EST-derived Microsatellite Research Articles

Genetic diversity and differentiation of nine populations of the hard clam (Meretrix petechialis) assessed by EST-derived microsatellites

Abstract Background Meretrix petechialis is one of the commercially important marine bivalves. In this study, we selected six highly polymorphic EST-derived microsatellite markers to assess the genetic diversity and population differentiation on nine wild populations of Meretrix petechialis. Results The number of alleles detected per locus ranged from 4 to 30 (mean NA = 27.5) with a total of 165 alleles. The mean value of observed and expected heterozygosities varied from 0.717 to 0.861 and from 0.797 to 0.856, respectively. Meanwhile, the result of Neighbor-joining and overall FST = 0.214 (P Conclusions GX population has high genetic diversity among individual, and there are certain differences in genetic characteristics among different populations. This study will provide a basis for the domestication and cultivation of genetic diversity of M. petechialis population and the protection of clam germplasm resources. How to cite Xu Q, Zheng J, Yan X, et al. Genetic diversity and differentiation of nine populations of the hard clam (Meretrix petechialis) assessed by EST-derived microsatellites. Electron J Biotechnol 2020;48. https://doi.org/10.1016/j.ejbt.2020.09.003

Identification of Growth-related EST-derived Microsatellite Marker in Nile Tilapia (Oreochromis niloticus)

This study aimed at investigating growth-related, EST-derived microsatellite markers in Nile tilapia (O. niloticus). The link between alleles and genotypes of these makers was particularly examined, as well as the growth performances of the samples of offspring produced from the broodstocks of 5 different fishery stations in Thailand. A total of 25 families of offspring were reared to observe the growth performance for a period of 83 days. Fish samples in this study were divided into two categories according to average body weight: fast (+SD) and slow (-SD) growth. Then, the fins of the fish samples were employed to perform DNA analysis. From the fast and slow growth evaluations, 3 families, namely F1, F24 (from Chumphon) and F11 (from Petchaburi), were chosen for the association analysis. A total of 30 fishes with 5 samples from fast and slow growth of each family were utilized. Nineteen EST-derived microsatellite markers were used to genotype 30 DNA samples of the fast and slow growth fishes. Out of 19 loci, 14 loci (i.e., OMO392, OMO051, OMO097, OMO072, OMO327, OMO277, OMO122, OMO193, OMO198, OMO200, OMO335, OMO374, OMO049, and OMO069) were found polymorphic. Another 4 loci (i.e., OMO059, OMO068, OMO315, and OMO337) were observed as monomorphic. Based on the genotype data, there appeared to indicate a strong, significant relationship between allele and growth of the A3 allele of OMO392 locus in the fast growth group. However, no significant genotypes regarding 19 EST-microsatellite were found to be related with growth. This study suggests that the identified allele A3, which has designated the growth hormone related EST-derived microsatellite primer OMO392, can potentially be used to facilitate marker-assisted selection regarding the fast growth of O. niloticus.

Multiplex and characterization of new EST-derived microsatellite and transferability among five Eucalyptus species (Myrtaceae)

Multiplex and characterization of new EST-derived microsatellite and transferability among five Eucalyptus species (Myrtaceae)

Cross-taxon transferability of sugarcane expressed sequence tags derived microsatellite (EST-SSR) markers across the related cereal grasses

Sugarcane is a crop of economic importance providing sugar and bioenergy resource which has a substantial contribution to the national GDP of India as well as other countries. Genomes of the several grasses have co-evolved which share large-scale synteny and genome collinearity; hence it is possible to employ EST-SSR markers of one grass species for genetic study of other related species. A set of sixty potentially informative EST-derived microsatellite (SSR) markers were employed to examine their cross-taxa transferability across the limit of taxonomic boundaries of different grass genomes. The high (96.6%) rate of SSRs cross-transferability observed in genus Saccharum followed by Erianthus (93.3%), Narenga (88.3%), Sclerostachya (86.3%), and Miscanthus (83.3%). Relatively lower rates of cross amplification were observed in other cereal crops including Sorghum bicolor, Pennisetum glaucum, Zea mays, Triticum aestivum, Oryza sativa, and Avena sativa. Cross-transferable EST-SSR markers were utilized to evaluate phylogenetic relationships among genus Saccharum, closely related genera of Saccharum complex, and other allied cereal grasses. The polymorphic information content values ranged from 0.35 to 0.95, with an average of 0.71. Such a high rates of cross-taxa transferability of the EST-SSRs make them a potent tool for genetic studies and comparative genomics across wide evolutionary distances within and across genera along with technologically simplicity and cost effectiveness. EST-SSRs will facilitate to devise potent tools for genetic improvement through advance molecular breeding of such crops for high grain productivity and better quality. They will especially be useful for the orphan grass species, which contribute a major part of the global food security.

