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.     

A highly accurate, single PCR reaction for parentage assignment in Senegal sole based on eight informative microsatellite loci

From a battery of microsatellite markers (100 loci), recently identified by our group, we have selected eight for parentage assignment in Senegal sole ( Solea senegalensis ). This tool is based on microsatellite loci obtained from four genomic DNA libraries and one cDNA library. Within the eight loci (six from anonymous genomic DNA sequences and two located in expressed sequence tags of known genes), we have found, in an analysis of a reproductive broodstock, between nine and 16 alleles. The expected heterozygosity was between 0.616 and 0.860. In addition, we have optimized the polymerase chain reaction (PCR) conditions to amplify all loci simultaneously in a single multiplex PCR reaction, and we have tested three lots of male and female (five to six individuals) and three offspring (50–60 larvae each). The use of the eight microsatellite loci, the possibility of amplifying them in a single PCR reaction and the high value of the exclusion probability (0.9992) make this multiplex PCR method a unique tool for parentage assignment.
Finally, analysing one meiotic gynogenetic progeny, we have determined the relative distance of six of these loci to the centromere, and we have also found that all of them are unlinked. All these characteristics confer this tool with a high accuracy for parentage studies and genetic population analyses of Senegal sole.     

Genetic differentiation between Zhe oyster Crassostrea plicatula and pacific oyster Crassostrea gigas populations in China assessed by microsatellite analysis

ABSTRACT:   Genetic differentiation and relationships between Crassostrea plicatula and Crassostrea gigas populations from China were studied by means of the microsatellite technique. Seven loci were used to screen five populations each collected from C. plicatula and C. gigas . All loci showed high polymorphism for all populations, as observed in average number of alleles per locus (19.1–28.1), and average expected heterozygosity (0.891–0.954). Significant departures from Hardy–Weinberg equilibrium due to heterozygote deficiency were observed over most populations at each locus and were best explained by null alleles. F _ST values showed significant genetic differentiation between C. plicatula and C. gigas populations. According to the neighbor-joining tree constructed on the basis of the genetic distance ( D _A), the ten populations fell into two distinct groups ( C. plicatula and C. gigas groups), and the results of principal coordinate analysis and assignment tests also supported the neighbor-joining clustering. The outcomes presented here suggested that the microsatellite markers have great potential for differentiating C. plicatula from C. gigas populations. The information obtained in this study has important implications for the suitable management and conservation of these genetic resources in China.     

High microsatellite genetic variability of the stone loach, Barbatula barbatula, in anthropogenically disturbed watercourses

Abstract  Genetic variation within and among stone loach, Barbatula barbatula L., populations inhabiting anthropogenically degraded watercourses in Flanders (northern part of Belgium) was assessed using five microsatellite markers. High levels of genetic diversity were observed at all sampling sites, (MNA: 6.2–11.2; H _O: 0.64–0.75; H _E: 0.67–0.85). Estimates of the effective population size varied between 1535 and 3021 individuals and there were no indications of recent severe bottlenecks. Significant genetic differentiation was observed among sites belonging to different river systems and drainage basins. These results suggest human activities, such as pollution and river engineering, have not impacted significantly on genetic variability in the stone loach populations investigated. It is possible that this lack of genetic erosion may be attributed to species-specific characteristics such as pollution tolerance and ecological flexibility.     

Is the ballan wrasse (Labrus bergylta) two species? Genetic analysis reveals within‐species divergence associated with plain and spotted morphotype frequencies

The ballan wrasse (Labrus bergylta) is a marine fish belonging to the family Labridae characterized by 2 main morphotypes that occur in sympatry: spotty and plain. Previous studies have revealed differences in their life‐history traits, such as growth and maturation; however, the genetic relationship between forms is presently unknown. Using 20 recently developed microsatellite markers, we conducted a genetic analysis of 41 and 48 spotty and plain ballan wrasse collected in Galicia (northwest Spain). The 2 morphotypes displayed highly significant genetic differences to each other (F_ST = 0.018, P < 0.0001). A similar degree of genetic differentiation (F_ST = 0.025, P < 0.0001) was shown using the STRUCTURE clustering approach with no priors at K = 2. In this case, the frequency of spotty and plain morphotypes was significantly different (χ² = 9.46, P = 0.002). It is concluded that there is significant genetic heterogeneity within this species, which appears to be highly associated with the spotty and plain forms, but not completely explained by them. Given the previously demonstrated biological differences between morphotypes, and the present genetic analyses, we speculate about the convenience of a taxonomic re‐evaluation of this species.     

