Expressed Sequence Tag-derived Simple Sequence Repeat Research Articles

New linkage maps for the Cupressaceae species Chamaecyparis obtusa and Thujopsis dolabrata var. hondae and comparison with the linkage map of Cryptomeria japonica

ABSTRACT Comparative mapping studies, which can determine whether the genome structure differs among genera, have yet to be conducted in Cupressaceae. This study analyzed three important forestry species in Japan: Cryptomeria japonica, Chamaecyparis obtusa, and Thujopsis dolabrata var. hondae. To date, linkage maps have only been constructed for C. japonica. We constructed the first linkage maps for C. obtusa and T. dolabrata var. hondae using double digest restriction-site associated DNA sequencing (ddRAD-Seq) data, expressed sequence tag-derived simple sequence repeat (EST-SSR) markers, and single-nucleotide polymorphism (SNP) markers designed from homologous unigenes obtained from RNA-seq data. The lengths of the linkage maps were 1,835 cM (1,981 markers) for C. obtusa and 1,833 cM (1,295 markers) for T. dolabrata var. hondae. A total of 11 distinct linkage groups were identified in these maps, corresponding to the haploid number of Cupressaceae. Using four published linkage maps for C. japonica, we then constructed a composite linkage map with 7,784 markers. Finally, the three linkage maps were compared using 207 SNP markers designed from homologous unigenes. Although most markers were mapped to the same LG despite representing different species, indicating strong synteny. The high level of interspecific and intergeneric synteny and macro-collinearity detected in Cupressaceae were similar to those of other trees. We also detected three major chromosomal rearrangements between but not within subfamilies, which may have been caused by the taxonomic distance. This is the first example of comparative mapping for Cupressaceae species.

Virulence and molecular genetic diversity, variation, and evolution of the Puccinia triticina population in Hebei Province of China from 2001 to 2010.

Wheat leaf rust, caused by Puccinia triticina, is one of the most important fungal diseases of wheat in China. However, little is known about the dynamic changes of population structure and genetic diversity of P. triticina during a period of time. In this study, 247 isolates of P. triticina collected from Hebei Province from 2001 to 2010 were tested on 36 Thatcher near-isogenic lines for virulence diversity and detected by 21 pairs of Expressed Sequence Tag derived Simple Sequence Repeat (EST-SSR) primers for genetic diversity. A total of 204 isolates were successfully identified as 164 races, and THTT, THST, PHRT, THTS, and PHTT were the most common races in Hebei Province from 2001 to 2010. The cluster analysis based on virulence showed that P. triticina has a rich virulence polymorphism, which had a certain correlation with the years, while the cluster analysis based on EST-SSR showed that the genetic diversity of the P. triticina population was significantly different between years in Hebei Province from 2001 to 2010. In addition, the population structure of P. triticina may have changed greatly in 2007 and 2009, which was significantly different from that of 2001-2006 on either virulence or genetic characteristics. The variation frequency of the population structure had an increasing trend during this period. From 2001 to 2010, there was a certain degree of gene flow among the P. triticina populations. No significant correlation was found between virulence and molecular polymorphism. The genetic differentiation analysis of the 10 tested populations (each year as a population) showed that the coefficient of genetic differentiation (Gst) was 0.27, indicating that there was a certain genetic differentiation among or within populations of P. triticina in Hebei Province. The genetic variation within populations (73.08%) was higher than that among populations (26.92%), which indicated that the genetic variations were mainly found within populations. Our study provides the foundation for a better understanding of the population structure change and genetic diversity of P. triticina over a period in Hebei Province of China.

Transcriptome analysis and development of EST-SSR markers in Anoectochilus emeiensis (Orchidaceae).

Anoectochilus emeiensis K. Y. Lang, together with other Anoectochilus species, has long been used as the main source of many traditional Chinese medicines. Owing to the shortcomings of molecular markers, the study of the genetic diversity and medicinal component synthesis mechanism of the endemic Anoectochilus species has been delayed. In this study, I carried out a transcriptome analysis of A. emeiensis. A total of 78,381 unigenes were assembled from 64.2 million reads, and 47,541 (60.65%) unigenes were matched to known proteins in the public databases. Then, 9284 expressed sequence tag-derived simple sequence repeats (EST-SSRs) were identified, and the frequency of SSRs in the A. emeiensis transcriptome was 9.88%. Trinucleotide repeats (3699, 39.84%) were the most common type, followed by dinucleotide (3251, 35.02%) and mononucleotide (1750, 18.85%) repeats. Based on the SSR sequence, 6683 primer pairs were successfully designed, 40 primer pairs were randomly selected, and 10 primer pairs were identified as polymorphic loci from 186 individuals of A. emeiensis. The EST-SSR markers examined in this study will be informative for future population genetic studies of A. emeiensis.

