Abundance, marker development and genetic mapping of microsatellites from unigenes in Brassica napus

Rapeseed (Brassica napus) is the second most important oil crop in the world after soybean. The repertoire of simple sequence repeat (SSR) markers for rapeseed is limited and warrants a search for a larger number of polymorphic SSRs for germplasm characterization and breeding applications. In this study, a total of 5,310 SSR-containing unigenes were identified from a set of 46,038 B. napus unigenes with an average density of one SSR every 5.75?kb. A set of 1,000 expressed sequence tag (EST)-SSR markers with repeat length ??18?bp were developed and tested for their ability to detect polymorphism among a panel of six rapeseed varieties. Of these SSR markers, 776 markers detected clear amplification products, and 511 displayed polymorphisms among the six varieties. Of these polymorphic markers, 195 EST-SSR markers, corresponding to 233 loci, were integrated into an existing B. napus linkage map. These EST-SSRs were randomly distributed on the 19 linkage groups of B. napus. Of the mapped loci, 166 showed significant homology to Arabidopsis genes. Based on the homology, 44 conserved syntenic blocks were identified between B. napus and Arabidopsis genomes. Most of the syntenic blocks were consistent with the duplication and rearrangement events identified previously. In addition, we also identified three previously unreported blocks in B. napus. A subset of 40 SSRs was used to assess genetic diversity in a collection of 192 rapeseed accessions. The polymorphism information content of these markers ranged from 0.0357 to 0.6753 with an average value of 0.3373. These results indicated that the EST-SSR markers developed in this study are useful for genetic mapping, molecular marker-assisted selection and comparative genomics.     

Isolation of EST-derived microsatellite markers for genotyping the A and B genomes of wheat

Genetic variation present in 64 durum wheat accessions was investigated by using three sources of microsatellite (SSR) markers: EST-derived SSRs (EST-SSRs) and two sources of SSRs isolated from total genomic DNA. Out of 245 SSR primer pairs screened, 22 EST-SSRs and 20 genomic-derived SSRs were polymorphic and used for genotyping. The EST-SSR primers produced high quality markers, but had the lowest level of polymorphism (25%) compared to the other two sources of genomic SSR markers (53%). The 42 SSR markers detected 189 polymorphic alleles with an average number of 4.5 alleles per locus. The coefficient of similarity ranged from 0.28 to 0.70 and the estimates of similarity varied when different sources of SSR markers were used to genotype the accessions. This study showed that EST-derived SSR markers developed in bread wheat are polymorphic in durum wheat when assaying loci of the A and B genomes. A minumum of ten EST-SSRs generated a very low probability of identity (0.36×10⁻¹²) indicating that these SSRs have a very high discriminatory power. EST-SSR markers directly sample variation in transcribed regions of the genome, which may enhance their value in marker-assisted selection, comparative genetic analysis and for exploiting wheat genetic resources by providing a more-direct estimate of functional diversity. Received: 19 December 2000 / Accepted: 17 April 2001     

转录组测序揭示油桐脂肪酸的合成途径

油桐是我国重要的木本油料植物,过去对油桐的研究主要集中于栽培和常规育种,与油桐种仁油脂合成相关的分子机理研究还未见报道。本研究采用转录组测序（RNA-Seq）技术,比较了油桐种子油脂合成3个不同时期的转录组,获得了大量差异表达的Unigene序列。通过GO（GeneOntology）分类和代谢途径富集性分析将这些差异表达的Unigene归类于包含脂肪酸生物合成途径在内的128个代谢途径。随后将脂肪酸生物合成途径中的54个Unigene序列在KEGG（KyotoEncyclopediaofGenesandGenomes）数据库中进行比对,获得了14个关键酶的同源蛋白质。本研究通过对编码这些同源蛋白质的基因在油桐种子油脂合成期的表达模式进行分析,以期为油桐油脂合成,尤其是桐酸合成机理的解析提供理论参考,并为进一步的理论研究和油桐的遗传改良提供潜在的基因资源,从而提高油桐的单位面积产量。     

Development of Gene-Based SSR Markers in Rice Bean (Vigna umbellata L.) Based on Transcriptome Data

