Development of EST-SSR markers in castor bean (Ricinus communis L.) and their utilization for genetic purity testing of hybrids

Expressed sequence tag (EST) databases offer opportunity for the rapid development of simple sequence repeat (SSR) markers in crops. Sequence assembly and clustering of 57?895 ESTs of castor bean resulted in the identification of 10?960 unigenes (6459 singletons and 4501 contigs) having 7429 SSRs. On an average, the unigenes contained 1 SSR for every 1.23?kb of unigene sequence. The identified SSRs mostly consisted of dinucleotide (62.4%) and trinucleotide (33.5%) repeats. The AG class was the most common among the dinucleotide motifs (68.9%), whereas the AAG class (25.9%) was predominant among the trinucleotide motifs. A total of 611 primer pairs were designed for the SSRs, having repeat length more than or equal to 20 nucleotides, of which a set of 130 markers were tested and 92 of these yielding robust amplicons were analyzed for their utility in genetic purity assessment of castor bean hybrids. Nine markers were able to detect polymorphism between the parental lines of nine commercial castor bean hybrids (DCH-32, DCH-177, DCH-519, GCH-2, GCH-4, GCH-5, GCH-6, GCH-7, and RHC-1), and their utility in genetic purity testing was demonstrated. These novel EST-SSR markers would be a valuable addition to the growing molecular marker resources that could be used in genetic improvement programmes of castor bean.     

Development of genomic simple sequence repeat markers in opium poppy by next-generation sequencing

Opium poppy (Papaver somniferum L.) is an important pharmaceutical crop with very few genetic marker resources. To expand these resources, we sequenced genomic DNA using pyrosequencing technology and examined the DNA sequences for simple sequence repeats (SSRs). A total of 1,244,412 sequence reads were obtained covering 474 Mb. Approximately half of the reads (52 %) were assembled into 166,724 contigs representing 105 Mb of the opium poppy genome. A total of 23,283 non-redundant SSRs were identified in 18,944 contigs (11.3 % of total contigs). Trinucleotide and tetranucleotide repeats were the most abundant SSR repeats, accounting for 49.0 and 27.9 % of all SSRs, respectively. The AAG/TTC repeat was the most abundant trinucleotide repeat, representing 19.7 % of trinucleotide repeats. Other SSR repeat types were AT-rich. A total of 23,126 primer pairs (98.7 % of total SSRs) were designed to amplify SSRs. Fifty-three genomic SSR markers were tested in 37 opium poppy accessions and seven Papaver species for determination of polymorphism and transferability. Intraspecific polymorphism information content (PIC) values of the genomic SSR markers were intermediate, with an average 0.17, while the interspecific average PIC value was slightly higher, 0.19. All markers showed at least 88 % transferability among related species. This study increases sequence coverage of the opium poppy genome by sevenfold and the number of opium poppy-specific SSR markers by sixfold. This is the first report of the development of genomic SSR markers in opium poppy, and the genomic SSR markers developed in this study will be useful in diversity, identification, mapping and breeding studies in opium poppy.     

Development of EST-SSR markers through data mining and their use for genetic diversity study in Indian accessions of Jatropha curcas L.: a potential energy crop

