Isolation and characterization of nine microsatellite markers from Coffea arabica L., showing wide cross‐species amplifications

Genetic improvement of coffee (Coffea arabica L.) is constrained by low genetic diversity and lack of genetic markers, suitable screening tools, information on the genetic make‐up of available gene pool and long generation time. In this context, use of DNA markers such as microsatellites that provide high genetic‐resolution becomes highly desirable. Here, we report the development of nine new microsatellite markers from partial genomic library of an elite variety of Coffea arabica. The developed microsatellites revealed robust cross‐species amplifications in 17 related species of coffee, and their Polymorphic Information Content varied from 0 to 0.6, 0 to 0.78 and 0.67 to 0.90 for the arabica, robusta genotypes and species representatives, respectively. The data thus suggest their potential use as genetic markers for assessment of germplasm diversity and linkage analysis of coffee.     

微卫星标记及其在蚜虫种群生物学研究中的应用

微卫星（SSR）是以1～6个碱基为重复单位组成的简单串联重复序列,具有丰度高、多态性高、共显性遗传、选择中性和可重复性好等优点。微卫星标记技术可分为三种类型：基于杂交的SSR指纹,基于PCR的SSR指纹和单位点SSR的PCR扩增。前两者不需要知道基因组序列信息,属于多位点标记;后者则需要前期工作,属于单位点标记,即通常所指的微卫星标记。到目前为止,共在14种蚜中筛选到141个微卫星位点,并发表了相关的引物序列,这给今后的相关研究提供了丰富的共享资源。业已证明微卫星引物在邻近种之间有一定的通用性。微卫星标记严格遵守孟德尔遗传规律,已经被用来推断某些蚜虫有性生殖的情况。同种蚜虫中可能同时存在有性系和无性系,微卫星研究证明有性系比无性系有更高的遗传多样性,无性系通常表现出杂合过剩和连锁不平衡。蚜虫的迁飞规律适合用微卫星标记加以研究,已有的研究显示在具有高度迁飞特性的种类中,地理种群间的遗传分化程度低,基因频率的相似性高。存在广泛分布的相同的多位点基因型是迁飞性蚜虫的另一个重要特征。在我国,微卫星应用于蚜虫生物学研究的工作还较少,鉴于该种标记的优良特性和巨大的潜力,本文建议今后相关的研究应该首先考虑微卫星标记。     

Effect of microsatellite selection on individual and population genetic inferences: an empirical study using cross‐specific and species‐specific amplifications

Although whole‐genome sequencing is becoming more accessible and feasible for nonmodel organisms, microsatellites have remained the markers of choice for various population and conservation genetic studies. However, the criteria for choosing microsatellites are still controversial due to ascertainment bias that may be introduced into the genetic inference. An empirical study of red deer (Cervus elaphus) populations, in which cross‐specific and species‐specific microsatellites developed through pyrosequencing of enriched libraries, was performed for this study. Two different strategies were used to select the species‐specific panels: randomly vs. highly polymorphic markers. The results suggest that reliable and accurate estimations of genetic diversity can be obtained using random microsatellites distributed throughout the genome. In addition, the results reinforce previous evidence that selecting the most polymorphic markers leads to an ascertainment bias in estimates of genetic diversity, when compared with randomly selected microsatellites. Analyses of population differentiation and clustering seem less influenced by the approach of microsatellite selection, whereas assigning individuals to populations might be affected by a random selection of a small number of microsatellites. Individual multilocus heterozygosity measures produced various discordant results, which in turn had impacts on the heterozygosity‐fitness correlation test. Finally, we argue that picking the appropriate microsatellite set should primarily take into account the ecological and evolutionary questions studied. Selecting the most polymorphic markers will generally overestimate genetic diversity parameters, leading to misinterpretations of the real genetic diversity, which is particularly important in managed and threatened populations.     

