Length Polymorphisms of Simple Sequence Repeat DNA in Soybean

The objective of this work was to ascertain the presence and degree of simple sequence repeat (SSR) DNA length polymorphism in the soybean [Glycine max (L.) Merr.]. A search of GenBank revealed no (CA)n or (GT)n SSRs with n greater than 8 in soybean. In contrast, 5 (AT)n and 1 (ATT)n SSRs with n ranging from 14 to 27 were detected. Polymerase chain reaction (PCR) primers to regions flanking the six SSR loci were used in PCR amplification of DNA from 43 homozygous soybean genotypes. At three loci, amplification produced one PCR product per genotype and revealed 6, 7 and 8 product length variants (alleles) at the three loci, respectively. F1 hybrids between parents carrying different alleles produced two PCR products identical to the two parents. Codominant segregation of alleles among F2 progeny was demonstrated at each locus. A soybean DNA library was screened for the presence of (CA/GT)n SSRs. Sequencing of positive clones revealed that the longest such SSR was (CA)9. Thus, (CA)n SSRs with n of 15 or more are apparently much less common in soybean than in the human genome. In contrast to humans, (CA)n SSRs will probably not provide an abundant source of genetic markers in soybean. However, the apparent abundance of long (AT)n sequences should allow this SSR to serve as a source of highly polymorphic genetic markers in soybean.     

In silico polymorphic novel SSR marker development and the first SSR-based genetic linkage map in pistachio

Simple sequence repeats (SSRs) are co-dominant markers, and are very useful in constructing consensus maps in heterozygous perennial plant species like pistachio. Pistacia vera L. is the only cultivated species in the genus Pistacia. It is dioecious with a haploid chromosome count of n =?15. Saturated genetic linkage maps can be a reference to identify markers linked to economically important phenotypic traits that could be useful for early breeding and selection programs. Therefore, this study aimed to develop polymorphic SSR markers in silico and to construct the first SSR-based genetic linkage map in pistachio. The DNA sequences of three cultivars (Siirt, Ohadi, and Bagyolu) of P. vera and one genotype belonging to P. atlantica (Pa-18) were obtained by next-generation sequencing, and 625 polymorphic SSR loci were identified from 750 screened in silico polymorphic SSR primer pairs. The novel SSRs were used to construct SSR-based genetic linkage maps in pistachio along with published SSRs in Siirt × Bagyolu F1 population. Most (71.4%) of the SSRs were common markers that were used to construct consensus and parental maps spanning 15 linkage groups (LGs). A total of 384, 317, and 341 markers were mapped in the consensus, female, and male genetic maps with total lengths of 1511.3, 1427.0, and 1453.4 cM, respectively. The large number of SSR markers discovered and the first SSR-based genetic linkage map constructed in this study will be useful for anchoring loci for map integration, and will facilitate marker-assisted selection efforts for important horticultural traits in the genus Pistacia.     

Mining and characterization of EST derived microsatellites in Curcuma longa L

Turmeric (Curcuma longa L.) (Family: Zingiberaceae) is a perennial rhizomatous herbaceous plant often used as a spice since time immemorial. Turmeric plants are also widely known for its medicinal applications. Recently EST-derived SSRs (Simple sequence repeats) are a free by-product of the currently expanding EST (Expressed Sequence Tag) databases. SSRs have been widely applied as molecular markers in genetic studies. Development of high throughput method for detection of SSRs has given a new dimension in their use as molecular markers. A software tool SciRoKo was used to mine class I SSR in Curcuma EST database comprising 12953 sequences. A total of 568 non-redundant SSR loci were detected with an average of one SSR per 14.73 Kb of EST. Furthermore, trinucleotide was found to be the most abundant repeat type among 1-6-nucleotide repeat types. It accounted for 41.19% of the total, followed by the mononucleotide (20.07%) and hexanucleotide repeats (15.14%). Among all the repeat motifs, (A/T)n accounted for the highest proportion followed by (AGG)n. These detected SSRs can be greatly used for designing primers that can be used as markers for constructing saturated genetic maps and conducting comparative genomic studies in different Curcuma species.     

AFLP-based assessment of genetic diversity among nine alfalfa germplasms using bulk DNA templates.