Development of EST-derived microsatellite markers to investigate the population structure of sparganum - the causative agent of zoonotic sparganosis.

The plerocercoid (sparganum) of Spirometra erinaceieuropaei is the main aetiological agent of human sparganosis. To improve the current knowledge on S. erinaceieuropaei evolution, we performed multi-locus microsatellite typing of sparganum isolates from China for the first time. All available expressed sequence tag (EST) sequences for the Spirometra were downloaded from the GenBank. The identification and localization of microsatellites in ESTs was accomplished by MISA. Based on the selected microsatellites, the genetic structure of 64 sparganum isolates collected from 11 geographical locations in southwest China were investigated through principal component analysis, STRUCTURE analysis and neighbour-joining clustering. A total of 522 non-redundant ESTs containing 915 simple sequence repeats were identified from 12 481 ESTs screened. Five primer pairs were finally selected. Using these loci, a total of 12 alleles were detected in 64 sparganum isolates. Little variability was observed within each of geographical population, especially among isolates derived from Kunming of Yunnan (YN-KM) province. Both STRUCTURE analysis and the clustering analysis supported that two genotypes existed among the sparganum isolates from southwest China. In conclusion, five microsatellite markers were successfully developed, and sparganum population was observed to harbour low genetic variation, further investigation with deeper sampling was needed to elucidate the population structure.

Characterization of leaf transcriptome, development and utilization of unigenes-derived microsatellite markers in sugarcane (Saccharum sp. hybrid).

Sugarcane (Saccharum species hybrid) is the major source of sugar (> 80% sugar) in the world and is cultivated in more than 115 countries. It has recently gained attention as a source of biofuel (ethanol). Due to genomic complexity, the development of new genomic resources is imperative in understanding the gene regulation and function, and to fine tune the genetic improvementof sugarcane. In this study, a cDNA library was constructed from mature leaves so as to develop ESTs resources which were further compared with nucleotide and protein databases to explore the functional identity of sugarcane genes. The non-redundant ESTs (unigenes) were categorized into 18 metabolic functions. The major categories were bioenergetics and photosynthesis (4%), cell metabolism (5%), development related protein (3%), membrane-related, mobile genetic elements (5%), signal transduction (2%), DNA (1%), RNA (1%) and protein (2%) metabolism, other metabolic processes (3%), transcription factors (1%), transport (4%) and proteinsrelated to stress/defense (4%). From 540 unique ESTs, 212 simple sequence repeats were identified, of which 206 were from 463 singlets and six were mined from 77 contig sequences. A total of 540 unique EST sequences were used for SSR search of which 97 (17.9%) contained specified SSR motifs, generating 212 unique SSRs. The genes characterized in this study and the EST-derived microsatellite markers identified from the cDNA library will enrich genomic resources for association- and linkage-mapping studies in sugarcane.

Development of highly transferable microsatellites for Panax ginseng (Araliaceae) using whole-genome data.

Premise of the study:Highly transferable expressed sequence tag (EST) microsatellites were developed for Panax ginseng (Araliaceae), one of the most celebrated traditional Chinese medicines and an endangered species in East Asia, using whole-genome data.Methods and Results:Twenty-one EST microsatellites were characterized from next-generation sequencing and were composed of di- and trinucleotide repeats. Polymorphisms and genetic diversity were evaluated for 45 accessions of three ginseng landraces. The number of alleles for each locus ranged from one to five among the landraces, and the polymorphism information content varied from 0.0000 to 0.6450. These microsatellites were also tested for congeneric amplification with P. notoginseng, P. stipuleanatus, P. quinquefolius, P. bipinnatifidus, and the closely related species Aralia elata.Conclusions:These novel EST-derived microsatellite markers will facilitate further population genetic studies of the genera Panax and Aralia.