High gene flow on a continental scale in the polyandrous Kentish plover Charadrius alexandrinus

Gene flow promotes genetic homogeneity of species in time and space. Gene flow can be modulated by sex‐biased dispersal that links population genetics to mating systems. We investigated the phylogeography of the widely distributed Kentish plover Charadrius alexandrinus. This small shorebird has a large breeding range spanning from Western Europe to Japan and exhibits an unusually flexible mating system with high female breeding dispersal. We analysed genetic structure and gene flow using a 427‐bp fragment of the mitochondrial (mtDNA) control region, 21 autosomal microsatellite markers and a Z microsatellite marker in 397 unrelated individuals from 21 locations. We found no structure or isolation‐by‐distance over the continental range. However, island populations had low genetic diversity and were moderately differentiated from mainland locations. Genetic differentiation based on autosomal markers was positively correlated with distance between mainland and each island. Comparisons of uniparentally and biparentally inherited markers were consistent with female‐biased gene flow. Maternally inherited mtDNA was less structured, whereas the Z‐chromosomal marker was more structured than autosomal microsatellites. Adult males were more related than females within genetic clusters. Taken together, our results suggest a prominent role for polyandrous females in maintaining genetic homogeneity across large geographic distances.     

Isolation and characterization of 15 microsatellite loci in the Korean goral (Nemorhaedus caudatus)

Microsatellites have been applied in a variety of fields, including conservation genetics. Species‐specific microsatellites are considered as more powerful genetic markers to generate an accurate genetic composition of a species itself. Accordingly, we characterized 15 polymorphic microsatellite loci from Korean goral, Nemorhaedus caudatus, one of the most endangered species in South Korea. The new markers should benefit future studies of the endangered species of other Asian gorals and their relatives for the study of genetic diversity and potential conservation management.     

Mining online genomic resources in Anolis carolinensis facilitates rapid and inexpensive development of cross-species microsatellite markers for the Anolis lizard genus

Online sequence databases can provide valuable resources for the development of cross-species genetic markers. In particular, mining expressed tag sequences (EST) for microsatellites and developing conserved cross-species microsatellite markers can provide a rapid and relatively inexpensive method to develop new markers for a range of species. Here, we adopt this approach to develop cross-species microsatellite markers in Anolis lizards, which is a model genus in evolutionary biology and ecology. Using EST sequences from Anolis carolinensis, we identified 127 microsatellites that satisfied our criteria, and tested 49 of these in five species of Anolis (carolinensis, distichus, apletophallus, porcatus and sagrei). We identified between 8 and 25 new variable genetic markers for five Anolis species. These markers will be a valuable resource for studies of population genetics, comparative mapping, mating systems, behavioural ecology and adaptive radiations in this diverse lineage.     

High degree of transferability of 86 newly developed zebra finch EST-linked microsatellite markers in 8 bird species

High-resolution analysis for population genetic and functional studies requires the use of large numbers of polymorphic markers. The recent increase of available genetic tools is facilitated by the use of publicly available expressed sequence tag (EST) sequence databases that are a valuable resource for identifying gene-linked markers. In the present study, we applied bioinformatics analyses to identify microsatellite markers present in EST sequences from a zebra finch (Taeniopgia guttata) EST database and we explore the success of cross-species amplification of EST-linked microsatellite markers in 7 passerine and 1 nonpasserine species. Eighty-six zebra finch EST-linked microsatellite loci were screened for polymorphism revealing a high amplification success rate and adequate levels of polymorphism (33.3-51%) for relatively closely related species, whereas success decreased in the most distantly related species to zebra finch. EST-linked microsatellites appear to be more highly transferable between taxa than anonymous microsatellites as they revealed higher amplification and polymorphism success between different families indicating that they will be a useful source of gene-linked polymorphic markers in a broad range of avian species.     

Genome‐wide microsatellite characterisation and marker development in Chenopodium quinoa

Microsatellites, as the tracts of repetitive DNA, are an essential constituent of the plant genome that holds important evolutionary significance, and have been extensively used to develop molecular makers for genetic analysis. To understand the microsatellite dynamics of quinoa genome and its relatives, in this study we performed a genome‐wide analysis of microsatellites in five Amaranthaceae species using available genome sequences. The results demonstrated that the microsatellites of the five Amaranthaceae species were characterised by relatively high proportions of mono‐, di‐ and trinucleotide repeats with A/T rich motifs, implying conservative organisation and composition of microsatellites in this family. Furthermore, a significant negative correlation between microsatellite frequencies and GC contents (r = ?.87) were observed. In total, 533,961 (89.57%) and 542,601 (89.86%) microsatellite loci could be used to develop simple sequence repeat (SSR) molecular markers, of which 7,178 were found to be polymorphic between the two sequenced quinoa cultivars, QQ74 and Real Blanca, through in silico PCR analysis. Finally, 15 SSR markers were randomly selected to validate their polymorphism across 12 quinoa accessions by wet‐lab PCR amplification. The newly developed genome‐wide SSR markers provide a useful resource for population genetics, gene mapping and molecular breeding studies in quinoa and beyond.     

