Assessing genetic diversity of cotton cultivars using genomic and newly developed expressed sequence tag-derived microsatellite markers

Estimations of genetic diversity and of relationships between varieties are crucial for cotton breeding. The genetic diversity of 59 core cotton cultivars, most of which were collected from China's main cotton-growing areas, was analyzed based on genomic and newly developed expressed sequence tag-derived microsatellite markers, using total DNA extracted from fresh leaf tissue. Three hundred and two fragments were detected, of which 255 were polymorphic. The number of amplification products generated by each primer varied from 2 to 14, with a mean of 5.08 bands/primer. The polymorphism information content was 0.50 to 0.90, with a mean of 0.80. The genetic similarity coefficients were calculated and dendrograms were constructed by the unweighted pair group with arithmetic mean method; the resulting distance matrix gave a dendrogram with four main clusters. Some cultivars with similar pedigrees could be clustered. For example, Zhong206 and Shanmian4, both derived from Deltapine15, were clustered. The genetic similarity coefficient of the 59 core cultivars ranged from 0.53 to 0.99, with a mean of 0.72, indicating that there was a relatively high level of genetic variation.     

Genetic analysis of salt-marsh sedge Carex scabrifolia Steud. populations using newly developed microsatellite markers

Nine microsatellite loci were isolated and characterized from the clonal salt-marsh sedge Carex scabrifolia, and genetic diversities within four populations were analyzed. The number of alleles per locus ranged from 2 to 7, with an average of 4.7. The observed and expected heterozygosities ranged from 0.000 to 1.000 and 0.000 to 0.679, respectively. In two populations, almost all polymorphic loci showed a significant excess of heterozygotes due to their high clonal reproduction and low number of genotypes (genets) composing the populations. Thus, the number of identical genets varied greatly among populations and ranged from 1 to 28, irrespective of population size (i.e., population area and ramet number). When attempting to conserve and restore clonal plants such as C. scabrifolia, it is important to preserve sufficient genetic diversity within a population, which can be assessed using genetic markers such as the simple sequence repeat markers described here.     

Analysis of tetraploid Elymus species using wheat microsatellite markers and RAPD markers.

An analysis of Amplification fragment polymorphism of DNA from 27 accessions of 19 tetraploid Elymus species was carried out using 18 wheat microsatellite (WMS) primer pairs and 10 decamer primers. Ten WMS primer pairs produced multiple polymorphism on all accessions tested. Two independent phenograms, one based on WMS-PCR and one on RAPDs, separated the 19 tetraploid species into two main groups, viz., the SH genome species group and the SY genome species group. The results coincide with the genomic classification of these species and hence support previous studies showing that Elymus is not a monophyletic genus. The assays indicated that accessions within a species cluster together, which concurs with the morphological classification. Interspecific and intraspecific polymorphisms were detected by the WMS-PCR and RAPD analyses. Variation was observed among accessions of Elymus caninus. The WMS-PCR detected a much higher level of polymorphism than the RAPD analysis. WMSs seem to be more efficient markers than RAPD markers for studying the population diversity of Elymus species. The potential of cross-species amplification of microsatellite markers as an additional source for genetic analysis and applications in Elymus is discussed in the context of these results.     

Genetically diversified populations in the loach Misgurnus anguillicaudatus inferred from newly developed microsatellite markers

The loach Misgurnus anguillicaudatus is a freshwater fish which include natural polyploids and clones besides most bisexually reproducing diploids. Recent genetic studies using allozymes differentiated the populations in northern region of Hokkaido from other populations in Japan. Here, we developed 23 new sets of polymerase chain reaction primers to detect polymorphic microsatellites loci and tested them in 20 individuals from Memanbetsu town (northern Hokkaido) and Kita village (southern Hokkaido). These markers can be potentially diagnostic for identification of two populations.     

Evolutionary factors affecting the cross‐species utility of newly developed microsatellite markers in seabirds

Microsatellite loci are ideal for testing hypotheses relating to genetic segregation at fine spatio‐temporal scales. They are also conserved among closely related species, making them potentially useful for clarifying interspecific relationships between recently diverged taxa. However, mutations at primer binding sites may lead to increased nonamplification, or disruptions that may result in decreased polymorphism in nontarget species. Furthermore, high mutation rates and constraints on allele size may also with evolutionary time, promote an increase in convergently evolved allele size classes, biasing measures of interspecific genetic differentiation. Here, we used next‐generation sequencing to develop microsatellite markers from a shotgun genome sequence of the sub‐Antarctic seabird, the thin‐billed prion (Pachyptila belcheri), that we tested for cross‐species amplification in other Pachyptila and related sub‐Antarctic species. We found that heterozygosity decreased and the proportion of nonamplifying loci increased with phylogenetic distance from the target species. Surprisingly, we found that species trees estimated from interspecific F_ST provided better approximations of mtDNA relationships among the studied species than those estimated using D_C, even though F_ST was more affected by null alleles. We observed a significantly nonlinear second order polynomial relationship between microsatellite and mtDNA distances. We propose that the loss of linearity with increasing mtDNA distance stems from an increasing proportion of homoplastic allele size classes that are identical in state, but not identical by descent. Therefore, despite high cross‐species amplification success and high polymorphism among the closely related Pachyptila species, we caution against the use of microsatellites in phylogenetic inference among distantly related taxa.     

