Development of genomic simple sequence repeat markers from an enriched genomic library of grass pea (Lathyrus sativus L.)

Simple sequence repeat (SSR) or microsatellite markers are a valuable tool for several purposes such as evaluation of genetic diversity, fingerprinting, marker‐assisted selection and breeding. In this study, a SSR genomic enriched library was developed in Lathyrus sativus (grass pea) by affinity capture of restriction fragments to biotinylated microsatellite oligonucleotides. About 400 randomly selected clones were sequenced, and SSRs were present in approximately 30% of them. Clones contained 75%, 9% and 16% of simple, interrupted and compound SSRs, respectively. Of the 10 SSRs tested, 7 primer pairs produced clearly distinguishable DNA banding patterns. Successively, SSR primer pairs were successfully tested to reveal polymorphism in a set of four different grass pea germplasm accessions. The transferability of SSR markers was high among three related species of Lathyrus, namely Lathyrus cicera, Lathyrus ochrus and Lathyrus tingitanus, and the legume crop, Pisum sativum. These results indicate that the novel SSR markers are informative and will be useful and convenient for genetic analysis in grass pea and related species.     

用于区别不同棉花品种基因组特征的微卫星位点筛选

保守性强、重复性好、多态性高的微卫星位点可被有效用于构建作物DNA条形码。选取目前生产上主要推广种植、代表不同来源系统的12个棉花品种作为微卫星位点筛选材料,参考我室构建的四倍体栽培棉种种间高密度遗传图谱信息,从376对覆盖全基因组的SSR引物中,筛选出51对引物可扩增出带型清晰且多态性高的微卫星位点。这些引物在12个供试品种中共产生155个等位位点,每对引物揭示的等位基因位点在2~7之间,平均值为3.04。参照微卫星位点的染色体定位和多态信息,在每条染色体上选择一个多态性相对高的SSR位点,其相应的26对SSR引物被推荐为构建棉花品种DNA条形码的一套首选引物,并初步应用于12个品种的DNA条形码编制。其余25对引物作为候选引物。使用该套引物扩增出的微卫星位点可用于大量棉花品种DNA条形码构建,为棉花品种真实性和纯度的分子鉴定奠定基础。     

Development of EST‐SSR markers for diversity and breeding studies in opium poppy

All publicly available opium poppy expressed sequence tag (EST) sequences, totalling 20 885, were assembled into unigenes and examined for simple sequence repeats (SSRs). Nearly 19% of the 14 957 unigenes contained SSRs with 4% harbouring more than one SSR. Average density of the SSRs was 1 SSR per 3.6 kb of non‐redundant EST sequence. Trinucleotide SSRs were most frequently identified (39%), and many of the most prevalent motifs were AT‐rich. Flanking primers were designed for 86% of the SSRs and 67 primer pairs were tested on 37 opium poppy accessions and seven related species. All markers were transferable to the related species. Polymorphism information content (PIC) values for the markers were intermediate for comparisons within opium poppy (average of 0.27) and slightly higher for comparisons across species (average of 0.29). The markers were found to be useful for diversity analysis as they successfully distinguished among Turkish opium poppy accessions and land races.     

Identification and characterization of EST-SSRs in finger millet (<Emphasis Type="Italic">Eleusine coracana</Emphasis> (L.) Gaertn.)

In recent years, microsatellites have become the most used markers for studying population genetic diversity. The increased availability of the DNA sequences has given the possibility to develop EST-derived SSR markers. A total of 1,927 ESTs of Eleusine coracana available in the NCBI database were mined for SSRs. Di-nucleotides are the most occurring motifs accounting for more than 50% of the repeats, of which GA was the most abundant motif and tetra-nucleotides are the least occurring motifs. Of the 132 markers identified, 30 primer pairs based were synthesized. SSR markers were used for variety discrimination and genetic assessment in 15 finger millet accessions; 20 primers showed polymorphism and 13 primers were identified as having a PIC value above 0.5. On the basis of the distribution of these polymorphic alleles, the 15 accessions were classified into two groups. This study has demonstrated the potential of EST-derived SSR primer pairs in finger millet. These primers will serve as valuable source for further breeding programs.     

