Expressed Sequence Tags-simple Sequence Repeats Markers Research Articles

Analyses of genetic diversity and population structure of anchote (Coccinia abyssinica (Lam.) Cogn.) using newly developed EST-SSR markers

Anchote (Coccinia abyssinica (Lam.) Cogn.) is a perennial root crop belonging to Cucurbitaceae family. It is endemic to Ethiopia and distributed over wide range of agro-ecologies. For further improvement and efficient conservation of this crop, characterization of its genetic diversity and its pattern of distribution is a vitally important step. Expressed sequence tags-simple sequence repeats (EST-SSRs) markers were developed from publicly available watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai] ESTs in the GenBank database. Among those novel markers, eight were polymorphic and subsequently used for genetic diversity and population structure analyses of 30 anchote accessions collected from western Ethiopia. A total of 24 alleles were obtained across the eight polymorphic loci and 30 accessions that revealed moderate level of genetic diversity in this minor crop. Among the eight loci, locus CA_06 was the most informative with six alleles and polymorphic information content (PIC) of 0.76. The accessions showed about threefold variation in terms of genetic diversity, with expected heterozygosity (He) ranging from 0.15 (accession An) to 0.44 (accession Dg). Other accessions with higher genetic diversity include Ar and Gu (He = 0.43 and 0.41, respectively). Analysis of molecular variance (AMOVA) revealed that the variation within accessions and among accessions accounted for 84.7% and 15.3% of the total variation, respectively. The study revealed low but significant population differentiation in this crop with no clear pattern of population structure. The EST-SSR markers developed in this study are the first of their kind for anchote and can be used for characterization of its wider genetic resources for conservation and breeding purposes.

VfODB: a comprehensive database of ESTs, EST-SSRs, mtSSRs, microRNA-target markers and genetic maps in Vicia faba.

Faba bean (Vicia faba) is an essential food and fodder legume crop worldwide due to its high content of proteins and fibres. Molecular markers tools represent an invaluable tool for faba bean breeders towards rapid crop improvement. Although there have historically been few V. faba genome resources available, several transcriptomes and mitochondrial genome sequence data have been released. These data in addition to previously developed genetic linkage maps represent a great resource for developing functional markers and maps that can accelerate the faba bean breeding programmes. Here, we present the Vicia faba Omics database (VfODB) as a comprehensive database integrating germplasm information, expressed sequence tags (ESTs), expressed sequence tags-simple sequence repeats (EST-SSRs), and mitochondrial-simple sequence repeats (mtSSRs), microRNA-target markers and genetic maps in faba bean. In addition, KEGG pathway-based markers and functional maps are integrated as a novel class of annotation-based markers/maps. Collectively, we developed 31 536 EST markers, 9071 EST-SSR markers and 3023 microRNA-target markers based on V. faba RefTrans V2 mining. By mapping 7940 EST and 2282 EST-SSR markers against the KEGG pathways database we successfully developed 107 functional maps. Also, 40 mtSSR markers were developed based on mitochondrial genome mining. On the data curation level, we retrieved 3461 markers representing 12 types of markers (CAPS, EST, EST-SSR, Gene marker, INDEL, Isozyme, ISSR, RAPD, SCAR, RGA, SNP and SSR), which mapped across 18 V. faba genetic linkage maps. VfODB provides two user-friendly tools to identify, classify SSR motifs and in silico amplify their targets. VfODB can serve as a powerful database and helpful platform for faba bean research community as well as breeders interested in Genomics-Assisted Breeding.

Morphological and Genetic Diversity of Sea Buckthorn (Hippophae rhamnoides L.) in the Karakoram Mountains of Northern Pakistan