Characterization of newly developed expressed sequence tag-derived microsatellite markers revealed low genetic diversity within and low connectivity between European Saccharina latissima populations

The kelp Saccharina latissima is a species of high ecological and economic importance. We developed a novel set of S. latissima-specific genetic markers that will find applications in conservation biology, biodiversity assessment, and commercial exploitation of this macroalga. Thirty-two expressed sequence tag (EST)-derived microsatellite markers (SSRs) were developed and characterized in this study using publically available EST sequences. Twenty-seven percent of the 7064 analyzed ESTs contained repeat motifs, and polymerase chain reaction (PCR) amplification primers were designed for 96 selected loci. Fifty-one (53 %) of the primer pairs amplified their target loci, of which 32 (33 %) were polymorphic within a sample of 96 S. latissima sporophytes collected from six localities distributed along the European Atlantic coast from Southern Brittany (France) to Spitzbergen (Norway). The 32 loci harbored moderate levels of polymorphism with 2–13 alleles per locus (mean 5.4). The 25 loci that were retained for population genetic analyses revealed substantial genetic differentiation among the European populations (pairwise F ST values ranging from 0.077 to 0.562) that did not follow any pattern of isolation by distance. In addition, within-population genetic diversity was generally low (Hs < 0.323). Two non-mutually exclusive hypotheses were proposed to explain this low diversity pattern: (1) lower variability of the EST-derived microsatellites compared to the random distribution of SSRs developed from genomic DNA since the former are frequently located in coding regions, which are generally less variable, or (2) reduced effective population size of S. latissima. The particularly high genetic differentiation between the French and Scandinavian S. latissima populations is in agreement with the reported ecotypic differentiation, which may reflect an important resource for genetic improvement. The pattern of genetic diversity revealed in this study thus suggests that care should be taken to avoid the transfer of strains between different geographic regions.

Application of EST-derived microsatellite markers for analysis of genetic variation in tall fescue and its comparison with morphological markers

Analysis of genetic diversity in germplasm collections is an important component of crop improvement programs. This study was conducted to analyze genetic variation and to classify tall fescue genotypes based on phenotypic evaluation and EST-SSR molecular markers. Twenty-five genotypes were assessed based on phenotypic and 42 EST-SSR molecular markers according to a completely randomized block design with three replications during eight years (2007–2014). Results indicated that the effect of year, genotype and their interaction were significant for all of the measured traits. Both morphological and molecular assessments showed considerable genetic variation among genotypes. The estimates of broad-sense heritability (h2b) were moderate to high (h2b = 42.1–78.4) for the traits studied. Based on EST-SRR analysis, a total number of 229 alleles were detected with an average of 4.58 alleles per marker. Average PIC value was 0.49 with a range of 0.014 for NFA140 to 0.95 for NFA047. Phenotypic evaluations and EST-SSR molecular marker classified genotypes into 3 and 7 clusters, respectively which mainly supported geographical origins. The general correspondence was observed between morphological and molecular classification. Therefore, combining the molecular markers with morphological responses could be more beneficial to describe genetic variation and distinguish superior genotypes for future breeding programs.

Genetic variation of Central European oaks: shaped by evolutionary factors and human intervention?