Detailed insight into genetic diversity of the Old Kladruber horse substructure in comparison to the Lipizzan breed

The aim of the present study was to analyse the genetic subdivision of the Old Kladruber horse population compared to the historically close Lipizzan breed and to estimate genetic relatedness between them. A set of 13 microsatellites was used for genotyping a total of 270 Old Kladruber horses representing grey and black colour varieties and 418 Lipizzan horses from Slovak and Slovenian studs. The proportion of obtained heterozygosity indicates no major loss of genetic diversity within them. At the individual level across analysed populations, the formation of clusters in respect to breed’s origin and particular studs was observed. The Wright’s F_ST and genetic distances indicated genetic segregation of both colour varieties at the intraspecific level of the Kladruber breed. Moreover, the membership probability outputs showed that the frequencies of alleles varied across the three main regions represented by both Old Kladruber varieties and Lipizzan, depending on breeding history and strategy of studs.     

Potato cultivar identification using simple sequence repeats markers (SSR)

Summary Identification of potato cultivars is currently based on phenotypic characters. Crop inspections are needed at different stages and the increasing number of cultivars means the process is becoming more and more complex. Molecular markers are a possible complementary tool to identify potato cultivars and to rapidly check the identity of seeds lots. In this study 286 potato cultivars produced in France were characterized by Polymerase Chain Reaction (PCR) using Simple Sequence Repeats (SSR) markers. Sequential amplifications with 4 to 5 of the chosen SSR markers enabled complete discrimination between all the cultivars. The patterns were registered in a database and the procedure is now used routinely in France.     

Population structure of Phytophthora infestans collected on potato and tomato in Italy

Late blight caused by the oomycete Phytophthora infestans is a disease of potato and tomato of worldwide relevance and is widespread throughout Europe and the Mediterranean region. While pathogen populations in northern Europe have been sampled and characterized for many years, the genetic structure of populations from southern Europe, including Italy, has been less studied. Between 2018 and 2019, we collected 91 samples of P. infestans from potato and tomato crops in Italy, Algeria, and Tunisia on FTA cards and genotyped them using 12-plex microsatellites. These samples were compared to genotypes of P. infestans previously collected within the framework of the EuroBlight network and from published sources. Four clonal lineages were identified: 13_A2 (Blue 13), 2_A1, 23_A1, and 36_A2. Two other isolates collected could not be matched to any currently known clonal lineage. The 13_A2 and 36_A2 lineages were found exclusively in southern Italy and Algeria, while 2_A1 was only found in Algeria. This is the first report of the 36_A2 lineage in Italy. Two isolates from Solanum nigrum were 13_A2, suggesting this weed host could be a reservoir of inoculum. The 23_A1 lineage was found widely on infected tomato crops in Italy and is the same as the lineage US-23 that is widespread in North America. Differences in genotypes across the country suggests that there may be different sources of introduction into Italy, possibly via infected seed tubers from other countries in Europe, tubers for consumption from North Africa, or tomatoes.     

文昌鸡群体遗传多样性的微卫星分析

利用10对来自鸡不同染色体的微卫星引物序列,对文昌鸡46个样本（雌雄各半）进行了遗传多样性检测。结果表明：该群体平均多态信息含量为0．808,平均观察杂合度为0．351,平均期望杂合度为0．841,平均等位基因14．6个,表明海南文昌鸡群体遗传变异较大,遗传多样性较为丰富。研究结果为文昌鸡品种资源合理保护与开发利用提供了科学依据。