Sea buckthorn (Hippophae rhamnoides L.) is a dioecious, wind-pollinated shrub growing in Eurasia including the Karakoram Mountains of Pakistan (Gilgit-Baltistan territory). Contrary to the situation in other countries, in Pakistan this species is heavily underutilized. Moreover, a striking diversity of berry colors and shapes in Pakistan raises the question: which varieties might be more suitable for different national and international markets? Therefore, both morphological and genetic diversity of sea buckthorn were studied to characterize and evaluate the present variability, including hypothetically ongoing domestication processes. Overall, 300 sea buckthorn individuals were sampled from eight different populations and classified as wild and supposedly domesticated stands. Dendrometric, fruit and leaf morphometric traits were recorded. Twelve EST-SSRs (expressed sequence tags-simple sequence repeats) markers were used for genotyping. Significant differences in morphological traits were found across populations and between wild and village stands. A significant correlation was found between leaf area and altitude. Twenty-two color shades of berries and 20 dorsal and 15 ventral color shades of leaves were distinguished. Mean genetic diversity was comparatively high (He = 0.699). In total, three distinct genetic clusters were observed that corresponded to the populations’ geographic locations. Considering high allelic richness and genetic diversity, the Gilgit-Baltistan territory seems to be a promising source for selection of improved germplasm in sea buckthorn.

Cross-species transferability of microsatellite markers from Fusarium oxysporum for the assessment of genetic diversity in Fusarium udum

Expressed sequence tags (ESTs) are the source of simple sequence repeats (SSRs) that can be used to develop molecular markers for the study of polymorphism and genetic diversity. In the present investigation, 30 EST simple sequence repeats (SSR) primer sets derived from three formae speciales of Fusarium oxysporum: melonis (Fom), cucumerium (Foc), and lycopersici (Fol) – were tested for transferability to Fusarium udum. The majority of SSR loci contain trinucleotide (63.70%) while fewer contain di- (27.41%), tetra- (5.64%) and penta-nucleotide (3.22%) repeats. The number of alleles at these SSR loci ranged from one to three, with an average of 1.4 alleles per locus. CAG (24.19%) and AC (16.93%) were the most abundant motifs identified. Three markers (FomSSR-8, FolSSR-2 and FolSSR-4) were found highly informative for genetic characterization of F. udum and very useful in distinguishing the polymorphism rate of the markers at specific locus; however, polymorphic information content (PIC) was maximum (0.597) in FocSSR-7. In terms of cross species transferability, 70% of the primer sets of Fom-SSR and Fol-SSR and 30% of the Foc-SSR produced an amplicon in F. udum isolates. To the best of our knowledge, this is the first set of EST SSR markers developed and assessed for the variability, genetic analysis and evolutionary relationships of the F. udum population.

Development of expressed sequence tags-simple sequence repeats (EST-SSRs) for Musa and their applicability in authentication of a Musa breeding population

Maintaining identity of clones is essential in breeding programs. New EST-SSR markers have been developed for banana and used to screen a diploid population for clonal identity. A total of 410 primer pairs were designed from an EST database, validated using polyacrylamide gel electrophoresis (PAGE) and a subset was optimized for accurate genotyping on a capillary genetic analyzer. Combining PAGE and capillary electrophoresis, about 44% of the designed primers were informative in the diploid population. The majority of markers produced two alleles as expected in a diploid population. However, some showed three to four alleles, possibly indicating closely-related members of gene families. Screening of field samples using SSR markers revealed genotype identity issues in the target population. The present study demonstrates the applicability of SSRs in the establishment of parentage and relatedness between accessions. The newly-developed SSRs will be valuable tools in the understanding of Musa genetics, in marker-trait associations, thereby enhancing the effectiveness of breeding programs. Key words : EST, SSRs, markers, Musa, breeding.

Identification of selected apricot cultivars using RAPD and EST-SSR markers

Random amplified polymorphic DNA (RAPD) and expressed sequence tags–simple sequence repeats (EST-SSR) are useful for cultivar identification. In this study, we applied a new approach using RAPD fingerprints to distinguish 34 apricot cultivars based on the optimization of RAPD by choosing 11-nucleotide (nt) primers and strict screening PCR annealing temperature. We further compared the discriminating ability of EST-SSR markers using the same number of primer pairs. The results showed that this new approach can clearly utilize and record the fingerprints generated from various RAPD primers during cultivar identification, and a cultivar identification diagram (CID) could be constructed. The CID approach can be used for efficient identification of apricot cultivars, providing information to separate groups of cultivars. The reliability and efficiency of this method was checked and verified. Five RAPD primers clearly distinguished 34 apricot cultivars, but five EST-SSR primer pairs could only identify 14 cultivars. To the best of our knowledge, this new approach is the most efficient and reliable method for identification of plant varieties by RAPD or EST-SSR markers, and will be very helpful in identification of plant cultivars, for cultivar rights protection and will also be beneficial in the nursery industry due to early identification of seedlings.