Development and Characterization of Expressed Sequence-tagged Simple Sequence Repeat Markers for Denphal-type Dendrobium Cultivars and Transferability to Dendrobium Species

Denphal-type Dendrobium is the most popular orchid for cut flower and potted plant trade. To improve commercial traits, many novel cultivars have been produced through hybridization by commercial breeders. However, the genetic relationship of most cultivars is unclear, thus hindering the progress of Denphal-type Dendrobium breeding programs. Therefore, the development of molecular markers is encouraged to identify different cultivars. In this study, based on the transcriptome database of the Denphal-type Dendrobium ‘Red Bull’, the polymorphisms expressed sequence tag-derived simple sequence repeat (EST-SSR) were developed from 100 pairs EST-SSRs that were randomly selected from the EST-SSR database. The genetic relationship of 42 Denphal-type Dendrobium cultivars was analyzed according to the developed EST-SSRs. Then, the transferability of EST-SSRs was analyzed by performing a relationship analysis of 40 Dendrobium species. The results showed that a total of 5174 potential EST-SSR markers were identified with 4486 unigene sequences, and 5289 primer pairs were successfully designed. Of the selected 100 pairs of EST-SSRs, a total of 86 pairs produced the expected polymerase chain reaction products of the primary screening, 58 pairs produced the expected fragment size, and 20 pairs showed polymorphisms. Furthermore, the dendrogram of 42 cultivars showed that at a genetic distance of 0.15, the cultivars collected were grouped into five clusters of three major clusters and two minor clusters; all these clusters had the same characters of each cluster. The transferability analysis showed that 18 of the 20 EST-SSR markers among the 40 Dendrobium species were polymorphic. Overall, this study developed EST-SSR markers and will be valuable to facilitating genetic diversity in Denphal-type Dendrobium cultivars and Dendrobium species.

Development of EST-SSR markers for an endangered plant species, Camellia fascicularis (Theaceae)

AbstractThe plant Camellia fascicularis, belonging to family Theaceae, has high ornamental and medicinal value, and rare gene resources for genetic improvement of Camellia crops, but is currently threatened with extinction because of the unique and extremely small wild populations. Molecular markers have clarified the wild plant species’ genetic diversity structure, new gene resources and relationship with crops. This will be beneficial for conservation of these valuable crop-related wild species and crop improvement. In this study, we identified 95,979 microsatellite loci from 155,011 transcriptome unigenes, and developed 14 polymorphic expressed sequence tag-derived simple sequence repeat (EST-SSR) microsatellite markers for C. fascicularis. The number of alleles (Na) per locus was 2–8 with a mean of 4.86. The genetic diversity of 40 individuals from four natural populations of C. fascicularis was analysed using these polymorphic markers. The number of alleles (Na) for EST-SSR ranged from 2 to 5, with the expected heterozygosities (He) and observed heterozygosities (Ho) in all loci ranging from 0.183 to 0.683, and from 0.201 to 0.700, respectively, implying a rich genetic variation present in wild C. fascicularis populations. Moreover, the phylogenetic analysis among four populations, using the 14 EST-SSR markers developed in this study, grouped 40 individuals into three groups, which coincide with their geographic distribution. These results showed that 14 EST-SSR markers are available for the analysis of genetic variation in C. fascicularis populations and genetic improvement of new Camellias cultivars by interspecific hybridization, and are beneficial for conservation of the endangered species.

Novel Expressed Sequence Tag-Derived and Other Genomic Simple Sequence Repeat Markers Revealed Genetic Diversity in Ethiopian Finger Millet Landrace Populations and Cultivars.

