The evolving technology of DNA fingerprinting and its application to fisheries and aquaculture

In 1985, Alec Jeffreys reported the development of multilocus DNA fingerprinting by Southern blot-detection of hypervariable minisatellites or variable number of tandem repeat (VNTR) loci. This technology found immediate application to various forensic and scientific problems, including fisheries and aquaculture. By 1989, however, it was recognized by many researchers that inherent problems exist in the application of multilocus fingerprinting to large sample sizes as might occur in fisheries and aquaculture genetic studies. As such, individual VNTRs were cloned for single-locus DNA fingerprinting. Although single-locus fingerprinting ameliorates many of the problems associated with multilocus DNA fingerprinting, it suffers from the problem that electrophorectic anomalies of band migration within and between gels necessitates binning of alleles, thus underestimating genetic variability in a given population. Amplification of microsatellite loci by the polymerase chain reaction, however, solved many of the problems of Southern blot-based DNA fingerprinting. Moreover, microsatellites exhibit attributes that make them particularly suitable as genetic markers for numerous applications in aquaculture and fisheries research: (1) they are abundant in the genome; (2) they display varying levels of polymorphism; (3) alleles exhibit codominant Mendelian inheritance; (4) minute amounts of tissue are required for assay (e.g., dried scales or otoliths); (5) loci are conserved in related species; (6) potential for automated assay. Recent innovations in DNA fingerprinting technology developed over the past 5 years are discussed with special emphasis on microsatellites and their application to fisheries and aquaculture, e.g., behavioural and population genetics of wild species, and selection and breeding programmes for aquaculture broodstock.     

微卫星DNA在进化中的研究进展

在过去的十年中,微卫星已经变成最流行的基因标记之一。尽管微卫星分析已有广泛的应用,然而关于微卫星DNA变异动力学的完整的画面才刚刚浮现。文章将从微卫星的起源、微卫星进化模式的推断、DNA复制的滑动是微卫星DNA变异的主要机制、影响微卫星突变率的因素、微卫星的长度分布和微卫星的频率分布等方面综述有关微卫星进化动力学方面的研究进展。     

On the Structural Differences Between Markers and Genomic AC Microsatellites

AC microsatellites have proved particularly useful as genetic markers. For some purposes, such as in population biology, the inferences drawn depend on the quantitative values of their mutation rates. This, together with intrinsic biological interest, has led to widespread study of microsatellite mutational mechanisms. Now, however, inconsistencies are appearing in the results of marker-based versus non-marker-based studies of mutational mechanisms. The reasons for this have not been investigated, but one possibility, pursued here, is that the differences result from structural differences between markers and genomic microsatellites. Here we report a comparison between the CEPH AC marker microsatellites and the global population of AC microsatellites in the human genome. AC marker microsatellites are longer than the global average. Controlling for length, marker microsatellites contain on average fewer interruptions, and have longer segments, than their genomic counterparts. Related to this, marker microsatellites show a greater tendency to concentrate the majority of their repeats into one segment. These differences plausibly result from scientists selecting markers for their high polymorphism. In addition to the structural differences, there are differences in the base composition of flanking sequences, marker flanking regions being richer in C and G and poorer in A and T. Our results indicate that there are profound differences between marker and genomic microsatellites that almost certainly affect their mutation rates. There is a need for a unified model of mutational mechanisms that accounts for both marker-derived and genomic observations. A suggestion is made as to how this might be done.Reviewing Editor: Dr. Magnto Nordborg     

Mutational Dynamics of Microsatellites

Microsatellites are a ubiquitous class of simple repetitive DNA sequences, which are widespread in both eukaryotic and prokaryotic genomes. The use of microsatellites as polymorphic DNA markers has considerably increased both in the number of studies and in the number of organisms, primarily for genetic mapping, studying genomic instability in cancer, population genetics, forensics, conservation biology, molecular anthropology and in the studies of human evolutionary history. Although simple sequence repeats have been extensively used in studies encompassing varied areas of genetics, the mutation dynamics of these genome regions is still not well understood. The present review focuses on the mutational dynamics of microsatellite DNA with special reference to mutational mechanisms and their role in microsatellite evolution.     

