Development and Characterization of EST-SSR Markers for Juniperus squamata (Cupressaceae), an ecologically important conifer in Asian mountains

AbstractJuniperus squamata, an endemic conifer of Asia, is an important shrub ecologically and economically. Yet little is known about its genetic diversity and population structure due to lacking of highly polymorphic molecular markers. In this study, expressed sequence tag microsatellite markers (EST-SSR) were developed for Juniperus squamata. Illumina HiSeq data were used to reconstruct the transcriptome of this species by de novo assembly. Based on this transcriptome, 18 SSR markers were designed and successfully amplified. Just one locus was eliminated due to its detection of null alleles and the remaining 17 loci were polymorphic, generating five to 14 alleles per locus in J. squamata. Markers cross-amplification tests were successful in two closely related species of J. squamata. These markers will serve as a basis for further studies to assess the genetic diversity and population structure of J. squamata. As well, they could be useful in promoting sustainable forest management strategies for this species in the face of global climate change.

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Isolation and characterization of polymorphic microsatellite markers in Toxicodendron vernicifluum

Czech Journal of Genetics and Plant Breeding ◽

10.17221/183/2016-cjgpb ◽

2018 ◽

Vol 54 (No. 1) ◽

pp. 17-25 ◽

Cited By ~ 2

Author(s):

D.-D. Vu ◽

T.T.-X. Bui ◽

T.H.-N. Nguyen ◽

S.N.M. Shah ◽

N.-H. Vu ◽

...

Keyword(s):

Ssr Markers ◽

Expressed Sequence Tag ◽

De Novo ◽

Average Length ◽

Polymorphic Information Content ◽

Shaanxi Province ◽

Illumina Hiseq ◽

Tissue Samples ◽

Isolation And Characterization ◽

Toxicodendron Vernicifluum

A total 20 074 230 sequencing reads were generated by Illumina HiSeq<sup>™ </sup>2500 from three different Toxicodendron vernicifluum tissue samples. In total, 48 693 unigenes with an average length of 703.34 bp were obtained by de novo assembly. 3392 potential EST-SSRs (expressed sequence tag-simple sequence repeat) were identified as potential molecular markers from unigenes with lengths exceeding 1 kb. A total of 80 pairs of PCR primers were randomly selected to validate the assembly quality and develop EST-SSR markers from genomic DNA. Of these primer pairs, 14 primer pairs successfully amplified DNA fragments and detected significant amounts of polymorphism within the lacquer tree population in Langao, Shaanxi province, China. There were high genetic diversities (number of alleles per locus (A) = 2.93, polymorphic information content (PIC) = 0.53, observed heterozygosity (Ho) = 0.62 and expected heterozygosity (He) = 0.85) in the lacquer tree natural population. The four loci were significantly deviated from Hardy-Weinberg equilibrium. These results suggested high homozygosity in the population and low or deficiency in heterozygosity (inbreeding coefficient (Fis) = 0.27). These polymorphic EST-SSR markers will provide the base for further studies of genetic structure and breeding in T. vernicifluum.

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EST-SSR markers identification based on RNA-sequencing and application in Schisandra chinensis

10.21203/rs.3.rs-17573/v1 ◽

2020 ◽

Author(s):

Guangli Shi ◽

Zhenxing Wang ◽

Dan Sun ◽

Susu Zhang ◽

Jianhui Guo ◽

...