Finger millet (Eleusine coracana (L.) Geartn.) is a self-pollinating amphidiploid crop cultivated with minimal input for food and feed, as well as a source of income for small-scale farmers. To efficiently assess its genetic diversity for conservation and use in breeding programs, polymorphic DNA markers that represent its complex tetraploid genome have to be developed and used. In this study, 13 new expressed sequence tag-derived simple sequence repeat (EST-SSR) markers were developed based on publicly available finger millet ESTs. Using 10 polymorphic SSR markers (3 genomic and 7 novel EST-derived), the genetic diversity of 55 landrace accessions and 5 cultivars of finger millet representing its major growing areas in Ethiopia was assessed. In total, 26 alleles were detected across the 10 loci, and the average observed number of alleles per locus was 5.6. The polymorphic information content (PIC) of the loci ranged from 0.045 (Elco-48) to 0.71 (UGEP-66). The level of genetic diversity did not differ much between the accessions with the mean gene diversity estimates ranging only from 0.44 (accession 216054) to 0.68 (accession 237443). Similarly, a narrow range of variation was recorded at the level of regional states ranging from 0.54 (Oromia) to 0.59 (Amhara and Tigray). Interestingly, the average gene diversity of the landrace accessions (0.57) was similar to that of the cultivars (0.58). The analysis of molecular variance (AMOVA) revealed significant genetic variation both within and among accessions. The variation among the accessions accounted for 18.8% of the total variation (FST = 0.19; P < 0.001). Similarly, significant genetic variation was obtained among the geographic regions, accounting for 6.9% of the total variation (P < 0.001). The results of the cluster, principal coordinate, and population structure analyses suggest a poor correlation between the genetic makeups of finger millet landrace populations and their geographic regions of origin, which in turn suggests strong gene flow between populations within and across geographic regions. This study contributed novel EST-SSR markers for their various applications, and those that were monomorphic should be tested in more diverse finger millet genetic resources.

Non-linear genetic diversity and notable population differentiation caused by low gene flow of bermudagrass [Cynodon dactylon (L.) Pers.] along longitude gradients.

BackgroundEnvironmental variation related to ecological habitat is the main driver of plant adaptive divergence. Longitude plays an important role in the formation of plant population structure, indicating that environmental differentiation can significantly shape population structure.MethodsGenetic diversity and population genetic structure were estimated using 105 expressed sequence tag-derived simple sequence repeat (EST-SSR) loci. A total of 249 C. dactylon (L.) Pers. (common bermudagrass) individuals were sampled from 13 geographic sites along the longitude (105°57′34″–119°27′06″E).ResultsThere was no obvious linear trend of intra-population genetic diversity along longitude and the intra-population genetic diversity was not related to climate in this study. Low gene flow (Nm = 0.7701) meant a rich genetic differentiation among populations of C. dactylon along longitude gradients. Significantly positive Mantel correlation (r = 0.438, P = 0.001) was found between genetic distance and geographical interval while no significant partial Mantel correlation after controlling the effect of mean annual precipitation, which indicated geographic distance correlated with mean annual precipitation affect genetic distance. The genetic diversity of C. dactylon with higher ploidy level was higher than that with lower ploidy level and groups of individuals with higher ploidy level were separated further away by genetic distance from the lower ploidy levels. Understanding the different genetic bases of local adaptation comparatively between latitude and longitude is one of the core findings in the adaptive evolution of plants.

De novo transcriptome assembly and mining of EST-SSR markers in Gloriosa superba

Gloriosa superba is an economical source of pharmaceutical colchicine, which is a mitotic poison used to treat gout, cancer and inflammatory diseases. It is important to study the genetic variations in this plant, but the progress is impeded due to limited number of molecular markers. In this study, we developed the expressed sequence tag-derived simple sequence repeat (EST-SSR) markers from the transcriptome sequence of the leaf samples of three different ecotypes of G. superba. De novo assembly was performed on these sequencing data to generate a total of 65,579 unigenes and 38,200 coding sequences (CDSs). These CDSs were annotated using NCBI Nr protein database, gene ontology terms and KEGG pathways. Differential gene expression was studied to yield differences in these ecotypes at the molecular level. Finally, a total of 14,672 potential EST-SSRs were identified from these unigenes, among which the dinucleotide (5754, 39.22%) and trinucleotide (5421, 36.95%) repeats were most abundant types followed by mononucleotides (3213, 21.83%). The most frequent motifs were CT/GA (1392, 9.48%), AG/TC (1219, 8.31%), and GA/CT (1146, 7.82%) among the dinucleotide repeats and CCG/ CGG (1487, 10.13%), AGG/CCT (1421, 9.68%), AGC/CTG (697, 4.75%) and AAG/CTT (621, 4.23%) among the trinucleotide repeats. Polymorphism study using a random set of 20 newly developed EST-SSRs revealed polymorphic information content value ranging from 0 to 0.5926 with an average of 0.4021. The large-scale ESTs developed in the current study will be useful as a genomic resource for further investigation of the genetic variations in this species.

