Development and characterization of a new set of 164 polymorphic EST‐SSR markers for diversity and breeding studies in rubber tree (Hevea brasiliensis Müll. Arg.)

Despite its economic importance and recent genome release, the need for molecular tools for Hevea brasiliensis is high. In the frame of a disease resistance study, EST sequences were retrieved from public database or generated by sequencing SSH libraries. Sequences were trimmed and microsatellite motifs searched using an ad hoc bioinformatic pipeline, and pairs of primers for the amplification of candidate markers were generated. We found a total of 10 499 unigenes from both sources of sequences, and 673 microsatellites motifs were detected using the default parameters of the pipeline. Two hundred sixty‐four primer pairs were tested and 226 (85.6%) successfully amplified. Out of the amplified candidate markers, 164 exhibited polymorphism. Relationships based on dendrograms using simple matching index and diversity statistics based on EST‐SSRs were compared with Genomic SSRs, showing the potentialities of EST‐derived microsatellites for resistance studies but also for population genetics approaches.     

The development and use of microsatellite markers for genetic analysis and plant breeding with emphasis on bread wheat

In recent years, a variety of molecular markers, based on microsatellites or simple sequence repeats (SSRs) have become the markers of choice, thus necessitating their development and use in a variety of plant systems. In this review, the basic principles underlying different hybridization-based (oligonucleotide fingerprinting) and PCR based approaches (STMS, MP-PCR, AMP-PCR/ ISSR/ ASSR, RAMPs/ dRAMPs, SAMPL), making use of microsatellites, have been outlined. Different methods for enrichment of genomic libraries for microsatellites have also been outlined. Relevant literature on the subject, giving a summary of results obtained using each approach, has been reviewed and critically discussed. The review also includes a discussion on literature, which deals with the use of microsatellites in genome mapping, gene tagging, DNA fingerprinting, characterization of germplasm and cytogenetics research. Special emphasis has been laid on the genome of bread wheat, where the work done in the authors' own laboratory has also been briefly reviewed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Comparative study of the discriminating capacity and effectiveness of AFLP, STMS and EST markers in assessing genetic relationships among evergreen azaleas

The application of amplified fragment length polymorphism (AFLP), sequence tagged microsatellite site (STMS) and expressed sequence tag (EST) markers to study the genetic relationships in an evergreen azalea gene pool was investigated. STMS and EST markers revealed a higher genetic distance detection capacity than AFLPs, which, nevertheless, were the most efficient marker system due to their highest polymorphism detection capacity. Similarity matrices showed weak, yet significant, correlations when Mantel's test was applied. To assess the usefulness of the overall information provided by these marker data for establishing phylogenetic relationships and horticultural classification, cluster analysis was performed. The joint AFLP, STMS and EST data were demonstrated to be remarkably effective for group discrimination and phylogenetic studies. The use of these polymerase chain reaction marker systems is discussed in terms of the choice of appropriate marker techniques for different aspects of evergreen azalea germplasm evaluation.     

Microsatellite markers: an overview of the recent progress in plants

In recent years, molecular markers have been utilized for a variety of applications including examination of genetic relationships between individuals, mapping of useful genes, construction of linkage maps, marker assisted selections and backcrosses, population genetics and phylogenetic studies. Among the available molecular markers, microsatellites or simple sequence repeats (SSRs) which are tandem repeats of one to six nucleotide long DNA motifs, have gained considerable importance in plant genetics and breeding owing to many desirable genetic attributes including hypervariability, multiallelic nature, codominant inheritance, reproducibility, relative abundance, extensive genome coverage including organellar genomes, chromosome specific location and amenability to automation and high throughput genotyping. High degree of allelic variation revealed by microsatellite markers results from variation in number of repeat-motifs at a locus caused by replication slippage and/or unequal crossing-over during meiosis. In spite of limited understanding of the functions of the SSR motifs within the plant genes, SSRs are being widely utilized in plant genome analysis. Microsatellites can be developed directly from genomic DNA libraries or from libraries enriched for specific microsatellites. Alternatively, microsatellites can also be found by searching public databases such as GenBank and EMBL or through cross-species transferability. At present, EST databases are an important source of candidate genes, as these can generate markers directly associated with a trait of interest and may be transferable in close relative genera. A large number of SSR based techniques have been developed and a quantum of literature has accumulated regarding the applicability of SSRs in plant genetics and genomics. In this review we discuss the recent developments (last 4–5 years) made in plant genetics using SSR markers.     

