Mining of SSR markers from Expressed Sequence Tags of bamboo species

With the ever increasing number of Expressed Sequence Tags (ESTs) from various sequencing projects, ESTs have become valuable and first-hand source of in-silico mining of simple sequence repeats (SSR) markers. We examined a total of 3419 EST sequences from three bamboo species, namely, Phyllostachys edulis, Bambusa oldhamii and Dendrocalamus sinicus for the presence of di- to hexa- microsatellites. The frequency of SSR containing ESTs varied from 5.36% in B. oldhamii to 13.05% in P. edulis. No SSRs were found in D. sinicus. Tri-nucleotide repeats (49.34%) were most frequent in P. edulis, while not much comparable difference in repeats was found in B. oldhamii. Flanking primer pairs were also designed in-silico for the sequences containing SSRs and their position on the genome hypothesized using similarity searching. SSRs located in open reading frame (ORF) were given functional annotation using Gene Ontology. Polymorphic SSRs were also detected using new pipeline- polySSR. Polymorphism level was very low (2.43%) and the position of the polymorphic SSRs was determined. The development of SSRs and the study of polymorphism will help in the further study of intra- and inter- gene flow, genetic structure, variability, linkage mapping and evolutionary relationships in bamboo.     

Isolation and characterization of microsatellite markers in an endangered species Dracaena cambodiana (Liliaceae)

?Premise of the study: The development of microsatellite primers in the endangered species Dracaena cambodiana will be the foundation for genetic and conservation studies of D. cambodiana and several Dracaena species. ?Methods and Results: A total of 26 microsatellite markers were developed in Chinese populations of D. cambodiana, using the Fast Isolation by AFLP of Sequences Containing Repeats (FIASCO) protocol. Among them, sixteen primer pairs generated polymorphic loci (fourteen of them successfully amplified in other four Dracaena species) and ten primer pairs produced monomorphic loci. ?Conclusions: These microsatellite markers could be used in the further investigation of population genetics of D. cambodiana and other Dracaena species.     

Development of 29 microsatellite markers for Osmanthus fragrans (Oleaceae), a traditional fragrant flowering tree of China

? Premise of the study: Microsatellite markers were developed for a traditional fragrant flowering tree of China, Osmanthus fragrans, to investigate the genetic diversity of its wild populations and to facilitate the classification and identification of O. fragrans cultivars. ? Methods and Results: Using the fast isolation by AFLP of sequences containing repeats (FIASCO) protocol, 29 primer sets were identified in two wild populations. All primer pairs displayed polymorphism. The number of alleles per locus ranged from two to eight, with a mean of 3.9. The expected and observed heterozygosities ranged from 0.125 to 0.932 and from 0.083 to 0.917, respectively. The transferability of the 29 primer pairs was tested on O. serrulatus, O. delavayi, and O. yunnanensis (three individuals for each species). Eighteen (62.1%), 16 (55.2%), and 21 (72.4%) of them were successfully amplified in O. serrulatus, O. delavayi, and O. yunnanensis, respectively. ? Conclusions: These markers will facilitate further studies on the population genetics of O. fragrans and the classification and identification of O. fragrans cultivars.     

Development and use of chloroplast microsatellites in Phaseolus spp. and other legumes

Chloroplast microsatellites (cpSSRs) provide a powerful tool to study the genetic variation and evolution of plants. We have investigated the usefulness of 39 primer pairs tagging cpSSR loci on a set of eight different genera of Leguminosae (Papilionoideae subfamily) and five species belonging to the genus Phaseolus . Thirty-six 'universal' primer pairs were retrieved from the literature, one was re-designed and a further two were designed de novo . The cpSSR loci analysed were highly polymorphic across the individuals examined. Twenty-seven primer pairs were polymorphic in the overall sample, 18 within Phaseolus , and 16 in both P. vulgaris and P. coccineus . Analysis of the plastome sequences of four Leguminosae species (obtained from GenBank) showed that in the loci targeted by universal primer pairs: (i) the originally tagged cpSSRs can be lost; (ii) other cpSSRs can be present; and (iii) polymorphism arises not only from differences in the numbers of cpSSR repeats, but often from other insertion/deletion events. Multilocus linkage disequilibrium analysis suggests that homoplasy is not a major problem in our dataset, and principal component analysis indicates intelligible relationships among the species considered. Our study demonstrates that this set of chloroplast markers provides a useful tool to study the diversity and the evolution of several legumes, and particularly P. vulgaris and P. coccineus .     