微卫星多态性检测技术及其在保护遗传学中的应用

微卫星是一种新型的分子遗传标记,具有只需极少量样品即可进行PCR扩增等优点,使之极其适合于保护遗传化学研究。阐释了微卫星的结构、微卫星多态检测技术的原理,简要论述了微卫生多态检测技术的优点及其应用,并介绍了一种分离动物微卫星位点的富集方法。     

Targeted development of a microsatellite marker associated with a true loose smut resistance gene in barley (Hordeum vulgare L.)

Microsatellite markers have many of the properties of an ideal marker, but development of microsatellite markers is tedious, time-consuming and expensive. In the past few years, great efforts have been made to develop, map and utilize microsatellite markers in various crops. It is still a major challenge to find a microsatellite marker associated with an economically important trait. In the present study we report on the targeted development of a microsatellite marker to a barley disease resistance gene. The method includes the following steps: (1) pooling DNA samples from a segregating population based on the principle of bulked-segregant analysis; (2) digesting the pooled DNAs and ligating adaptors; (3) selectively amplifying and identifying polymorphic microsatellites; and (4) developing primers for the microsatellite associated with the targeted trait. Using this method, a microsatellite marker associated with the true loose smut resistance gene (Un8) in the Harrington × TR306 doubled-haploid population was identified. This marker showed polymorphism in four breeding populations segregating for true loose smut resistance. In three of these populations, genetic distance between the microsatellite and the true loose smut resistance gene varied from 8.6 to 10.3 cM. Polymorphism of the microsatellite was tested among three disease resistant lines and 21 susceptible cultivars. Fourteen to eighteen of the 21 susceptible cultivars exhibited a polymorphism for the microsatellite with respect to at least one of the disease-resistant lines. This method for the targeted development of microsatellite markers should have widespread applicability and should efficiently provide highly polymorphic markers for use in breeding programs.     

鲸类保护遗传学研究进展

简要介绍了mtDNA的PCR直接测序、微卫星microsatellite DNA分型、组织相容性复合体（major histocompatibility complex,MHC)分析等DNA分子标记技术在鲸类遗传变异、种群结构、进化历史、个体识别、亲缘鉴定及系统分类等保护遗传学领域的应用。     

Comparison of microsatellite and single nucleotide polymorphism markers for the genetic analysis of a Galloway cattle population

Highly informative genetic markers are essential for efficient management of cattle populations, as well as for food safety. After a decade of domination by microsatellite markers, a new type of genetic marker, single nucleotide polymorphism (SNP), has recently appeared on the scene. In the present study, the exclusion power of both kinds of markers with regards to individual identification and parental analysis was directly compared in a Galloway cattle population. Seventeen bovine microsatellites were distributed in three incremental marker sets (10, 14 and 17 microsatellite markers) and used for cattle genotyping. A set of 43 bovine SNP was used for genotyping the same cattle population. The accuracy of both kinds of markers in individual identification was evaluated using probability of identity estimations. These were 2.4 x 10(-8) for the 10 microsatellite set, 2.3 x 10(-11) for the 14 microsatellite set, and 1.4 x 10(-13) for the 17 microsatellite marker set. For the 43 SNP markers, the estimated probability of identity was 5.3 x 10(-11). The exclusion power of both kinds of markers in parental analysis was evaluated using paternity exclusion estimations, and, in addition to this, by estimation of the parental exclusion probability in 18 Galloway family trios. Paternity exclusion was estimated to be over 99% for microsatellites, and approx. 98% for SNP. Both, microsatellite and SNP sets of markers showed similar parental exclusion probabilities.     

Microsatellite DNA in fishes

For the last 30 years, attempts have been made to discriminate among fish populations by using molecular markers. Although some techniques have proved successful in certain circumstances, the consistent trend to newer markers among fishery geneticists highlights the general lack of resolving power observed with older technologies. The last decade has seen the increasing use of satellite DNA in investigations of genetic variability and divergence. Applications to fish and fisheries-related issues initially concentrated on minisatellite single-locus probes. Although minisatellites have successfully addressed a number of fishery-related questions, this class of satellite DNA has not been widely adopted by fishery geneticists. Most of the current research effort is concentrated on another class of satellite DNA called microsatellites. The large interest in microsatellite loci is largely due to the very high levels of variability that have been observed and the ability to investigate this variation using PCR technology. The isolation and application of microsatellites to research fields as diverse as population genetics, parentage analyses and genome mapping are reviewed. Despite the undisputed advantages that the marker possesses, there are a number of potential problems associated with investigating variation at microsatellite loci. Statistical considerations (e.g. appropriate sample sizes, number of loci and the mutation model assumptions on which the estimate is based) have not been considered in detail yet and the problems are often exacerbated in fish species, as some species show very large numbers of alleles at microsatellite loci. These issues and others, e.g. null alleles, are reviewed and possible solutions are proposed     