Novel polymorphic microsatellite markers developed in the cotton bollworm Helicoverpa armigera （Lepidoptera：Noctuidae）

A novel set of five polymorphic di- or trinucleofide microsatellite loci suitable for population genetic study were developed from an enriched genomic library for the pest insect cotton bollworm, Helicoverpa armigera, and cross-amplifiability of these and other published loci was tested in a closely related species, the tobacco budworm, H.assulta. The expected heterozygosity at these loci ranges from 0.62 to 0.91 in the cotton bollworm. The observed allele numbers varies from 4 to 12 in the limited number of individuals tested. Although a large proportion of cloned microsatellite sequences are present in multi-copy in the cotton bollworm genome, the overwhelming majority of the finalized polymorphic diallelic loci are tri-nucleotide microsatellites - an unexpected outcome, which should facilitate subsequent genotyping analysis.     

Identification of microsatellite markers in the rye genome

The rye genomic library, which consists of DNA fragments in the range of 0.5–1.1 kb, was screened for the presence of tri-and tetranucleotide and compound microsatellites. Of the 1,600,000 clones analysed, 102 clones were positive and 41 were suitable for SSR primer pair design. Twenty-six primer pairs amplified specific products, and six of them were capable of detecting polymorphism among 30 rye accessions of different genetic backgrounds. Using a set of Chinese Spring-Imperial wheat-rye addition lines, it was possible to locate 3 newly identified microsatellites on chromosomes 3R, 4R and 7R.     

Characterization of newly developed SSLP markers for the rat

We have isolated more than 12,000 clones containing microsatellite sequences, mainly consisting of (CA)n dinucleotide repeats, using genomic DNA from the BN strain of laboratory rat. Data trimming yielded 9636 non-redundant microsatellite sequences, and we designed oligonucleotide primer pairs to amplify 8189 of these. PCR amplification of genomic DNA from five different rat strains yielded clean amplification products for 7040 of these simple-sequence-length-polymorphism (SSLP) markers; 3019 markers had been mapped previously by radiation hybrid (RH) mapping methods (Nat Genet 22, 27–36, 1998). Here we report the characterization of these newly developed microsatellite markers as well as the release of previously unpublished microsatellite marker information. In addition, we have constructed a genome-wide linkage map of 515 markers, 204 of which are derived from our new collection, by genotyping 48 F₂ progeny of (OLETFxBN)F₂ crosses. This map spans 1830.9 cM, with an average spacing of 3.56 cM. Together with our ongoing project of preparing a whole-genome radiation hybrid map for the rat, this dense linkage map should provide a valuable resource for genetic studies in this model species. Received: 8 July 1999 / Accepted: 3 December 1999     

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.     

Additional microsatellite markers for mouse genome mapping

Mouse sequence information from the EMBL and GenBank databases, published sequences and genomic clones have been analyzed for simple repetitive elements or microsatellites. Each microsatellite has been amplified by the polymerase chain reaction (PCR) as a single locus marker. PCR primers were designed from unique sequence flanking each repeat. Size variation of PCR products less than 750 base pairs (bp) between mouse strains has been determined using ethidium bromide-stained acrylamide or agarose gels. A further 74 newly characterized microsatellites are presented in this paper, bringing to 185 the total we have analyzed. Of these, 157/185 (85%) have more than one allele, 143/178 (80%) vary in length between C57BL/6J and Mus spretus, and 82/168 (49%) vary between DBA/2J and C57BL/6J. Microsatellites provide informative single locus probes for linkage analysis in the construction of a genetic map of the mouse genome.     

