Isolation and characterization of polymorphic simple sequence repeat loci in Armillaria ostoyae

A modified hybridization strategy was used to construct a microsatellite enriched library from DNA of Armillaria ostoyae, a serious root pathogen on pine. Sequence characterization of 19 random clones revealed 12 distinct loci harbouring a repetitive motif. Primer design from the flanking regions allowed for their development as polymerase chain reaction based markers. Polymorphic assessment at both the population and global levels revealed levels of variation useful for genetic studies. The level of cross‐species amplification observed with closely related Armillaria species was high, raising the possible exploitation of these primers across the genus.     

Development of 16 polymorphic simple sequence repeat markers for Lycoris longituba from expressed sequence tags

Lycoris longituba is a tulip-like ornamental plant in China. We report on the data mining of L. longituba expressed sequence tags (ESTs) to generate simple sequence repeat (SSRs) markers. Eighteen EST-SSRs were isolated and validated for 32 individuals. These markers will be valuable for studying the genetic structure and diversity of populations for L. longituba.     

Development of simple sequence repeat markers for the soybean rust fungus, Phakopsora pachyrhizi

Twenty-four simple sequence repeat markers were developed for Phakopsora pachyrhizi, a fungal pathogen of soybean (Glycine max) and other legumes. All 24 of the loci were evaluated on 28 isolates of P. pachyrhizi. Twenty-one loci were polymorphic, with allelic diversity ranging from two to eight alleles, and null alleles were observed for eight of the 24 loci. A preliminary screen with the closely related species, P. meibomiae, indicated that these primer pairs are specific to P. pachyrhizi.     

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.     

Development of multiplex sets of simple sequence repeat DNA markers covering the soybean genome

Multiplexing involves the analysis of several markers in a single gel lane that is based on the allele size range of marker loci. Multiplex SSR marker analysis is conducted with primers that are labeled with one of three dyes. The development of an SSR multiplex system requires estimates of the allele size range of markers to strategize primer labeling and for grouping markers into multiplex sets. A method is presented that describes the development of multiplex sets of SSR markers in soybean (Glycine max (L.) Merr.) by the selective placement of primer sites and by the analysis of diverse germplasm. Primer sites were placed at specific distances from the SSR to adjust the allele size range of marker loci. The analysis of pooled DNA samples comprising diverse soybean genotypes provided robust estimates of the allele size range of marker loci that enabled the development of multiplex sets. Eleven multiplex sets comprising 74 SSR markers distributed across the 20 linkage groups of soybean were developed. Multiplex sets constructed from the analysis of diverse soybean germplasm should have a wide range of genotyping applications. The procedures used in this study were systematic and rapid and should be applicable for multiplex development in any species with SSR marker technology.     

Fifteen polymorphic simple sequence repeat markers from expressed sequence tags of Liriodendron tulipifera

We developed and evaluated simple sequence repeat (SSR) markers derived from expressed sequence tags (ESTs) of Liriodendron tulipifera. Characteristics of 15 EST‐SSR loci were investigated using 33 L. tulipifera individuals. The number of alleles per locus ranged from two to five. The expected and observed heterozygosities ranged from 0.216 to 0.751 and from 0.182 to 0.97, respectively. These loci were further tested for their cross‐species transferability to Liriodendron Chinense. Because of their high level of polymorphism and transferability, our 15 single‐locus EST‐SSR markers will be valuable tools for research on mating system, population genetics and systemic evolution of Liriodendron.     

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.     

Identification of 11 polymorphic simple sequence repeat loci in the phytopathogenic fungus Fusarium pseudograminearum as a tool for genetic studies

Simple sequence repeat (SSR) markers for Fusarium pseudograminearum with 2 to 3 bp repeat motifs were identified by screening the genome database of the related species Fusarium graminearum. Twelve SSRs amplified single loci in both F. graminearum and F. pseudograminearum. Forty F. pseudograminearum and six F. graminearum individual isolates were screened to determine levels of polymorphism, with all SSRs displaying three to 14 alleles across all isolates. Eleven SSRs were polymorphic across F. pseudograminearum isolates tested proving the usefulness of genome databases of closely related species in identifying genetic markers.     

