Microsatellite markers for the wood decay fungus Phlebiopsis gigantea

Nine polymorphic microsatellite markers were developed for the wood-decay basidiomycete Phlebiopsis gigantea, which is used commercially as a biocontrol agent for annosum root disease on conifers. Microsatellite sequences were isolated from repeat-enriched genomic libraries. Primers flanking these sequences were screened on P. gigantea isolates from Europe and North America. The number of alleles per locus ranged from 5 to 15, and gene diversity ranged from 0.72 to 0.90. These markers should be useful for studies of P. gigantea natural population structure and for making predictions about the impact of P. gigantea application in conifer forests.     

Isolation and characterization of EST‐derived microsatellite loci from the fungal wheat pathogen Phaeosphaeria nodorum

Eleven polymorphic microsatellite loci and one minisatellite locus originating from expressed sequence tag (EST) libraries of Phaeosphaeria (syn. Stagonospora) nodorum were isolated and characterized. The satellite markers were used to genotype isolates from field populations collected in China, North America and South Africa. The number of alleles per locus ranged from two to 15. Genotype diversity ranged from 87.5 to 95.3 and gene diversity from 0.1 to 0.8. The variable levels of polymorphism within and among populations of P. nodorum renders these 12 satellite loci ideal markers for population genetic analysis of P. nodorum.     

Microsatellite markers for Spergularia media (L.) C. Presl. (Caryophyllaceae) and their cross-species transferability

We developed 11 polymorphic microsatellite markers for Spergularia media (Caryophyllaceae), a perennial halophyte of coastal salt meadows and continental areas of western Eurasia. The number of alleles per locus observed in a single population of 20 individuals from the German North Sea coast ranged from 3 to 20. Observed and expected heterozygosities ranged from 0.200 to 0.850 and from 0.278 to 0.936 respectively. Observed heterozygosities were lower than expected heterozygosities at all loci, presumably as a consequence of inbreeding. All markers cross-amplified in the closely related S. salina. Of these, nine were polymorphic in the heterologous species. Only two primer pairs generated PCR products in S. diandra.     

Characterization of polymorphic microsatellite markers for Microcyclus ulei,causal agent of South American leaf blight of rubber trees

South American leaf blight caused by the ascomycete Microcyclus ulei is the most harmful disease of the rubber tree in Latin America and a potential threat to Asiatic and African natural rubber production. Until now, the variability of this fungus was assessed through observation of pathogenicity of isolates on a range of rubber tree clones with known resistance reactions. The present study describes the process used to design 11 microsatellite markers and evaluates their usefulness in detecting genetic polymorphism. Nine of these markers were polymorphic among six isolates from Brazil (with two to three alleles per locus) and five markers were polymorphic among four isolates from French Guiana (with two to four alleles per locus).     

Fifty‐three polymorphic microsatellite loci in the chestnut blight fungus,Cryphonectria parasitica

We report on 53 microsatellite loci for use in population genetic or linkage mapping studies in Cryphonectria parasitica. In 40 isolates collected from throughout the Northern Hemisphere, the number of alleles per locus ranged from two to 14 (mean 5.17) with gene diversity values ranging from 0.049 to 0.859 (mean 0.437). Samples from Asia were more diverse than those from Europe and North America. Most of the markers (48 of 53) were developed from an expressed sequence tag library, and hence, offer the opportunity to examine population structure or provide genome location information for specific expressed genes vs. anonymous genomic regions.     

Ten microsatellite markers for the freshwater diatom Sellaphora capitata

Ten polymorphic microsatellite markers were developed for the benthic freshwater diatom Sellaphora capitata and tested on 40 isolates from a Belgian pond. Genotyping was very successful (95%). The number of alleles per locus ranged from three to 12 (mean 6.6) and expected heterozygosities from 0.2 to 0.86 (mean 0.67). This is the first time that microsatellite markers have been developed for a freshwater or benthic diatom.     

Isolation and characterization of first microsatellite markers for the noble crayfish, <Emphasis Type="Italic">Astacus astacus</Emphasis>

Noble crayfish (Astacus astacus) is listed as vulnerable by the IUCN Red List of Threatened Species in Europe. However, very little is known about genetic diversity and structuring of noble crayfish populations, mainly because of the lack of informative genetic markers. We describe the isolation and characterization of the first microsatellite markers for this species, which were obtained by screening 4,000 recombinant clones. Eight loci revealed polymorphisms in a panel of 172 individuals from seven populations in Northern Europe. Number of alleles per locus ranged from two to 10 (average 4.4) and heterozygosity levels among populations varied from 0 to 0.80 for H _o and from 0 to 0.72 for H _e.     