Linkage map construction and mapping QTL for cotton fibre quality using SRAP, SSR and RAPD

Tetraploid cotton is one of the most extensively cultivated species. Two tetraploid species, Gossypium hirsutum L. and G. barbadense L., dominate the world's cotton production. To better understand the genetic basis of cotton fibre traits for the improvement of fibre quality, a genetic linkage map of tetraploid cotton was constructed using sequence‐related amplified polymorphisms (SRAPs), simple sequence repeats (SSRs) and random amplified polymorphic DNAs (RAPDs). A total of 238 SRAP primer combinations, 368 SSR primer pairs and 600 RAPD primers were used to screen polymorphisms between G. hirsutum cv. Handan208 and G. barbadense cv. Pima90 which revealed 749 polymorphic loci in total (205 SSRs, 107 RAPDs and 437 SRAPs). Sixty‐nine F₂ progeny from the interspecific cross of ‘Handan208’בPima90’ were genotyped with the 749 polymorphic markers. A total of 566 loci were assembled into 41 linkage groups with at least three loci in each group. Twenty‐eight linkage groups were assigned to corresponding chromosomes by SSR markers with known chromosome locations. The map covered 5141.8 cM with a mean interlocus space of 9.08 cM. A × test for significance of deviations from the expected ratio (1: 2: 1 or 3: 1) identified 135 loci (18.0%) with skewed segregation, most of which had an excess of maternal parental alleles. In total, 13 QTL associated with fibre traits were detected, among which two QTL were for fibre strength, four for fibre length and seven for micronaire value. These QTL were on nine linkage groups explaining 16.18‐28.92% of the trait variation. Six QTL were located in the A subgenome, six QTL in the D subgenome and one QTL in an unassigned linkage group. There were three QTL for micronaire value clustered on LG1, which would be very useful for improving this trait by molecular marker‐assisted selection.     

BAC-derived SSR markers chromosome locations in cotton

Bacterial artificial chromosome (BAC) libraries with large DNA fragment inserts have rapidly become the preferred choice for physical mapping. BAC-derived microsatellite or simple sequence repeats (SSRs) markers facilitate the integration of physical maps with genetic maps. The objective of this research was to identify chromosome locations of the BAC-derived SSR markers in tetraploid cotton. A total of 192 SSR primer pairs were derived from BAC clones of an Upland cotton genetic standard line TM-1 (Gossypium hirsutum L.). Metaphor agarose gel electrophoresis results revealed 76 and 59 polymorphic markers between TM-1 and 3–79 (G. barbadense) or G. tomentosum, respectively. Using deletion analysis method, we assigned 39 markers out of the 192 primer pairs to 17 different chromosomes or chromosome arms. Among them, 19 and 17 markers were localized to A-subgenomes (chromosome 1–13) and D-subgenomes (chromosome 14–26), respectively. The subgenome status for the remaining three markers remained unclear due to their two potential chromosome locations achieved by tertiary monosomic stocks deletion analysis. Chromosomal assignment of these BAC-derived SSR markers will help in integrating physical and cotton genetic linkage maps and thus facilitate positional candidate gene cloning, comparative genome analysis, and the coordination of chromosome-based genome sequencing project in cotton. Disclaimer: Mention of trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product by USDA, ARS and does not imply its approval to the exclusion of other products or vendors that may also be suitable. The U.S. Government’s right to retain a non-exclusive, royalty-free license in and to any copyright is acknowledged.     