Evaluation of genetic diversity and construction of DNA fingerprinting in Polygonatum Mill. based on EST-SSR and SRAP molecular markers.

Polygonatum sibiricum is widely consumed as a traditional Chinese herb and edible plant in China. Despite its nutritional and medical values, research on Polygonatum Mill. has been scarce, particularly as far as its genetic diversity is concerned. In this study, fourteen expressed sequence tag-derived simple sequence repeat (EST-SSR) and seven sequence-related amplified polymorphism (SRAP) markers were used to evaluate the genetic diversity in fifty Polygonatum Mill. accessions. The EST-SSRs and SRAPs produced 173 (90.58%) and 113 (93.39%) polymorphic bands, respectively. Unweighted Pair-Group Method Analysis (UPGMA) based on the combined data matrices of EST-SSRs and SRAPs divided the fifty Polygonatum Mill. accessions into fourteen groups. In addition, accessions of P. cyrtonema Hua obtained from Anhui and Zhejiang provinces were clustered according to their geographic origin. Furthermore, some accessions were gathered together based on species, such as P. kingianum Coll. et Hemsl, P. punctatum Royle ex Kunth, P. odoratum (Mill.) Druce, and P. sibiricum Red., and bootstrap analysis for clustering fully supported the grouping of the accessions. The Analysis of Molecular Variance (AMOVA) results revealed higher variation within populations (95%) rather than among populations (5%), indicating that Polygonatum Mill. has a low genetic differentiation between populations, and Principal Coordinate Analysis (PCoA) greatly supported the results of cluster analysis and AMOVA analysis. Finally, five markers which could produce abundant and stable bands were used to construct DNA fingerprinting database of Polygonatum Mill.. Our results demonstrated the utility of both EST-SSR and SRAP markers to successfully evaluate and identify Polygonatum Mill..

DNA sequence-based mapping and comparative genomics of the St genome of Pseudoroegneria spicata (Pursh) Á. Löve versus wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.).

Bluebunch wheatgrass (referred to as BBWG) [Pseudoroegneria spicata (Pursh) Á. Löve] is an important rangeland Triticeae grass used for forage, conservation, and restoration. This diploid has the basic St genome that occurs also in many polyploid Triticeae species, which serve as a gene reservoir for wheat improvement. Until now, the St genome in diploid species of Pseudoroegneria has not been mapped. Using a double-cross mapping populations, we mapped 230 expressed sequence tag derived simple sequence repeat (EST-SSR) and 3468 genotyping-by-sequencing (GBS) markers to 14 linkage groups (LGs), two each for the seven homologous groups of the St genome. The 227 GBS markers of BBWG that matched those in a previous study helped identify the unclassified seven LGs of the St sub-genome among 21 LGs of Thinopyrum intermedium (Host) Barkworth & D.R. Dewey. Comparisons of GBS sequences in BBWG to whole-genome sequences in bread wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) revealed that the St genome shared a homology of 35% and 24%, a synteny of 86% and 84%, and a collinearity of 0.85 and 0.86, with ABD and H, respectively. This first-draft molecular map of the St genome will be useful in breeding cereal and forage crops.

Genetic Diversity and Population Structure of Bermudagrass [Cynodon dactylon (L.) Pers.] along Latitudinal Gradients and the Relationship with Polyploidy Level

Understanding the population genetic pattern and process of gene flow requires a detailed knowledge of how landscape characteristics structure populations. Although Cynodon dactylon (L.) Pers. (common bermudagrass) is widely distributed in the world, information on its genetic pattern and population structure along latitudinal gradients is limited. We tried to estimate the genetic diversity and genetic structure of C. dactylon along a latitudinal gradient across China. Genetic diversity among different ploidy levels was also compared in the study. The material used consisted of 296 C. dactylon individuals sampled from 16 geographic sites from 22°35′ N to 36°18′ N. Genetic diversity was estimated using 153 expressed sequence tag-derived simple sequence repeat (EST-SSR) loci. Higher within-population genetic diversity appeared at low-latitude, as well as having positive correlation with temperature and precipitation. The genetic diversity increased with the ploidy level of C. dactylon, suggesting polyploidy creates higher genetic diversity. No isolation by distance and notable admixture structure existed among populations along latitudes. Both seed dispersal (or vegetative organs) and extrinsic pollen played important roles for gene flow in shaping the spatial admixture population structure of C. dactylon along latitudes. In addition, populations were separated into three clusters according to ploidy levels. C. dactylon has many such biological characters of perennial growth, wind-pollination, polyploidy, low genetic differentiation among populations, sexual and asexual reproduction leading to higher genetic diversity, which gives it strong adaptability with its genetic patterns being very complex across all the sampled latitudes. The findings of this study are related to landscape population evolution, polyploidy speciation, preservation, and use of bermudagrass breeding.