Microsatellite markers for the grapevine pathogen, Eutypa lata

We isolated and characterized nine polymorphic microsatellite markers for Eutypa lata, a fungal pathogen responsible for Eutypa dieback of grapevine, in populations from two California vineyards (24 isolates per vineyard). Allele frequency ranged from two to 11 alleles per locus and haploid gene diversity ranged from 0.33 to 0.83. All samples comprised unique haplotypes. Our results suggest that there is sufficient allelic polymorphism to estimate fine-scale spatial structure, and to identify possible sources of inoculum from habitats outside of vineyards.     

Fourteen polymorphic microsatellite DNA markers for the human malaria parasite Plasmodium vivax

We have optimized a set of 14 polymorphic microsatellite markers for the human malaria parasite Plasmodium vivax, all of them consisting of either tri‐ or tetranucleotide repeats. These markers, whose polymerase chain reaction amplification conditions are identical, were used to screen 25 parasite isolates from malaria‐endemic areas in Sri Lanka. The total number of alleles per locus ranged between 6 and 13 (average, 7.8), and expected heterozygosity ranged from 0.627 to 0.913 (average, 0.790). These markers are now being used to characterize the population structure of P. vivax in other endemic areas.     

Development of polymorphic microsatellite markers for the fungal tree pathogen Cryphonectria eucalypti

Polymorphic microsatellite DNA markers were developed from a single spore isolate of Cryphonectria eucalypti collected from a Eucalyptus stem canker in South Africa. Markers were obtained using the enrichment technique known as fast isolation by AFLPs of sequences containing repeats (FIASCO). Ten polymorphic markers were isolated, of which, two were discarded due to their high polymorphism in the flanking region. The mean number of alleles produced by the remaining eight markers from 20 isolates was 7.25, and alleles per locus ranged from four to 12. The markers will be used to study populations of C. eucalypti.     

Isolation and characterization of polymorphic microsatellite markers for Alternaria alternata

Alternaria alternata is of major significance as a food and feed contaminant and is able to produce a range of mycotoxins that may elicit adverse effects in both animals and humans. We describe the isolation and characterization of five microsatellite markers for studying A. alternata. Marker polymorphism was screened in 64 isolates of A. alternata. The number of alleles per locus ranged from eight to 24, and allelic diversity ranged from 0.425 to 0.882. These markers will be useful in the study of relationships and population genetics amongst isolates of A. alternata.     

Fifty‐two polymorphic microsatellite loci in the rust fungus,Cronartium quercuum f.sp. fusiforme

We report on 52 microsatellite markers for use in Cronartium quercuum f.sp. fusiforme. The markers were developed from di‐, tri‐, and tetranucleotide repeat‐enriched genomic libraries. In 46 isolates collected from two natural populations in the southeastern USA, the number of alleles per locus ranged from two to 20 (mean 6.94) with gene diversity values ranging from 0.043 to 0.933 (mean 0.537). The markers should prove highly useful for genetic ‘fingerprinting’ of single‐spore isolates commonly used in host–pathogen gene interaction studies, as marker loci for linkage mapping studies, and for examining fine‐scale population genetic structure in natural populations of the fungus.     

Isolation and characterization of microsatellites in the woody shrub, Banksia sphaerocarpa var. caesia (Proteaceae)

Microsatellite markers were developed for the Australian bird-pollinated woody shrub Banksia sphaerocarpa var. caesia to study gene flow among populations in a highly fragmented landscape. Eight loci were developed, and in a sample of 40 individuals from one population, the number of alleles per locus ranged from five to 21 and observed heterozygosities ranged from 0.385 to 0.914. All eight loci showed independent inheritance. Analysis of open-pollinated progeny arrays confirmed Mendelian inheritance at seven loci, while null alleles were suspected at the remaining locus.     

鸭茅种质遗传变异及亲缘关系的SSR分析

利用SSR标记技术对来自世界5大洲的53份鸭茅(Dactylis glomerata L.)材料的遗传变异及亲缘关系进行了研究。用筛选的15对引物对53份材料的DNA进行PCR扩增, 获得下述结果: (1)15个位点共检测到127个等位基因, 每个位点等位基因变幅为5～12个, 平均为8.5个, 多态性位点率 (P)平均为95.21%; 多态性信息含量(PIC)范围为0.30(A04C24) 到 0.44 (A01F24), 平均为0.36; (2)材料间遗传相似系数(GS)范围在0.43到0.94之间; 地理类群间的GS值在0.73到0.91间, 其中亚洲(P,90.55%)和欧洲(P,86.61%)类群遗传多样性丰富, 表明供试鸭茅种质具有丰富的遗传变异; (3)根据研究结果进行聚类分析和主成分分析, 可将53份鸭茅材料分成5大类, 来自相同洲的材料能聚为一类, 呈现出一定的地域性分布规律。     