Oak species (Quercus spp.) in Central Europe grow on a relatively wide range of sites. Due to the economic importance of oak for its wood and other products, oak forests have long been managed by humans. This raises the question whether adaptation and/or human activities—especially the moving of propagules—have left their footprints on the genetic variation of oak populations. To address this question, we focused on the Upper Rhine Valley, a densely populated area today that was settled by humans early on. Here, the three most common native Central European oak species can be found. We studied their genetic variation across a large number of oak stands, growing on different sites and having different silvicultural histories, using neutral and EST-derived microsatellite markers. At the interspecific level, we showed that Quercus robur is relatively well delimited, while Quercus petraea and Quercus pubescens are more closely related. Natural hybridization might explain the increased genetic introgression between these two species. Within species, we found a low differentiation among populations of Q. robur and Q. petraea. In spite of forest fragmentation, we detected no spatial genetic barriers. However, we found that populations of Q. pubescens, a species with a marginal distribution in the study area were spatially structured. Genetic drift but also unidirectional introgressive hybridization with Q. petraea may account for this. Regarding the question of adaptation, we considered soil flooding, texture, drainage, and calcium carbonate in the upper horizons as physiologically important site condition variables. But with multivariate statistics, we could not find any significant effects of these parameters on genetic differentiation. Although there was no evidence for natural selection due to adaptation in stands of Q. robur, we demonstrated that age had a significant effect on their genetic variation and that stands established after the end of the Second World War had higher genetic diversity. We interpret these findings as being the result of an increase in large-scale transfers of reproductive materials during this time period and discuss arguments supporting this hypothesis. Finally, we consider the implications of these results for forest management.

Analysis of expressed sequence tags (ESTs) from a normalized cDNA library and isolation of EST simple sequence repeats from the invasive cotton mealybug Phenacoccus solenopsis.

The cotton mealybug, Phenacoccus solenopsis Tinsley, is a serious and invasive pest. At present, genetic resources for studying P. solenopsis are limited, and this negatively affects genetic research on the organism and, consequently, translational work to improve management of this pest. In the present study, expressed sequence tags (ESTs) were analyzed from a normalized complementary DNA library of P. solenopsis. In addition, EST-derived microsatellite loci (also known as simple sequence repeats or SSRs) were isolated and characterized. A total of 1107 high-quality ESTs were acquired from the library. Clustering and assembly analysis resulted in 785 unigenes, which were classified functionally into 23 categories according to the Gene Ontology database. Seven EST-based SSR markers were developed in this study and are expected to be useful in characterizing how this invasive species was introduced, as well as providing insights into its genetic microevolution.

Short note: Development, characterization and cross-amplification of eight EST-derived microsatellites in Salix

Abstract A set of eight simple sequence repeat (SSR) markers were developed from 707 Salix expressed sequence tags (ESTs) deposited in GenBank. Each of the EST-SSR amplicons was identical to the original EST, with sequence identity 60.90-96.03% and presence of the expected repeat motifs. Of the eight EST-SSR loci, five were polymorphic among 14 individuals of S. eriocephala, with the number of alleles per locus (Na), observed heterozygosity (Ho), expected heterozygosity (He) and polymorphic information content (PIC) being 2-7 (mean 4.8), 0.29-0.85 (mean 0.65), 0.25-0.84 (mean 0.65) and 0.21-0.78 (mean 0.58), respectively. High rates of crossspecies/ genus amplification were also observed within fourteen different species. The primer sequences for the eight EST-SSRs have been deposited in the Probe database of GenBank (IDs Pr031820546 - Pr031820553). The EST-SSRs developed herein would be a valuable addition of functional markers for genetics and breeding applications in a wide range of Salix species.

Novel polymorphic EST-derived microsatellite markers for the red-listed five needle pine, Pinus dabeshanensis

Pinus dabeshanensis is an endangered five needle pine endemic to mid-South of China. This pine finds its very limited natural occurrence in the Dabie mountains area with altitudes ranging 900–1400 m. We identified 3,516 SSR loci from the transcriptome assembly of the congeneric species P. lambertiana, and evaluated 671 loci we found. Finally, 28 novel polymorphic microsatellite markers were developed for P. dabeshanensis. Markers were characterized using 36 individuals, representing all existing populations from across the species geographic range. Species level analysis of these 28 polymorphic markers revealed the mean number per locus was 2.786 (range 2–6) and mean observed and expected heterozygosities were 0.332 (range 0.000–1.000) and 0.316 (range 0.033–0.691), respectively. This study provides the first set of microsatellite markers for the conservation genetics which in turn will facilitate conservation management for this pine.