Characterization of microsatellite markers,their transferability to orphan legumes and use in determination of genetic diversity among chickpea (Cicer arietinum L.) cultivars

The microsatellite or simple sequence repeat (SSR) marker is the most preferred marker because of its many desirable properties. It is important to increase the genic and genomic resources particularly in legumes because the SSR markers currently available in chickpea, pigeonpea, horsegram, blackgram, and cowpea are very limited. In the present study, 201 pairs of SSR markers comprising of 172 genic and 29 genomic SSRs were screened against 11 chickpea genotypes, among which 153 produced monomorphic and 48 produced polymorphic bands. The polymorphic information content ranged from 0.152 to 0.373 for both genic and genomic SSRs. Among the polymorphic markers, two-three alleles were detected for genic and two-four alleles for genomic SSRs. A unique banding pattern could be found for all the genotypes within 48 polymorphic SSR markers and cultivar specific markers could be identified for seed purity test. We have also studied the ability of chickpea genic and genomic SSRs to amplify distantly related but important legumes viz., horsegram, blackgram, cowpea, pigeonpea, and soybean. Out of 201 chickpea SSR primer pairs, 66.7% in blackgram, 62.2% in horsegram, 61.7% in redgram, 54.7% in cowpea, and 62.7% in soybean produced amplification. The transferability of about 60.0% of the chickpea SSRs to distantly related legumes could be considered successful. In the present study, 134, 133, 126, 124, and 110 new SSR primers for blackgram, horsegram, soybean, redgram, and cowpea pulse crops, respectively, were identified. It is an important addition to the already available genomic resources in these crops. In addition, among genic primer pairs, 12 in horsegram, three in soybean, 13 in redgram, and eight in cowpea, and among genomic primer pairs, two in horsegram and four in redgram were polymorphic even in the two-three genotypes tested indicating their potentially for application in genetic studies and mapping.     

Genic microsatellite markers for discrimination of spinach cultivars

A set of simple sequence repeat (SSR or microsatellite) markers was used to discriminate a collection of 33 Spinacia oleracea hybrid cultivars from seven different breeding stations all over the world. All SSR markers were genic microsatellite markers located in coding or non-coding regions of genes of known function. Cluster analysis based on 13 of the SSR markers showed that the spinach hybrids grouped into three clusters. The first two clusters consisted of European spinach types, which were well discriminated according to their origin from different breeding stations. The third cluster was a mixture of Asian as well as European types of spinach. Subclusters in this group did not reflect differences in morphology, earliness or company origin. The data show that genic microsatellites are a powerful tool for discrimination of spinach cultivars.     

Development and utilization of genomic and genic microsatellite markers in Assam tea (Camellia assamica ssp. assamica) and related Camellia species

Microsatellite or simple sequence repeat (SSR) markers are valuable tools for many purposes, such as phylogenetic, fingerprinting and molecular breeding studies. However, such marker resources are unavailable in Assam tea (Camellia assamica ssp. assamica; Masters). With an objective to enrich the repertoire of microsatellite markers in traditional tea, 185 novel microsatellite (150 genomic and 35 genic) markers were identified from (GA)n‐enriched genomic libraries and public expressed sequence data in Assam tea. High‐quality 0.412‐Mb non‐redundant (NR) genomic data set derived from nucleotide sequencing of 1297 (GA)n‐enriched genomic positive clones and 2723 unigenes (1.33 Mb) predicted from 10 803 random public expressed sequence tags (ESTs) in C. assamica ssp. assamica were utilized for identification of genomic and genic microsatellite markers, respectively. The average number of alleles and polymorphic information content (PIC) recorded for the newly developed SSR markers were 6.17 and 0.398, respectively. The average observed (H_o) and expected (H_e) heterozygosity varied from 0.626 to 0.697, respectively. These markers were found to be highly transferable (74.5–100%) to cultivated (C. sinensis, C. assamica ssp. lasiocalyx) and five wild Camellia species. Genetic diversity coefficient detected a high level of divergence in 24 cultivated tea accessions (69.3%). Phylogenetic analysis revealed that major groupings were broadly in accordance with taxonomic classification of tea, and all the wild Camellia species remained as an out‐group. The high polymorphic content coupled with high rate of cross‐transferability demonstrates wider applicability of novel microsatellite markers in genotyping, genetic diversity, genome mapping and evolutionary studies in various Camellia species.     

The art of attrition: development of robust oat microsatellites

Microsatellite or simple sequence repeat (SSR) markers are important tools for genetic analyses, especially those targeting diversity. The primary objective of this study was to develop robust oat‐based microsatellite markers from newly enriched genomic libraries to expand on a relatively small existing oat SSR toolbox. Microsatellite motifs characterized by (CA/GT), (AAT/TTA), (ATG/TAC) and (CATC/GTAG) repeats were targeted for enrichment. Preliminary screening showed that 90% of clones from the (CA/GT) and 79% of the clones from the (ATG/TAC) libraries contained repeats, while < 11% of the clones from (AAT/TTA) and (CATC/GTAG) libraries contained repeats. Subsequent sequencing of 1536 clones from the (CA/GT) and (ATG/TAC) libraries resulted in 539 and 578 SSRs for which primers were designed, respectively. A total of 246 SSRs were polymorphic across 11 oat lines. One hundred and twenty‐five of the markers produced highly reproducible assays that interrogated 369 alleles at 193 loci. Of these, 79 robust assays interrogated 146 codominant alleles. These markers will be useful for a wide range of genetic analyses in oat including assessment of diversity and marker‐assisted breeding.     

