Identification and characterization of (GA/CT) n - microsatellite loci from Quercus petraea

In this study a size selected genomic library from Quercus petraea was screened for (GA/CT)_n-microsatellite sequences. The resulting loci were analysed by PCR for their usefulness as molecular markers in Q. petraea and Q. robur. 17 out of 52 tested primer pairs resulted in the amplification of a polymorphic single-locus pattern. The number of alleles found per locus varied from 6 to 16. Combining the genetic variation observed for the characterized loci provides a unique genotype for all the individuals tested. Using intraspecific controlled crosses of Q. robur trees Mendelian inheritance could be shown for five loci.     

Development of polymorphic simple sequence repeat markers for Pisolithus microcarpus

Six polymorphic simple sequence repeat (SSR) markers were developed for the ectomycorrhizal fungus Pisolithus microcarpus. A polymerase chain reaction (PCR)‐based technique was used in which random amplified polymorphic DNA (RAPD) fingerprints were probed with labelled SSR oligonucleotides by southern hybridization. The number of alleles per locus ranged from two to nine with expected heterozygosity values from 0.33 to 0.76. These loci will be potentially useful for genetic structure and gene flow studies of P. microcarpus populations. Cross‐species amplification with Pisolithus albus isolates at all loci was also observed.     

Identification of microsatellite sequences in Vitis riparia and their applicability for genotyping of different Vitis species.

A Vitis riparia genomic library was screened for the presence of (GA)n simple sequence repeats (SSR) and 18 primer pairs yielding amplification products of the expected size were designed. Heterologous amplification with the primer pairs in related species (V. rupestris, V. berlandieri, V. labrusca, V. cinerea, V. aestivalis, V. vinifera, and interspecific hybrids) was successful in most primer-species combinations. Therefore, the new markers are applicable to the genotyping of a range of Vitis species. Variations in the SSR flanking sequence were detected between and within the species. The degree of polymorphism and performance of the markers were determined in up to 120 individuals of V. vinifera. Four of fifteen alleles per locus were detected and expected heterozygosity ranged between 0.37 and 0.88. Null alleles were shown to be present at two loci by a lack of heterozygous individuals and by transmission of the null alleles in a controlled cross. Regular Mendelian inheritance is indicated for all but one loci by a preliminary segregation analysis in 36 offspring. Thirteen of the markers were found suitable for the genotyping of grapevines (V. vinifera).     

Comparative sequencing of a microsatellite locus reveals size homoplasy within and between european oak species (<Emphasis Type="Italic">Quercus</Emphasis> spp.)

Microsatellites (simple sequence repeats [SSRs]) are highly variable molecular markers that are a rich and readily assayed source of variation for population genetic studies. Cross-amplification between closely related species is possible when there are no (or few) sequence differences in the primer binding sites. The occurrence of nonhomologous fragments of the same size (size homoplasy) is a contraint of microsatellites. Size homoplasy can be caused by insertions/deletions (indels) in SSR flanking regions. We found that size variation in locus ssrQZAG9 is due to different repeat numbers of the SSR motifs but also to indels in SSR flanking regions. Indels were found within species belonging to sectionsRobur andCerris of genusQuercus and also between species of the 2 sections. In sectionRobur (Quercis robur L.,Quercus petraea [Matt.] Liebl.,Quercus pubescens Willd.), we detected rare alleles with an indel of 57 bp or 62 bp followed by a smaller indel of 12 bp in the SSR flanking regions. These alleles show a size range overlapping with that of alleles amplified inQuercus cerris L. (sectionCerris). Multiple alignments with sequences of sectionRobur revealed the same SSR repeat motif but multiple indels in SSR flanking regions inQ. cerris. We discuss the effects of size homoplasy of SSR loci for the study of interspecific gene flow and on estimates of population differentiation.     

Microsatellites in Aureliana fasciculata var. fasciculata (Solanaceae), a shrub that inhabits the Atlantic Rainforest

? Premise of the study: Microsatellite primers were developed in Aureliana fasciculata var. fasciculata, a shrub that inhabits the Atlantic Rainforest, to investigate the intraspecific genetic structure of the species. ? Methods and Results: Nine pairs of microsatellite primers were designed that were based on a simple sequence repeat (SSR)-enriched library. Seven loci were amplified successfully. Two populations of A. fasciculata var. fasciculata were genotyped and all loci were polymorphic, with two to five alleles per locus per population detected. Cross-species amplification was achieved in other members of the genus and members of the sister genus Athenaea. ? Conclusions: The development of these microsatellite markers will contribute to future population genetic studies in A. fasciculata var. fasciculata. Cross-amplification among other species in the genus and in the sister genus Athenaea might be a useful tool to establish patterns of genetic variability within the Atlantic Rainforest.     