Exploring genetic variability within lentil (Lens culinaris Medik.) and across related legumes using a newly developed set of microsatellite markers

Lentil (Lens culinaris Medik.) is an economically important grain legume, yet the genetic and genomic resources remain largely uncharacterized and unexploited in this crop. Microsatellites have become markers of choice for crop improvement applications. Hence, simple sequence repeat (SSR) markers were developed for lentil through the construction of genomic library enriched for GA/CT motifs. As a result 122 functional SSR primer pairs were developed from 151 microsatellite loci and validated in L. culinaris cv. Precoz. Thirty three SSR markers were utilized for the analysis of genetic relationships between cultivated and wild species of Lens and related legumes. A total of 123 alleles were amplified at 33 loci ranging from 2–5 alleles with an average of 3.73 alleles per locus. Polymorphic information content (PIC) for all the loci ranged from 0.13 to 0.99 with an average of 0.66 per locus. Varied levels of cross genera transferability were obtained ranging from 69.70 % across Pisum sativum to 12.12 % across Vigna radiata. The UPGMA based dendrogram was able to establish the uniqueness of each genotype and grouped them into two major clusters clearly resolving the genetic relationships within lentil and related species. The new set of SSR markers reported here were efficient and highly polymorphic and would add to the existing repertoire of lentil SSR markers to be utilized in molecular breeding. Moreover, the improved knowledge about intra- and inter-specific genetic relationships would facilitate germplasm utilization for lentil improvement.     

The construction of a tetraploid cotton genome wide comprehensive reference map

Integration of multiple genomic maps provides a higher density of markers and greater genome coverage, which not only facilitates the identification and positioning of QTLs and candidate genes, but it also provides a basic structure for the genome sequence assembly. However, the diversity in markers and populations used in individual mapping studies limits the ability to fully integrate the available data. By concentrating on marker orders rather than marker distances, published map data could be used to produce a comprehensive reference map (CRM) that includes a majority of known markers with optimally estimated order of those markers across the genome. In this study, a tetraploid cotton genome-wide CRM was constructed from 28 public cotton genetic maps. The initial CRM contained 7,424 markers and represented over 93% of the combined mapping information from the 28 individual maps. The current output is stored and displayed through CottonDB (http://www.cottondb.org), the public cotton genome database.     

Newly developed polymorphic microsatellite markers for frogs of the genus Ascaphus

Thirteen polymorphic microsatellite loci were identified and developed for the coastal tailed frog, Ascaphus truei, from sites within the Olympic Peninsula of Washington, USA. These tetranucleotide repeat loci were highly variable, averaging 19 alleles per locus and expected heterozygosity of 0.91. In addition, these loci cross-amplify in the sister species, Ascaphus montanus. These markers will prove useful in identifying fine-scale genetic structure, as well as provide insight into the evolution and conservation of this group across fragmented landscapes.     

Fully informative microsatellite markers for the tetraploid plant Eritrichium nanum (Boraginaceae)

We characterized eight polymorphic, codominant nuclear microsatellite loci in the tetraploid plant Eritrichium nanum. The different allelic configurations occurring under tetrasomic inheritance were fully resolved at all loci. Two natural populations showed high observed heterozygosities, which were in agreement with Hardy-Weinberg expectations. There was no evidence of genetic linkage disequilibrium for any pair of loci. The results suggest that these microsatellite markers are useful for mating system and population genetic analyses in high-alpine E. nanum.     

Genetic profiling of the critically endangered palm species Medemia argun using newly developed chloroplast DNA markers

Background: Medemia argun is a rare wild palm tree species. Its global existence is assumed to include the main population of about 1000 trees in the Nubian Desert of Sudan and some scattered individuals in southern Egypt. The species had previously been assumed to be extinct, but then reported to be extant about 20 years ago.

Aims: To assess genetic variation and explore population genetic structure of M. argun, through development and analysis of microsatellite markers.

Methods: The genome sequence mining approach was applied in order to identify microsatellites in the chloroplast genome of Bismarckia nobilis, a species closely related of M. argun. A set of 49 markers were designed, and their characteristics are now provided. Seven chloroplast DNA markers were developed for use in the genetic characterisation of this threatened species.

Results: Five markers were found polymorphic in M. argun, which enabled the assessment of the genetic diversity of the species. Significant genetic differentiation was observed among generations and collection sites, accompanied by low genetic variation. The seven markers developed were polymorphic among the wild relatives Hyphaene thebaica and Borassus aethiopum.

Conclusions: This is the first study to report molecular markers for M. argun. Our results suggest that the genetic consequences of population fragmentation in M. argun are beginning to be evident.     

Characterization of tomato SSR markers developed using BAC-end and cDNA sequences from genome databases

We developed nearly 700 non-redundant 2- or 3-base simple sequence repeat (SSR) markers from tomato using sequence data obtained from open genome databases. Among various types of core motifs, AT was most abundant in SSRs derived from cDNAs (~53%) and bacterial artificial chromosome (BAC) ends (~72%). There was a positive correlation between the rate of detection of polymorphic alleles (heterozygosity value; Hv) and the repeat number of the core motif in all markers showing polymorphisms among at least one pair of six cultivars or lines tested (r = 0.566**). The average Hv of BAC-end-derived SSR markers (~0.5) was higher than that of cDNA-derived markers (~0.3). These characteristics of BAC-end-derived SSRs are useful for genetic studies using closely related cultivars and lines. However, BAC-end-derived SSRs tended to cluster in centromeric regions (~80%). A scheme for the construction of a high-density linkage map of tomato is discussed.     

