd doubled haploid lines for which a framework (FW) map comprising a total of 339 SSR, restriction fragment length polymorphism (RFLP) and amplified fragment length polymorphism (AFLP) markers was available. We added an equal number of DArT markers to this data set and also incorporated 71 sequence tagged microsatellite (STM) markers. A comparison of logarithm of the odds (LOD) scores, call rates and the degree of genome coverage indicated that the quality and information content of the DArT data set was comparable to that of the combined SSR/RFLP/AFLP data set of the FW map.

Development and genetic mapping of sequence-tagged microsatellites (STMs) in bread wheat (Triticum aestivum L.)

The density of SSRs on the published genetic map of bread wheat (Triticum aestivum L.) has steadily increased over the last few years. This has improved the efficiency of marker-assisted breeding and certain types of genetic research by providing more choice in the quality of SSRs and a greater chance of finding polymorphic markers in any cross for a chromosomal region of interest. Increased SSR density on the published wheat genetic map will further enhance breeding and research efforts. Here, sequence-tagged microsatellite profiling (STMP) is demonstrated as a rapid technique for the economical development of anonymous genomic SSRs to increase marker density on the wheat genetic map. A total of 684 polymorphic sequence-tagged microsatellites (STMs) were developed, and 380 were genetically mapped in three mapping populations, with 296 being mapped in the International Triticeae Mapping Initiative W7984 x Opata85 recombinant inbred cross. Across the three populations, a total of 479 STM loci were mapped. Several technological advantages of STMs over conventional SSRs were also observed. These include reduced marker deployment costs for fluorescent-based SSR analysis, and increased genotyping throughput by more efficient electrophoretic separation of STMs and a high amenability to multiplex PCR.

Characterization of novel sugarcane expressed sequence tag microsatellites and their comparison with genomic SSRs

AbstractMicrosatellites or simple sequence repeats (SSRs) are one of the most suitable markers for genome analysis as they have great potential to aid breeders to develop new improved sugarcane varieties. The development of SSR derived from expressed sequence tags (EST) opens new opportunities for genetic investigations at a functional level. In the present work, the polymorphism obtained with a subset of 51 EST–SSRs derived from sucest was compared with those generated by 50 genomic SSRs (gSSR) in terms of number of alleles, polymorphism information content, discrimination power and their ability to establish genetic relationships among 18 sugarcane clones including three Saccharum species (S. officinarum, S. barberi, S. sinense). The majority of EST–SSRs loci had four to six alleles in contrast to the seven to nine observed for the gSSRs loci. Approximately, 35% of the gSSRs had PIC values around 0.90 in contrast to 15% of the EST–SSRs. However, the mean discrimination power of the two types of SSR did not differ significantly as much as the average genetic similarity (GS) based on Dice coefficient. The correlation between GS of the two types of SSRs was high (r = 0.71/P = 0.99) and significant. Although differences were observed between dendrograms obtained with each SSR type, both were in good agreement with pedigree information. The S. officinarum clone IJ76‐314 was grouped apart from the other clones evaluated. The results here demonstrate that EST–SSRs can be successfully used for genetic relationship analysis, extending the knowledge of genetic diversity of sugarcane to a functional level.

Development of sequence tagged microsatellites (STMs) for the barley scald pathogen Rhynchosporium secalis

AbstractA rapid and cost efficient technique was developed and used to generate 168 sequence tagged microsatellites (STMs) in the barley scald pathogen Rhynchosporium secalis. Sixty‐two STMs, amplifying 66 loci, revealed a high level of polymorphism among a diverse set of 16 Australian isolates. Each locus revealed two to nine alleles (average 4 ± 1.82), and a gene diversity measure of 0.54 was obtained. This technique not only halved the cost of marker development compared to traditional methods, but substantially reduced the cost of performing fluorescence‐based microsatellite assays. These STMs provide a powerful tool for genetic studies in R. secalis.

Development of microsatellite markers and analysis of intraspecific genetic variability in chickpea (Cicer arietinum L.)

Paucity of polymorphic molecular markers in chickpea (Cicer arietinum L.) has been a major limitation in the improvement of this important legume. Hence, in an attempt to develop sequence-tagged microsatellite sites (STMS) markers from chickpea, a microsatellite enriched library from the C. arietinum cv. Pusa362 nuclear genome was constructed for the identification of (CA/GT)n and (CT/GA)n microsatellite motifs. A total of 92 new microsatellites were identified, of which 74 functional STMS primer pairs were developed. These markers were validated using 9 chickpea and one C. reticulatum accession. Of the STMS markers developed, 25 polymorphic markers were used to analyze the intraspecific genetic diversity within 36 geographically diverse chickpea accessions. The 25 primer pairs amplified single loci producing a minimum of 2 and maximum of 11 alleles. A total of 159 alleles were detected with an average of 6.4 alleles per locus. The observed and expected heterozygosity values averaged 0.32 (0.08-0.91) and 0.74 (0.23-0.89) respectively. The UPGMA based dendrogram was able to distinguish all the accessions except two accessions from Afghanistan establishing that microsatellites could successfully detect intraspecific genetic diversity in chickpea. Further, cloning and sequencing of size variant alleles at two microsatellite loci revealed that the variable numbers of AG repeats in different alleles were the major source of polymorphism. Point mutations were found to occur both within and immediately upstream of the long tracts of perfect repeats, thereby bringing about a conversion of perfect motifs into imperfect or compound motifs. Such events possibly occurred in order to limit the expansion of microsatellites and also lead to the birth of new microsatellites. The microsatellite markers developed in this study will be useful for genetic diversity analysis, linkage map construction as well as for depicting intraspecific microsatellite evolution.