General vs. local differentiation between two closely related white oak species

The geographical distribution of species differentiation throughout the natural range of two sympatric, closely related species of oaks, Quercus petraea (Matt) Liebl. and Quercus robur L., was investigated. By sampling species in pairs in different European regions, from Spain to Poland and Romania, the differentiation between the two species could be subdivided into general and local differentiation. Nine sequence characterized amplified region (SCAR) markers corresponding to genomic regions which discriminate the two species were analysed using single-strand conformation polymorphism (SSCP) or classical electrophoresis (double-stranded DNA on agarose gel) on PCR products providing, respectively, codominant and dominant markers. Similar levels of gene diversity ( H _E) within the two species were observed, varying generally from 0.3 to 0.5 for dominant markers and from 0.5 to 0.86 for codominant markers. SSCP loci exhibited numerous alleles that were differently involved in species differentiation. The geographical distribution of species differentiation is heterogeneous between the regions, the north-east populations exhibiting higher differentiation than the others. For most loci, general differentiation was higher than local differentiation and was interpreted as the result of historical causes, selection pressures, and intra- and interspecific gene flow.     

Development and characterization of simple sequence repeat (SSR) markers and their use in determining relationships among Lycopersicon esculentum cultivars

The simple sequence repeat (SSR) or microsatellite marker is currently the preferred molecular marker due to its highly desirable properties. The aim of this study was to develop and characterize more SSR markers because the number of SSR markers currently available in tomato is very limited. Five hundred DNA sequences of tomato were searched for SSRs and analyzed for the design of PCR primers. Of the 158 pairs of SSR primers screened against a set of 19 diverse tomato cultivars, 129 pairs produced the expected DNA fragments in their PCR products, and 65 of them were polymorphic with the polymorphism information content (PIC) ranging from 0.09 to 0.67. Among the polymorphic loci, 2-6 SSR alleles were detected for each locus with an average of 2.7 alleles per locus; 49.2% of these loci had two alleles and 33.8% had three alleles. The vast majority (93.8%) of the microsatellite loci contained di- or tri-nucleotide repeats and only 6.2% had tetra- and penta-nucleotide repeats. It was also found that TA/AT was the most frequent type of repeat, and the polymorphism information content (PIC) was positively correlated with the number of repeats. The set of 19 tomato cultivars were clustered based on the banding patterns generated by the 65 polymorphic SSR loci. Since the markers developed in this study are primarily from expressed sequences, they can be used not only for molecular mapping, cultivar identification and marker-assisted selection, but for identifying gene-trait relations in tomato.     

Development and transferability of apricot and grape EST microsatellite markers across taxa

EST microsatellite markers were developed in apricot (Prunus armeniaca L.) and grape (Vitis vinifera L.). cDNA libraries from either apricot leaves or grape roots were used in an enrichment procedure for GA and CA repeats. The transferability of EST simple sequence repeat (SSR) markers from apricot and grapevine to other related and unrelated species was examined. Overall, grape primers amplified products in most of the Vitaceae accessions while the apricot primers amplified polymorphic alleles only in closely related species of the Rosaceae. In this taxonomic family, ten EST SSR loci were tested, and one single primer pair, PacB22, was amplified across species and sections in the Prunoideae and Maloideae. Sequencing of EST SSR loci in other species and genera confirmed a higher level of conservation in the microsatellite motif and flanking regions in the Vitaceae compared to the Rosaceae. Two distinct fragments of the PacB22 locus amplified across the Malus and Pyrus genera; however, while the coding region was highly conserved, the microsatellite repeat motif was no longer present. The banding pattern was explained by base substitution and insertion/deletion events in the intronic region of PacB22. This study includes the determination of the degree of polymorphism detected among species and genera in two unrelated taxonomic families and the evaluation of the information provided by the microsatellite repeats and the flanking regions.     

Polymorphic microsatellites developed by cross-species amplifications in common pheasant breeds

Genetic variability was analysed in two common breeds of pheasant (Phasianus colchicus L. 1758) by means of cross-species amplifications of microsatellite loci: 154 chicken, Gallus gallus and 32 turkey, Meleagris gallopavo, primers were tested for amplification of pheasant DNA. Thirty-six primers (25 specific for chicken and 11 for turkey) amplified pheasant DNA. Fifteen markers yielded specific products and were tested for polymorphism. Eight of them (55%) were polymorphic, with an average polymorphism of two alleles per locus. Specific polymerase chain reaction (PCR) products were sequenced; repeats were found in 11 of the 15 markers, although only two loci showed the same repeat and could be homologous to chicken ones.     