Integration of trinucleotide microsatellites into a linkage map of Citrus

 We report the successful assignment of the first seven microsatellite markers to the Citrus RFLP and isozyme map. A total of 14 microsatellite primer pairs were developed and tested for amplification and product-length polymorphism within a population of plants previously used for linkage-map construction. In each case, the successfully assigned microsatellite mapped to the termini of a different linkage group indicating a widespread distribution throughout the genome. Analysis of allele segregation revealed that two of nine microsatellites displayed a significant deviation from expected ratios (P>0.5). This was compared with other marker types within Citrus and a similar proportion of skewed loci was also found to be present. The analysis of two markers was complicated by the non-amplification of an inherited null allele within the mapping population. The successful integration of microsatellites into the genetic map of Citrus demonstrates the utility of this marker type for genetic analysis within wide intergeneric plant crosses. Received: 16 September 1996 / Accepted: 18 October 1996     

微卫星DNA与生化标记分析对长爪沙鼠群体遗传分析的比较

目的比较生化标记和微卫星DNA标记方法对长爪沙鼠群体遗传分析的可靠性。方法应用27个生化位点和13个微卫星DNA位点,采用已建立的生化标记和微卫星DNA标记分析方法对国内2个长爪沙鼠群体进行遗传分析,计算并比较两种方法测得的各群体遗传参数。结果生化基因位点中有13个位点在整体中呈现遗传多态性,多态率为48.1%;微卫星位点中有11个位点在整体中表现出多态性,多态率均为84.6%。两种方法测得的平均有效等位基因数趋于一致,微卫星DNA的多态位点百分率和平均杂合度均明显高于生化标记方法。但生化标记和微卫星DNA检测对两个长爪沙鼠群体的遗传多样性差异反映一致,所反映的群体平衡状况也基本一致。结论生化标记分析和微卫星DNA方法均可较好地反映长爪沙鼠群体遗传结构。     

Mutation and evolution of microsatellite loci in Neurospora

The patterns of mutation and evolution at 13 microsatellite loci were studied in the filamentous fungal genus Neurospora. First, a detailed investigation was performed on five microsatellite loci by sequencing each microsatellite, together with its nonrepetitive flanking regions, from a set of 147 individuals from eight species of Neurospora. To elucidate the genealogical relationships among microsatellite alleles, repeat number was mapped onto trees constructed from flanking-sequence data. This approach allowed the potentially convergent microsatellite mutations to be placed in the evolutionary context of the less rapidly evolving flanking regions, revealing the complexities of the mutational processes that have generated the allelic diversity conventionally assessed in population genetic studies. In addition to changes in repeat number, frequent substitution mutations within the microsatellites were detected, as were substitutions and insertion/deletions within the flanking regions. By comparing microsatellite and flanking-sequence divergence, clear evidence of interspecific allele length homoplasy and microsatellite mutational saturation was observed, suggesting that these loci are not appropriate for inferring phylogenetic relationships among species. In contrast, little evidence of intraspecific mutational saturation was observed, confirming the utility of these loci for population-level analyses. Frequency distributions of alleles within species were generally consistent with the stepwise mutational model. By comparing variation within species at the microsatellites and the flanking-sequence, estimated microsatellite mutation rates were approximately 2500 times greater than mutation rates of flanking DNA and were consistent with estimates from yeast and fruit flies. A positive relationship between repeat number and variance in repeat number was significant across three genealogical depths, suggesting that longer microsatellite alleles are more mutable than shorter alleles. To test if the observed patterns of microsatellite variation and mutation could be generalized, an additional eight microsatellite loci were characterized and sequenced from a subset of the same Neurospora individuals.     