Development of microsatellite markers in potato and their transferability in some members of Solanaceae

We have developed thirty new microsatellite markers in potato by screening genomic libraries and ESTs. Genomic libraries of potato cultivar Kufri Bahar were screened for sequences containing microsatellite motifs GA, GT, ACA, ATC, GAA, TAA and GATA. Using flanking sequences, PCR primers were designed for microsatellites identified from genomic libraries and ESTs. Sixteen new primer pairs from genomic libraries and fourteen from ESTs along with seven previously published primer pairs amplified PCR products in the selected genotypes comprising of 65 Solanum tuberosum lines and 14 other species of the potato gene pool. Neighbor-joining tree based on genetic distance matrix developed using microsatellite markers successfully distinguished all these genotypes in the expected size range. Seventeen microsatellites could also be cross-amplified in at least one of the five members of solanaceae, namely tomato, eggplant, pepper, petunia and tobacco. The new microsatellite markers obtained in this study will be useful in various genetic and taxonomic studies in potato and related genomes.     

Genetic structure of Araucaria angustifolia (Araucariaceae) populations in Brazil: implications for the in situ conservation of genetic resources

The distribution of the genetic variation within and among natural populations of A. ANGUSTIFOLIA growing in different regions in Brazil was assessed at microsatellite and AFLP markers. Both markers revealed high gene diversity ( H = 0.65; AR = 9.1 for microsatellites and H = 0.27; P = 77.8 % for AFLPs), moderate overall differentiation ( RST = 0.13 for microsatellites and FST = 0.10 for AFLPs), but high divergence of the northernmost, geographically isolated population. In a Bayesian analysis, microsatellite data suggested population structure at two levels: at K = 2 and at K = 3 in agreement to the geographical distribution of populations. This result was confirmed by the UPGMA dendrogram based on microsatellite data (bootstrap support > 95 %). Non-hierarchical AMOVA revealed high variation among populations from different A POSTERIORI defined geographical groups. The genetic distance between sample locations increased with geographical distance for microsatellites ( R = 0.62; P = 0.003) and AFLPs ( R = 0.32; P = 0.09). This pattern of population differentiation may be correlated with population history such as geographical isolation and postglacial colonization of highlands. Implications of the population genetic structure for the conservation of genetic resources are discussed.     

Microsatellite marker identification using genome screening and restriction-ligation

We have identified a fast and easy method for finding microsatellite markers that utilizes genome screening with inter-simple sequence repeat (ISSR) primers to detect microsatellite regions and to obtain sequence information flanking one side of the microsatellites and a restriction-ligation technique with a specific adaptor to allow sequence walking to obtain sequence information flanking the other side of the microsatellites. Two main alternatives of the method (with or without cloning) are presented. We successfully utilized the method when identifying microsatellite markers for 21 bryophyte species, three algal species, and for the raccoon dog. The proportion of polymorphic markers equaled 95%. We observed that microsatellites are commonly found within the sequenced ISSR amplification products (54% in the present study), in which case specific primers can be identified for the microsatellite without a further restriction-ligation step. It is evident that the DNA regions amplified by ISSR markers commonly represent microsatellite hotspots. We propose that the identified method and the knowledge of the common presence of additional microsatellite repeats within ISSR amplification products are especially attractive to researchers who conduct small-scale microsatellite identification, such as researchers in population genetics and conservation biology.     

A standard panel of microsatellites for Asian seabass (Lates calcarifer)

Microsatellites are the most popular markers for parentage assignment and population genetic studies. To meet the demand for international comparability for genetic studies of Asian seabass, a standard panel of 28 microsatellites has been selected and characterized using the DNA of 24 individuals from Thailand, Malaysia, Indonesia and Australia. The average allele number of these markers was 10.82 ± 0.71 (range: 6–19), and the expected heterozygosity averaged 0.76 ± 0.02 (range: 0.63–1.00). All microsatellites showed Mendelian inheritance. In addition, eight standard size controls have been developed by cloning a set of microsatellite alleles into a pGEM‐T vector to calibrate allele sizes determined by different laboratories, and are available upon request. Seven multiplex PCRs, each amplifying 3–5 markers, were optimized to accurately and rapidly genotype microsatellites. Parentage assignment using 10 microsatellites in two crosses (10 × 10 and 20 × 20) demonstrated a high power of these markers for revealing parent‐sibling connections. This standard set of microsatellites will standardize genetic diversity studies of Asian seabass, and the multiplex PCR sets will facilitate parentage assignment.     

