Development Of EST-SSR Markers Research Articles

EST-SSR marker development based on de novo transcriptomic assembly of Aesculus chinensis Bunge

EST-SSR marker development based on de novo transcriptomic assembly of Aesculus chinensis Bunge

Transcriptome analysis and development of EST-SSR markers in the mushroom Auricularia heimuer

Auricularia heimuer, the third most frequently cultivated edible mushroom species worldwide, has high medicinal value. However, a shortage of molecular marker hinders the efficiency and accuracy of genetic breeding efforts for A. heimuer. High-throughput transcriptome sequencing data are essential for gene discovery and molecular markers development. This study aimed to clarify the distribution of SSR loci across the A. heimuer transcriptome and to develop highly informative EST-SSR markers. These tools can be used for phylogenetic analysis, functional gene mining, and molecular marker-assisted breeding of A. heimuer. This study used Illumina high-throughput sequencing technology to obtain A. heimuer transcriptome data. The results revealed 37,538 unigenes in the A. heimuer transcriptome. Of these unigenes, 24,777 (66.01%) were annotated via comparison with the COG, Pfam, and NR databases. Overall, 2510 SSRs were identified from the unigenes, including 6 types of SSRs. The most abundant type of repeats were trinucleotides (1425, 56.77%), followed by mononucleotides (391, 15.58%) and dinucleotides (456, 18.17%). Primer pairs for 102 SSR loci were randomly designed for validity confirmation and polymorphism identification; this process yielded 53 polymorphic EST-SSR markers. Finally, 13 pairs of highly polymorphic EST-SSR primers were used to analyze the genetic diversity and population structure of 52 wild A. heimuer germplasms, revealing that the 52 germplasms could be divided into three categories. These results indicated that SSR loci were abundant in types, numbers, and frequencies, providing a potential basis for germplasm resource identification, genetic diversity analysis, and molecular marker-assisted breeding of A. heimuer.

Development of EST-SSR Markers and Population Genetic Analysis of Hemsleya zhejiangensis, an Endangered Species Endemic to Eastern China

Development of EST-SSR Markers and Population Genetic Analysis of Hemsleya zhejiangensis, an Endangered Species Endemic to Eastern China

Characterization and development of transcriptome-derived novel EST-SSR markers to assess genetic diversity in Chaetomium globosum.

The online version contains supplementary material available at 10.1007/s13205-023-03794-7.

Development of EST-SSR Markers and Genetic Diversity Analysis Among Wild Pistachio Species

Development of EST-SSR Markers and Genetic Diversity Analysis Among Wild Pistachio Species

Development and characterization of EST-SSR markers in Rhodomyrtus tomentosa Hassk. based on transcriptome

Development and characterization of EST-SSR markers in Rhodomyrtus tomentosa Hassk. based on transcriptome

Development of EST-SSR markers based on transcriptome sequencing for germplasm evaluation of 65 lilies (Lilium).

Lilium genus consists of approximately 100 species and numerous varieties, many of which are interspecific hybrids, which result in a complicated genetic background. The germplasm identification, genetic relationship analysis, and breeding of Lilium rely on exploiting genetic information among different accessions. Hence, an attempt was made to develop new EST-SSR markers and study the molecular divergence among 65 genotypes of Lilium. A total of 5509 EST-SSRs were identified from the high-throughput sequencing database of L. 'Elodie'. After primer screening, six primer pairs with the most abundant polymorphic bands were selected from 100 primer pairs. Combined with the other 10 reported SSR primers, a total of 16 pairs detected genetic information with an average PIC value of 0.7583. The number of alleles per locus varied from four to 33, the expected heterozygosity varied from 0.3289 to 0.9231, and the observed heterozygosity varied from 0.2857 to 0.5122. Based on the phylogenic results, 22 Asiatic hybrids (A), seven Longiflorum × Asiatic hybrids (LA), as well as two native species were grouped. Eighteen Oriental hybrids (O) and nine Oriental × Trumpet (OT) hybrids, four native species, one LO, and one LL (L. pardalinum × L. longiflorum) variety were grouped. Two major clusters were reported and a large number of genotypes were grouped based on UPGMA and STRUCTURE analysis methods. The PIC value as well as other parameters revealed that the EST-SSR markers selected were informative. In addition, the clustering pattern displayed better agreement with the cultivar's pedigree. The newly identified SSRs in this study provides molecular markers for germplasm characterization and genetic diversity for Lilium.