Jatropha curcas L. is gaining importance as a potential energy crop. However, lack of sufficient numbers of molecular markers hinder current research on crop improvement in Jatropha. The expressed sequences tags (EST) sequences deposited in public databases, offers an excellent opportunity to identify simple sequence repeats (SSRs) through data mining, for further research on molecular breeding. In the present study 42,477 ESTs of J. curcas were screened, out of which 5,673 SSRs were identified with 48.8 % simple (excluding mononucleotide repeats) and 52.2 % compound repeat motifs. Amongst these repeat motifs, dinucleotide repeats were abundant (26.5 %), followed by trinucleotide (23.1 %) and tetranucleotide repeats (0.8 %). From these microsatellites, 32 EST-SSR (genic microsatellite) primer pairs were designed. These primers were used to analyze the genetic diversity among 42 accessions collected from different parts of India. Out of the 32 EST-SSR primers, 24 primer pairs exhibited polymorphism among the genotypes, with amplicons varying from one to eight, giving an average of 2.33 alleles per polymorphic marker. Polymorphic information content value ranged from 0.02 to 0.5 with an average of 0.402 indicating moderate level of informativeness within these EST-SSRs markers. The EST-SSR markers developed here will serve as a valuable resource for genetic studies, like linkage mapping, diversity analysis, quantitative trait locus/association mapping, and molecular breeding. The current study also revealed low diversity in the screened Indian Jatropha germplasm. Therefore, the future efforts must be made to broaden the gene pool of Jatropha for the creation of genetic diversity that can be further used for crop improvement through breeding.     

Development of EST-SSR markers and their utility in revealing cryptic diversity in safflower (Carthamus tinctorius L.)

With the aim of developing additional genomic resources in safflower, a set of 41,011 ESTs of safflower were mined for the presence of SSRs. 18,773 SSR containing ESTs (SSR-ESTs) were identified and were analyzed to remove redundant sequences leading to identification of 8,810 non-redundant SSR-ESTs (categorized into 6104 singletons and 2,706 contigs) having 13,085 non-redundant SSRs. The average number of non-redundant SSRs per EST was 0.32 and they predominantly consisted of dinucleotide (57.7 %), and trinucleotide (37.7 %) repeat motifs. 500 primer pairs were designed for the non-redundant EST-SSRs of which, 151 were tested. 60 markers which gave robust amplicons, were validated in a set of 19 Carthamus lines. A subset of EST-SSR markers, having average polymorphism information content (PIC) ≥0.4 could precisely elucidate the pedigree relatedness among these lines. Further, these markers exhibited high cross-species transferability to five other wild species of Carthamus. The markers reported here would be a valuable addition to existing safflower marker resources aiding in hastening its improvement.     

Genome-wide identification of simple sequence repeats and development of polymorphic SSR markers for genetic studies in tea plant (<Emphasis Type="Italic">Camellia sinensis</Emphasis>)

The tea plant (Camellia sinensis (L.) O. Kuntze) is one of the most popular non-alcoholic beverage crops worldwide. The availability of complete genome sequences for the Camellia sinensis var. ‘Shuchazao’ has provided the opportunity to identify all types of simple sequence repeat (SSR) markers by genome-wide scan. In this study, a total of 667,980 SSRs were identified in the ~?3.08 Gb genome, with an overall density of 216.88 SSRs/Mb. Dinucleotide repeats were predominant among microsatellites (72.25%), followed by trinucleotide repeats (15.35%), while the remaining SSRs accounted for less than 13%. The motif AG/CT (49.96%) and AT/TA (40.14%) were the most and the second most abundant among all identified SSR motifs, respectively; meanwhile, AAT/ATT (41.29%) and AAAT/ATTT (67.47%) were the most common among trinucleotides and tetranucleotides, respectively. A total of 300 primer pairs were designed to screen six tea cultivars for polymorphisms of SSR markers using the five selected repeat types of microsatellite sequences. The resulting 96 SSR markers that yielded polymorphic and unambiguous bands were further deployed on 47 tea cultivars for genetic diversity assessment, demonstrating high polymorphism of these SSR markers. Remarkably, the dendrogram revealed that the phylogenetic relationships among these tea cultivars are highly consistent with their genetic backgrounds or places of origin. The identified genome-wide SSRs and newly developed SSR markers will provide a powerful means for genetic researches in tea plant, including genetic diversity and evolutionary origin analysis, fingerprinting, QTL mapping, and marker-assisted selection for breeding.     

