Sequence-tagged microsatellite profiling (STMP): a rapid technique for developing SSR markers

We describe a technique, sequence-tagged microsatellite profiling (STMP), to rapidly generate large numbers of simple sequence repeat (SSR) markers from genomic or cDNA. This technique eliminates the need for library screening to identify SSR-containing clones and provides an ~25-fold increase in sequencing throughput compared to traditional methods. STMP generates short but characteristic nucleotide sequence tags for fragments that are present within a pool of SSR amplicons. These tags are then ligated together to form concatemers for cloning and sequencing. The analysis of thousands of tags gives rise to a representational profile of the abundance and frequency of SSRs within the DNA pool, from which low copy sequences can be identified. As each tag contains sufficient nucleotide sequence for primer design, their conversion into PCR primers allows the amplification of corresponding full-length fragments from the pool of SSR amplicons. These fragments permit the full characterisation of a SSR locus and provide flanking sequence for the development of a microsatellite marker. Alternatively, sequence tag primers can be used to directly amplify corresponding SSR loci from genomic DNA, thereby reducing the cost of developing a microsatellite marker to the synthesis of just one sequence-specific primer. We demonstrate the utility of STMP by the development of SSR markers in bread wheat.     

PERMANENT GENETIC RESOURCES: Development of microsatellite markers for the guava rust fungus, Puccinia psidii

We developed and characterized 15 polymorphic microsatellite markers present in the genome of the guava rust fungus, Puccinia psidii. The primers for these microsatellite markers were designed by sequencing clones from a genomic DNA library enriched for a simple sequence repeat (SSR) motif of (AG). All these 15 primer pairs successfully amplified DNA fragments from a sample of 22 P. psidii isolates, revealing a total of 71 alleles. The observed heterozygosity at the 15 loci ranged from 0.05 to 1.00. The SSR markers developed would be useful for population genetics study of the rust fungus.     

Sequence tagged microsatellite profiling (STMP): improved isolation of DNA sequence flanking target SSRs

Sequence tagged microsatellite profiling (STMP) enables the rapid development of large numbers of co-dominant DNA markers, known as sequence tagged microsatellites (STMs). Each STM is amplified by PCR using a single primer specific to the conserved DNA sequence flanking the microsatellite repeat in combination with a universal primer that anchors to the 5′-ends of the microsatellites. It is also possible to convert STMs into conventional microsatellite, or simple sequence repeat (SSR), markers that are amplified using a pair of primers flanking the repeat sequence. Here, we describe a modification of the STMP procedure to significantly improve the capacity to convert STMs into conventional SSRs and, therefore, facilitate the development of highly specific DNA markers for purposes such as marker-assisted breeding. The usefulness of this technique was demonstrated in bread wheat.     

茶树EST-SSRs分布特征及引物开发

为了在茶树中开发EST-SSRs功能性标记,利用生物信息学方法对NCBI网上公开的3288奈茶树（Camellia subebsus）ESTs序列进行EST-SSRs特征分析。剔除冗余序列,得到非冗余序列2083条。在非冗余序列中发现含不同重复基元SSRs的EST序列有385条,共486个EST-SSRs,平均相隔2．10kb出现1个SSR。在2～6bp的重复基元中,二核苷酸重复基元的SSRs出现频率最高（51．97％）,其次是三核苷酸（19．55％）。对所有的重复基元类型进行统计分析发现,所占比例最高的是AG／CT（47．74％）,其次分别是AT／TA（4．73％）和AAG／CTT（4．73％）。利用Prime5软件,设计了206对EST-SSRs引物,随机选用72对引物进行SSR扩增,发现31对引物可以扩增出条带,其中29对引物具有多态性,多态性比率为93．5％。这些EST-SSRs将有助于茶树基因组学方面的研究。     