棉花非冗余性EST-SSR新标记的开发及其评价

利用Clustal X等软件对公共数据库现有的393 753条棉花EST序列分析,得到349 815条非冗余EST序列,借助自主开发的SSRmine软件共发掘SSR位点11 372个,分布于10 507条EST中,EST-SSR的频率是3%,平均相隔21 kb出现一个SSR。在2~6 bp的重复基元中,三核苷酸和六核苷酸分别占34.1%、40.6%,二、三、四、五和六核苷酸基序分别以AG/CT、AAG/CTT、AAAT/ATTT、AAAAG/CTTTT和AAAAAG/CTTTTT的类型最多。利用去冗余的且在亚洲棉、陆地棉、海岛棉中没有被开发过的410条EST序列设计开发了200对非冗余性SSR引物,利用自主开发的SSRD软件通过SSR引物序列下载、预处理、Blastn、提取相似性分值≥81%的引物编号、提取引物冗余对、冗余引物写成一行等6个步骤去除来源于自身部分同源序列以及与CMD释放的不同棉种相似性SSR引物,得到了非相似性引物,定名为CRIXXX (CRI即Cotton Research Institute)。并分别选用棉花12个种的代表性材料对其中100对进行引物功效评价,包括多态信息含量(polymorphism information content, PIC)及引物通用性研究。结果显示,从自主开发的100对SSR引物筛选出56对均能在12份材料间扩增出稳定明显的条带, 其中多态性引物35对,多态率占35%。引物的PIC变幅为0.097~0.888,平均为0.482;1对海岛棉EST-SSR引物在12份材料间的通用性为100%,25对亚洲棉引物通用性为81%,74对陆地棉引物通用性为80.1%。     

Mapping resistance gene analogs (RGAs) in cultivated tetraploid cotton using RGA-AFLP analysis

Diseases cause significant losses in cotton production throughout the US Cotton Belt. Growing resistant cultivars can significantly improve cotton yields and effectively reduce production inputs. Disease resistance (R) genes have been isolated in numerous plant species and the R genes with domains of nucleotide binding sites (NB) and leucine rich repeats (LRR) represent the largest R gene family. Degenerate primers designed based on conserved motifs of plant disease resistance genes were used alone or in combination with AFLP primers to analyze disease resistance gene analogs (RGAs) in a recombinant inbred line (RIL) population of Pima (Gossypium barbadense) 3–79 and Upland cotton (G. hirsutum) line NM 24016. Eighty-eight polymorphic RGA markers were amplified by 8 pairs of RGA degenerate primers, while 131 polymorphic RGA-AFLP markers were produced from six pairs of RGA-AFLP primer combinations. Of the 219 polymorphic RGA and RGA-AFLP markers that were identified, 212 were assigned to 18 chromosomes and linkage groups based on existing SSR markers that are on known chromosomes. However, the RGA and RGA-AFLP markers are not evenly distributed among chromosomes in that 189 RGA and RGA-AFLP markers (88%) are assigned onto three “giant” chromosomes, i.e., C6, C12, and C15, suggesting RGA clusters in the cotton genome. Several RGA and RGA-AFLP markers were mapped to the same linkage group carrying a root-knot nematode resistance gene. The identification and mapping of RGA and RGA-AFLP markers provide a framework to facilitate marker-assisted selection of disease resistance in cotton breeding and to understand the physical relationship of cotton resistance genes.     

Genetic relationships of Brassica vegetables determined using database derived simple sequence repeats

Sequence databases were screened to identify simple sequence repeats (SSRs) in Brassica oleracea sequences. A total of 512 B. oleracea DNA sequences were screened and 43 potential SSRs were identified. Thirty-six primer pairs were designed to amplify target sequences. Of the 36 primer pairs, six failed to amplify fragments of expected sizes, and 17 primer pairs failed to generate polymorphisms. Thirteen SSRs were used to assess genetic similarity between 54 B. oleracea cultivars, belonging to 3 variteal groups (cabbage, cauliflower, and broccoli). Pairwise genetic similarities were calculated for cultivars, and a dendrogram of relationships was produced. All cabbage cultivars were distinguished from each other and clustered in two separate groups. Five cauliflower cultivars could not be distinguished with SSR markers used in the study. Three broccoli cultivars clustered with cauliflower cultivars, and two cauliflower cultivars grouped with broccoli cultivars. The varietal group with the narrowest genetic variation in the study was cauliflower (B. oleracea var. botrytis) followed by broccoli (B. oleracea var. italica) and cabbage (B. oleracea var. capitata) groups. Polymorphism information content (PIC) values and number of alleles produced per marker ranged between 0.25 to 0.86 and 1 to 8, respectively, for database derived SSR markers.     