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.     

微卫星跨物种交叉PCR扩增的一个新问题:扩增非同源产物

微卫星已被广泛应用于群体遗传学、生态学和进化生物学研究。然而,一些物种微卫星尚未克隆。为了节省时间和经费,研究人员往往使用一个物种已发表的微卫星引物扩增其近缘物种的微卫星。该研究对属于3个不同科(Clariidae、Heteropneustidae 和Pimelodidae)的7个鲶鱼物种的微卫星跨物种PCR扩增产物进行了序列分析,研究发现扩增非同源（non-orthologous）产物是微卫星跨物种PCR扩增的一个新问题。该研究共采用4对胡子鲶微卫星座位引物对7个鲶鱼物种进行了跨物种PCR扩增。对获得的204个PCR产物的序列分析结果表明,两对微卫星座位引物扩增了所有7个物种的同源特异产物。而其他两个座位的引物扩增了特异但非同源的多态产物,对近缘物种的扩增也获得类似结果。另外,除胡子鲶等位基因大小异源同型(size homoplasy)的特征不明显外,其他物种在3个微卫星座位都具有这一非常明显的特征。这些数据表明,微卫星跨物种间交叉扩增能产生非同源产物;等位基因大小异源同型与微卫星座位本身有关,而与物种间的亲缘关系无明显的相关性。微卫星跨物种扩增产生的非同源产物和等位基因大小异源同型将使系统发育、群体遗传学和进化研究明显复杂化。因此,在应用微卫星跨物种交叉扩增数据以前,最好对跨物种交叉扩增产物进行测序验证。     

Comparative analysis of microsatellite loci in chicken and turkey.

Cross-species amplification of 520 chicken microsatellite markers was tested by polymerase chain reaction with genomic DNA of the turkey (Meleagris gallopavo). Each primer pair was tested at six different combinations of annealing temperature and MgCl2 concentration. A total of 280 (54%) of the primer pairs produced amplification products. The majority of these products were similar, if not identical in size to those expected based on the fragment sizes of the corresponding chicken loci. Structure of the dinucleotide repeat and flanking sequences was examined for 13 turkey fragments (amplified with chicken primers) and 5 chicken fragments (amplified with turkey primers). Sequence analysis found a wide array of mutations between species in addition to differences in repeat length. To estimate the usefulness of the amplified loci for genetic mapping in the turkey, allelic polymorphism was determined for 57 of the 280 amplified loci. A total of 20 of 57 markers (35%) were polymorphic with an average of 1.4 alleles per locus. The results of this study suggest that approximately 20% of the chicken microsatellite markers will be useful for mapping the turkey genome.     

The conversion of RFLP markers to allele specific amplicons linked to QTLs governing malting quality in barley

Application of marker-assisted selection with RFLP based markers has been constrained by high cost and time requirements in situations involving a large number of plants. RFLP markers mapped on a Harrington/TR306 population have been identified elsewhere as linked to quantitative trait loci (QTL) governing malting quality. The probes ABG610, ABC622, as well as probes for the Nar1, Amy1 and Nar7 were sequenced and locus specific primers developed. These locus specific primers were applied to genomic DNA from both Harrington and TR306. Sequence analysis of the resultant monomorphic fragments revealed sequence divergence for the Xabg610, Xabc622, Amy1 and Nar1 loci, but not for the Nar7 locus. Application of a set of Hor2 primers to genomic DNA from the barley lines Harrington and TR306 led to the direct amplification of codominant alleles. Allele-specific primers were designed based on the sequence divergence identified among the Xabg610, Xabc622 and Nar1 alleles. Amplification conditions were optimized for each of these alleles such that only the favourable allele from Harrington was amplified. The usefulness of these primers for selecting Harrington alleles was demonstrated by their failure to amplify the corresponding alleles from the lines, Sterling, Stella and WM872. The Amy1 allele-specific amplicon was only capable of differentiating this locus between Harrington and TR306. The conversion of these markers into PCR amplifiable, allele-specific amplicons would greatly facilitate their application to barley breeding programs.     

Microsatellite DNA markers in Populus tremuloides.