Improving commercial utilization of perennial Medicago collections requires developing approaches that can rapidly and accurately characterize genetic diversity among large numbers of populations. This study evaluated the potential of using amplified fragment length polymorphism (AFLP) DNA markers, in combination with DNA bulking over multiple genotypes, as a strategy for high-throughput characterization of genetic distances (D) among alfalfa (Medicago sativa L.) accessions. Bulked DNA templates from 30 genotypes within each of nine well-recognized germplasms (African, Chilean, Flemish, Indian, Ladak, Medicago sativa subsp. falcata, Medicago sativa subsp. varia, Peruvian, and Turkistan) were evaluated using 34 primer combinations. A total of 3754 fragments were identified, of which 1541 were polymorphic. The number of polymorphic fragments detected per primer combination ranged from 20 to 85. Pairwise D estimates among the nine germplasms ranged from 0.52 to 1.46 with M. sativa subsp. falcata being the most genetically dissimilar. Unweighted pair-group method arithmetic average (UPGMA) analysis of the marker data produced two main clusters, (i) M. sativa subsp. sativa and M. sativa subsp. varia, and (ii) M. sativa subsp. falcata. Cluster-analysis results and D estimates among the Chilean, Peruvian, Flemish, and M. sativa subsp. varia germplasms supported the hypothesis that Peruvian was more similar to original Spanish introductions into Central and South America than Chilean. Hierarchical arrangement of the nine germplasms was supported by their respective geographic, subspecific, and intersubspecific hybrid origins. Subsets of as few as seven highly informative primer pairs were identified that produced comparable D estimates and similar heirarchical arrangements compared with the complete dataset. The results indicate that use of primer-pair subsets for AFLP analysis of bulk DNA templates could serve as a high-throughput system for accurately characterizing genetic diversity among large numbers of alfalfa populations.     

Simple sequence repeat analyses of interspecific hybrids and MAALs of <Emphasis Type="Italic">Oryza </Emphasis><Emphasis Type="Italic">officinalis</Emphasis> and <Emphasis Type="Italic">Oryza sativa</Emphasis>

Wild rice is a valuable resource for the genetic improvement of cultivated rice (Oryza sativa L., AA genome). Molecular markers are important tools for monitoring gene introgression from wild rice into cultivated rice. In this study, Simple sequence repeat (SSR) markers were used to analyze interspecific hybrids of O. sativa-O. officinalis (CC genome), the backcrossing progenies and the parent plants. Results showed that most of the SSR primers (335 out of 396, 84.6%) developed in cultivated rice successfully amplified products from DNA samples of wild rice O. officinalis. The polymorphism ratio of SSR bands between O. sativa and O. officinalis was as high as 93.9%, indicating differences between the two species with respect to SSRs. When the SSR markers were applied in the interspecific hybrids, only a portion of SSR primers amplified O. officinalis-specific bands in the F(1) hybrid (52.5%), BC(1) (52.5%), and MAALs (37.0%); a number of the bands disappeared. Of the 124 SSR loci that detected officinalis-specific bands in MAAL plants, 96 (77.4%) showed synteny between the A and C-genomes, and 20 (16.1%) showed duplication in the C-genome. Sequencing analysis revealed that indels, substitution and duplication contribute to the diversity of SSR loci between the genomes of O. sativa and O. officinalis.     

Characterization of 215 simple sequence repeat markers in creeping bentgrass (Agrostis stolonifera L.)

Creeping bentgrass (Agrostis stolonifera L.) is a versatile, cross-pollinated, temperate and perennial turfgrass species. It occurs naturally in a wide variety of habitats and is also cultivated on golf courses, bowling greens and tennis courts worldwide. Isozymes and amplified fragment length polymorphisms (AFLPs) have been used to determine genetic diversity, and restriction fragment length polymorphisms (RFLPs) and random amplified polymorphic DNA (RAPDs) were used to construct a genetic linkage map of this species. In the current report, we developed and characterized 215 unique genomic simple sequence repeat (SSR) markers in creeping bentgrass. The SSRs reported here are the first available markers in creeping bentgrass to date. Eight hundred and eighteen alleles were amplified by 215 SSR loci, an average of 3.72 alleles per locus. Fifty-nine per cent of those alleles segregated in a 1:1 Mendelian fashion (P > 0.05). Twenty-two per cent had a distorted segregation ratio (P ≤ 0.05). These SSR markers will be useful for assessing genetic diversity in creeping bentgrass and will be important for the development of genetic linkage maps and identifying quantitative trait loci. These markers could enhance breeding programmes by improving the efficiency of selection techniques.     