Mining and gene ontology based annotation of SSR markers from expressed sequence tags of Humulus lupulus

Humulus lupulus is commonly known as hops, a member of the family moraceae. Currently many projects are underway leading to the accumulation of voluminous genomic and expressed sequence tag sequences in public databases. The genetically characterized domains in these databases are limited due to non-availability of reliable molecular markers. The large data of EST sequences are available in hops. The simple sequence repeat markers extracted from EST data are used as molecular markers for genetic characterization, in the present study. 25,495 EST sequences were examined and assembled to get full-length sequences. Maximum frequency distribution was shown by mononucleotide SSR motifs i.e. 60.44% in contig and 62.16% in singleton where as minimum frequency are observed for hexanucleotide SSR in contig (0.09%) and pentanucleotide SSR in singletons (0.12%). Maximum trinucleotide motifs code for Glutamic acid (GAA) while AT/TA were the most frequent repeat of dinucleotide SSRs. Flanking primer pairs were designed in-silico for the SSR containing sequences. Functional categorization of SSRs containing sequences was done through gene ontology terms like biological process, cellular component and molecular function.     

Distribution and characterization of simple sequence repeats in Gossypium raimondii genome

Simple sequence repeats (SSRs) can be derived from the complete genome sequence. These markers are important for gene mapping as well as marker-assisted selection (MAS). To develop SSRs for cotton gene mapping, we selected the complete genome sequence of Gossypium raimondii, which consisted of 4447 non-redundant scaffolds. Out of 775.2 Mb sequence examined, a total of 136,345 microsatellites were identified with a density of 5.69 kb per SSR in the G. raimondii genome leading to development of 112,177 primer pairs. The distributions of SSRs in the genome were non-random. Among the different motifs ranging from 1 to 6 bp, penta-nucleotide repeats were most abundant (30.5%), followed by tetra-nucleotide repeats (18.2%) and di-nucleotide repeats (16.9%). Among all identified 457 motif types, the most frequently occurring repeat motifs were poly-AT/TA, which accounted for 79.8% of the total di-nt SSRs, followed by AAAT/TTTA with 51.5% of the total tetra-nucleotede. Further, 18,834 microsatellites were detected from the protein-coding genes, and the frequency of gene containing SSRs was 46.0% in 40,976 genes of G. raimondii. These genome-based SSRs developed in the present study will lay the groundwork for developing large numbers of SSR markers for genetic mapping, gene discovery, genetic diversity analysis, and MAS breeding in cotton.     

Genome-Wide Characterization of Simple Sequence Repeat (SSR) Loci in Chinese Jujube and Jujube SSR Primer Transferability

Chinese jujube (Ziziphus jujuba), an economically important species in the Rhamnaceae family, is a popular fruit tree in Asia. Here, we surveyed and characterized simple sequence repeats (SSRs) in the jujube genome. A total of 436,676 SSR loci were identified, with an average distance of 0.93 Kb between the loci. A large proportion of the SSRs included mononucleotide, dinucleotide and trinucleotide repeat motifs, which accounted for 64.87%, 24.40%, and 8.74% of all repeats, respectively. Among the mononucleotide repeats, A/T was the most common, whereas AT/TA was the most common dinucleotide repeat. A total of 30,565 primer pairs were successfully designed and screened using a series of criteria. Moreover, 725 of 1,000 randomly selected primer pairs were effective among 6 cultivars, and 511 of these primer pairs were polymorphic. Sequencing the amplicons of two SSRs across three jujube cultivars revealed variations in the repeats. The transferability of jujube SSR primers proved that 35/64 SSRs could be transferred across family boundary. Using jujube SSR primers, clustering analysis results from 15 species were highly consistent with the Angiosperm Phylogeny Group (APGIII) System. The genome-wide characterization of SSRs in Chinese jujube is very valuable for whole-genome characterization and marker-assisted selection in jujube breeding. In addition, the transferability of jujube SSR primers could provide a solid foundation for their further utilization.     