Rice bean (Vigna umbellata (Thunb.) Ohwi & Ohashi) is a warm season annual legume mainly grown in East Asia. Only scarce genomic resources are currently available for this legume crop species and no simple sequence repeat (SSR) markers have been specifically developed for rice bean yet. In this study, approximately 26 million high quality cDNA sequence reads were obtained from rice bean using Illumina paired-end sequencing technology and assembled into 71,929 unigenes with an average length of 986 bp. Of these unigenes, 38,840 (33.2%) showed significant similarity to proteins in the NCBI non-redundant protein and nucleotide sequence databases. Furthermore, 30,170 (76.3%) could be classified into gene ontology categories, 25,451 (64.4%) into Swiss-Prot categories and 21,982 (55.6%) into KOG database categories (E-value < 1.0E-5). A total of 9,301 (23.5%) were mapped onto 118 pathways using the Kyoto Encyclopedia of Genes and Genome (KEGG) pathway database. A total of 3,011 genic SSRs were identified as potential molecular markers. AG/CT (30.3%), AAG/CTT (8.1%) and AGAA/TTCT (20.0%) are the three main repeat motifs. A total of 300 SSR loci were randomly selected for validation by using PCR amplification. Of these loci, 23 primer pairs were polymorphic among 32 rice bean accessions. A UPGMA dendrogram revealed three major clusters among 32 rice bean accessions. The large number of SSR-containing sequences and genic SSRs in this study will be valuable for the construction of high-resolution genetic linkage maps, association or comparative mapping and genetic analyses of various Vigna species.     

Molecular characterization and genetic diversity analysis of soybean (Glycine max (L.) Merr.) germplasm accessions in India

Molecular characterization and genetic diversity among 82 soybean accessions was carried out by using 44 simple sequence repeat (SSR) markers. Of the 44 SSR markers used, 40 markers were found polymorphic among 82 soybean accessions. These 40 polymorphic markers produced a total of 119 alleles, of which five were unique alleles and four alleles were rare. The allele number for each SSR locus varied between two to four with an average of 2.97 alleles per marker. Polymorphic information content values of SSRs ranged from 0.101 to 0.742 with an average of 0.477. Jaccard’s similarity coefficient was employed to study the molecular diversity of 82 soybean accessions. The pairwise genetic similarity among 82 soybean accessions varied from 0.28 to 0.90. The dendrogram constructed based on genetic similarities among 82 soybean accessions identified three major clusters. The majority of genotypes including four improved cultivars were grouped in a single subcluster IIIa of cluster III, indicating high genetic resemblance among soybean germplasm collection in India.

Electronic supplementary material

The online version of this article (doi:10.1007/s12298-014-0266-y) contains supplementary material, which is available to authorized users.     

基于油桐种子3个不同发育时期转录组的油脂合成代谢途径分析

油桐是我国重要的木本油料植物, 过去对油桐的研究主要集中于栽培和常规育种, 与油桐种仁油脂合成相关的分子机理研究还未见报道。文章采用RNA-seq技术对油桐种子油脂合成的3个不同时期的转录组进行比较, 获得了大量差异表达的Unigene序列。在此基础上, 通过GO分类和Pathway富集性分析将这些差异表达Unigene归类于128个代谢途径, 其中包含与油脂合成相关的脂肪酸生物合成和甘油磷脂代谢途径。桐酸经脂肪酸生物合成途径合成后通过甘油磷脂代谢途径以桐油的形式贮存。将这两个代谢途径的Unigene序列在KEGG数据库中进行比对, 获得了一些关键酶的同源蛋白质。文章通过对编码这些同源蛋白质的基因在油桐种子油脂合成期的表达模式进行分析, 以期为油桐油脂合成, 尤其是桐酸合成机理的解析提供理论参考, 并为油桐的遗传改良提供潜在的基因资源, 从而提高油桐的单位面积产量。     

基于陆地棉茎尖转录组序列的genic-SSR标记的开发与评价

利用软件MISA和SSR Locator 对陆地棉（Gossypium hirsutum L.）Coker312茎尖转录组测序得到的73515条Unigene序列进行分析,查找到4507个SSR位点,分布于4039条转录本序列中,SSR出现频率为5.5%,非编码区的SSR位点要显著高于编码区（2853/1654）。SSR重复基元中,三核苷酸重复基元占主导地位（51.03%）,且AAG/CTT （20.08%）类型最多;其次是二核苷酸重复基元（28.76%）,主要为AG/CT（55.47%）。利用引物批量设计软件共开发1569对SSR引物,在陆地棉TM-1、海岛棉3-79 (G. barbadense L.)、阿非利加棉(G. herbaceum L.)、雷蒙德氏棉(G. raimondii Ulbrich.)四个棉种中进行初步评价,其中1117对引物能在四个棉种间扩增出稳定的条带,扩增率为71.2%。选择650对能稳定扩增出条带的引物,在涉及5个棉种（增加了亚洲棉（G. arboreum L.））的13份材料中进一步筛选,83对引物能在13份材料中扩增出特异性条带, PIC变幅在0.121～0.648之间,平均值为0.422,其中80对引物能在7份陆地棉材料中扩增出特异性条带,平均PIC值为0.336;挑选5对在本实验室作图亲本鲁棉研22和鲁原343间有明显多态的引物进行连锁分析,其中4对成功连锁到本实验室构建的遗传图谱上。本研究丰富了棉属genic-SSR的数量,为棉属遗传作图、性状关联分析提供了更多引物选择。     