Transcriptomic sequencing analysis, development, and validation of EST-SSR markers in reed canary grass

Transcriptomic sequencing analysis, development, and validation of EST-SSR markers in reed canary grass

Expression Profiles of Microsatellites in Fruit Tissues of Akebia trifoliata and Development of Efficient EST-SSR Markers

Akebia trifoliata, a member of the family Lardizabalaceae, has high exploitation potential for multiple economic purposes, so genetic improvements to meet requirements for commercial demand are needed. However, this progress is largely impeded by a lack of effective selection markers. In this study, we obtained 271.49 Gb of clean transcriptomic data from 12 samples (three tissues at four developmental stages) of A. trifoliata fruit. We identified 175,604, 194,370, and 207,906 SSRs from the de novo assembled 416,363, 463,756, and 491,680 unigene sequences obtained from the flesh, seed, and rind tissues, respectively. The profile and proportion of SSR motifs expressed in each fruit tissue and developmental stage were remarkably similar, but many trinucleotide repeats had differential expression levels among different tissues or at different developmental stages. In addition, we successfully designed 16,869 functional EST-SSR primers according to the annotated unigenes. Finally, 94 and 72 primer pairs out of 100 randomly selected primer pairs produced clear bands and polymorphic bands, respectively. These results were also used to elucidate the expression profiles of different tissues at various stages. Additionally, we provided a set of effective, polymorphic, and reliable EST-SSR markers sufficient for accelerating the discovery of metabolic and pathway-specific functional genes for genetic improvement and increased commercial productivity.

De novo transcriptome assembly and development of EST-SSR markers for Pterocarpus santalinus L. f. (Red sanders), a threatened and endemic tree of India

The endemic and precious tree Pterocarpus santalinus L. f. (Red sanders) is a drought hardy species for conservation in peninsular India due to its high risk of illegal timber harvest. It is only found in Eastern Ghats of India, and has become threatened owing to overexploitation of its valuable timber. The development of genomic resources, particularly simple sequence repeat (SSR) markers, is essential for strict implementation of in situ conservation measures and application of DNA information based red sanders genetic resource management. However, a lack of genomic data and efficient molecular markers limit the study of its spatial and temporal population genetic structure, identification of diversity hotspots and tree improvement. The current study aims at comprehensive molecular characterization of red sanders and the somatic chromosome counts, flow cytometry and EST-SSR analyses. The results revealed that red sanders is diploid with 2n = 20 and the 2C genome size was 0.7872 ± 0.0561 pg for the first time in this species. A total of 3128 EST-SSRs were detected based on 25,854 de novo assembled unigenes from transcriptome data and primer sets designed for 1953 SSRs. Fifty-nine EST-SSR markers were evaluated for polymorphism in the natural populations of red sanders and 13 were found to be suitable for genetic analysis. Two major transcription factor families bHLH and ERF, responsible for abiotic stress and secondary metabolite synthesis were analysed which would provide the foundation for further research on production of medicinally important biocompounds.

Inflorescence Transcriptome Sequencing and Development of New EST-SSR Markers in Common Buckwheat (Fagopyrum esculentum).

Common buckwheat (Fagopyrum esculentum M.) is known for its adaptability, good nutrition, and medicinal and health care value. However, genetic studies of buckwheat have been hindered by limited genomic resources and genetic markers. In this study, Illumina HiSeq 4000 high-throughput sequencing technology was used to sequence the transcriptome of green-flower common buckwheat (Gr) with coarse pedicels and white-flower Ukrainian daliqiao (UD) with fine pedicels. A total of 118,448 unigenes were obtained, with an average length of 1248 bp and an N50 of 1850 bp. A total of 39,432 differentially expressed genes (DEGs) were identified, and the DEGs of the porphyrins and chlorophyll metabolic pathway had significantly upregulated expression in Gr. Then, a total of 17,579 sequences containing SSR loci were detected, and 20,756 EST-SSR loci were found. The distribution frequency of EST-SSR in the transcriptome was 17.52%, and the average distribution density was 8.21 kb. A total of 224 pairs of primers were randomly selected for synthesis; 35 varieties of common buckwheat and 13 varieties of Tartary buckwheat were verified through these primers. The clustering results well verified the previous conclusion that common buckwheat and Tartary buckwheat had a distant genetic relationship. The EST-SSR markers identified and developed in this study will be helpful to enrich the transcriptome information and marker-assisted selection breeding of buckwheat.