Chloroplast, mitochondrial, and nuclear microsatellites from the southern Appalachian hornwort, Nothoceros aenigmaticus (Dendrocerotaceae)

? Premise of the study: New microsatellite primers were developed for testing genetic differentiation within Nothoceros aenigmaticus and their potential use in other Nothoceros species. The microsatellites are designed to investigate partitioning of genetic variation in a taxon with a peculiar sex allopatry in the southern Appalachian Mountains and relationships with conspecific sexual populations from Mexico. ? Methods and Results: We used two methods for microsatellite development: an enriched library and second-generation shotgun sequence reads. From these two methods, a total of nine primer pairs were selected and tested on 89 southern Appalachian N. aenigmaticus accessions, nine Mexican accessions, and 16 N. vincentianus accessions. Three mitochondrial loci were recovered from the enriched library method and six loci from 454 shotgun sequencing: three were from the chloroplast and three from the nucleus. The primers amplified repeats with two to 20 alleles per locus. ? Conclusions: New microsatellite primers were developed for testing genetic differentiation within N. aenigmaticus and potentially for use in other Nothoceros species. We present one of the first reports of highly polymorphic mitochondrial microsatellites in plants.     

巨龙竹种下不同变异类型的RAPD分析

巨龙竹 (Dendrocalamussinicus)其种下具有 2个明显不同的变异类型 ,即“通直型”和“歪脚型”。本文利用 2 0个随机引物对 5 6份巨龙竹材料进行RAPD标记。首先用RAPDis tance软件计算各个体间的相似性系数 ,再用NTSYS软件对巨龙竹 5 6个样品以UPGMA方法进行聚类 ,聚类图中有 2个明显的分枝 ,一为“通直型” ,一为“歪脚型” ,结果表明巨龙竹表型的差异与遗传因素密切相关。同时用POPGENE软件和AMOVA软件分析表明 :巨龙竹种下不同变异类型的遗传变异较大 ,具有丰富的遗传多样性 ;歪脚类型的遗传多样性高于通直类型 ;巨龙竹变异类型群体之间及群体之内均出现显著的分化。     

Isolation and characterization of 52 polymorphic EST-SSR markers for Callitris columellaris (Cupressaceae)

?Premise of the study: We developed simple sequence repeat (SSR) markers from expressed sequence tags (ESTs) for Callitris columellaris sensu lato (s.l.) to elucidate population genetic structure and detect outlier loci by genome scan. ?Methods and Results: mRNA from an individual seedling was subjected to cDNA synthesis and then de novo pyrosequencing. Two hundred and nineteen primer pairs bordering sequence regions were designed from the obtained sequence data. In total, 52 showed polymorphism within 16 individuals representative of the species' entire range, with the number of alleles per locus and expected heterozygosity ranging from two to 10 and 0.06 to 0.84, respectively. ?Conclusions: The EST-SSR markers developed in this study will be useful for evaluating the range-wide genetic structure of C. columellaris s.l. and detecting outlier loci under selection, as well as providing useful markers to investigate the conservation genetics and reproductive ecology of the species.     

Development of EST-SSR markers for Elaeocarpus photiniifolia (Elaeocarpaceae), an endemic taxon of the Bonin Islands

? Premise of the study: Expressed sequence tag (EST)-derived microsatellite markers were developed for Elaeocarpus photiniifolia, an endemic taxon of the Bonin Islands. ? Methods and Results: Initially, a complementary DNA (cDNA) library was constructed by de novo pyrosequencing of total RNA extracted from a seedling. A total of 267 primer pairs were designed from the library. Of the 48 tested loci, 25 loci were polymorphic among 41 individuals representing the entire geographical range of the species, with the number of alleles per locus and expected heterozygosity ranging from two to 14 and 0.09 to 0.86, respectively. Most loci were transferable to a related species, E. sylvestris. ? Conclusions: The developed markers will be useful for evaluating the genetic structure of E. photiniifolia.     