Development and characterization of tomato SSR markers from genomic sequences of anchored BAC clones on chromosome 6

Simple sequence repeat motifs are abundant in plant genomes and are commonly used molecular markers in plant breeding. In tomato, currently available genetic maps possess a limited number of simple sequence repeat (SSR) markers that are not evenly distributed in the genome. This situation warrants the need for more SSRs in genomic regions lacking adequate markers. The objective of the study was to develop SSR markers pertaining to chromosome 6 from bacterial artificial chromosome (BAC) sequences available at Solanaceae Genomics Network. A total of 54 SSR primer pairs from 17 BAC clones on chromosome 6 were designed and validated. Polymorphism of these loci was evaluated in a panel of 16 genotypes comprising of Solanum lycopersicum and its wild relatives. Genetic diversity analysis based on these markers could distinguish genotypes at species level. Twenty-one SSR markers derived from 13 BAC clones were polymorphic between two closely related tomato accessions, West Virginia 700 and Hawaii 7996 and were mapped using a recombinant inbred line population derived from a cross between these two accessions. The markers were distributed throughout the chromosome spanning a total length of 117.6 cM following the order of the original BAC clones. A major QTL associated with resistance to bacterial wilt was mapped on chromosome 6 at similar location of the reported Bwr-6 locus. These chromosome 6-specific SSR markers developed in this study are useful tools for cultivar identification, genetic diversity analysis and genetic mapping in tomato.     

Characterization of dinucleotide and trinucleotide EST-derived microsatellites in the wheat genome

Over the past decade microsatellites or simple sequence repeats (SSRs) have attracted a considerable amount of attention from researchers. The aim of the present paper was to analyse expressed sequence tag-derived SSR (EST-SSR) marker variability in wheat and to investigate the relationships between the number and type of repeat units and the level of microsatellite polymorphism. Two hundred and forty-one new EST-SSR markers available in a public database () were characterized in eight durum wheat cultivars (Svevo, Ciccio, Primadur, Duilio, Meridiano, Claudio, Latino, Messapia), two accessions of Triticum turgidum var. dicoccoides (MG4343, MG29896), one accession of T. turgidum var. dicoccum (MG5323) and in the common wheat cv. Chinese Spring. Of these, 201 primer pairs (83.4%) amplified PCR products successfully, while the remaining 40 (16.6%) failed to amplify any product. Of the EST-SSRs analysed, 45.2% of the primer pairs amplified one or two PCR products. Multiple discrete PCR products were observed among both di- and trinucleotide EST-SSR markers (31.2 and 40.5%, respectively). Markers based on dinucleotide microsatellites were more polymorphic than those based on trinucleotide SSRs in the 12 wheat genotypes tested (68.9 and 52.7%, respectively). An average of 2.5 alleles for dinucleotide and 2.0 alleles for trinucleotide SSRs was observed. The data reported in the present work indicate the presence of a significant relationship between motif sequence types and polymorphism. The primer set based on the AG repeat motif showed the lowest percentage of polymorphism (55.0%), while the primer set based on the AC repeat motif showed t he highest percentage (85.0%). Among trinucleotide SSRs, the AGG microsatellite markers showed the highest percentage of polymorphism (70.0%), and the ACG motif the lowest value (25.0%). The characterization of these new EST-SSR markers and the results of our studyon the effect of repeat number and type of motifs could have important applications in the genetic analysis of agronomically important traits, quantitative trait locus discovery and marker-assisted selection.     

High‐throughput development of SSR marker candidates and their chromosomal assignment in rye (Secale cereale L.)

Shotgun survey sequences of flow‐sorted individual rye chromosomes were data mined for the presence of simple sequence repeats (SSRs). For 787,850 putative SSR loci, a total of 358,660 PCR primer pairs could be designed and 51,138 nonredundant SSR marker candidates were evaluated by in silico PCR. Of the 51,138 SSR primer candidates, 1,277 were associated with 1,125 rye gene models. A total of 2,112 of the potential SSR markers were randomly selected to represent about equal numbers for each of the rye chromosomes, and 856 SSRs were assigned to individual rye chromosomes experimentally. Potential transferability of rye SSRs to wheat and barley was of low efficiency with 4.3% (2,189) and 0.4% (223) of rye SSRs predicted to be amplified in wheat and barley, respectively. This data set of rye chromosome‐specific SSR markers will be useful for the specific detection of rye chromatin introgressed into wheat as well as for low‐cost genetic and physical mapping in rye without the need for high‐tech equipment.     