Comparative transcriptome analysis and simple sequence repeat marker development for two closely related Isodon species used as ‘Xihuangcao’ herbs

Purpose : To facilitate the molecular identification of original plants, resolve taxonomic problems and identify standards for ‘Xihuangcao’-based products on the market. Methods : A transcriptomic analysis of two closely related species, i.e., Isodon serra (Maxim.) (IS) and I. lophanthoides (Buch.-Ham. ex D. Don) Hara, was conducted by using the Illumina HiSeq 2500 platform, and expressed sequence tag-derived simple sequence repeat (EST-SSR) markers were developed based on these transcriptomes. Results : In total, 149,650 and 103,221 contigs were obtained, with N50 values of 1,400 and 1,516, from the IS and I. lophanthoides RNA-Seq datasets, respectively. These contigs were clustered into 107,777 and 68,220 unigenes, which were functionally annotated to identify the genes involved in therapeutic components. In total, 14,138 and 11,756 EST-SSR motifs were identified, and of these motifs, 7,453 and 6,428 were used to design primers for IS and I. lophanthoides, respectively. After PCR verification and fluorescence-based genotyping, 24 SSR markers with bright bands, high polymorphism, and single amplification were obtained and used to identify closely related Isodon species/varieties. Conclusion : These data could help herbal scientists identify high-quality herbal plants and provide a reference for genetic improvement and population genetic and phylogenetic studies investigating ‘Xihuangcao’ herbs. Keywords : Xihuangcao, Transcriptome, EST-SSR, Molecular markers

Assessment of genetic diversity in IranianAsparagus spp.related to garden asparagus

Asparagus is a large genus comprising three subgenera (Asparagus, Protasparagus and Myrsiphyllum) with species distributed in Africa, Asia, Australia and Europe. The southwest of Asia includes the natural distribution of several species of this genus. Also, the proposed center of origin of garden asparagus (A. officinalis) is located in this region. The genetic diversity and the phylogenetic relationships among twelve diploid and polyploid (4x, 8x and 10x) Asparagus accessions (7 A. officinalis + 3 A. verticillatus + 1 A. persicus + 1 A. breslerianus), collected from different sites of Iran, were assessed in this study. Around ten plants per accession were analyzed using a set of six expressed sequence tag-derived simple sequence repeats (EST-SSRs) primer pairs. An average of 23 alleles per marker was detected in the studied collection, and an octoploid A. officinalis showed the highest number of alleles (62 alleles total). A cluster analysis was carried out with the alleles matrix (1 = presence, 0 = absence). In the dendrogram obtained, all individuals from each accession were grouped together and the different accessions were clearly separated. In addition, three main groups were obtained: a first group was formed by both A. verticillatus and A. persicus populations, a second one by diploid A. officinalis, and the third group joined the polyploid A. officinalis (4x, 8x and 10x) and the octoploid A. breslerianus. A high genetic variability was observed in this study, which could be used to broaden the genetic base of garden asparagus.

Evaluation of the Genetic Diversity and Differentiation of Black Locust (Robinia pseudoacacia L.) Based on Genomic and Expressed Sequence Tag-Simple Sequence Repeats.