Evaluating the potential of barley and wheat microsatellite markers for genetic analysis of<Emphasis Type="Italic"> Elymus trachycaulus</Emphasis> complex species

The potential of barley and wheat microsatellite markers for genetic analysis of Elymus trachycaulus complex species was evaluated. A set of 25 barley and 3 wheat microsatellite markers were tested for their ability to cross-amplify DNA from four accessions of E. trachycaulus and two accessions Pseudoroegneria spicata. Thirteen barley (52%) and two (68%) wheat primer pairs successfully amplified consistent products from both E. trachycaulus and P. spicata species. Four of the 15 successful primer pairs produced visible polymorphisms among the accessions tested. A higher successful rate of cross-species amplification of barley and wheat microsatellite markers in E. trachycaulus and P. spicata was found in this study. These primer pairs are now available for use as markers in genetic analysis of E. trachycaulus complex species. Our results suggest that publicly available wheat and barley microsatellite markers are a valuable resource for the genetic characterization of wild Triticeae species.     

Development of microsatellite markers for the neotropical tree species Dipteryx alata (Fabaceae)

? Premise of the study: Microsatellite markers were developed for the population genetic analyses of the neotropical tree Dipteryx alata (Fabaceae). ? Methods and Results: Microsatellites were developed from a genomic shotgun library. Polymorphism at each microsatellite loci was analyzed based on 94 individuals from three populations. Eight loci amplified successfully and presented one to 10 alleles, and expected heterozygosities ranged from 0.097 to 0.862. Four loci also amplified in Pterodon emarginatus and presented similar polymorphism. ? Conclusion: The eight microsatellite primer pairs are potentially suitable for population genetic studies and successfully amplified in another Fabaceae species.     

Isolation and characterization of microsatellite markers for a worldwide invasive weed, Chromolaena odorata (Asteraceae)

? PREMISE OF THE STUDY: Microsatellite loci were isolated and characterized for a worldwide invasive weed Chromolaena odorata (Asteraceae) to elucidate the population genetic structure and invasive history. ? MetHODS AND RESULTS: A total of 14 microsatellite primer pairs were developed using the Fast Isolation by AFLP of Sequences Containing repeats (FIASCO) protocol, and their polymorphism was assessed in two natural populations of C. odorata from Mexico and Trinidad and Tobago. Eleven loci showed polymorphism and eight of these loci were successfully amplified in Ageratina adenophora, another invasive weed related to C. odorata. ? CONCLUSIONS: These microsatellite markers are useful for investigating the population genetic structure and the history of range expansion of these invasive species.     

Portable microsatellite primers for Ficus (Moraceae)

? Premise of the study: Highly portable microsatellite primers were developed for Ficus to facilitate investigation of genetic structure of complete regional floras using a single set of markers. ? Methods and Results: Pyrosequencing of five species of Ficus produced a library of 5723 potential primers. Potential primers found in at least two species and presenting identical annealing temperatures were tested on a set of five additional Ficus species. A set of 20 primer pairs producing well-defined and easily readable peaks was retained and tests showed their potential utility for analyzing population genetic structure of 24 Ficus species from Taiwan. Numbers of alleles per locus ranged from one to six in the least variable species and from one to 17 in the most variable species. ? Conclusions: The results indicate that our set of primers can be used to analyze polymorphism and compare levels of polymorphism among Ficus species.     