濒危珍稀植物半枫荷转录组中SSR位点分析

分析半枫荷转录组中的SSR位点信息,并设计简单重复序列(SSR)引物,以期为半枫荷EST-SSR分子标记提供有力工具。利用MISA工具筛选了半枫荷转录组测序获得的77629条Unigenes,对其SSR位点信息进行了分析;在此基础上利用Primer 3.0设计SSR引物,并随机选择50对SSR引物对4株不同来源的半枫荷进行多态性扩增分析。在半枫荷的转录组中,共找到15041个SSRs,分布于10669条Unigenes,SSR位点发生频率为13.74%,含多个位点的序列数为3114,占SSRs位点总数的29.19%,以复合形式出现的位点数2044个,占SSRs位点总数的19.16%,SSRs的平均距离是3.2 kb。SSRs位点中二碱基重复是主要类型,占总SSRs的42.17%;其次是单碱基重复基序(38.25%)SSRs。所包含的重复基元中,单碱基重复基元A/T(5572),二碱基重复基元AG/CT(4845)是优势重复基元类型,分别占总SSRs的37.05%、32.21%。利用Primer 3共设计出28590对SSR引物。随机选择50对引物进行PCR扩增,其中44对(88.0%)扩增出清晰、可重复的条带,15对(34.1%)扩增条带表现出多态性。半枫荷转录组SSR位点出现频率高,类型丰富;大量的SSR为其遗传多样性分析、分子标记辅助育种和遗传图谱构建提供了丰富的候选分子标记。     

基于转录组测序的槜李EST-SSR标记开发

为开发槜李EST-SSR标记,本研究利用MISA软件筛选了槜李花转录组测序获得的35584条Unigenes,对其SSR信息进行分析后,利用Primer Premier 3.0软件设计EST-SSR引物,并随机选取40对SSR引物对12个李品种进行EST-SSR引物筛选及多态性分析。结果发现,在槜李花转录组中共搜索到个10791个SSR位点,分布于8433条Unigenes,SSR发生频率为23.70%,平均每3.71 kb含有1个SSR;SSR重复基元中二核苷酸重复出现频率最高,占总SSR数量的52.98%,其次为三核苷酸重复(占24.00%)和单核苷酸重复(占20.95%);二核苷酸重复基元以AG/CT为主(85.95%),三核苷酸重复基元以AAG/CTT为主(31.24%)。利用Primer Premier 3.0软件共设计出9870对候选引物,随机选择40对引物对12个李品种进行SSR引物筛选及多态性分析。40对引物均能扩增出预期大小的条带,有效扩增效率为100%,40对引物中有5对引物在12个李品种中表现出多态性。本研究开发的EST-SSR标记可为李属植物遗传多样性分析提供丰富的候选标记,同时可为槜李发育相关功能基因定位、遗传图谱构建、及分子标记辅助育种等研究提供帮助。     

基于桑葚转录组测序的SSR位点分析

采用转录组测序技术对三个时期的桑葚进行转录组测序,建立桑葚的EST数据库。基于生物信息学方法分析桑葚转录组数据库的简单重复序列位点。从51895条Unigenes中共检索出23641条Unigenes含有44867个简单重复序列位点,共计252种基序类型。在所有基序类型中,以A/T与AT/AT型出现次数最多。单核苷酸基序类型与二核苷酸基序类型出现频率最高,共计75.69%。基序长度以10~20 bp为主,重复次数集中5~18次。设计27149对引物,随机选择20对引物,通过PCR验证,在4个桑树品种中展现出良好的重现性与通用性。桑葚转录组SSR位点类型丰富,具备开发出适宜于果桑选育的分子标记的潜力。     

Development and utilization of novel SSRs in foxtail millet [Setaria italica (L.) P. Beauv.]