Understanding the genetic diversity and differentiation of the genetic resources of a species is important for the effective use and protection of forest tree resources. Ex situ development is a common method for the protection of genetic diversity and an essential resource for users who require ready access to a species’ germplasm. In this study, we collected seeds of black locust (Robinia pseudoacacia L.) from 19 provenances, covering most of its natural distribution; we randomly selected 367 tender leaves with well-grown and different maternal strains from this group for further analysis. Forty-eight simple sequence repeat (SSR) primers were successfully selected from 91 pairs of SSR primers using native-deformation polyacrylamide gel electrophoresis. In addition, we identified identical genotypes among all individuals and evaluated the quality of the markers. From this, 35 loci were confirmed for analyses of genetic diversity and differentiation of the black locust provenances, which contained 28 expressed sequence tag-derived simple sequence repeats (EST-SSRs) and 7 genomic DNA-derived simple sequence repeats (G-SSRs). We observed high genetic diversity among the native black locust provenances, from which Wright’s fixation index and molecular variance suggested that a majority of the genetic differentiation variation could be attributed to within-provenance differences. The genetic distance and identity results indicated that geographic distance was not a dominating factor influencing the distribution of black locust. This is the first study to evaluate provenance genetic variation in native black locust samples using two types of SSR markers, which provides a comprehensive theoretical basis for ex situ conservation and utilization of genetic resources, with an emphasis on breeding applications.

De Novo Transcriptome Assembly and Population Genetic Analyses for an Endangered Chinese Endemic Acer miaotaiense (Aceraceae).

Acer miaotaiense (P. C. Tsoong) is a rare and highly endangered plant in China. Because of the lack of genomic information and the limited number of available molecular markers, there are insufficient tools to determine the genetic diversity of this species. Here, 93,305 unigenes were obtained by multiple assembled contigs with a transcriptome sequencing program. Furthermore, 12,819 expressed sequence tag-derived simple sequence repeat (EST-SSR) markers were generated, 300 were randomly selected and synthesized, 19 primer pairs were identified as highly polymorphic (average number of alleles (Na) = 8, expected heterozygosity (He) = 0.635, polymorphism information content (PIC) = 0.604) and were further used for population genetic analysis. All 261 samples were grouped into two genetic clusters by UPGMA, a principal component analyses and a STRUCTURE analyses. A moderate level of genetic differentiation (genetic differentiation index (Fst) = 0.059–0.116, gene flow = 1.904–3.993) among the populations and the major genetic variance (81.01%) within populations were revealed by the AMOVA. Based on the results, scientific conservation strategies should be established using in situ and ex situ conservation strategies. The study provides useful genetic information for the protection of precious wild resources and for further research on the origin and evolution of this endangered plant and its related species.

Development and linkage mapping of novel sex-linked markers for marker-assisted cultivar breeding in pistachio (Pistacia vera L.)

The dioecious character of Pistacia vera L (the pistachio tree) limits its breeding capacity. Thus, early stage selection of males can save time, labor, and land. This study aimed to develop sex-linked single nucleotide polymorphism (SNP) markers, together with expressed sequence tag-derived simple sequence repeats (EST-SSRs), to determine position of the sex locus in pistachio by constructing a linkage map of its sex chromosome for the first time. Nine novel sex-linked SNP markers were successfully identified by SNaPshot minisequencing analysis of 25 SNP loci from 17 restriction site-associated DNA (RAD) reads in 309 individuals. All nine markers were heterozygous in females and homozygous in males supporting a ZW/ZZ sex determination system in pistachio. A total of 105 segregating SSRs and sex-linked markers were used to identify the sex chromosome and the position of the sex locus through analysis of a Siirt × Bagyolu F1 population with 122 progenies. Of these 105 markers, four common and four paternal SSRs were mapped onto the sex chromosome, along with the phenotypic sex locus and sex-linked markers. The resulting consensus map had a total length of 65.19 cM. The sex locus and sex-linked SNP markers were located in the center of the chromosome at a distance of 31.86 and 31.92 cM, respectively. This study presents valuable information about the sex chromosome and sex locus position as well as novel polymorphic EST-SSRs and nine sex-linked SNP markers in pistachio.