五种索科线虫RAPD亲缘关系分析

采用RAPD技术构建了索科线虫4属5种的指纹图谱。从47个引物中筛选出12个稳定性好、多态性高的引物,共扩增出161条谱带,其中150条谱带具遗传多态性,占93·17%。所获片段长度大小为200~3200bp,单个引物扩增的条带数在11~16之间,平均为13·42条。采用RAPDistance软件及MEGA程序,计算Nei氏相似系数和遗传距离,建立UPGMA和NJ聚类图,两个聚类图拓扑结构相同,将5种索科线虫分为两大分支:同属于蚊幼寄生罗索属线虫的食蚊罗索线虫(Romanomermisculicivorax)与武昌罗索线虫(R.wuchangensis)亲缘关系最近,先聚在一起,再与同翅目(Homoptera)寄生长沙多索线虫(Agamermischang-shaensis)聚为一支;鳞翅目(Lepidoptera)寄生中华卵索线虫(Ovomermissinensis)和同翅目寄生两索属线虫(Amphimermissp·)亲缘关系较近,两者聚为一支。5种索线虫属内种间的遗传距离较小,食蚊罗索线虫与武昌罗索线虫之间遗传距离仅为0·1789;而属间遗传距离较大,在0·4471~0·5488之间。上述结果表明:RAPD技术可以应用于索科线虫亲缘关系的分析,能够反映出不同线虫间的遗传差距,从而成功地进行属、种的分类及进化问题研究。     

Microsatellite markers for genome analysis in Brassica. I. development in Brassica napus and abundance in Brassicaceae species

One hundred and twenty one microsatellites were identified by screening a λ phage library of Brassica napus. The distribution of these microsatellites within Brassicaceae species was estimated using 81 locus-specific primer pairs. Most of them (83%) amplified fragments either from Brassica oleracea or Brassica campestris, or from both species, whereas less than 30% detected loci in Brassica nigra. The same was true (30–35%) for more-distantly related crucifer species such as Diplotaxis ssp., Brassica tournefortii, Sinapis alba, Raphanus sativus and Eruca sativa. Only 16 microsatellite-specific primer pairs (19.8%) amplified fragments from Arabidopsis thaliana. Moreover, 61 of the primer pairs detecting 198 polymorphisms were used to estimate the extent of genetic diversity among 32 Brassica napus varieties and breeding lines. On average, four alleles per locus were observed. The spring and winter types of oilseed rape could be clearly distinguished by using the microsatellite markers in a cluster analysis. The results demonstrated the high efficiency of these markers for monitoring genetic diversity. Received: 14 April 2000 / Accepted: 3 July 2000     

Microsatellite loci for Gossypium davidsonii (Malvaceae) and other D-genome, Sonoran Desert endemic cotton species

? Premise of the study: Microsatellite primers previously developed for domesticated cotton (Gossypium hirsutum; tetraploid) were screened for their utility in investigating genetic structure and gene flow within G. davidsonii and five other wild, Mexican, D-genome cotton species (all diploid). ? Methods and Results: We screened 50 microsatellite primer pairs from the Cotton Marker Database, identifying 10 loci as polymorphic within G. davidsonii. In genotyping approximately 200 individuals from four populations, we found that the number of alleles per locus ranged from seven to 17, and mean observed and expected heterozygosity ranged from 0.145 to 0.492 and from 0.436 to 0.734, respectively. We genotyped six to 20 individuals from each of the remaining species, finding these 10 loci to cross-amplify in all cases and in most cases to be polymorphic. ? Conclusions: These markers may be useful for further investigation of population genetics of G. davidsonii and other wild D-genome cotton species.     

Development of a highly efficient callus induction and plant regeneration system for Dendrocalamus sinicus using hypocotyls as explants

Plant Cell, Tissue and Organ Culture (PCTOC) - Dendrocalamus sinicus is the largest bamboo species in the world. To aid its rapid propagation and explore its desired traits, we identified the...     