Development of simple sequence repeat (SSR) markers for the assessment of gene flow between sea beet (Beta vulgaris ssp. maritima) populations

Molecular markers can be used to estimate gene flow indirectly by monitoring the relative frequency of alleles in adjacent populations. Sea beet (Beta vulgaris ssp. maritima) is a wild plant species found along the coastlines of many European countries and is closely related to cultivated beets. A set of six simple sequence repeat (SSR) markers that are polymorphic in UK populations have been developed for sea beet to assess the problems of indirect measurement of gene flow in these populations.     

Development and characterization of polymorphic markers for the sap-stain fungus Ophiostoma quercus

Eight polymorphic markers were developed from South African isolates of Ophiostoma quercus. The genome was screened for repeat regions using the fast isolation by amplified fragment length polymorphism of sequences containing repeats protocol and 20 de novo primer pairs flanking putative microsatellite regions were designed. Eight loci were optimized and their polymorphisms evaluated by sequencing. The repeat and flanking regions were highly polymorphic containing both indels and base-pair substitutions revealing a total of 46 alleles in 14 isolates and an average heterozygosity of 0.68. Substantial sequence variability makes these markers useful for genotyping populations in order to calculate diversity and monitor global movement of O. quercus.     

Development of simple sequence repeat markers for Japanese isolates of Magnaporthe grisea

We developed nine simple sequence repeat (SSR) markers useful for differentiating Japanese isolates of Magnaporthe grisea through a bioinformatic approach. Repeat sequences in the genome of M. grisea were identified by a Tandem Repeat Finding program. Length polymorphisms at 28 loci were examined, nine of which were selected on the basis of detected polymorphisms. These nine SSR markers showed a Nei's gene diversity ranging from 0.23 to 0.91 among 48 field isolates of two natural populations. These SSR markers are well suited for M. grisea epidemiology and population genetics.     

Development and characterization of simple sequence repeat DNA markers for Zelkova serrata

We developed 14 microsatellite loci from an enriched genomic DNA library of a broad‐leaved deciduous tree, Zelkova serrata. Of 198 clones from the library, 112 contained microsatellite repeat regions. The M13‐tailed primer method was used for economy. Sequence‐specific primer pairs were designed for 58 of 76 candidate clones. Fourteen of these primer pairs successfully amplified polymorphic single loci among 34 individuals collected from the Kanto breeding region in Japan. The expected heterozygosity for the 14 microsatellite markers ranged from 0.378 to 0.876, suggesting that these will prove valuable for breeding and ecological studies on Z. serrata.     

Eleven polymorphic simple sequence repeat markers from expressed sequence tags of Pacific oyster Crassostrea gigas EST database

Oyster (Crassostrea gigas) is widely distributed in coastal areas of world. We developed and evaluated simple sequence repeat (SSR) markers from expressed sequence tags (ESTs) of C. gigas and to amplify EST-SSR in C. gigas. Characteristics of 11 EST-SSR loci were investigated using 45 C. gigas individuals. The number of alleles per locus ranged from two to thirteen. The observed heterozygosity (H _o) ranged from 0.0889 to 0.7333 and the expected heterozygosity (H _e) ranged from 0.0859 to 0.8981. Because of their high level of polymorphism, our 11 single-locus EST-SSR markers will be valuable tools for research on mating system, population genetics and systemic evolution of oyster in the future.     

Release of a reducing substance by the ectomycorrhizal fungi Pisolithus tinctorius and Paxillus involutus

Culture solutions of the ectomycorrhizal fungi Pisolithus tinctorius and Paxillus involutus are shown to reduce a high valency oxide of manganese at a range of pH values from 3.0 to 13.0. Manganese reduction in unmoculated culture media was confined to pH values below 5.0 and above 10.0. The results are consistent with the release of a reducing substance from the fungal mycelium.     