MICROSATELLITE GENOTYPING OF SINGLE CELLS OF THE DINOFLAGELLATE SPECIES LINGULODINIUM POLYEDRUM (DINOPHYCEAE): A NOVEL APPROACH FOR MARINE MICROBIAL POPULATION GENETIC STUDIES1

In recent years, two new approaches have been introduced in genetic studies of phytoplankton species. One is the application of highly polymorphic microsatellite markers, which allow detailed population genetic studies; the other is the development of methods that enable the direct genetic characterization of single cells as an alternative to clonal cultures. The aim of this study was to combine these two approaches in a method that would allow microsatellite genotyping of single phytoplankton cells, providing a novel tool for high‐resolution population genetic studies. The dinoflagellate species Lingulodinium polyedrum (F. Stein) J. D. Dodge was selected as a model organism to develop this novel approach. The method we describe here is based on several key developments: (i) a simple and efficient DNA extraction method for single cells, (ii) the characterization of microsatellite markers for L. polyedrum, (iii) a protocol for the species identification of single cells through the analysis of partial rRNA gene sequences, and (iv) a two‐step multiplex PCR protocol for the simultaneous amplification of microsatellite markers and partial rRNA gene sequences from single cells. Our protocol allowed the amplification of up to six microsatellite loci together with either the complete ITS1‐5.8S‐ITS2 region or a partial 18S region of the ribosomal gene of L. polyedrum from single motile cells and resting cysts. This article describes and evaluates the developed approach and discusses its significance for population genetic studies of L. polyedrum and other phytoplankton species.     

Seventy‐five EST‐linked Atlantic salmon (Salmo salar L.) microsatellite markers and their cross‐amplification in five salmonid species

An Atlantic salmon (Salmo salar L.) expressed sequence tag (EST) database consisting of 58 146 ESTs was screened for microsatellite sequences. Subsequent development of 75 polymorphic EST‐associated microsatellite markers in this species is described together with cross‐species amplification results of 133 gene‐associated tandem repeat markers in five salmonid species (Salmo trutta, Oncorhynchus mykiss, Salvelinus aplinus, Thymallus thymallus, Coregonus lavaretus). The number of alleles among EST‐linked microsatellites in Atlantic salmon ranged from two to 41 with an average of 12 alleles per locus. Cross‐species amplification resulted in detection of a total of 111 polymorphic locus‐species combinations (12–32 loci per species).     

Detecting population structure in a high gene-flow species,Atlantic herring (Clupea harengus): direct,simultaneous evaluation of neutral vs putatively selected loci

In many marine fish species, genetic population structure is typically weak because populations are large, evolutionarily young and have a high potential for gene flow. We tested whether genetic markers influenced by natural selection are more efficient than the presumed neutral genetic markers to detect population structure in Atlantic herring (Clupea harengus), a migratory pelagic species with large effective population sizes. We compared the spatial and temporal patterns of divergence and statistical power of three traditional genetic marker types, microsatellites, allozymes and mitochondrial DNA, with one microsatellite locus, Cpa112, previously shown to be influenced by divergent selection associated with salinity, and one locus located in the major histocompatibility complex class IIA (MHC-IIA) gene, using the same individuals across analyses. Samples were collected in 2002 and 2003 at two locations in the North Sea, one location in the Skagerrak and one location in the low-saline Baltic Sea. Levels of divergence for putatively neutral markers were generally low, with the exception of single outlier locus/sample combinations; microsatellites were the most statistically powerful markers under neutral expectations. We found no evidence of selection acting on the MHC locus. Cpa112, however, was highly divergent in the Baltic samples. Simulations addressing the statistical power for detecting population divergence showed that when using Cpa112 alone, compared with using eight presumed neutral microsatellite loci, sample sizes could be reduced by up to a tenth while still retaining high statistical power. Our results show that the loci influenced by selection can serve as powerful markers for detecting population structure in high gene-flow marine fish species.     