Microsatellite primers in the Peltigera dolichorhiza complex (lichenized ascomycete, Peltigerales)

? Premise of the study: Microsatellite primers were developed for the lichen-forming fungus Peltigera dolichorhiza to investigate partitioning of genetic variation in a widespread, morphologically and chemically variable taxon likely to represent a complex of cryptic lineages, including P. neopolydactyla. ? Methods and Results: Using next generation shotgun sequence reads, 331 primer pairs were designed to amplify microsatellite sequences from an African accession of P. dolichorhiza. Eleven primer pairs representing the longest repeat units identified were tested on 15 P. dolichorhiza accessions from Africa (incl. Réunion), South America, Papua New Guinea, and on two accessions of P. neopolydactyla from North America. The primers amplified di-, tri, tetra-, and pentanucelotide repeats with 3-8 alleles per locus. All individuals represent distinct multiloci genotypes. ? Conclusions: These results indicate the utility of the new microsatellite primers for testing genetic differentiation within the widespread complex of P. dolichorhiza.     

Evidence for a low level of genomic specificity of sequence-tagged-sites in Stylosanthes

Genome-specific DNA markers are of great value in many applications. Recent work on different plants and animal species indicated that PCR- (polymerase chain reaction) based genetic marker systems using specific primers are highly genome-specific. To test the genome specificity of sequence-tagged-sites (STSs) as genetic markers in Stylosanthes, 20 pairs of primers were generated. Fifteen were from randomly selected single-copy Pstl genomic clones, and the other five were from two known gene sequences. These primer pairs were analysed against a set of 24 genotypes representing 12 different Stylosanthes species. Thirteen of these primer pairs amplified successfully. Overall, there was a low level of genome specificity, suggesting a low degree of genomic divergence within this group of Stylosanthes species. Of the 312 entries (24 genotypes by 13 primer pairs), PCR amplifications were unsuccessful (little or no products) in only 16 cases. The number of banding patterns detected by each of these primer pairs varied from 2 to 12 with an average pair-wise polymorphism of 44.3%. The level of intraspecific variation detected on normal agarose gels was only 3.8%. Further evidence that diploid S. hamata and diploid S. humilis are progenitors of tetraploid S. hamata and that S. viscosa is a progenitor of S. scabra, was obtained.     

The development and evaluation of consensus chloroplast primer pairs that possess highly variable sequence regions in a diverse array of plant taxa

Although universal or consensus chloroplast primers are available, they are limited by their number and genomic distribution. Therefore, a set of consensus chloroplast primer pairs for simple sequence repeats (ccSSRs) analysis was constructed from tobacco (Nicotiana tabacum L.) chloroplast sequences. These were then tested for their general utility in the genetic analysis of a diverse array of plant taxa. In order to increase the number of ccSSRs beyond that previously reported, the target sequences for SSR motifs was set at A or T (n 7) mononucleotide repeats. Each SSR sequence motif, along with ±200-bp flanking sequences from the first of each mononucleotide base repeat, was screened for homologies with chloroplast DNA sequences of other plant species in GenBank databases using BLAST search procedures. Twenty three putative marker loci that possessed conserved flanking sequence spans were selected for consensus primer pair construction using commercially available computer algorithms. All primer pairs produced amplicons after PCR employing genomic DNA from members of the Cucurbitaceae (six species) and Solanaceae (four species). Sixteen, 22 and 19 of the initial 23 primer pairs were successively amplified by PCR using template DNA from species of the Apiaceae (two species), Brassicaceae (one species) and Fabaceae (two species), respectively. Twenty of 23 primer pairs were also functional in three monocot species of the Liliaceae [onion (Allium cepa L.) and garlic (Allium sativum L.)], and the Poaceae [oat (Avena sativa L.)]. Sequence analysis of selected ccSSR fragments suggests that ccSSR length and sequence variation could be useful as a tool for investigating the genetic relationships within a genus or closely related taxa (i.e., tribal level). In order to provide for a marker system having significant coverage of the cucumber chloroplast genome, ccSSR primers were strategically "recombined" and named recombined consensus chloroplast primers (RCCP) for PCR analysis. Successful amplification after extended-length PCR of 16 RCCP primer pairs from cucumber (Cucumis sativus L.) DNA suggested that the amplicons detected are representative of the cucumber chloroplast genome. These RCCP pairs, therefore, could be useful as an initial molecular tool for investigation of traits related to a chloroplast gene(s) in cucumber, and other closely related species.Communicated by C. Möllers     

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