Development of EST-SSR markers based on transcriptome and its validation in ginger (Zingiber officinale Rosc.).

Ginger (Zingiber officinale Rosc.) is an economically important and valuable spice crop around the world. It is used as food, spice, condiment, and medicine. A considerable extent of genetic diversity in ginger occurs in the Western Ghats and North-Eastern India. However, genetic diversity studies at the molecular level in ginger is limited due to limited availability of genetic and genomic information. In the present study, for the first time, we have identified and validated expressed sequence tag (EST)-simple sequence repeat (SSR) markers from ginger. We obtained 16,790 EST-SSR loci from 78987 unigenes, and 4597 SSR loci in the predicted 76929 coding sequences from RNA-Seq assembled contigs of ginger through Illumina paired-end sequencing. Gene ontology results indicate that the unigenes with SSR loci participate in various biological processes such as metabolism, growth, and development in ginger. One hundred and twenty-five primer pairs were designed from unigenes and coding sequences. These primers were tested for PCR optimization, characterization, and amplification and identified 12 novel EST-SSR markers. Twelve flanking polymorphic EST-SSR primers were validated using 48 ginger genotypes representing North-Eastern India and different eco-geographical adaptations by PCR amplification and allele sizing through capillary electrophoresis. Twelve EST-SSR primers generated a total of 111 alleles with an average of 9.25 alleles per locus and allele sizes ranging between 115-189bp. This study implies that the SSR markers designed from transcriptome sequences provides ample EST-SSR resources, which are helpful for genetic diversity analysis of Zingiberaceae species and molecular verification of ginger genotypes.

Development of EST-SSR markers and association analysis of floral scent in tree peony

Tree peony (Paeonia Sect. Moutan) is widely grown as an ornamental plant and is known for its ‘National Beauty and Heavenly Fragrance’. However, the genetic improvement of floral scent traits is fundamentally limited by the paucity of genomic marker resources. Given the large-scale development of microsatellite markers using transcriptome sequences, we conducted an association analysis to identify microsatellite markers associated with main floral components. A total of 8,443 EST-SSRs were identified, with an average of one SSR per 15.55 kb. Di-nucleotide repeats were the most plentiful repeat type, followed by tri-, tetra-, penta- and hexa-nucleotide repeats. Among 969 synthesized primer pairs, 499 were successfully amplified and 136 showed polymorphism which exhibited relatively high levels of transferability were observed across 12 individuals in the genus Paeonia, thus expanding their utility. Subsequently, 31 polymorphic EST-SSR markers were selected for the analysis of genetic diversity and population structure, resulting in 176 alleles; the polymorphism information content (PIC) varied from 0.31 to 0.87. According to the population structure analysis, the association varieties were divided into three subgroups. Finally, single marker-trait association analysis based on mixed linear model (MLM) identified 29 significant associations, involving five floral components with 18 SSRs and a low proportion of phenotypic variance ranging from 4.57 to 23.46%. The newly developed markers were valuable resources that could provide a powerful genomic tool for genetic studies and accelerate the process of molecular marker-assisted breeding of tree peony floral scent.

Development of high quality EST-SSR markers in Rhododendron obtusum Hort. ex Wats. and their use in determining relationships among Rhododendron cultivars

Development of high quality EST-SSR markers in Rhododendron obtusum Hort. ex Wats. and their use in determining relationships among Rhododendron cultivars

Development and Characterization of Novel EST-SSR Markers in Masson Pine (Pinus massoniana) Based on Transcriptome Data

Development and Characterization of Novel EST-SSR Markers in Masson Pine (Pinus massoniana) Based on Transcriptome Data

Development of Novel Polymorphic EST-SSR Markers from the Cranberry Fruit Transcriptome.