Development of EST-derived microsatellite markers in Ophiorrhiza pumila (Rubiaceae) and their application in congeners

In East and Southeast Asian islands, forest area is decreasing and population size of multiple Ophiorrhiza species (Rubiaceae) is diminishing. Here we report the first expressed sequence tag-derived microsatellite markers, which can be applied to wide range of species of the genus. We developed 38 tri- and tetra-nucleotide microsatellite markers based on nonredundant unigenes of O. pumila and tested polymorphism of nine markers using the phylogenetically distant congener O. japonica (n = 96). The number of alleles ranged from 4 to 9. Observed and expected heterozygosities ranged 0.044–0.631 and 0.096–0.674, respectively. The polymorphic information content ranged 0.094–0.626. No null allele, significant deviation from Hardy–Weinberg equilibrium, or significant linkage disequilibrium was observed. Additional tests of cross-species amplification with three other congeners were successful in the nine markers. These markers will be used to investigate genetic diversity and differentiation of island populations of multiple Ophiorrhiza species to build conservation plans.

Effects of silvicultural practices on genetic diversity and population structure of white spruce in Saskatchewan

Forest harvesting and renewal practices using clearcut harvesting followed by artificial and natural regeneration (NR) may impact genetic diversity in subsequent forest tree populations. Plantations (PL) and phenotypic selections may exhibit lower genetic diversity than natural old growth (OG) and naturally-regenerated young populations because they may have a narrow genetic base. We used ten (six EST and four genomic) microsatellite loci, to reassess genetic impacts of silvicultural practices in white spruce (Picea glauca), previously assessed by using 51 RAPD markers by Rajora (1999). Allelic diversity at the genomic microsatellite loci was about three times higher than at the EST-derived microsatellite loci. Although the trends for microsatellite genetic diversity among different stands types were similar to that observed for RAPD markers, with natural OG stands showing the highest and tree improvement selections the lowest allelic and genotypic genetic diversity, no significant differences were observed for microsatellite genetic diversity among OG, young NR, PL and open-pollinated progeny of first-generation phenotypic selections (SEL). The inbreeding levels and genetic differentiation among populations within OG, NR and PL were also similar. However, phenotypic selections had somewhat different genetic constitution as they showed the highest genetic distances with OG, NR and SEL. On the other hand, the lowest genetic distances were observed between the OG and NR stands, which also had similar levels of genetic diversity. Our study suggests no significant negative impacts of harvesting and alternative reforestation practices on microsatellite genetic diversity in white spruce and calls for using more than one marker type in assessing the genetic impacts of silvicultural practices in forest trees.

Development and characterization of EST-derived microsatellite makers for Manila clam (Ruditapes philippinarum)

Twenty-five polymorphic microsatellite makers were developed and characterized from expressed sequence tag sequence of the Manila clam, Ruditapes philippinarum. The number of alleles at each locus ranged from 3 to 20 with an average of seven alleles per locus. The observed and expected heterozygosity varied from 0.081 to 0.730 and from 0.127 to 0.926, respectively. Thirteen loci were found deviate significantly from Hardy–Weinberg equilibrium. These microsatellite loci will be useful for further studies on the population structure and genetic variation of this species.

Summer Squash Identification by High-Resolution-Melting (HRM) Analysis Using Gene-Based EST–SSR Molecular Markers

Cucurbita pepo (squash, pumpkin, gourd), a worldwide cultivated vegetable of American origin, is extremely variable in fruit characteristics. Most of its widely grown commercial types are known as summer squashes and belong to the elongated forms of C. pepo ssp. pepo (Cocozelle, Vegetable marrow and Zucchini groups). Here, we have integrated the high-resolution-melting (HRM) analysis method with expressed sequence tags–simple sequence repeat (EST–SSR) marker genotyping, in order to facilitate the identification of 36 summer squash landraces originated from Greece. The six EST–SSR loci used were informative and generated a unique melting curve profile of EST-derived microsatellites for each accession allowing their comparison and classification. Moreover, HRM was highly informative, as by using only four microsatellite markers we were able to discriminate 36 summer squash landraces and by using six EST–SSRs. We were able to construct a highly informative and discriminative dendrogram where the 36 genotypes were classified in six distinct clusters. Furthermore, we acquired information about the genes containing the EST–SSRs using bioinformatics tools. We found that the EST–SSRs used in this study were hybridizing to genes involved in stress response to heavy metals and biotic stresses or the production of flavonoids or symporters of important nitrogen sources, like xanthine and uric acid amongst others. The results presented here suggest that the panel of EST–SSR markers used in combination with HRM analysis could be useful in a variety of applications, like squash biodiversity assessment but most importantly in managing squash germplasm to improve breeding programs.