Molecular breeding in the genus Musa: a strong case for STMS marker technology

Musa species are among the tallest monocotyledons and include major food-producing species. The principal cultivars, derived from two major species Musa acuminata (‘A’ genome) and Musa balbisiana (‘B’ genome), are polyploid hybrids (mainly AAA, AAB and ABB triploids), medium to highly sterile, parthenocarpic and clonally propagated. Bananas and plantains are crops to which molecular breeding is expected to have a positive impact. In order to better understand banana genetics, more knowledge has to be accumulated about the complex genome structure of hybrids and cultivars. Therefore, the aim of our work is to develop molecular markers that are codominant, reliable, universal, highly polymorphic and that are applicable to collaborative Musa germplasm genotyping and mapping. Two size-selected genomic libraries have been screened for the presence of simple sequence repeats (SSR). Our data demonstrate that SSR are readily applicable to the study of Musa genetics. Our comprehensive analyses of a significant number of banana sequence tagged microsatellite sites (STMS) will add to our knowledge on the structure and phylogeny of genomes of the Musa species, and suggest that microsatellites be used as anchor markers for a banana genetic core map. Additional markers, such as e.g. CAPS have also been tested in order to increase the detection of polymorphisms exceeding that revealed by STMS technology. The utility of PCR-derived markers for collaborative genetic analyses of the banana genome, and the transferability of 'streamlined’ laboratory techniques and data analysis to Developing Countries are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

EST-derived microsatellites as a rich source of molecular markers for oats

Polymerase chain reaction-based microsatellite markers are valuable tools for molecular breeding because of their co-dominant inheritance and their applicability for high-throughput analysis. Being still very limited for oats, their number has to be increased significantly to cover the entire genome. For these purposes, a set of 7031 recently published expressed sequence tags (ESTs) was screened for microsatellites with dinucleotide, trinucleotide and tetranucleotide repeat motifs. Subsequent in silico analysis resulted in the development of 216 primer pairs for Avena EST-derived microsatellite loci ( AME ). Using a sample set of 12 oat lines, 107 of 195 functional primers could be assayed as polymorphic. The marker variability averaged out at three alleles per locus and a polymorphic information content (PIC) value of 0.42. This variability documents their suitability for molecular oat-breeding purposes. Finally, 51 of the AME loci could be placed within the known reference map of 'Kanota' × 'Ogle' that previously contained only 12 microsatellite loci. Thus, a remarkable enhancing of the number of mapped oat microsatellite loci could be achieved.     

Development, characterization and utilization of microsatellite markers in pigeonpea

Pigeonpea is a major legume of the semi‐arid tropics that has been neglected in terms of molecular breeding. The objectives of this study were to develop microsatellite markers and evaluate their potential for use in pigeonpea genetics and breeding. Two hundred and eight microsatellite loci were isolated by screening a non‐enriched partial genomic library. Primers were designed for 39 microsatellite loci, 20 of which amplified polymerase chain reaction products of the expected size. Nineteen of the primer pairs were polymorphic amongst 15 cultivated and nine wild pigeonpea accessions providing evidence for cross‐species transferability within the genus Cajanus. A total of 98 alleles were detected at the 19 polymorphic loci with an average of 4.9 alleles per locus. The observed heterozygosity ranged from 0.17 to 0.80 with a mean of 0.42 per locus. Less allelic variation (31 alleles) was observed within the cultivated species than across the wild species (92 alleles). The diversity analysis readily distinguished all wild relatives from each other and from the cultivated germplasm. Development of more microsatellites is recommended for future genomic studies in pigeonpea.     

Application of microsatellites from maize to teosinte and other relatives of maize

Teosinte comprises different Zea species (Zea mays, Zea diploperennis, Zea perennis, Zea luxurians) that can be crossed with cultivated maize (Z. mays ssp. mays). Nine microsatellites from maize were applied to different teosinte species in order to evaluate their usefulness in markerbased exploitation of these genetic resources. The same microsatellites were tested with rye, barley, and sorghum as potential molecular markers for these species. Almost all microsatellite × teosinte combinations yielded polymerase chain reaction (PCR) fragments in the range of cultivated maize. Using an F₂ population of a cross between maize inbred A188 and an individual of Zea mays ssp. mexicana, amplification products for maize and teosinte originated from the same genomic location for each of nine microsatellites investigated. PCR fragments of reduced intensity were generally obtained by applying maize microsatellites to rye, barley and sorghum. Polymorphisms among accessions within teosinte (sub)species occurred frequently. In contrast, no polymorphisms were obtained within rye, barley, and sorghum. Hence, application of maize microsatellites to teosinte for fingerprinting or marker-assisted introgression of genomic regions from teosinte into cultivated maize appears promising.     