Mapping of simple sequence repeat (SSR) DNA markers in diploid and tetraploid alfalfa

Cultivated alfalfa (Medicago sativa) is an autotetraploid. However, all three existing alfalfa genetic maps resulted from crosses of diploid alfalfa. The current study was undertaken to evaluate the use of Simple Sequence Repeat (SSR) DNA markers for mapping in diploid and tetraploid alfalfa. Ten SSR markers were incorporated into an existing F₂ diploid alfalfa RFLP map and also mapped in an F₂ tetraploid population. The tetraploid population had two to four alleles in each of the loci examined. The segregation of these alleles in the tetraploid mapping population generally was clear and easy to interpret. Because of the complexity of tetrasomic linkage analysis and a lack of computer software to accommodate it, linkage relationships at the tetraploid level were determined using a single-dose allele (SDA) analysis, where the presence or absence of each allele was scored independently of the other alleles at the same locus. The SDA diploid map was also constructed to compare mapping using SDA to the standard co-dominant method. Linkage groups were generally conserved among the tetraploid and the two diploid linkage maps, except for segments where severe segregation distortion was present. Segregation distortion, which was present in both tetraploid and diploid populations, probably resulted from inbreeding depression. The ease of analysis together with the abundance of SSR loci in the alfalfa genome indicated that SSR markers should be a useful tool for mapping tetraploid alfalfa. Received: 10 September 1999 / Accepted: 11 November 1999     

Construction of two genetic linkage maps in cultivated tetraploid alfalfa (<Emphasis Type="Italic">Medicago sativa</Emphasis>) using microsatellite and AFLP markers

Background  

Alfalfa (Medicago sativa) is a major forage crop. The genetic progress is slow in this legume species because of its autotetraploidy and allogamy. The genetic structure of this species makes the construction of genetic maps difficult. To reach this objective, and to be able to detect QTLs in segregating populations, we used the available codominant microsatellite markers (SSRs), most of them identified in the model legume Medicago truncatula from EST database. A genetic map was constructed with AFLP and SSR markers using specific mapping procedures for autotetraploids. The tetrasomic inheritance was analysed in an alfalfa mapping population.     

Characterization of comparative genome‐derived simple sequence repeats for acanthopterygian fishes

Simple sequence repeats (SSRs) have become one of the most popular molecular markers for population genetic studies. The application of SSR markers has often been limited to source species because SSR loci are too labile to be maintained in even closely related species. However, a few extremely conserved SSR loci have been reported. Here, we tested for the presence of conserved SSR loci in acanthopterygian fishes, which include over 14 000 species, by comparing the genome sequences of four acanthopterygian fishes. We also examined the comparative genome‐derived SSRs (CG‐SSRs) for their transferability across acanthopterygian fishes and their applicability to population genetic analysis. Forty‐six SSR loci with conserved flanking regions were detected and examined for their transferability among seven nonacanthopterygian and 27 acanthopterygian fishes. The PCR amplification success rate in nonacanthopterygian fishes was low, ranging from 2.2% to 21.7%, except for Lophius litulon (Lophiiformes; 80.4%). Conversely, the rate in most acanthopterygian fishes exceeded 70.0%. Sequencing of these 46 loci revealed the presence of SSRs suitable for scoring while fragment analysis of 20 loci revealed polymorphisms in most of the acanthopterygian fishes. Population genetic analysis of Cottus pollux (Scorpaeniformes) and Sphaeramia orbicularis (Perciformes) using CG‐SSRs showed that these populations did not deviate from linkage equilibrium or Hardy–Weinberg equilibrium. Furthermore, almost no loci showed evidence of null alleles, suggesting that CG‐SSRs have strong resolving power for population genetic analysis. Our findings will facilitate the use of these markers in species in which markers remain to be identified.     