Identification and validation of novel EST-SSR markers in olives

The olive (Olea europaea L.) is a leading oil crop in the Mediterranean area. Limited information on the inheritance of agronomic significant traits hinders progress in olive breeding programs, which encourages the development of markers linked to the traits. In this study, we report on the development of 46 olive simple sequence repeat (SSR) markers, obtained from 577,025 expressed sequence tags (ESTs) in developing olive fruits generated in the framework of the Slovenian national olive transcriptome project. Sequences were de novo assembled into 98,924 unigenes, which were then used as a source for microsatellites searching. We identified 923 unigenes that contained 984 SSRs among which dinucleotide SSRs (36 %) were the most abundant, followed by tri- (33 %) and hexa- (21 %) nucleotides. Microsatellite repeat motif GA (37 %) was the most common among dinucleotides, while microsatellite repeat motif GAA was the most abundant trinucleotide SSR motif (16 %). Gene ontology annotations could be assigned to 27 % of the unigenes. A hundred and ten expressed sequence tag-derived-simple sequence repeats (EST-SSRs) with annotated genes were selected for primer designing and finally, 46 (42 %) polymorphic EST-SSRs were successfully amplified and used to validate genetic diversity among 24 olive varieties. The average number of alleles per locus, observed heterozygosity, expected heterozygosity, and polymorphic information content were 4.5, 0.649, 0.604 and 0.539, respectively. Twenty-seven EST-SSRs showed good diversity properties and were recommended for further olive genome investigation.

Genetic Diversity in Vegetable and Grain Type Soybean Genotypes Identified using Morphological Descriptor and EST-SSR Markers

Genetic Diversity in Vegetable and Grain Type Soybean Genotypes Identified using Morphological Descriptor and EST-SSR Markers Thirty four soybean genotypes were evaluated for morphological, quality traits and genetic parameters. Correlation and path coefficients were studied for some traits. Analysis of variance and mean performance for various traits revealed significant differences diversity between all the genotypes for traits studied. The genetic diversity among the soybean genotypes was analyzed using ten expressed sequence tag derived simple sequence repeat (ESTSSR) markers. A total of 27 alleles were detected from 334 amplicons with an average of 2.7 alleles per locus. The polymorphism information content (PIC) values of EST-SSR markers ranged from 0.334 to 0.837 with an average of 0.559. These grain and vegetable soybean genotypes could be divided into 7 subgroups based on similarity matrix and arithmetic average (UPGMA) cluster with no correlation between genetic and morphological diversity. The analysis clearly indicated that even with the EST-SSR primers, reliable estimation of genetic diversity among the population could be obtained...

Development and Characterization of 1,906 EST-SSR Markers from Unigenes in Jute (Corchorus spp.).

Jute, comprising white and dark jute, is the second important natural fiber crop after cotton worldwide. However, the lack of expressed sequence tag-derived simple sequence repeat (EST-SSR) markers has resulted in a large gap in the improvement of jute. Previously, de novo 48,914 unigenes from white jute were assembled. In this study, 1,906 EST-SSRs were identified from these assembled uingenes. Among these markers, di-, tri- and tetra-nucleotide repeat types were the abundant types (12.0%, 56.9% and 21.6% respectively). The AG-rich or GA-rich nucleotide repeats were the predominant. Subsequently, a sample of 116 SSRs, located in genes encoding transcription factors and cellulose synthases, were selected to survey polymorphisms among12 diverse jute accessions. Of these, 83.6% successfully amplified at least one fragment and detected polymorphism among the 12diverse genotypes, indicating that the newly developed SSRs are of good quality. Furthermore, the genetic similarity coefficients of all the 12 accessions were evaluated using 97 polymorphic SSRs. The cluster analysis divided the jute accessions into two main groups with genetic similarity coefficient of 0.61. These EST-SSR markers not only enrich molecular markers of jute genome, but also facilitate genetic and genomic researches in jute.

Short communication. Employment of molecular markers to develop tetraploid “supermale” asparagus from andromonoecious plants of the landrace ‘Morado de Huétor’

The aim of this work was the development of new “supermale” genotypes retaining the highest genetic diversity possible of the tetraploid asparagus landrace ‘Morado de Huetor’. The elite andromonoecious male HT664 of the ‘Morado de Huetor’ asparagus landrace and the andromonoecious hybrid male HC027, obtained by crossing between this landrace and a commercial cultivar of Asparagus officinalis, were selected for self-pollination to produce possible “supermales” with genes of ‘Morado de Huetor’ (SMHT). To confirm the hybrid nature of HC027, we characterized this genotype with EST-SSR (Expressed Sequence Tag-derived Simple Sequence Repeats) markers. We also adopted the sex-linked marker Asp1-T7 for sex determination in ‘Morado de Huetor’ and the resulting hybrids between this landrace and other commercial cultivars. Asp1-T7 marker was used for the selection and genotyping of SMHT. “All-male” cultivars with a different genetic background can be generated by crossing females with these new “supermale” genotypes, and the agronomical traits of these new cultivars would be very different from the “all-male” cultivars currently available in the market, making them extremely interesting for asparagus breeding programs.