磁珠富集法开发北柴胡多态性简单序列重复标记

目的:利用磁珠富集法分离北柴胡微卫星序列,以开发北柴胡微卫星引物,获得有多态性的简单序列重复(SSR)标记。方法:用生物素标记的混合探针(AC)15、(AG)15、(MAB)12和两端连接已知序列人工接头的北柴胡基因组DNA酶切片段混和后与磁珠杂交,构建微卫星序列富集的小片段插入文库;利用接头引物分别与生物素探针引物Biotin-(AC)15、Biotin-(AG)15、Biontin-(MAB)12形成3个组合,用PCR方法对文库进行初步筛选;对可能的阳性克隆子进行测序复筛,选取微卫星侧翼序列足够长的序列设计引物,用荧光标记的基因分型技术以栽培柴胡种质为材料分析其多态性。结果:开发了5对多态性SSR标记,它们在5份柴胡栽培种质中共扩增出30.70个多态性等位基因,平均每条引物可以扩增出6.14个多态性等位基因;观察等位基因数最多13个,最少3个;有效等位基因数最多11.4个,最少1.6个。同时分析了4对EST-SSR引物,比较了2种SSR标记扩增结果。结论:磁珠富集法是开发柴胡多态性SSR标记的有效方法。     

An improved technique for isolating codominant compound microsatellite markers

An approach for developing codominant polymorphic markers (compound microsatellite (SSR) markers), with substantial time and cost savings, is introduced in this paper. In this technique, fragments flanked by a compound SSR sequence at one end were amplified from the constructed DNA library using compound SSR primer (AC)₆(AG)₅ or (TC)₆(AC)₅ and an adaptor primer for the suppression-PCR. A locus-specific primer was designed from the sequence flanking the compound SSR. The primer pairs of the locus-specific and compound SSR primers were used as a compound SSR marker. Because only one locus-specific primer was needed for design of each marker and only a common compound SSR primer was needed as the fluorescence-labeled primer for analyzing all the compound SSR markers, this approach substantially reduced the cost of developing codominant markers and analyzing their polymorphism. We have demonstrated this technique for Dendropanax trifidus and easily developed 11 codominant markers with high polymorphism for D. trifidus. Use of the technique for successful isolation of codominant compound SSR markers for several other plant species is currently in progress.     

花生微卫星标记的研究进展

近年来花生微卫星标记的开发取得了一定的进展, 初步揭示了花生在DNA水平上的遗传多样性。花生微卫星标记的开发途径主要包括通过构建小片段基因组文库开发基因组SSR标记, 根据花生EST序列开发EST-SSR标记, 根据豆科植物序 列信息和SSR标记开发花生SSR标记, 将SSR标记与其它分子标记结合开发新的DNA标记, 以及基于SSR核心序列开发ISSR标记。花生微卫星标记主要应用于遗传多样性研究、遗传图谱与品种指纹图谱构建以及分子标记辅助育种等领域。本文综述了花生SSR标记开发研究的进展及应用。     

GinMicrosatDb: a genome-wide microsatellite markers database for sesame (<Emphasis Type="Italic">Sesamum indicum</Emphasis> L.)

Molecular breeding in sesame is still at infancy due to limited number of microsatellite markers available and the low level of polymorphism exhibited by them. Therefore, whole genome sequencing was used for development of microsatellite markers so as to ensure availability of substantial number of polymorphic markers for use in marker assisted breeding programs. Whole genome sequencing of sesame variety ‘Swetha’ was done using Illumina paired-end sequencing and Roche 454 shotgun sequencing technologies (GCA_000975565.1 in GenBank). ‘GinMicrosatDb’, a genome-wide microsatellite marker database has been developed using the whole genome sequence data of sesame variety ‘Swetha’. The database consists of microsatellites localized on both linkage groups and scaffolds with their genomic co-ordinates. It provides five sets of forward and reverse primers for each of the microsatellite loci along with the flanking sequences, primer GC content, product size and melting temperature etc. The distribution of microsatellites can be viewed and selected through a genome browser as well as through a physical map. The newly identified microsatellite markers are expected to help sesame breeders in developing marker tags for traits of economic importance thereby bringing about greater efficiency in marker-assisted selection programs.     

花生微卫星标记的研究进展

近年来花生微卫星标记的开发取得了一定的进展,初步揭示了花生在DNA水平上的遗传多样性。花生微卫星标记的开发途径主要包括通过构建小片段基因组文库开发基因组SSR标记,根据花生EST序列开发EST-SSR标记,根据豆科植物序列信息和SSR标记开发花生SSR标记,将SSR标记与其它分子标记结合开发新的DNA标记,以及基于SSR核心序列开发ISSR标记。花生微卫星标记主要应用于遗传多样性研究、遗传图谱与品种指纹图谱构建以及分子标记辅助育种等领域。本文综述了花生SSR标记开发研究的进展及应用。     