豚鼠基因组26个多态性微卫星标记的筛选

目的筛选豚鼠基因组的多态性微卫星标记,为豚鼠遗传质量控制及基因定位等工作奠定基础。方法采用磁珠富集法和豚鼠基因组数据库筛选法获取微卫星位点序列,通过分析和初步筛选,挑选部分候选位点,根据其序列设计引物,对5种不同来源的豚鼠基因组DNA标本进行PCR扩增,以期获得多态性分子标记。结果本实验采用磁珠富集法共获得微卫星序列304个,设计引物125对,最终获得多态性位点1个,暂未发现多态性的特异性位点17个;用数据库筛选法共获得微卫星序列292个,设计并合成相应引物178对,最终发现多态性位点25个,暂未发现多态性的特异性位点28个。结论本实验获得26个多态性微卫星标记,45个潜在的候选标记,为微卫星标记在豚鼠遗传质量监测及突变基因定位等工作的应用奠定了基础。     

Single-copy,species-transferable microsatellite markers developed from loblolly pine ESTs

Microsatellites, or simple sequence repeats (SSRs), are usually regarded as the markers of choice in population genetics research because they exhibit high variability. The development cost of these markers is usually high. In addition, microsatellite primers developed for one species often do not cross-amplify in related species, requiring separate development for each species. However, microsatellites found in expressed sequence tags (ESTs) might better cross-amplify as they reside in or near conserved coding DNA. In this study, we identified 14 Pinus taeda (loblolly pine) EST-SSRs from public EST databases and tested for their cross-species transferability to P. contorta ssp. latifolia, P. ponderosa, and P. sylvestris. As part of our development of a P. contorta microsatellite set, we also compared their transferability to that of 99 traditional microsatellite markers developed in P. taeda and tested on P. contorta ssp. latifolia. Compared to traditional microsatellites, EST-SSRs had higher transfer rates across pine species; however, the level of polymorphism of microsatellites derived from ESTs was lower. Sequence analyses revealed that the frequencies of insertions/deletions and base substitutions were lower in EST-SSRs than in other types of microsatellites, confirming that EST-SSRs are more conserved than traditional SSRs. Our results also provide a battery of 23 polymorphic, robust microsatellite primer pairs for lodgepole pine.Communicated by O. Savolainen     

Six newly developed microsatellite markers of <Emphasis Type="Italic">Laccaria amethystina</Emphasis>, using an improved CSSR approach

Microsatellite markers were established by an improved combined simple sequence repeat (CSSR) approach for the ectomycorrhizal basidiomycete Laccaria amethystina. Six markers delivered codominant polymorphic results, with up to six different alleles. They were tested with DNA originating from sporocarps and ectomycorrhizal root tips from silver fir (Abies alba), collected in the northern Black Forest. Sporocarps and ectoymcorrhizae exhibited similar allelic profiles, without any distorting influence of the host tree species. Results were compared to allelic profiles delivered by three published Asian markers. Allelic frequencies of the new markers showed a higher resolution of individuals within the population community, but partially a lower grade of heterozygosity. Possible reasons are discussed, and further questions addressed.     

Analysis of microsatellite markers in the genome of the plant pathogen Ceratocystis fimbriata

Ceratocystis fimbriata sensu lato represents a complex of cryptic and commonly plant pathogenic species that are morphologically similar. Species in this complex have been described using morphological characteristics, intersterility tests and phylogenetics. Microsatellite markers have been useful to study the population structure and origin of some species in the complex. In this study we sequenced the genome of C. fimbriata. This provided an opportunity to mine the genome for microsatellites, to develop new microsatellite markers, and map previously developed markers onto the genome. Over 6000 microsatellites were identified in the genome and their abundance and distribution was determined. Ceratocystis fimbriata has a medium level of microsatellite density and slightly smaller genome when compared with other fungi for which similar microsatellite analyses have been performed. This is the first report of a microsatellite analysis conducted on a genome sequence of a fungal species in the order Microascales. Forty-seven microsatellite markers have been published for population genetic studies, of which 35 could be mapped onto the C. fimbriata genome sequence. We developed an additional ten microsatellite markers within putative genes to differentiate between species in the C. fimbriata s.l. complex. These markers were used to distinguish between 12 species in the complex.