Markers for eight new microsatellite DNA or simple sequence repeat (SSR) loci were developed and characterized in trembling aspen (Populus tremuloides) from a partial genomic library. Informativeness of these microsatellite DNA markers was examined by determining polymorphisms in 38 P. tremuloides individuals. Inheritance of selected markers was tested in progenies of controlled crosses. Six characterized SSR loci were of dinucleotide repeats (two perfect and four imperfect), and one each of trinucleotide and tetranucleotide repeats. The monomorphic SSR locus (PTR15) was of a compound imperfect dinucleotide repeat. The primers of one highly polymorphic SSR locus (PTR7) amplified two loci, and alleles could not be assigned to a specific locus. At the other six polymorphic loci, 25 alleles were detected in 38 P. tremuloides individuals; the number of alleles ranged from 2 to 7, with an average of 4.2 alleles per locus, and the observed heterozygosity ranged from 0.05 to 0.61, with an average of 0.36 per locus. The two perfect dinucleotide and one trinucleotide microsatellite DNA loci were the most informative. Microsatellite DNA variants of four SSR loci characterized previously followed a single-locus Mendelian inheritance pattern, whereas those of PTR7 from the present study showed a two-locus Mendelian inheritance pattern in controlled crosses. The microsatellite DNA markers developed and reported here could be used for assisting various genetic, breeding, biotechnology, genome mapping, conservation, and sustainable forest management programs in poplars.     

SSR markers for Quercus suber tree identification and embryo analysis.

Three Quercus simple sequence repeat (SSR) markers were amplified by polymerase chain reaction (PCR) from nuclear DNA extracts of trees and in vitro-induced haploid embryos from anther cultures of Quercus suber L. These markers were sufficiently polymorphic to identify 10 of 12 trees located in two Spanish natural areas. The same loci have been analyzed in anther-derived haploid embryos showing the parental tree allele segregation. All the alleles were present in the haploid progeny. The presence of diverse alleles in embryos derived from the same anther demonstrated that they were induced on multiple microspores or pollen grains and they were not clonally propagated. Also, diploid cultures and mixtures of haploid-diploid tissues were obtained. The origin of such cultures, either somatic or gametic, was elucidated by SSR markers. All the embryos showed only one allele, corroborating a haploid origin. Allelic composition of the haploid progeny permitted parental identification among all analyzed trees.     

基于磁珠富集法的绣线菊SSR分子标记分离与筛选

微卫星序列（SSR）具有多态性高、共显性遗传等特点,是一种极具价值的分子遗传标记。采用磁珠富集法从高山绣线菊基因组DNA中分离和筛选SSR标记。高山绣线菊基因组经限制性内切酶Mse I酶切后与接头连接,并与生物素标记SSR探针（AC）15和（AG）15杂交,然后通过链霉亲和素磁珠富集、洗脱、PCR扩增、克隆,完成微卫星文库构建。利用载体通用引物和探针序列引物进行PCR扩增,筛选重组克隆并测序,获得112条序列。随机挑选其中60条序列设计的引物,经初期筛选获得多态性引物16对。用所得16对引物对4个居群92个个体的蒙古绣线菊和高山绣线菊进行PCR扩增。统计分析PCR产物的毛细管电泳结果,发现4个居群的平均等位基因数、平均期望杂合度及平均观测杂合度都比较高。64个数据系列（4个居群×16个位点）中的26个显著偏离HardyWeinberg平衡,推测可能由于无效等位基因的存在所引起。分析显示研究开发的16对多态性SSR引物可以用于后续遗传多样性、物种进化与亲缘关系等方面研究,丰富了绣线菊遗传多样性研究的分子标记。     

Development of reliable PCR-based markers linked to downy mildew resistance genes in lettuce

Summary Sequence characterized amplified regions (SCARs) were derived from eight random amplified polymorphic DNA (RAPD) markers linked to disease resistance genes in lettuce. SCARs are PCR-based markers that represent single, genetically defined loci that are identified by PCR amplification of genomic DNA with pairs of specific oligonucleotide primers; they may contain high-copy, dispersed genomic sequences within the amplified region. Amplified RAPD products were cloned and sequenced. The sequence was used to design 24-mer oligonucleotide primers for each end. All pairs of SCAR primers resulted in the amplification of single major bands the same size as the RAPD fragment cloned. Polymorphism was either retained as the presence or absence of amplification of the band or appeared as length polymorphisms that converted dominant RAPD loci into codominant SCAR markers. This study provided information on the molecular basis of RAPD markers. The amplified fragment contained no obvious repeated sequences beyond the primer sequence. Five out of eight pairs of SCAR primers amplified an alternate allele from both parents of the mapping population; therefore, the original RAPD polymorphism was likely due to mismatch at the primer sites.     