Analysis of simple sequence repeats (SSRs)dynamics in fungus Fusarium graminearum

The abundance and inherent potential for variations in simple sequence repeats (SSRs) or microsatellites resulted in valuable source for genetic markers in eukaryotes. We describe the organization and abundance of SSRs in fungus Fusarium graminearum (causative agent for Fusarium head blight or head scab of wheat). We identified 1705 SSRs of various nucleotide repeat motifs in the sequence database of F. graminearum. It is observed that mononucleotide repeats (62%) were most abundant followed by di- (20%) and trinucleotide repeats (14%). It is noted that tetra-, penta- and hexanucleotide repeats accounted for only 4% of SSRs. The estimated frequency of Class I SSRs (perfect repeats ≥20 nucleotides) was one SSR per 124.5 kb, whereas the frequency of Class II (perfect repeats >10 nucleotides and ≫20 nucleotides) was one SSR per 25.6 kb. The dynamics of SSRs will be a powerful tool for taxonomic, phylogenetic, genome mapping and population genetic studies as SSR based markers show high levels of allelic variation, codominant inheritance and ease of analysis.     

Novel and Stress Relevant EST Derived SSR Markers Developed and Validated in Peanut

With the aim to increase the number of functional markers in resource poor crop like cultivated peanut (Arachis hypogaea), large numbers of available expressed sequence tags (ESTs) in the public databases, were employed for the development of novel EST derived simple sequence repeat (SSR) markers. From 16424 unigenes, 2784 (16.95%) SSRs containing unigenes having 3373 SSR motifs were identified. Of these, 2027 (72.81%) sequences were annotated and 4124 gene ontology terms were assigned. Among different SSR motif-classes, tri-nucleotide repeats (33.86%) were the most abundant followed by di-nucleotide repeats (27.51%) while AG/CT (20.7%) and AAG/CTT (13.25%) were the most abundant repeat-motifs. A total of 2456 EST-SSR novel primer pairs were designed, of which 366 unigenes having relevance to various stresses and other functions, were PCR validated using a set of 11 diverse peanut genotypes. Of these, 340 (92.62%) primer pairs yielded clear and scorable PCR products and 39 (10.66%) primer pairs exhibited polymorphisms. Overall, the number of alleles per marker ranged from 1-12 with an average of 3.77 and the PIC ranged from 0.028 to 0.375 with an average of 0.325. The identified EST-SSRs not only enriched the existing molecular markers kitty, but would also facilitate the targeted research in marker-trait association for various stresses, inter-specific studies and genetic diversity analysis in peanut.     

Development of polymorphic microsatellite markers in the medicinal plant Gardenia jasminoides (Rubiaceae)

A total of 12,960 simple sequence repeats (SSR) motifs were identified in the genome of the medicinal plant Gardenia jasminoides using Illumina-based EST sequences. Among the SSRs, mono-nucleotides were the most abundant (56.7%), followed by di- (19%) and tri-nucleotides (16.4%). AG/TC (60.2%), TTC/AAG (25.8%), and TTTC/GAAA (35.9%) repeat motifs were the most abundant of the di-, tri- and tetra-nucleotide motifs, respectively. Subsequently, twenty-five allelic, polymorphic primer pairs were identified and tested in 153 individuals from five natural populations of G. jasminoides. The number of alleles per polymorphic locus (A) ranged from two to eight. Observed and expected heterozygosity varied from 0.095 to 0.857 and 0.182 to 0.832, respectively. The PIC values for each locus ranged from 0.171 to 0.792. These new polymorphic EST-SSR markers will be useful for further genetic studies on this economically important plant.     