Chromosomal assignment of microsatellite loci in cotton

Microsatellite markers or simple sequence repeats (SSRs) represent a new class of genetic markers for cotton (Gossypium sp.). Sixty-five SSR primer pairs were used to amplify 71 marker loci and genotype 13 monosomic and 27 monotelodisomic cotton cytogenetic stocks. Forty-two SSR loci were assigned to cotton chromosomes or chromosome arms. Thirty SSRs were not located to specific chromosomes in this study. Nineteen marker loci were shown to occur on the A subgenome and 11 on the D subgenome by screening accessions of G. herbaceum (2n = 2x = 26 = 2A1) and G. raimondii (2n = 2x = 26 = 2D5). The aneuploid stocks proved to be very powerful tools for localizing SSR markers to individual cotton chromosomes. Multiplex PCR bins of the SSR primers and semiautomated detection of the amplified products were optimized in this experiment. Thirteen multiplex PCR bins were optimized to contain an average of 4 SSR primer pairs per bin. This provides a protocol for high-throughput genotyping of cotton SSRs that improves the efficiency of genetic mapping and marker-assisted programs utilizing SSR markers.     

Allelic diversity of simple sequence repeats among elite inbred lines of cultivated sunflower.

Simple sequence repeat (SSR) markers were developed for cultivated sunflower (Helianthus annuus L.) from the DNA sequences of 970 clones isolated from genomic DNA libraries enriched for (CA)n,, (CT)n, (CAA)n, (CATA)n, or (GATA)n. The clones harbored 632 SSRs, of which 259 were unique. SSR markers were developed for 130 unique SSRs by designing and testing primers for 171 unique SSRs. Of the total, 74 SSR markers were polymorphic when screened for length polymorphisms among 16 elite inbred lines. The mean number of alleles per locus was 3.7 for dinucleotide, 3.6 for trinucleotide, and 9.5 for tetranucleotide repeats and the mean polymorphic information content (PIC) scores were 0.53 for dinucleotide, 0.53 for trinucleotide, and 0.83 for tetranucleotide repeats. Cluster analyses uncovered patterns of genetic diversity concordant with patterns produced by RFLP fingerprinting. SSRs were found to be slightly more polymorphic than RFLPs. Several individual SSRs were significantly more polymorphic than RFLP and other DNA markers in sunflower (20% of the polymorphic SSR markers had PIC scores ranging from 0.70 to 0.93). The newly developed SSRs greatly increase the supply of sequence-based DNA markers for DNA fingerprinting, genetic mapping, and molecular breeding in sunflower; however, several hundred additional SSR markers are needed to routinely construct complete genetic maps and saturate the genome.     

Genome mapping of white clover (<Emphasis Type="Italic">Trifolium repens</Emphasis> L.) and comparative analysis within the Trifolieae using cross-species SSR markers

Allotetraploid white clover (Trifolium repens L.), a cool-season perennial legume used extensively as forage for livestock, is an important target for marker-assisted breeding. A genetic linkage map of white clover was constructed using simple sequence repeat (SSR) markers based on sequences from several Trifolieae species, including white clover, red clover (T. pratense L.), Medicago truncatula (Gaertn.) and soybean (Glycine max L.). An F₁ population consisting of 179 individuals, from a cross between two highly heterozygous genotypes, GA43 and Southern Regional Virus Resistant, was used for genetic mapping. A total of 1,571 SSR markers were screened for amplification and polymorphism using DNA from two parents and 14 F₁s of the mapping population. The map consists of 415 loci amplified from 343 SSR primer pairs, including 83 from white clover, 181 from red clover, 77 from M. truncatula, and two from soybean. Linkage groups for all eight homoeologous chromosome pairs of allotetraploid white clover were detected. Map length was estimated at 1,877 cM with 87% genome coverage. Map density was approximately 5 cM per locus. Segregation distortion was detected in six segments of the genome (homoeologous groups A1, A2, B1, B2, C1, and D1). A comparison of map locations of markers originating from white clover, red clover, and alfalfa (M. sativa L.) revealed putative macro-colinearity between the three Trifolieae species. This map can be used to link quantitative trait loci with SSR markers, and accelerate the improvement of white clover by marker-assisted selection and breeding. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

An expressed sequence tag SSR map of tetraploid alfalfa (Medicago sativa L.)