Characterization,development and mapping of Unigene-derived microsatellite markers in sorghum [<Emphasis Type="Italic">Sorghum bicolor</Emphasis> (L.) Moench]

Molecular variation within known genes controlling specific functions provide candidate gene-based markers which are tightly linked with the trait of interest. Unigene-derived microsatellite markers, with their unique identity and positions, offer the advantage of unraveling variation in the expressed component of the genome. We characterized ≥12-bp-long microsatellite loci from 13,899 unique sequences of sorghum [Sorghum bicolor (L.) Moench] available in the NCBI unigene database for their abundance and possible use in sorghum breeding. Analysis of 12,464 unigenes (≥200-bp) using MISA software identified 14,082 simple sequence repeats (SSRs) in 7,370 unigenes, from which 1,519 unigene SSR markers were developed. The average frequency of SSR was 1 per1.6 kb and 1.0 per 1.1 unigene; hexamers followed by trimers were found in abundance, of which 33.3% AT-rich and CCG repeats were the most abundant. Of the 302 unigene SSRs tested, 60 (19.8%) were polymorphic between the two parents, M35-1 and B35 of a recombinant inbred line (RIL) mapping population. A mapping population consisting of 500 RILs was developed using the above two parents, and a subset of random 245 RILs was used for genotyping with polymorphic SSRs. We developed a linkage map containing 231 markers, of which 228 (174 genomic and 54 genic) were microsatellites and three were morphological markers. Markers were distributed over 21 linkage groups, and spanned a genetic distance of 1235.5 cM. This map includes 81 new SSRs, of which 35 (21 unigene and 14 genomic) were developed in the present study and 46 from other studies. The order of the SSR markers mapped in the present study was confirmed physically by BLAST search against the whole-genome shotgun sequence of sorghum. Many unigene sequences used for marker development in this study include genes coding for important regulatory proteins and functional proteins that are involved in stress-related metabolism. The unigene SSR markers used together with other SSR markers to construct the sorghum genetic map will have applications in studies on comparative mapping, functional diversity analysis and association mapping, and for quantitative trait loci detection for drought and other agronomically important traits in sorghum.     

Genetic assessment of safflower (Carthamus tinctorius L.) collection with microsatellite markers acquired via pyrosequencing method

A genetic evaluation of safflower germplasm collections derived from different geographical regions and countries will provide useful information for sustainable conservation and the utilization of genetic diversity. However, the molecular marker information is limited for evaluation of genetic diversity of safflower germplasm. In this study, we acquired 509 putative genomic SSR markers for sufficient genome coverage using next‐generation sequencing methods and characterized thirty polymorphic SSRs in safflower collection composed of 100 diverse accessions. The average allele number and expected heterozygosity were 2.8 and 0.386, respectively. Analysis of population structure and phylogeny based on thirty SSR profiles revealed genetic admixture between geographical regions contrary to genetic clustering. However, the accessions from Korea were genetically conserved in distinctive groups in contrast to other safflower gene pool. In conclusion, these new genomic SSRs will facilitate valuable studies to clarify genetic relationships as well as conduct population structure analyses, genetic map construction and association analysis for safflower.     

Development of Juglans Regia SSR Markers by Data Mining of the EST Database

Walnut (Juglans regia), an economically important woody plant, is widely cultivated in temperate regions for its timber and nutritional fruits. Despite abundant studies in germplasm, systemic molecular evaluations of walnut are sparsely reported mainly due to the limited molecular markers available. Expressed sequence tags (EST) provide a valuable resource for developing simple sequence repeat (SSR) markers. In this study, a total of 5,025 walnut ESTs (covering 16.41 Mb) were retrieved from the National Center for Biotechnology Information database. The SSR motifs were then analyzed by the SSRHunter software. In total, 398 SSRs were obtained with an average frequency of 1/4.08 kb. Dinucleotide (di-) repeat motifs accounted for 69.85% of all SSRs, followed by trinucleotide (tri-) with a frequency of 27.64%, while low frequency (2.51%) of tetranucleotide (tetra-) to hexanucleotide (hexa-) was observed. Meanwhile, GCA and TC motifs were prevalent among di- and tri- loci, respectively. Subsequently, a total of 123 primer pairs were designed from the non-redundant SSR-containing unigenes with the selection threshold of SSR length set to 10 bp or more. To examine the efficiency of candidate markers, seven DNA pools were collected from geographically different accessions. Results demonstrated that 41 SSR primer sets could generate high polymorphic amplification products (33.3%), and these polymorphic loci were mainly located in the 3′-untranslated region. Annotation analysis revealed that only two of these 41 loci were located inside open reading frames of characterized proteins (E ≤ 1E−30).     