Characterization and Genetic Relationships of Wild Species and Old Garden Roses Based on Microsatellite Analysis

Six polymorphic sequence-tagged microsatellite sites (STMSs) were used to characterize 65 accessions of old garden roses [OGRs (Rosa L. spp.)] from seven botanical sections and 13 horticultural groups. Aims of the study were to define the genetic profiles of accessions and to provide information useful for the classification and pedigree reconstruction of OGRs. In roses, a precise botanical classification is difficult due to repeated hybridization carried out in breeding; OGRs are classified in horticultural groups on the basis of their original parentage or of their morphological traits. A total of 82 alleles were detected at six loci. The number of alleles per locus ranged from six to 21, with an average of 13.7 alleles per locus. A dendrogram was constructed by cluster analysis, displaying the relative genetic similarities between species' accessions, hybrids, and cultivars. Cluster analysis grouped the genotypes into seven major clusters that were substantially consistent with their classification into botanical sections and horticultural groups. Several hypotheses of apportionment of accessions to horticultural groups were evaluated on the basis of the relative position in the dendrogram of the analyzed individuals. Results demonstrated that DNA analyses can contribute to drawing the botanic classification of rose accessions, improving the genetic knowledge on the background of modern rose, and providing the basis for breeding programs.

Genetic Diversity in Australian Populations of Puccinia graminis f. sp. avenae

ABSTRACT Sequence-tagged microsatellite profiling was used to develop 110 microsatellites for Puccinia graminis f. sp. tritici (causal agent of wheat stem rust). Low microsatellite polymorphism was exhibited among 10 pathogenically diverse P. graminis f. sp. tritici isolates collected from Australian cereal growing regions over a period of at least 70 years, with two polymorphic loci detected, each revealing two alleles. Limited cross-species amplification was observed for the wheat rust pathogens, P. triticina (leaf rust) and P. striiformis f. sp. tritici (stripe rust). However, very high transferability was revealed with P. graminis f. sp. avenae (causal agent of oat stem rust) isolates. A genetic diversity study of 47 P. graminis f. sp. avenae isolates collected from an Australia-wide survey in 1999, and a historical group of 16 isolates collected from Australian cereal growing regions from 1971 to 1996, revealed six polymorphic microsatellite loci with a total of 15 alleles. Genetic analysis revealed the presence of several clonal lineages and subpopulations in the pathogen population, and wide dispersal of identical races and genotypes throughout Australian cereal-growing regions. These findings demonstrated the dynamic population structure of this pathogen in Australia and concur with the patterns of diversity observed in pathogenicity studies.

Identification of microsatellite markers from Cicer reticulatum: molecular variation and phylogenetic analysis

Microsatellite sequences were cloned and sequenced from Cicer reticulatum, the wild annual progenitor of chickpea (C. arietinum L.). Based on the flanking sequences of the microsatellite motifs, 11 sequence-tagged microsatellite site (STMS) markers were developed. These markers were used for phylogenetic analysis of 29 accessions representing all the nine annual Cicer species. The 11 primer pairs amplified distinct fragments in all the annual species demonstrating high levels of sequence conservation at these loci. Efficient marker transferability (97%) of the C. reticulatum STMS markers across other species of the genus was observed as compared to microsatellite markers from the cultivated species. Variability in the size and number of alleles was obtained with an average of 5.8 alleles per locus. Sequence analysis at three homologous microsatellite loci revealed that the microsatellite allele variation was mainly due to differences in the copy number of the tandem repeats. However, other factors such as (1) point mutations, (2) insertion/deletion events in the flanking region, (3) expansion of closely spaced microsatellites and (4) repeat conversion in the amplified microsatellite loci were also responsible for allelic variation. An unweighted pairgroup method with arithmetic averages (UPGMA)-based dendrogram was obtained, which clearly distinguished all the accessions (except two C. judaicum accessions) from one another and revealed intra- as well as inter-species variability in the genus. An annual Cicer phylogeny was depicted which established the higher similarity between C. arietinum and C. reticulatum. The placement of C. pinnatifidum in the second crossability group and its closeness to C. bijugum was supported. Two species, C. yamashitae and C. chorassanicum, were grouped distinctly and seemed to be genetically diverse from members of the first crossability group. Our data support the distinct placement of C. cuneatum as well as a revised classification regarding its placement.