Relative information content of polymorphic microsatellites and mitochondrial DNA for inferring dispersal and population genetic structure in the olive sea snake, Aipysurus laevis

Polymorphic microsatellites are widely considered more powerful for resolving population structure than mitochondrial DNA (mtDNA) markers, particularly for recently diverged lineages or geographically proximate populations. Weaker population subdivision for biparentally inherited nuclear markers than maternally inherited mtDNA may signal male-biased dispersal but can also be attributed to marker-specific evolutionary characteristics and sampling properties. We discriminated between these competing explanations with a population genetic study on olive sea snakes, Aipysurus laevis. A previous mtDNA study revealed strong regional population structure for A. laevis around northern Australia, where Pleistocene sea-level fluctuations have influenced the genetic signatures of shallow-water marine species. Divergences among phylogroups dated to the Late Pleistocene, suggesting recent range expansions by previously isolated matrilines. Fine-scale population structure within regions was, however, poorly resolved for mtDNA. In order to improve estimates of fine-scale genetic divergence and to compare population structure between nuclear and mtDNA, 354 olive sea snakes (previously sequenced for mtDNA) were genotyped for five microsatellite loci. F statistics and Bayesian multilocus genotype clustering analyses found similar regional population structure as mtDNA and, after standardizing microsatellite F statistics for high heterozygosities, regional divergence estimates were quantitatively congruent between marker classes. Over small spatial scales, however, microsatellites recovered almost no genetic structure and standardized F statistics were orders of magnitude smaller than for mtDNA. Three tests for male-biased dispersal were not significant, suggesting that recent demographic expansions to the typically large population sizes of A. laevis have prevented microsatellites from reaching mutation-drift equilibrium and local populations may still be diverging.     

Microsatellite DNA library for Caiman latirostris

New genetic markers were characterized for the broad-snouted caiman (Caiman latirostris) by constructing libraries enriched for microsatellite DNA. Construction and characterization of these libraries are described in the present study. One microsatellite marker was developed from a (ACC-TGG)(n)enriched microsatellite DNA library, and 12 microsatellite markers were developed from a (AC-TG)(n)enriched microsatellite DNA library. These markers were tested in wild-caught animals, and these tests resulted in ten new polymorphic microsatellites for C. latirostris.     

In silico whole-genome EST analysis reveals 2322 novel microsatellites for the silver-lipped pearl oyster, Pinctada maxima

Molecular stock improvement techniques such as marker assisted selection have great potential in accelerating selective breeding programmes for animal production industries. However, the discovery and application of trait/marker associations usually requires a large number of genome-wide polymorphic loci. Here, we present 2322 unique microsatellites for the silver-lipped pearl oyster, Pinctada maxima, a species of aquaculture importance throughout the Indo-Australian Archipelago for production of the highly valued South Sea pearl. More than 1.2 million Roche 454 expressed sequence tag (EST) reads were screened for microsatellite repeat motifs. A total of 12,604 sequences contained either a di, tri, tetra, penta or hexa microsatellite repeat motif (n ≥ 6), with 6435 of these sequences having sufficient flanking regions for primer development. All identified microsatellites with designed primers were condensed into 2322 unique clusters (i.e., unique loci) of which 360 were shown to be polymorphic based on multiple sequence reads with different repeat motifs. Genotyping of five microsatellite loci demonstrated that in silico evaluation of polymorphism levels was a very useful method for identification of polymorphic loci, with the variation uncovered being a lower bound. Gene Ontology annotations of sequences containing microsatellites suggest that most are derived from a diverse array of unique genes. This EST derived microsatellite database will be a valuable resource for future studies in genetic map construction, diversity analysis, quantitative trait loci analysis, association mapping and marker assisted selection, not only for P. maxima, but also closely related species within the genus Pinctada.     

Heterologous nuclear and chloroplast microsatellite amplification and variation in tea, Camellia sinensis.

The advantage of the cross transferability of heterologous chloroplast and nuclear microsatellite primers was taken to detect polymorphism among 24 tea (Camellia sinensis (L.) O. Kuntze) genotypes, including both the assamica and the sinensis varieties. Primer information was obtained from the closely related Camellia japonica species for four nuclear microsatellites, and from Nicotiana tabaccum for seven universal chloroplast microsatellites. All of the nuclear microsatellite loci tested generated an expected DNA fragment in tea, revealing between three and five alleles per locus. Four out of the seven chloroplast microsatellites primers amplified positively, and of these only one was polymorphic with three alleles, which is in agreement with the conserved nature of chloroplast microsatellites at the intraspecific level. A factorial correspondence analysis carried out on all genotypes and nuclear microsatellite alleles separated the assamica and sinensis genotypes into two groups, thus demonstrating the value of these markers in establishing the genetic relationship between tea varieties. Genetic diversity measured with nuclear microsatellites was higher than that measured with other types of molecular markers, offering prospects for their use in fingerprinting, mapping, and population genetic studies, whereas polymorphisms detected at a cpSSR locus will allow the determination of plastid inheritance in the species.     