EST-SSR markers derived from Laminaria digitata and its transferable application in Saccharina japonica

The public availability of numerous expressed sequence tag (EST) enables EST-based SSR (simple sequence repeat) markers to be widely used for genetics and breeding studies. In the present study, EST-SSR markers were developed from ESTs of Laminaria digitata and were transferred to the non-congeneric species Saccharina japonica. Among the 2,668 non-redundant ESTs, 83 (3.1%) ESTs containing SSR were identified totally, with an average of one SSR per 13.6 kb. Analysis of SSR motifs revealed that the trinucleotide and tetranucleotide were major motifs, accounted for 44.58% and 16.87%, respectively. Based on the 83 ESTs containing SSR, we designed 45 pairs of primers in the flanking regions of the SSR, of which 13 pairs showed polymorphism in a wild S. japonica population, and the mean alleles per locus was 3.6 (ranging from 2 to 6). The observed (Ho) and expected (He) heterozygosities of these EST-SSRs were 0.234–0.632 and 0.260–0.635, respectively. All loci were in Hardy–Weinberg equilibrium in the wild population and no linkage disequilibrium was detected among loci. The obtained EST-SSR markers can facilitate and promote related research such as ecological investigation, genetic diversity assessment and breeding practice of S. japonica as well.     

Exploitation of pepper EST–SSRs and an SSR-based linkage map

As genome and cDNA sequencing projects progress, a tremendous amount of sequence information is becoming publicly available. These sequence resources can be exploited for gene discovery and marker development. Simple sequence repeat (SSR) markers are among the most useful because of their great variability, abundance, and ease of analysis. By in silico analysis of 10,232 non-redundant expressed sequence tags (ESTs) in pepper as a source of SSR markers, 1,201 SSRs were found, corresponding to one SSR in every 3.8 kb of the ESTs. Eighteen percent of the SSR–ESTs were dinucleotide repeats, 66.0% were trinucleotide, 7.7% tetranucleotide, and 8.2% pentanucleotide; AAG (14%) and AG (12.4%) motifs were the most abundant repeat types. Based on the flanking sequences of these 1,201 SSRs, 812 primer pairs that satisfied melting temperature conditions and PCR product sizes were designed. 513 SSRs (63.1%) were successfully amplified and 150 of them (29.2%) showed polymorphism between Capsicum annuum ‘TF68’ and C. chinense ‘Habanero’. Dinucleotide SSRs and EST–SSR markers containing AC-motifs were the most polymorphic. Polymorphism increased with repeat length and repeat number. The polymorphic EST–SSRs were mapped onto the previously generated pepper linkage map, using 107 F₂ individuals from an interspecific cross of TF68 × Habanero. One-hundred and thirtynine EST–SSRs were located on the linkage map in addition to 41 previous SSRs and 63 RFLP markers, forming 14 linkage groups (LGs) and spanning 2,201.5 cM. The EST–SSR markers were distributed over all the LGs. This SSR-based map will be useful as a reference map in Capsicum and should facilitate the use of molecular markers in pepper breeding.Gibum Yi and Je Min Lee equally contributed to this work.     

Development and use of EST-SSR markers for assessing genetic diversity in the brown planthopper (Nilaparvata lugens Stål)

To assess genetic diversity in populations of the brown planthopper (Nilaparvata lugens St?l) (Homoptera: Delphacidae), we have developed and applied microsatellite, or simple sequence repeat (SSR), markers from expressed sequence tags (ESTs). We found that the brown planthopper clusters of ESTs were rich in SSRs with unique frequencies and distributions of SSR motifs. Three hundred and fifty-one EST-SSR markers were developed and yielded clear bands from samples of four brown planthopper populations. High cross-species transferability of these markers was detected in the closely related planthopper N. muiri. The newly developed EST-SSR markers provided sufficient resolution to distinguish within and among biotypes. Analyses based on SSR data revealed host resistance-based genetic differentiation among different brown planthopper populations; the genetic diversity of populations feeding on susceptible rice varieties was lower than that of populations feeding on resistant rice varieties. This is the first large-scale development of brown planthopper SSR markers, which will be useful for future molecular genetics and genomics studies of this serious agricultural pest.     

Development and analysis of EST-SSRs for flax (Linum usitatissimum L.)