Development and characterization of cDNA library based novel EST-SSR marker in radish (Raphanus sativus L.)

To develop novel expressed sequence tags-simple sequence repeats (EST-SSR) markers in radish, a taproot cDNA library was constructed. A total of 10,052 clones from the cDNA library were sequenced and a large number of high quality ESTs was generated. Among these ESTs, a set of 179 SSRs, distributing in 176 unigene sequences, were detected, of which, 50.84% were di-nucleotide, followed by tri- (29.61%), hexa- (9.49%), penta- (6.15%) and tetra-nucleotide (3.91%) repeats, respectively. GA/TC (25.69%) was the most frequent repeat type of all repeat types. Out of 125 primer pairs designed, 110 (88%) could generate unambiguous amplification products. Totally 28 EST-SSR primer pairs were selected for genetic diversity analysis in 32 radish genotypes. It was found that two to seven alleles could be detected with a mean of 3.5 per primer pair and the polymorphism information content (PIC) values of these primers ranged from 0.000 to 0.825 with an average of 0.500. The genotype data were analyzed with NTSYS-pc software, which grouped the 32 accessions into three main clusters at the similarity index value of 0.60, which was mainly in accordance with the different biological characterizations of the accessions. In addition, the 28 EST-SSR primer pairs were further used to test the transferability on 12 accessions from three genera of Raphanus, Brassica and Arabidopsis in Brassicaceae. The results showed that 18 primer pairs (64.2%) could produce target PCR bands in the 12 accessions. The EST-SSR markers developed herein represented a valuable resource for the genetic diversity analysis, genetic mapping and marker-assisted selection in radish.

Characterization of new polymorphic functional markers for sugarcane

Expressed sequence tags (ESTs) offer the opportunity to exploit single, low-copy, conserved sequence motifs for the development of simple sequence repeats (SSRs). The authors have examined the Sugarcane Expressed Sequence Tag database for the presence of SSRs. To test the utility of EST-derived SSR markers, a total of 342 EST-SSRs, which represent a subset of over 2005 SSR-containing sequences that were located in the sugarcane EST database, could be designed from the nonredundant SSR-positive ESTs for possible use as potential genic markers. These EST-SSR markers were used to screen 18 sugarcane (Saccharum spp.) varieties. A high proportion (65.5%) of the above EST-SSRs, which gave amplified fragments of foreseen size, detected polymorphism. The number of alleles ranged from 2 to 24 with an average of 7.55 alleles per locus, while polymorphism information content values ranged from 0.16 to 0.94, with an average of 0.73. The ability of each set of EST-SSR markers to discriminate between varieties was generally higher than the polymorphism information content analysis. When tested for functionality, 82.1% of these 224 EST-SSRs were found to be functional, showing homology to known genes. As the EST-SSRs are within the expressed portion of the genome, they are likely to be associated to a particular gene of interest, improving their utility for genetic mapping; identification of quantitative trait loci, and comparative genomics studies of sugarcane. The development of new EST-SSR markers will have important implications for the genetic analysis and exploitation of the genetic resources of sugarcane and related species and will provide a more direct estimate of functional diversity.

Polymorphic EST–SSR markers and their mode of inheritance in Fenneropenaeus chinensis

229 SSRs (simple sequence repeats) were identified among 10,443 ESTs (expressed sequence tags) of Chinese shrimp ( Fenneropenaeus chinensis). The average density of SSRs was one SSR per 19.1 kb of EST sequence screened. The dinucleotide repeats appeared to be the most abundant SSRs detected. Nine EST–SSR markers were detected polymorphisms of the thirty SSR primer pairs derived from F. chinensis ESTs. The number of alleles per locus ranged from 5 to 15, with an average of 9.1 alleles per locus. The observed heterozygosity of nine loci ranged from 0.47 to 0.87. These loci were used successfully for pedigree analysis in three families of Fenneropenaeus chinensis. Two of the nine microsatellite loci showed the existence of null alleles. Assuming the existence of null alleles at Fc07 and Fc14 loci, the allelic inheritance mode of the EST–SSR DNA markers ( Fc04, Fc06, Fc07, Fc10, Fc14, Fc18, Fc22, Fc24, and Fc27) was consistent with Mendelian segregation.