Predicting polymorphic EST‐SSRs in silico

The public availability of large quantities of gene sequence data provides a valuable resource of the mining of Simple Sequence Repeat (SSR) molecular genetic markers for genetic analysis. These markers are inexpensive, require minimal labour to produce and can frequently be associated with functionally annotated genes. This study presents the characterization of barley EST‐SSRs and the identification of putative polymorphic SSRs from EST data. Polymorphic SSRs are distinguished from monomorphic SSRs by the representation of varying motif lengths within an alignment of sequence reads. Two measures of confidence are calculated, redundancy of a polymorphism and co‐segregation with accessions. The utility of this method is demonstrated through the discovery of 597 candidate polymorphic SSRs, from a total of 452 642 consensus expressed sequences. PCR amplification primers were designed for the identified SSRs. Ten primer pairs were validated for polymorphism in barley and for transferability across species. Analysis of the polymorphisms in relation to SSR motif, length, position and annotation is discussed.     

Development and Characterization of Polymorphic EST-SSR and Genomic SSR Markers for Tibetan Annual Wild Barley

Tibetan annual wild barley is rich in genetic variation. This study was aimed at the exploitation of new SSRs for the genetic diversity and phylogenetic analysis of wild barley by data mining. We developed 49 novel EST-SSRs and confirmed 20 genomic SSRs for 80 Tibetan annual wild barley and 16 cultivated barley accessions. A total of 213 alleles were generated from 69 loci with an average of 3.14 alleles per locus. The trimeric repeats were the most abundant motifs (40.82%) among the EST-SSRs, while the majority of the genomic SSRs were di-nuleotide repeats. The polymorphic information content (PIC) ranged from 0.08 to 0.75 with a mean of 0.46. Besides this, the expected heterozygosity (He) ranged from 0.0854 to 0.7842 with an average of 0.5279. Overall, the polymorphism of genomic SSRs was higher than that of EST-SSRs. Furthermore, the number of alleles and the PIC of wild barley were both higher than that of cultivated barley, being 3.12 vs 2.59 and 0.44 vs 0.37. Indicating more polymorphism existed in the Tibetan wild barley than in cultivated barley. The 96 accessions were divided into eight subpopulations based on 69 SSR markers, and the cultivated genotypes can be clearly separated from wild barleys. A total of 47 SSR-containing EST unigenes showed significant similarities to the known genes. These EST-SSR markers have potential for application in germplasm appraisal, genetic diversity and population structure analysis, facilitating marker-assisted breeding and crop improvement in barley.     

Selection of highly informative and user-friendly microsatellites (SSRs) for genotyping of cultivated potato

Characterization of nearly 1,000 cultivated potato accessions with simple sequence repeats (SSRs; also referred to as microsatellites) has allowed the identification of a reference set of SSR markers for accurate and efficient genotyping. In addition, 31 SSRs are reported here for a potato genetic map, including new map locations for 24 of them. A first criterion for this proposed reference set was ubiquity of the SSRs in the eight landrace cultivar groups of the potato, Solanum tuberosum. All SSRs tested in the present study displayed the same allele phenotypes and allele size range in the diverse germplasm set as in the advanced potato cultivar germplasm in which they were originally discovered. Thirteen of 13 SSR products from all cultivar groups are shown to cross-hybridize with the corresponding SSR product of the source cultivar to ascertain sequence homology. Other important SSR selection criteria are quality of amplification products, locus complexity, polymorphic index content, and well-dispersed location on a potato genetic map. Screening of 156 SSRs allowed the identification of a highly informative and user-friendly set comprising 18 SSR markers for use in characterization of potato genetic resources. In addition, we have identified true- and pseudo-multiplexing SSRs for even greater efficiency.Communicated by F. Salamini     

Mining and charactering microsatellites in Cucumis melo expressed sequence tags from sequence database

Simple sequence repeats (SSRs) derived from expressed sequence tags (ESTs) are valuable markers because they represent transcribed regions and often have putative functions. We mined and characterized microsatellites in melon ESTs. Three hundred and eighty‐three SSR loci were identified in 309 of 3188 unigenes assembled by 5747 EST and mRNA sequences in GenBank with occurring frequency of 1/4.7 kb. Twenty‐two polymorphic EST‐SSR markers were developed with the mean allele number of 2.9 per locus and mean expected heterozygosity of 0.442. Amplification products were also detected by 15 pairs of primer in Cucumis sativus. Those informative EST‐SSR markers can be used in melon genetic improvement projects.