基于转录组测序信息的水生植物莕菜SSR标记开发

利用软件MISA对水生植物莕菜转录组测序所获得的79536条EST序列进行分析,共检测出12319个EST-SSR位点。在莕菜的EST-SSR中,二核苷酸和三核苷酸重复单元是主导类型,分别占总SSR的57.31%和30.87%,其中AG/CT、AAG/CTT分别是二、三重复单元类型的优势重复基元,分别占总SSR的29.76%和8.66%。随机挑选了130对EST-SSR引物对莕菜两个居群进行遗传多样性检测,结果发现：78对引物能扩增出清晰可分辨的条带,其中37对能成功检测出多态性,引物多态率为 47.44%。这些多态性引物共检测出114个等位基因,每个位点2~6个,平均3.08个。观测杂合度（H_o）及预期杂合度（H_e）分别在0.229~1.000和0.351~0.756之间,多态信息含量PIC值在0.286~0.698之间,平均达0.495。以上研究结果表明,通过莕菜转录组测序产生的EST数据来开发SSR标记是一种简单而高效的途径,这些新的SSR分子标记为研究莕菜的居群遗传多样性及其遗传结构提供了工具。     

Real-time PCR detection of Phaeomoniella chlamydospora and Phaeoacremonium aleophilum

Phaeomoniella chlamydospora and Phaeoacremonium aleophilum are the two main fungal causal agents of Petri disease and esca. Both diseases cause significant economic losses to viticulturalists. Since no curative control measures are known, proactive defensive measures must be taken. An important aspect of current research is the development of sensitive and time-saving protocols for the detection and identification of these pathogens. Real-time PCR based on the amplification of specific sequences is now being used for the identification and quantification of many infective agents. The present work reports real-time PCR protocols for identification of P. chlamydospora and P. aleophilum. Specificity was demonstrated against purified DNA from 60 P. chlamydospora isolates or 61 P. aleophilum isolates, and no amplification was obtained with 54 nontarget DNAs. The limits of detection (i.e., DNA detectable in 95% of reactions) were around 100 fg for P. chlamydospora and 50 fg for P. aleophilum. Detection was specific and sensitive for P. chlamydospora and P. aleophilum. Spores of P. chlamydospora and P. aleophilum were detected without the need for DNA purification. The established protocols detected these fungi in wood samples after DNA purification. P. chlamydospora was detectable without DNA purification and isolation in 67% of reactions. The detection of these pathogens in wood samples has great potential for use in pathogen-free certification schemes.     

Development and characterization of 12 compound microsatellite markers in <Emphasis Type="Italic">Platypus </Emphasis><Emphasis Type="Italic">quercivorus</Emphasis> (Murayama) (Coleoptera: Platypodidae)

Twelve compound microsatellite loci were isolated from the ambrosia beetle Platypus quercivorus. Every locus was polymorphic among 50 individuals from two localities, with two to six alleles per locus, without linkage disequilibrium. The observed and expected heterozygosities ranged from 0.140 to 0.740 and from 0.255 to 0.731, respectively. These markers will be available for population genetic studies and parentage analysis of this beetle.     

Development and characterization of 60 microsatellite markers in the abalone Haliotis diversicolor

The abalone, Haliotis diversicolor, is one of the most important mariculture species in southern China. We developed 60 new polymorphic microsatellite markers for H. diversicolor and characterized them in 30 individuals from a cultured population in Sanya, China. All 60 markers were found to be polymorphic. The number of alleles ranged from two to nine per locus, with an average of 4.12/locus. The expected and observed heterozygosities ranged from 0.10 to 0.88 and from 0.07 to 0.87, respectively. Forty-four loci were in Hardy-Weinberg equilibrium. These 44 microsatellite markers should be useful for genome mapping and population genetic studies.     

Isolation of polymorphic microsatellite loci from Eurasian woodcock (Scolopax rusticola) and their cross‐utility in related species

We isolated one trinucleotide and seven tetranucleotide microsatellite loci for the Eurasian woodcock (Scolopax rusticola). We describe polymerase chain reaction conditions and primers for the successful amplification of these loci and report the results obtained from their use in 42 specimens from two populations in Europe. The number of alleles per locus ranged from three to 15, observed heterozygosity was comprised between 0.11 and 1.00 and expected heterozygosity ranged between 0.10 and 0.91. Cross‐specific amplification experiments highlighted the potential usefulness of these molecular markers for the study of three related scolopacid waders.