Although the foxtail millet [Setaria italica (L.) P. Beauv.] is recently regarded as a model crop for studying functional genomics of biofuel grasses, its genetic improvement to some extent was limited due to the non‐availability of molecular markers, particularly the microsatellite markers and the saturated genetic linkage map. Considering this, we attempted to generate a significant number of microsatellite markers in cultivar ‘Prasad’. Two hundred and fifty‐six clones were sequenced to generate 41.82‐kb high‐quality sequences retrieved from genomic library enriched with dinucleotide repeat motifs. Microsatellites were identified in 194 (76%) of the 256 positive clones, and 64 primer pairs (pp) were successfully designed from 95 (49%) unique SSR‐containing clones. The 67.4% primer designing ability, 100% PCR amplification efficiency and 45.3% polymorphic potential in the parents of F₂ mapping population established the efficacy of genomic microsatellites. All the 64 microsatellite markers displayed high level of cross‐species amplification (~67%) in 10 millets and non‐millets species. These experimental findings suggest the utility and efficacy of SSRs in diverse genotyping applications, resolving QTLs, phylogenetic relationships and transferability in several important grass species.     

鹅掌楸EST-SSR引物开发及通用性分析

通过对6520条鹅掌楸EST序列进行检索,在364条ESTs中发现394个SSRs,鹅掌楸EST-SSRs平均密度为每8.5kb含有1个SSR;在检索出的SSRs中,二核苷酸重复单元的SSRs类型最多,占总数的61.9%。利用SSR-ESTs序列共设计176对EST-SSR引物,其中132对在鹅掌楸上有扩增产物,66对扩增出多态,多态性引物占所设计引物的36.9%。这批EST-SSR引物在物种之间具有较高的通用性。研究表明在鹅掌楸中表现多态的66对EST-SSR引物,85%在中国马褂木中有扩增,54%在白玉兰中有扩增。     

Comparison and development of EST–SSRs from two 454 sequencing libraries of Gossypium barbadense

EST–SSRs of Gossypium barbadense are mainly developed using traditional Sanger sequencing. However, due to the high cost and low throughput of Sanger sequencing, it is necessary to use high throughput sequencing technology for the development of more ESTs to more effectively analyze the structure and function of this species. In this study, a G. barbadense acc. 3–79 unnormalized fiber cDNA library (219.63 Mb) and a G. barbadense cv. Hai7124 normalized root cDNA library (204.61 Mb) were obtained by 454 sequencing. EST–SSRs were identified from the two libraries, and only 7,255 SSRs were obtained from the unnormalized library, with an average frequency of 1/31.00 kb. In contrast, 16,087 SSRs were obtained from the normalized library, with an average frequency of 1/13.02 kb. The frequencies of dinucleotides and tetranucleotides in the two libraries were very different. Comparing the two libraries, we found that a normalized cDNA library is more efficient for mining SSRs. Integrating the two libraries allowed the development of 1,129 EST–SSR markers, and 311 polymorphic loci were integrated into our interspecific BC₁ genetic linkage map. The mapping results showed that the distribution of EST–SSRs on sub-genomes and chromosomes was uneven; however, the distribution of the mapped G. barbadense EST–SSRs on homologous chromosomes was similar, with the exception of Chr05 versus Chr19 and Chr12 versus Chr26. This study provided new EST–SSR markers that will facilitate studies on cotton genetics and breeding.     

Development of pepper SSR markers from sequence databases

Simple sequence repeat (SSR) markers are potentiallyvaluable tools for plant breeding. The objectives ofthe work reported here were to search the EMBL andGenbank databases for the presence of SSR-containingsequences from the genus Capsicum, to assess thefrequency of different motifs, and to examine thepolymorphism of selected markers in a panel ofgenotypes, including 10 Capsicum spp. and 1tomato and 1 potato genotype. Fifty-eightmicrosatellites with different motifs were found inCapsicum sequences. A subset of twelve of thesewere selected for the polymorphism survey using PCRprimers flanking the SSR. Polymorphisms between Capsicum lines can be detected with 5 of these primerpairs. PCR products of the predicted size were alsoamplified with three primer pairs in potato and oneprimer pair in tomato. The study also showed thatshorter microsatellites could be valuable markers inCapsicum.