Characterization and comparison of microsatellites derived from repeat-enriched libraries and expressed sequence tags

The construction of high-density linkage maps for use in identifying loci underlying important traits requires the development of large numbers of polymorphic genetic markers spanning the entire genome at regularly spaced intervals. As part of our efforts to develop markers for rainbow trout (Oncorhynchus mykiss), we performed a comparison of allelic variation between microsatellite markers developed from expressed sequence tag (EST) data and anonymous markers identified from repeat-enriched libraries constructed from genomic DNA. A subset of 70 markers (37 from EST databases and 33 from repeat enriched libraries) was characterized with respect to polymorphism information content (PIC), number of alleles, repeat number, locus duplication within the genome and ability to amplify in other salmonid species. Higher PIC was detected in dinucleotide microsatellites derived from ESTs than anonymous markers (72.7% vs. 54.0%). In contrast, dinucleotide repeat numbers were higher for anonymous microsatellites than for EST derived microsatellites (27.4 vs.18.1). A higher rate of cross-species amplification was observed for EST microsatellites. Approximately half of each marker type was duplicated within the genome. Unlike single-copy markers, amplification of duplicated microsatellites in other salmonids was not correlated to phylogenetic distance. Genomic microsatellites proved more useful than EST derived microsatellites in discriminating among the salmonids. In total, 428 microsatellite markers were developed in this study for mapping and population genetic studies in rainbow trout.     

Genetic mapping of a new set of microsatellite markers in a reference common bean (Phaseolus vulgaris) population BAT93 x Jalo EEP558

The present study describes a new set of 61 polymorphic microsatellite markers for beans and the construction of a genetic map using the BAT93 x Jalo EEP558 (BJ) population for the purpose of developing a reference linkage map for common bean (Phaseolus vulgaris). The main objectives were to integrate new microsatellites on the existing framework map of the BJ population, and to develop the first linkage map for the BJ population based exclusively on microsatellites. Of the total of 264 microsatellites evaluated for polymorphism, 42.8% showed polymorphism between the genitors. An integrated map was created totaling 199 mapped markers in 13 linkage groups, with an observed length of 1358 cM and a mean distance between markers of 7.23 cM. For the map constructed exclusively with microsatellites, 106 markers were placed in 12 groups with a total length of 606.8 cM and average distance of 6.8 cM. Linkage group designation and marker order for BM microsatellites generally agreed with previous mapping, while the new microsatellites were well distributed across the genome, corroborating the utility of the BJ population for a reference map. The extensive use of the microsatellites and the availability of a reference map can help in the development of other genetic maps for common bean through the transfer of information of marker order and linkage, which will allow comparative analysis and map integration, especially for future quantitative trait loci and association mapping studies.     

Genetic diversity of loquat germplasm (Eriobotrya japonica (Thunb) Lindl) assessed by SSR markers.

Genetic relationships among 40 loquat (Eriobotrya japonica (Thunb) Lindl) accessions that originated from different countries and that are part of the germplasm collection of the Instituto Valenciano de Investigaciones Agrarias (IVIA) (Valencia, Spain) were evaluated using microsatellites. Thirty primer pairs flanking microsatellites previously identified in Malus x domestica (Borkh.) were assayed. Thirteen of them amplified polymorphic products and unambiguously distinguished 34 genotypes from the 40 accessions analyzed. Six accessions showing identical marker patterns were Spanish local varieties thought to have been derived from 'Algerie' by a mutational process very common in loquat species. A total of 39 alleles were detected in the population studied, with a mean value of 2.4 alleles per locus. The expected and observed heterozygosities were 0.46 and 51% on average, respectively, leading to a negative value of the Wright's fixation index (-0.20). The values of these parameters indicate a smaller degree of genetic diversity in the set of loquat accessions analyzed than in other members of the Rosaceae family. Unweighted pair-group method (UPGMA) cluster analysis, based on Nei's genetic distance, generally grouped genotypes according to their geographic origins and pedigrees. The high number of alleles and the high expected heterozygosity detected with SSR markers developed in Malus x domestica (Borkh.) make them a suitable tool for loquat cultivar identification, confirming microsatellite marker transportability among genera in the Rosaceae family.