A genetic map constructed from a population segregating for a trait of interest is required for QTL identification. The goal of this study was to construct a molecular map of tetraploid alfalfa (Medicago sativa.) using simple sequence repeat (SSR) markers derived primarily from expressed sequence tags (ESTs) and bacterial artificial chromosome (BAC) inserts of M. truncatula. This map will be used for the identification of drought tolerance QTL in alfalfa. Two first generation backcross populations were constructed from a cross between a water-use efficient, M. sativa subsp. falcata genotype and a low water-use efficient M. sativa subsp. sativa genotype. The two parents and their F₁ were screened with 1680 primer pairs designed to amplify SSRs, and 605 single dose alleles (SDAs) were amplified. In the F₁, 351 SDAs from 256 loci were mapped to 41 linkage groups. SDAs not inherited by the F₁, but transmitted through the recurrent parents and segregating in the backcross populations, were mapped to 43 linkage groups, and 44 of these loci were incorporated into the composite maps. Homologous linkage groups were joined to form eight composite linkage groups representing the eight chromosomes of M. sativa. The composite maps consist of eight composite linkage groups with 243 SDAs from M. truncatula EST sequences, 38 SDAs from M. truncatula BAC clone sequences, and five SDAs from alfalfa genomic SSRs. The total composite map length is 624 cM, with average marker density per composite linkage group ranging from 1.5 to 4.4 cM, and an overall average density of 2.2 cM. Segregation distortion was 10%, and distorted loci tended to cluster on individual homologues of several linkage groups. Electronic Supplementary Material Supplementary material is available for this article at     

Development, characterisation, and across-taxa utility of oil palm (Elaeis guineensis Jacq.) microsatellite markers.

The results of the development of oil palm (Elaeis guineensis Jacq.) microsatellite markers are given step by step, from the screening of libraries enriched in (GA)n, (GT)n, and (CCG)n simple-sequence repeats (SSRs) to the final characterisation of 21 SSR loci. Also published are primer sequences, estimates of allele size range, and expected heterozygosity in E. guineensis and in the closely related species E. oleifera, in which an optimal utility of the SSR markers was observed. Multivariate data analyses showed the ability of SSR markers to efficiently reveal the genetic-diversity structure of the genus Elaeis in accordance with known geographical origins and with measured genetic relationships based on previous molecular studies. High levels of allelic variability indicated that E. guineensis SSRs will be a powerful tool for genetic studies of the genus Elaeis, including variety identification and intra- or inter-specific genetic mapping. PCR amplification tests on a subset of 16 other palm species and allele-sequence data showed that E. guineensis SSRs are putative transferable markers across palm taxa. In addition, phenetic information based on SSR flanking region sequences makes E. guineensis SSR markers a potentially useful molecular resource for any researcher studying the phylogeny of palm taxa.     

Physical mapping of rRNA genes in Medicago sativa and M. glomerata by fluorescent in situ hybridization

Fluorescent in situ hybridization (FISH) was applied to diploid and tetraploid subspecies of alfalfa (Medicago sativa L.) to investigate the distribution of rRNA genes and to utilize the sites of 18S-5.8S-25S rDNA and 5S rDNA sequences as markers for studying the genome evolution within the species. Medicago glomerata Balb., the species considered to be the ancestor of alfalfa, was included in this study in order to obtain more information on the phylogenetics of alfalfa. Simultaneous in situ hybridization was performed with the probes pTa71 and pXVI labeled with digoxigenin and biotin, respectively. In the diploid taxa, M. glomerata, M. sativa ssp. coerulea Schmalh and ssp. falcata Arcangeli, the 18S-5.8S-25S rDNA sequences were mapped to two sites corresponding to the secondary constrictions of the nucleolar chromosome pair, while 5S rDNA appeared to be distributed in two pairs of sites. Chromosomes carrying 5S loci could be distinguished on the basis of their morphological characteristics. The number of rDNA sites detected in the tetraploid M. sativa ssp. falcata and ssp. sativa (L.) L. & L. were twice the number found in the respective diploid ssp. falcata and ssp. coerulea. The results of this study show that the distribution of ribosomal genes was maintained during the evolutionary steps from the primitive diploid to the cultivated alfalfa. Modifications of the number of rRNA loci were not observed. The importance of in situ hybridization for improving karyotype analysis in M. sativa L. is discussed.     