Background: Cranberry (Vaccinium macrocarpon Ait.) has high developmental prospects and great research value. Cranberry has a narrow genetic base, however, its morphological characteristics are not easily distinguishable. Besides, traditional breeding methods are limited, and breeding progress on cranberry cultivars has been slow.Objective: The objective of this study was to assess polymorphic EST-SSR markers developed from a cranberry fruit transcriptomic sequencing library to provide candidate EST-SSR sequences for future research on stress resistance breeding of cranberry.Materials and Methods: Thirteen cranberry accessions were used for EST-SSR analysis, and 16 accessions of other Vaccinium species were used to test primer transferability. Genomic DNA was extracted from young leaves of 6-year-old cranberry plants and subjected to PCR amplification. A binary matrix was established and analyzed in NTSYS-pc v.2.10e for calculation of the genetic similarity of cranberry cultivars and construction of a cluster dendrogram.Results: A total of 47 stress-resistance-related primer pairs were designed, of which 7 pairs showed polymorphism. The average number of effective alleles was 1.844, and the average expected heterozygosity was 0.455. The average transfer rate was 63.39%. Genetic similarity coefficients ranged from 0.28 to 1.00, with an average of 0.76. UPGMA clustering divided the 13 cranberry accessions into four groups at a genetic similarity of 0.74.Conclusions: The seven polymorphic EST-SSR markers were able to reveal genetic relationships among 13 cranberry accessions and can be used for future research on stress resistance breeding of cranberry.

EST-SSR marker development based on transcriptome sequencing and genetic analyses of Phoebe bournei (Lauraceae).

High-throughput sequencing of the Phoebe bournei transcriptome was performed, and novel SSR markers were identified. A total of 73,518 nonredundant unigenes were assembled and annotated by sequence similarity searching in diverse public databases. A total of 40,853 SSRs were identified from 73,518 unigenes. Twenty-three pairs of polymorphic EST-SSR markers were selected from 98 markers and used for genetic analyses in 75 individuals from three P. bournei populations. The 23 pairs of markers could detect abundant genetic information from the samples (PIC = 0.769), and cross-species amplification was successfully performed in other related species. Three populations had high level of genetic diversity (He = 0.658 in average), of which the population YS from Jiangxi province had the most abundant genetic diversity (He = 0.722). The results of genetic structure analyses showed that the population YS from Jiangxi province had obvious genetic differences from the other two populations, and the genetic information of the population SX from Fujian province was related to that of the population LC from Guangdong province and the population YS. The transcriptomic resources and EST-SSR markers are valuable tools not only for the ecological conservation of P. bournei but also for phylogenetic studies.

The Development of EST-SSR Markers from Transcriptome Sequencing and Genetic Diversity of Twenty Genotypes with High Yields of <i>Cornus wilsoniana</i>, an Important Wood Oil Plant

Oil plants are not only used as essential nutriments in food but also used broadly as ingredients of industrial products. Cornus wilsoniana Wanger is an important native woody oil plant with high-yield and high-oiliness characters in China. The fruit oil of Cornus wilsoniana not only could be used as equilibrated dietary oil, but also has hypolipidemic function and helps to overcome EFA deficiency. Genomic information is currently not available for Cornus wilsoniana , which will therefore affect its genetic improvement process. In this study, 8713 EST-SSRs were identified from the transcriptome sequencing of Cornus wilsoniana . Most of these EST-SSRs were composed of dimer and trimer repeats. The AG/CT motif is the most common dimeric EST-SSRs motif (53.5%), whereas the CG/CG (0.44%) microsatellites are present only at low abundances. Among the trimeric microsatellites, AAG/CTT (5.53%) and ATC/ATG (4.27%) are the most common motifs. There are no obvious dominant motifs among the tetra-, penta-, and hexa- nucleotide motifs. Fifteen pairs of EST-SSRs primers are developed with polymorphism and used for the genetic diversity analysis of twenty genotypes of Cornus wilsoniana with high fruit output. Allele number per locus ranges from 2 to6 with an average of 2.87, and the PIC value ranges from 0.05 to 0.58 with an average of 0.36. Average genetic diversity overall SSR loci for the 20 genotypes was 0.427, ranging from 0.049 to 0.659. All the loci are polymorphic and clearly distinguish the genotypes. Cluster analyze (NJ tree, UPGMA) identifies a similar pattern of variation. 15developed EST-SSRs are informative, codominant and reliable, and could be applied in the improvement programs of Cornus wilsoniana in the future.