Development and mapping of EST-derived simple sequence repeat markers for hexaploid wheat.

Expressed sequence tags (ESTs) are a valuable source of molecular markers. To enhance the resolution of an existing linkage map and to identify putative functional polymorphic gene loci in hexaploid wheat (Triticum aestivum L.), over 260,000 ESTs from 5 different grass species were analyzed and 5418 SSR-containing sequences were identified. Using sequence similarity analysis, 156 cross-species superclusters and 138 singletons were used to develop primer pairs, which were then tested on the genomic DNA of barley (Hordeum vulgare), maize (Zea mays), rice (Oryza sativa), and wheat. Three-hundred sixty-eight primer pairs produced PCR amplicons from at least one species and 227 primer pairs amplified DNA from two or more species. EST-SSR sequences containing dinucleotide motifs were significantly more polymorphic (74%) than those containing trinucleotides (56%), and polymorphism was similar for markers in both coding and 5' untranslated (UTR) regions. Out of 112 EST-SSR markers, 90 identified 149 loci that were integrated into a reference wheat genetic map. These loci were distributed on 19 of the 21 wheat chromosomes and were clustered in the distal chromosomal regions. Multiple-loci were detected by 39% of the primer pairs. Of the 90 mapped ESTs, putative functions for 22 were identified using BLASTX queries. In addition, 80 EST-SSR markers (104 loci) were located to chromosomes using nullisomic-tetrasomic lines. The enhanced map from this study provides a basis for comparative mapping using orthologous and PCR-based markers and for identification of expressed genes possibly affecting important traits in wheat.     

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.     

Polymorphic microsatellite markers of a Pisolithus sp. from a Eucalyptus plantation

We developed six polymorphic microsatellite markers for an ectomycorrhizal Pisolithus sp., which are associated with Eucalyptus camaldulensis in Thailand using a method starting with amplified fragments of intersimple sequence repeats (ISSR). The number of alleles per locus varied between two and five and expected heterozygosities ranged from 0.123 to 0.625. These loci are available for analysis of genetic structure and gene flow in Eucalyptus‐associated Pisolithus spp. populations.     

Development of simple sequence repeat markers for bermudagrass from its expressed sequence tag sequences and preexisting sorghum SSR markers

Bermudagrass (Cynodon spp.) is extensively cultivated for forage and turf in the the southern United States and in parts of Asia, Africa, southern Europe, Australia and South America. However, few simple sequence repeat (SSR) markers are available for bermudagrass genetics research. Accordingly, the objective of this study was to develop SSR markers in bermudagrass by transferring sorghum genomic SSR primers and by exploring bermudagrass expressed sequence tags (ESTs) from the National Center for Biotechnology Information (NCBI) database. The transferability of 354 tested sorghum SSRs was 57% to C. transvaalensis T577 (2n = 2x = 18), 27% to C. dactylon Tifton 10 (2n = 6x = 54) and 22% to Zebra (2n = 4x = 36). Among the transferred SSRs, 65 primer pairs generated reproducible SSR bands across the three genotypes. From 20,237 Cynodon ESTs at NCBI, 303 designed SSR primer pairs amplified target bands in at least one of C. dactylon var. aridus (2n = 2x = 18), C. transvaalensis T577, C. dactylon cv. Tifton 10, and C. dactylon var. dactylon Zebra. Of the effective EST SSRs, 230 primer pairs produced reproducible bands in all four genotypes. The study demonstrated that EST sequences and sorghum SSR primers are useful sources for the development of SSR markers for bermudagrass. The developed SSR markers will make a valuable contribution to the molecular identification of commercial cultivars, construction of genetic maps, and marker-assisted breeding in bermudagrass.