A second generation genetic linkage map for silver carp (Hypophthalmichehys molitrix) using microsatellite markers

In the study, we constructed a second generation genetic linkage map for silver carp (Hypophthalmichthys molitrix) using anonymous and EST-derived microsatellite markers in a mapping panel containing 156 “pseudo BC” progenies from two interspecific crosses between silver carp and bighead carp (Aristichthys nobilis). A total of 703 markers were ordered on 24 linkage groups (LGs) which are equal to chromosome numbers of the haploid genome of the species. The consensus map spanned 1561.1cM covering 93.1% of the silver carp genome with an average resolution of 2.2cM/locus. Length of LGs ranged from 42.1cM to 97.8cM (mean 65.0cM). Total number of markers on individual LG varied from 13 to 56 (mean 29.3). Estimated total length of the female map (1809.0cM) was 1.52 times longer than that of the male map (1188.5cM), and the recombination ratio between sexes (female vs. male) was 2.2, showing markedly higher recombination in the females. Percentage of distorted loci in the male map was obviously higher than that in the female map, and 5 segregation distortion regions were identified in the male linkage groups. This second generation genetic linkage map evidently extends previous genetic maps for silver carp, and provides a basis for such studies as quantitative trait locus mapping, comparative genomics and marker-assisted selection.

In silico identification and validation of microsatellite markers from onion EST sequences

SummaryMicrosatellites or simple sequence repeats (SSRs) play an important role in plant genetics and breeding. The development of microsatellites is a time-consuming and expensive process. In silico mining of microsatellites from expressed sequence tags (ESTs), which are available in electronic molecular databases, is a cheaper alternative. In the present study, we used a computational approach for mining SSRs from 20,159 ESTs in Allium cepa. These onion ESTs represented a total length of 13.2 Mb and were downloaded from the dbEST database of the National Center for Biotechnology Information (NCBI) and subjected to various pre-processing steps. The pre-processed ESTs were clustered, resulting in non-redundant unigenes. These unigenes were analysed for their SSR content and distribution. In all, 1,464 SSRs consisting of di-, tri-, tetra-, penta- and hexa-nucleotide repeats were mined from the non-redundant ESTs (contigs and singletons). Tri-nucleotide SSRs were the most abundant, followed by tetra-, di-, hexa- and penta-nucleotide SSRs. Among the tri-coding repeats, leucine and serine codons were more abundant. The SSR-containing sequences were annotated and grouped into their respective functional categories. The predominant functional group among the annotated unigenes was “metabolism”, followed by “transcription factors” and “transporter proteins”. Primer pairs could be designed for 1,092 SSR-containing sequences. Of these, 51 primer pairs were validated in the laboratory. A database has been developed to store the unigenes, primer pairs, putative annotations, and BLAST results. After validation, the EST-derived microsatellite (SSR) markers can be used in studies related to marker-assisted selection, detection of polymorphism, DNA fingerprinting, and diversity analysis in onion.

Development and characterization of EST-derived microsatellite makers for swimming crab, Portunus trituberculatus

In this study, sixteen polymorphic microsatellite makers were developed and characterized from expressed sequence tag sequence of the swimming crab, Portunus trituberculatus. The analysis of polymorphic was performed by using 30 specimens from the coastal waters of Xiangshan, Zhejiang province, China. The number of alleles at each locus ranged from 4 to 14 with an average of seven alleles per locus. The ranges of observed and expected heterozygosity were from 0.517–1.000 to 0.508–0.736, respectively. The polymorphism information content ranged from 0.375 to 0.710. Four loci were found deviate significantly from Hardy–Weinberg equilibrium. These microsatellite loci will be helpful for the genetic evaluation of P. trituberculatus enhancement.