Development of EST-SSR markers for genetic diversity analysis in coconut (Cocos nucifera L.).

Genetic improvement in coconut relies on exploiting the vast existing diversity among coconut accessions. Robust molecular markers are a pre-requisite for efficient characterization of genetic diversity. Microsatellites or simple sequence repeats (SSRs), mined from expressed sequence tags (ESTs), constitute an important resource for analysis of genetic diversity as they are abundant, polymorphic and represent function regions of the genome. We have identified a total of 318,528 putative EST-SSRs from 130,942 unigenes utilizing a leaf transcriptome dataset of coconut. Among the EST-SSRs, dinucleotide repeats were abundant (219,912; 69.04%) followed by trinucleotide (70,722; 22.2%) and tetra-nucleotide repeats (6281; 1.9%). Among the dinucleotide repeat motifs, the dominant repeat was AG/CT (35.87%), followed by AT/AT (18.59%), while the dominant trinucleotide repeat was AAG/CTT (4.59%). One hundred and twenty EST-SSR primer pairs were designed and utilized to amplify six DNA samples of coconut accessions. Fifty primers (41.7%) produced reproducible polymorphic fragments of expected sizes, from which a total of 10 primers were selected for the diversity assessment in 186 palms of 50 coconut accessions, comprising of 25 each of tall and dwarf accessions. A total of 137 alleles were detected with an average of 13.7 alleles per SSR locus. The number of alleles observed at each locus in the data set ranged from 7 to 22. All the loci showed 100% polymorphism with respect to the samples screened. The average observed heterozygosity was 0.46. The PIC values ranged from 0.79 (CnKGDEST129 and CnKGDEST100) to 0.91 (CnKGDEST117 andCnKGDEST122) with a mean value of 0.85, indicating the capacity of the EST-SSR markers to detect high levels of polymorphism. The cluster analysis revealed that accessions were generally clustered based on their relative similarity and irrespective of their geographic origins. The present study demonstrates the usefulness of transcriptome sequencing as a rapid and cost-effective methodology for the development of molecular markers. The EST-SSR markers generated through this study constitute useful and reliable tools for assessment of genetic diversity and marker-assisted selection in coconut.

Development of EST-SSR markers for Pongamia pinnata by transcriptome database mining: cross-species amplification and genetic diversity.

EST-SSR markers were developed from Pongamia pinnata transcriptome libraries. We have successfully utilised EST-SSRs to study the genetic diversity of Indian P. pinnata germplasms and transferability study on legume plants. P. pinnata is a non-edible oil, seed-bearing leguminous tree well known for its multipurpose benefits and acts as a potential source for medicine and biodiesel preparation. Moreover, the plant is not grazable by animal and wildly grown in different agro climatic condition of India. Recently, it is much used in reforestation and rehabilitation of marginal and coal mined land in different part of India. Due to increasing demand for cultivation, understanding of the genetic diversity is important parameter for further breeding and cultivation program. In this investigation, an attempt has been undertaken to develop novel EST-SSR markers by analyzing the assembled transcriptome from previously published Illumina libraries of P. pinnata, which is cross transferrable to legume plants. Twenty EST-SSR markers were developed from oil yielding and secondary metabolite biosynthesis genes. To our knowledge, this is the first EST-SSR marker based genetic diversity study on Indian P. pinnata germplasms. The genetic diversity parameter analysis of P. pinnata showed that the Gangetic plain and Eastern India are highly diverse compared to the Central Deccan and Western germplasms. The lowest genetic diversity in the Western region may be due to the pressure of lower precipitation, high-temperature stress and reduced groundwater availability. Nevertheless, the highest genetic diversity of Gangetic plain and Eastern India may be due to the higher groundwater availability, high precipitation, higher temperature fluctuations and growing by the side of glacier-fed river water. Thus, our study shows the evidence of natural selection on the genetic diversity of P. pinnata germplasms of the Indian subcontinent.