A set of 146,611 expressed sequence tags (ESTs) were generated from 10 flax cDNA libraries. After assembly, a total of 11,166 contigs and 11,896 singletons were mined for the presence of putative simple sequence repeats (SSRs) and yielded 806 (3.5%) non-redundant sequences which contained 851 putative SSRs. This is equivalent to one EST-SSR per 16.5 kb of sequence. Trinucleotide motifs were the most abundant (76.9%), followed by dinucleotides (13.9%). Tetra-, penta- and hexanucleotide motifs represented <10% of the SSRs identified. A total of 83 SSR motifs were identified. Motif (TTC/GAA)n was the most abundant (10.2%) followed by (CTT/AAG)n (8.7%), (TCT/AGA)n (8.6%), (CT/AG)n (6.7%) and (TC/GA)n (5.3%). A total of 662 primer pairs were designed, of which 610 primer pairs yielded amplicons in a set of 23 flax accessions. Polymorphism between the accessions was found for 248 primer pairs which detected a total of 275 EST-SSR loci. Two to seven alleles were detected per marker. The polymorphism information content value for these markers ranged from 0.08 to 0.82 and averaged 0.35. The 635 alleles detected by the 275 polymorphic EST-SSRs were used to study the genetic relationship of 23 flax accessions. Four major clusters and two singletons were observed. Sub-clusters within the main clusters correlated with the pedigree relationships amongst accessions. The EST-SSRs developed herein represent the first large-scale development of SSR markers in flax. They have potential to be used for the development of genetic and physical maps, quantitative trait loci mapping, genetic diversity studies, association mapping and fingerprinting cultivars for example. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

杂交兰转录组SSR信息分析及EST-SSR标记开发应用

该研究主要开发筛选适用于杂交兰的EST-SSR引物,为杂交兰种质资源评价和遗传变异研究等提供可靠的分子标记。该研究对杂交兰进行转录组高通量测序,挖掘SSR位点和开发EST-SSR标记,并对不同种质的遗传多样性进行分析。结果表明,从31724条杂交兰Unigene中检测出18603个SSR位点,SSR出现频率为58.64%;SSR位点中的主导类型是单核苷酸重复,占总SSR的65.10%,其次是二核苷酸(23.56%)和三核苷酸(10.76%)重复;优势重复基元为A/T、AG/CT、AT/AT和AAG/CTT,分别占总位点的64.72%、13.74%、8.19%和2.51%。利用Primer Premier 5.0共设计了565对SSR引物,从筛选出的64对有效扩增引物中随机选择28对引物,对40份杂交兰种质进行多态性验证与遗传关系分析,其中16对(占57.14%)引物表现出可重复的高多态性,平均多态信息量(PIC)达0.789。基于扩增的多态性SSR信息,40份种质资源可聚为4类,聚类结果与其遗传背景基本一致。该研究印证了转录组测序获得的Unigene是SSR标记开发的有效来源,开发的EST-SSR引物可为杂交兰及近缘种的良种鉴别、遗传图谱构建、分子标记辅助育种及功能基因挖掘等提供有价值的候选标记。     

基于转录组数据高通量发掘黄粉甲微卫星引物（英文）

黄粉虫Tenebrio molitor作为理想的模式研究生物, 虽然已围绕该昆虫在多个研究领域开展了诸多研究, 但是有关其分子和遗传方面的研究仍知之甚少。为此, 本研究基于前期构建的黄粉甲转录组数据库, 成功发掘获得1 249个微卫星序列。其中, 单碱基或三碱基序重复列最多, 分别占44.44%和41.15%; A/T型重复序列出现频率最高, 占42.70%。除单核苷酸重复序列外, 重复单元的重复次数以5次最多, 占30.90%。基于鉴定获得的微卫星序列, 共设计获得1 004对微卫星引物, 而且每对引物还设计了5对替代引物。研究获得的微卫星引物将有助于今后开展黄粉甲功能和比较基因组学方面的研究。