Detection of universal variable fragments as markers for genetic studies. A novel technology for DNA fingerprinting

A novel DNA technology enables the detection of universal variable fragments (UVF), thus revealing genetic variation without a priori sequence information. The detection of UVF markers is based on two amplifications of genomic DNA with the polymerase chain reaction. In the first amplification, two short oligonucleotide primers produce a large number of fragments. One primer is based on a microsatellite sequence, whereas the second primer can have any sequence. In the second amplification, the length of the primers is increased in order to decrease the number of amplicons. This enables the selection of polymorphic fragments. Restriction digestion can be used to further increase the number of polymorphisms. Until now, we have demonstrated UVF in several different species. In addition, with the present study we have contributed to the linkage map of the rabbit by localizing 11 UVF markers on different linkage groups. Mendelian inheritance was shown in this linkage study through a backcross of two inbred rabbit strains. The power of the UVF technique is based on the selection for microsatellite variation in combination with the detection of single-nucleotide polymorphisms. UVF thus offers the possibility of increasing the clustering of markers and localizing genes in species for which sequence information is either not present or only scarcely present.     

Direct sequencing of RAPD fragments using 3'-extended oligonucleotide primers and dye terminator cycle-sequencing.

Random amplified polymorphic DNA (RAPD) markers are used widely to develop high resolution genetic maps and for genome fingerprinting. Typically, single oligomers of approximately 10 nucleotides are used to PCR amplify characteristic RAPD marker fragments. We describe an efficient method for the direct end-sequencing of gel-purified RAPD fragments using one primer from a set of four 3'-terminal extended (A, T, C or G) oligonucleotides, identical to the RAPD primer but for the single nucleotide extension. Strand-specific DNA sequence could be independently read from each of the RAPD fragments without recourse to strand separation or fragment cloning. Informative RAPD fragments could be readily converted into mapped STS or SCAR loci using this technology. The 3'-extended primers may also be used to amplify independent genomic RAPD markers.     

桥式PCR，一种简易连接DNA标签序列的方法

MAST方法采用人工文库的DNA标签序列鉴定mRNA的可接近位点。大量的标签序列通过扩增和克隆测序达到阐明mRNA结合位点图。设计了单一引物的PCR,其引物在标签序列两端结合搭桥,在扩增中DNA标签序列在搭桥引物的作用下进行连接,连接的标签序列再克隆和测序。十几条这样的连接产物包含了上千条标签序列。该PCR方法简单、高效以用于高通量的方式对标签序列测序。     

Assessment of genetic diversity and phylogenetic relationship among grouper species Epinephelus spp. from the Saudi waters of the Arabian Gulf

Understanding of fish genetic characterization plays a vital role in the conservation and utilization of fish genetic resources of grouper species. The present study was carried out to assess the genetic diversity and phylogenetic relationships in five grouper species, Epinephelus spp. from eastern Saudi Arabian coast using two molecular marker systems, inter simple sequence repeat (ISSR) and microsatellite (SSR) markers. In total, 219 individuals grouper specimens (Epinephelus tauvina, E. coioides, E. bleekeri, E. malabaricus, and E. areolatus) were genotyped with 10 ISSR and 11 SSR selected primers. The ISSR produced 94 DNA fragments, of which 44 were polymorphic with an average of 2.13 fragment per primer. While SSR primers generated 107 alleles, all of them were polymorphic with an average 9.72 per primer. ISSR and SSR techniques demonstrated a high level of gene diversity and genetic distances illustrated by UPGMA dendrograms among the grouper species. The results proved that the SSR markers were highly informative and efficient in detecting genetic variability and relationships of the Epinephelus spp.     

荔枝SSR标记的研究

以无核荔枝A4号为实验材料,应用选择性扩增微卫星（SAM）法分离、克隆了100个简单序列重复（SSR）序列,其中88个非重复,可用。加上搜索数据库所获得的1个SSR序列,一共89个序列用于特异引物的设计。仅从71个序列的82个基因座设计出特异引物。合成41条特异引物(与5′锚定简并引物配对,个别相互配对),对其中的39个基因座进行检测。其中15对引物扩增出相应大小的片段,另外11对引物扩增出非预期片段。最后,以37个荔枝种质的基因组DNA为模板,从26对出带的引物中,筛选出多态性引物21对,获得了22个荔枝基因座特异性SSR标记。     