A polymorphic microsatellite in the 3'end of 'waxy' genes of wheat, Triticum aestivum

Potential polymorphism of an (AT)_N microsatellite at the 3’end of waxy genes in bread wheat was examined. Primers were designed from a published cDNA sequence of a wheat waxy gene. Polymerase chain reaction (PCR) amplification of genomic DNA from 135 mainly Australian cultivars revealed eight alleles on chromosome 7A. This polymorphic microsatellite is a potential codominant marker for the Wx-A1 locus in breeding programmes. A distinguishable fragment was also amplified from chromosome 7D. This fragment was absent where a plant was null for the waxy gene on chromosome 7D, being a dominant marker for the Wx-D1 locus. The primers were also useful for amplifying genomic DNA from barley, rye and triticale and can be used to detect potential polymorphism in these species.     

Discriminating durum wheat cultivars using highly polymorphic simple sequence repeat DNA markers

The winter type durum wheat varieties of Anatolia used in this study were differentiated for the first time by using simple sequence repeat (SSR) DNA markers or microsatellites. Seven microsatellite markers were used to distinguish four well‐adapted landrace selections, five cultivars and seven recently obtained advancing lines. The loci of seven microsatellites were all homozygous, but the WMS6 locus occurred with two alleles in all the genotypes. The genotypes were all distinguished from each other, with the number of alleles ranging from five to 13. The lowest and highest polymorphism information content (PIC) values were observed to be 0.609 and 0.872, respectively. Three markers alone, WMS6, WMS30 and WMS120, can distinguish all 16 genotypes. UPGMA dendogram, based on a similarity matrix by a simple matching coefficient algorithm, is in accordance with the available pedigree information.     

Isolation of two microsatellite markers from BAC clones of the Vf scab resistance region and molecular characterization of scab‐resistant accessions in Malus germplasm*

A polymerase chain reaction (PCR)‐based method was developed to isolate microsatellite markers from large‐insert genomic DNA clones of bacterial artificial chromosome (BAC) libraries. The method is fast and economic since it does not require subcloning. It was applied to isolate a microsatellite marker from a BAC clone of the chromosomal region containing the apple scab resistance gene Vf. The Vf gene of Malus floribunda 821 is the most widely used source of scab resistance in apple breeding. A second microsatellite was found on the extremity of a BAC clone flanking the Vf locus. The two microsatellites allowed the identification of the presence of the Vf gene in the scab‐resistant accessions M. micromalus SA573‐3, ‘Golden Gem’, M. prunifolia 19651 and MA 16 not previously known to carry this gene. They were also used to verify the correctness of the published genealogical tree of the Vf cultivar ‘Florina’, in which a probable mistake was identified. This analysis shows the importance of genotyping the Vf locus when choosing scab‐resistant germplasm as parents in breeding programmes.     

The use of microsatellite polymorphisms for the identification of Australian breeding lines of rice (Oryza sativa L.)

Ten microsatellite loci were analysed for 43 cultivars or breeding lines of rice. Polymorphism-Information-Content values ranged from 0.62 to 0.92. The microsatellite markers were found to be useful for cultivar identification and assessment of genetic relationships. Most of the cultivars could be uniquely identified by at least one microsatellite marker. Genetic heterogeneity was detected within rice samples by amplification of microsatellites from DNA extracted from multiple individual plants and also from bulked DNA preparations. This revised version was published online in July 2006 with corrections to the Cover Date.     

Identification and characterization of microsatellites in eggplant

The potential of microsatellite markers for use in genetic studies in eggplant, Solanum melongena, has been evaluated. A genomic library of eggplant was screened for GA and GT repeat motifs to isolate microsatellite clones. The frequency of each repeat motif in the eggplant genome was found to be every 3200 kb for GA repeats and every 820 kb for GT repeats. Sixty‐one per cent of GT repeats were found to directly flank AT repeats. A total of 37 polymerase chain reaction (PCR) primer pairs were designed, 23 of which amplified a single product or several products. The level of microsatellite polymorphism was evaluated by using S. melongena lines and related Solanum species. Two to six alleles per primer pair were displayed in the S. melongena lines and two to 13 alleles were displayed in the Solanum relatives. Seven microsatellites showed polymorphism between parental lines of the mapping population and segregated in a codominant Mendelian manner. These microsatellite loci were distributed throughout the linkage map.     

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

Microsatellites 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.