Conservation of microsatellites among tropical trees (Leguminosae)

Although microsatellites or simple sequence repeats (SSRs) have become a popular tool in genetic mapping and gene flow studies, their utility is limited due to paucity of information about DNA sequences in plants. We tested the utility of microsatellite markers characterized for the tropical tree Pithecellobium elegans as a genetic tool for related species. The results indicate that SSR loci are conserved among closely related species, and SSR primers developed for P. elegans could be successfully used as a genetic tool in several species of the tribe Ingeae. This study indicates that there is high potential for the transfer of SSR markers among closely related taxa, circumventing laborious cloning and screening procedures involved in characterizing SSR loci for many species.     

Isolation of EST-derived microsatellite markers for genotyping the A and B genomes of wheat

Genetic variation present in 64 durum wheat accessions was investigated by using three sources of microsatellite (SSR) markers: EST-derived SSRs (EST-SSRs) and two sources of SSRs isolated from total genomic DNA. Out of 245 SSR primer pairs screened, 22 EST-SSRs and 20 genomic-derived SSRs were polymorphic and used for genotyping. The EST-SSR primers produced high quality markers, but had the lowest level of polymorphism (25%) compared to the other two sources of genomic SSR markers (53%). The 42 SSR markers detected 189 polymorphic alleles with an average number of 4.5 alleles per locus. The coefficient of similarity ranged from 0.28 to 0.70 and the estimates of similarity varied when different sources of SSR markers were used to genotype the accessions. This study showed that EST-derived SSR markers developed in bread wheat are polymorphic in durum wheat when assaying loci of the A and B genomes. A minumum of ten EST-SSRs generated a very low probability of identity (0.36×10⁻¹²) indicating that these SSRs have a very high discriminatory power. EST-SSR markers directly sample variation in transcribed regions of the genome, which may enhance their value in marker-assisted selection, comparative genetic analysis and for exploiting wheat genetic resources by providing a more-direct estimate of functional diversity. Received: 19 December 2000 / Accepted: 17 April 2001     

基于RAPD的群体标记法分析苜蓿种质遗传多样性

苜蓿种质资源的分子遗传多样性分析对于种质资源保存和育种利用具有重要指导意义。本研究选用群体标记法对来自甘肃省的16个苜蓿品种的DNA进行RAPD扩增,旨在研究其遗传多样性,并在此基础上筛选品种特异性引物用于进一步的品种鉴定。依据16个苜蓿品种间的Roger’s遗传距离进行UPGMA聚类分析结果显示,品种间亲缘关系与其选育背景紧密相关,供试的16个品种被划为4个类群,其中,匍匐型品种Jindera与其他直立型品种差异显著,自成1个类群,引进品种和甘肃省内具有国外种质来源的育成品种被划为同1类群,而甘肃地方品种聚为2个类群。10个RAPD引物中有4个引物OPE4、OPE5、OPE6和OPE7分别检测到5个品种甘农3号、甘杂27、Jindera、陇东和Algonuin的特异性条带,可进一步用于开发设计特异性引物进行品种鉴定工作。以上结果进一步证实,利用RAPD标记研究苜蓿系谱发育关系和选择杂交亲本应采用群体标记法。     

An SSR genetic map of Sorghum bicolor (L.) Moench and its comparison to a published genetic map.

Sorghum bicolor (L.) Moench is an important grain and forage crop grown worldwide. We developed a simple sequence repeat (SSR) linkage map for sorghum using 352 publicly available SSR primer pairs and a population of 277 F2 individuals derived from a cross between the Westland A line and PI 550610. A total of 132 SSR loci appeared polymorphic in the mapping population, and 118 SSRs were mapped to 16 linkage groups. These mapped SSR loci were distributed throughout 10 chromosomes of sorghum, and spanned a distance of 997.5 cM. More important, 38 new SSR loci were added to the sorghum genetic map in this study. The mapping result also showed that chromosomes SBI-01, SBI-02, SBI-05, and SBI-06 each had 1 linkage group; the other 6 chromosomes were composed of 2 linkage groups each. Except for 5 closely linked marker flips and 1 locus (Sb6_34), the marker order of this map was collinear to a published sorghum map, and the genetic distances of common marker intervals were similar, with a difference ratio 相似文献