Isolation and characterization of microsatellites in trembling aspen (Populus tremuloides)

 We have identified, isolated, and characterized microsatellite/simple sequence repeat (SSR) loci in trembling aspen (Populus tremuloides) by screening partial genomic libraries. We have also examined the compatibility and use of the P. tremuloides SSR primers to resolve microsatellites in other Populus species. Fourteen microsatellites were identified from 1600 clones screened. The TC/AG microsatellites were the most abundant. A total of 29 alleles were detected in 36 P. tremuloides individuals at the four SSR loci (two each of di- and tri-nucleotide repeats) characterized. The number of alleles at the SSR loci ranged from 5 to 11, with an average of 7.25 alleles per locus, and the observed heterozygosity ranged from 0.19 to 0.82, with a mean of 0.46 per locus. Although the highest polymorphism was observed for a dinucleotide SSR locus, the trinucleotide SSR loci showed substantial polymorphism. There were 34 unique multilocus genotypes among the 36 P. tremuloides individuals examined, and 89% of the individuals had unique multilocus genotypes. Two pairs of SSR primers were successful in PCR, amplifying genomic DNA and resolving microsatellites of comparable size from Populus deltoides, P. nigra, P.×canadensis, and P. maximowiczii. The microsatellite DNA markers developed could be used for clonal fingerprinting, certification of controlled crosses, genome mapping, marker-assisted early selection, genetic diversity assessments, and conservation and sustainable management of poplar genetic resources. Received: 14 November 1997 / Accepted: 17 November 1997     

Isolation, characterisation and mapping of simple sequence repeat loci in potato

Solanum tuberosum L. DNA sequences containing simple sequence repeat (SSR) motifs were extracted from the EMBL database, cDNA and selectively enriched small-insert DNA libraries. Enrichment was achieved using either triplex affinity capture or single-strand hybridisation selection. One hundred and twelve primer pairs which successfully amplified products of the correct size from potato DNA were ultimately designed and synthesised. Ninety-eight of these revealed length polymorphisms in a panel of four diploid and two tetraploid clones, in agreement with the high information content of this class of markers which has been found in other species. All of the markers were assigned a quality score of 1–5 based on their potential usefulness. Eighty-nine loci from 65 of the primer pairs were located on two genetic linkage maps of potato by segregation analysis of the amplified alleles. Fifty-two of the SSRs were clearly single locus. The maps were aligned using 23 SSR primer pairs and 13 RFLP loci mapped in both populations. The markers described constitute a class which should replace Restriction Fragment Length Polymorphisms (RFLP) as the markers of choice for future genetic studies in potato. The sequences of the primers, together with other information on these markers are provided. Received: 12 January 1998 / Accepted: 25 March 1998     

Development and characterization of EST-derived simple sequence repeat (SSR) markers for pasture grass endophytes.

Fungal endophytes of the genus Neotyphodium are common in temperate pasture grass species and confer both beneficial and deleterious agronomic characteristics to their hosts. The aim of this study was to develop molecular markers based on simple sequence repeat (SSR) loci for the identification and assessment of genetic diversity among Neotyphodium endophytes in grasses. Expressed sequence tags (ESTs) from both Neptyphodium coenophialum and Neotyphodium lolii were examined, and unique SSR loci were identified in 9.7% of the N. coenophialum sequences and 6.3% of the N. lolii sequences. A variety of SSRs were present, although perfect trinucleotide repeat arrays were the most common. Primers were designed to 50 SSR loci from N. coenophialum and 57 SSR loci from N. lolii and were evaluated using 20 Neotyphodium and Epichlo? isolates. A high proportion of the N. coenophialum and N. lolii primers produced amplification products from the majority of isolates and most of these primers detected genetic variation. SSR markers from both N. coenophialum and N. lolii detected high levels of polymorphism between Neotyphodium and Epichlo? species, and low levels of polymorphism within N. coenophialum and N. lolii. SSR markers may be used in appropriate combinations to discriminate between species. Comparison with amplified fragment length polymorphism (AFLP) data demonstrated that the SSR markers were informative for the assessment of genetic variation within and between endophyte species. These markers may be used to identify endophyte taxa and to evaluate intraspecific population diversity, which may be correlated with variation for endophyte-derived agronomic traits.