Microsatellite characterization of Cimarron Uruguayo dogs

Various genetic markers, including microsatellites, have been used to analyze the genetic polymorphism and heterozygosity in canine breeds. In this work, we used nine microsatellite markers to investigate the genetic variability in Cimarron Uruguayo dogs, the only officially recognized native canine breed in Uruguay. DNA from 30 Cimarron Uruguayo dogs from northeastern and southern Uruguay was analyzed. The allelic frequencies for each microsatellite, the genetic variability and the consanguinity were calculated, as were the polymorphic information content (PIC) and the probability of exclusion (PE). All of the microsatellites studied were polymorphic. FH 2361, FH 2305 and PEZ 03 were the most informative, with PIC values > 0.7, in agreement with results for other canine breeds. The PE values for the markers were within the ranges previously described and were generally greater for microsatellites with higher PIC values. The heterozygosity value (0.649) was considered high since only nine microsatellites were analyzed. Compared with data for other breeds, the results obtained here indicate that Cimarron Uruguayo dogs have high genetic diversity.     

Characterization of novel microsatellites from Drosophila transversa

We investigated a partial genomic library of Drosophila transversa for microsatellites and developed 12 markers for genetic analyses. This is the first time that microsatellite primers from the quinaria species group have been described. Four loci were cross-amplified in D. phalerata. Nine out of the 12 microsatellite markers developed are likely to be on the X chromosome.     

Development and Characterization of RAPD and Microsatellite Markers for Genetic Variation Analysis in the Critically Endangered Yellow Catfish <Emphasis Type="Italic">Horabagrus nigricollaris</Emphasis> (Teleostei: Horabagridae)

Random-amplified polymorphic DNA (RAPD) and microsatellite markers were developed and used for the analysis of genetic variability in the critically endangered yellow catfish Horabagrus nigricollaris, sampled from the Chalakkudy River, Kerala, India. Eight RAPD and five microsatellite markers were detected to genotype the species. In RAPD, the 73 fragments were 20.55% polymorphic, whereas 4 polymorphic loci (80%) were obtained in microsatellites. In microsatellites, the number of alleles across the 5 loci was 1-5, and the range of heterozygosity was 0.25-0.5. The mean observed number of alleles was 2.4, and the effective number was 1.775 per locus. The average heterozygosity across all investigated samples was 0.29, indicating a significant deficiency of heterozygotes in this species. RAPD and microsatellite methods report a low degree of gene diversity and lack of genetic heterogeneity in the population of H. nigricollaris, emphasizing the need for fishery management, conservation, and rehabilitation of this species.     

Population genetics of the endangered Wattled Curassow (Crax globulosa,Cracidae, Aves) of the Colombian–Peruvian Amazon using DNA microsatellites and ND2 mitochondrial sequences

The Wattled Curassow (Crax globulosa, Cracidae, Aves) is a large bird living in the Western Amazon basin and a critically endangered species in the Colombian and in the Peruvian Amazon. We carried out the first population genetics analysis of this species employing six nuclear microsatellite markers and sequences of the mtND2 gene. The main results are as follows. (1) The levels of gene diversity were high for the overall population as well as for each of the three islands for both microsatellites and mtDNA. (2) A small amount of genetic differentiation among populations was found with both types of markers (F_ST = 0.027 for microsatellites and N_ST = 0.17 for mitochondrial sequences). (3) Using microsatellites, the Geneclass 2.0 software detected a low correct assignment of individuals to their respective populations. The Structure software only detected one gene pool for the entire area studied. These results are relevant for conservation efforts of this critically endangered species.     

Isolation of microsatellite loci from the scleractinian corals,Montastraea cavernosa and Porites astreoides

The ability to assess genetic variation is critical for determining genetic diversity and population structure. In corals, slow evolutionary rates in mitochondrial genomes have left allozymes as the only markers presently available to investigate patterns of intraspecific genetic variation. Characteristics of microsatellites render them more informative than allozymes for such analyses; however, few coral microsatellites are available. This study describes polymorphic microsatellite loci isolated from two scleractinian coral species. Most loci exhibit significant heterozygote deficiencies, likely due to nonrandom mating or Wahlund effects. These markers are being used to investigate gene flow among populations, providing insight into reef connectivity.