Use of bovine EST data and human genomic sequences to map 100 gene-specific bovine markers

A system to use bovine EST data in conjunction with human genomic sequence to improve the bovine linkage map over the entire genome or on specific chromosomes was evaluated. Bovine EST sequence was used to provide primer sequences corresponding to bovine genes, while human genomic sequence directed primer design to flank introns and produce amplicons of appropriate size for efficient direct sequencing. The sequence tagged sites (STS) produced in this way from the four sires of the MARC reference families were examined for single nucleotide polymorphisms (SNPs) that could be used to map the corresponding genes. With this approach, along with a primer/extension mass spectrometry SNP genotyping assay, 100 ESTs were placed on the bovine genetic linkage map. The first 70 were chosen at random from bovine EST–human genomic comparisons. An additional 30 ESTs were successfully mapped to bovine Chromosome 19 (BTA19), and comparison of the resulting BTA19 map to the position of the corresponding human orthologs on the HSA17 draft sequences revealed differences in the spacing and order of genes. Over 80% of successful amplicons contained SNPs, indicating that this is an efficient approach to generating EST-associated genetic markers. We have demonstrated the feasibility of constructing a linkage map based on SNPs associated with ESTs and the plausibility of utilizing EST, comparative mapping information, and human sequence data to target regions of the bovine genome for SNP marker development.     

SSR-based linkage map with new markers using an intraspecific population of common wheat

Simple sequence repeats (SSRs) are valuable molecular markers in many plant species. In common wheat (Triticum aestivum L.), which is characteristic of its large genomes and alloploidy, SSRs are one of the most useful markers. To increase SSR marker sources and construct an SSR-based linkage map of appropriate density, we tried to develop new SSR markers from SSR-enriched genomic libraries and the public database. SSRs having (GA)n and (GT)n motifs were isolated from enriched libraries, and di- and tri-nucleotide repeats were mined from expressed sequence tags (ESTs) and DNA sequences of Triticum species in the public database. Of the 1,147 primer pairs designed, 842 primers gave accurate amplification products, and 478 primers showed polymorphism among the nine wheat lines examined. Using a doubled haploid (DH) population from an intraspecific cross between Kitamoe and Münstertaler (KM), we constructed an SSR-based linkage map that consisted of 464 loci: 185 loci from genomic libraries, 65 loci from the sequence database including ESTs, 213 loci from the SSR markers already reported, and 1 locus of morphological marker. Although newly developed SSR loci were distributed throughout all chromosomes, clustering of them around putative centromeric regions was found on several chromosomes. The total length of the KM map spanned 3,441 cM and corresponded to approximately 86% genome coverage. The KM map comprised of 23 linkage groups because two gaps of over 50 cM distance remained on chromosome 6A. This is a first report of SSR-based linkage map using single intraspecific population of common wheat. This mapping result suggests that it becomes possible to construct linkage maps with sufficient genome coverage using only SSR markers without RFLP markers, even in an intraspecific population of common wheat. Moreover, the new SSR markers will contribute to the enrichment of molecular marker resources in common wheat.     

Characterization of the Kenaf (Hibiscus cannabinus) Global Transcriptome Using Illumina Paired-End Sequencing and Development of EST-SSR Markers

Kenaf (Hibiscus cannabinus L.) is an economically important natural fiber crop grown worldwide. However, only 20 expressed tag sequences (ESTs) for kenaf are available in public databases. The aim of this study was to develop large-scale simple sequence repeat (SSR) markers to lay a solid foundation for the construction of genetic linkage maps and marker-assisted breeding in kenaf. We used Illumina paired-end sequencing technology to generate new EST-simple sequences and MISA software to mine SSR markers. We identified 71,318 unigenes with an average length of 1143 nt and annotated these unigenes using four different protein databases. Overall, 9324 complementary pairs were designated as EST-SSR markers, and their quality was validated using 100 randomly selected SSR markers. In total, 72 primer pairs reproducibly amplified target amplicons, and 61 of these primer pairs detected significant polymorphism among 28 kenaf accessions. Thus, in this study, we have developed large-scale SSR markers for kenaf, and this new resource will facilitate construction of genetic linkage maps, investigation of fiber growth and development in kenaf, and also be of value to novel gene discovery and functional genomic studies.