Keyword(s):

Genetic Diversity ◽

Ssr Markers ◽

Genetic Linkage ◽

Expressed Sequence Tag ◽

Polymorphic Band ◽

Diversity Analysis ◽

Schisandra Chinensis ◽

Illumina Hiseq ◽

Genetic Diversity Analysis ◽

Predominant Type

Abstract Background: Schisandra chinensis, a climbing woody vine, is the best-known and representative genus of the Schisandraceae family which is an important plant in Chinese herbal medicine; however, the application of molecular breeding is restricted by the few genetic markers for this species. Results: In this study, we performed transcriptome sequencing of S. chinensis using the Illumina HiSeq platform to establish a library of expressed sequence tag-simple sequence repeat (EST-SSR) markers. A total of 59,786 unigenes were obtained and 6254 putative SSR sites were detected with a frequency of 10.46%. The predominant type of repeat motif was dinucleotide (35.71%), followed by trinucleotide (13.22%), hexanucleotide (0.50%), tetranucleotide (0.06%), and pentanucleotide (0.22%). We randomly selected 50 EST-SSR primer pairs and used 14 of these for genetic diversity analysis in 42 S. chinensis genotypes. All 42 accessions were successfully identified and formed four major clusters, indicating that the SSR markers can be used for genetic diversity analysis and genetic linkage map construction. In addition, using the polymorphic bands associated with 10 markers as DNA fingerprints, we generated a manual cultivar identification diagram that can distinguish between the 42 accessions, with different individuals identifiable based on polymorphic band patterns. Conclusion: S. chinensis transcriptome data is an effective resource for developing SSR markers. These results can provide a basis for the identification of S. chinensis accessions and construction of genetic linkage maps as part of future selective breeding and conservation efforts for this valuable plant.

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De novo assembly and transcriptome characterization of an endemic species of Vietnam, Panax vietnamensis Ha et Grushv., including the development of EST-SSR markers for population genetics

10.21203/rs.2.18797/v3 ◽

2020 ◽

Author(s):

Duy Dinh Vu ◽

Syed Noor Muhammad Shah ◽

Mai Phuong Pham ◽

Van Thang Bui ◽

Minh Tam Nguyen ◽

...

Keyword(s):

Genetic Diversity ◽

Genetic Structure ◽

Expressed Sequence Tag ◽

De Novo ◽

Average Length ◽

Species Conservation ◽

Group Method ◽

Arithmetic Means ◽

Illumina Hiseq ◽

Genetic Diversity And Structure

Abstract Background: Understanding the genetic diversity in threatened species that occur in forest remnants is necessary to establish efficient strategies for the species conservation, restoration and management. Panax vietnamensis Ha et Grushv. is medicinally important, endemic and endangered species of Vietnam. However, genetic diversity and structure of population is unknown due to lack of efficient molecular markers.Results: In this study, we employed Illumina HiSeq TM 4000 sequencing to analyze the transcriptomes of P. vietnamensis (roots, leaves and stems). A total of 23,741,783 raw reads were obtained and assembled, from which, 89,271 unigenes with an average length of 598.3191 nt were generated. During functional annotation, 31,686 unigenes were annotated in Gene Ontology categories, Kyoto Encyclopedia of Genes and Genomes pathways, Swiss-Prot database, and Nucleotide Collection (NR/NT) database. In addition, 11,343 expressed sequence tag-simple sequence repeat (EST-SSRs) were detected. From 7,774 primer pairs, 101 were selected for polymorphism validation, in which, 20 primer pairs were successfully amplified to DNA fragments and significant amounts of polymorphism was observed within population. The nine polymorphic microsatellite loci were used to analyze genetic diversity and structure of the natural populations. The obtained results revealed that the shows high levels of genetic diversity in populations, the average observed and expected heterozygosity were H O = 0.422 and H E = 0.479. During the Bottleneck analysis using TPM and SMM models (p < 0.01) shows that targeted population is significantly heterozygote deficient. This suggests sign of bottleneck in all populations. Genetic differentiation among populations was moderate (F ST = 0.133) and indicating limited gene flow (Nm = 1.63). Analysis of molecular variance (AMOVA) showed 63.17% of variation within individuals and 12.45% among populations. These results showed a moderate genetic structure of P. vietnamensis. STRUCTURE analysis and the unweighted pair-group method with arithmetic means (UPGMA) tree revealed strong genetic structure and two genetic clusters related to geographical distances, as well. Conclusion: Our study will assist conservators in future conservation management, breeding, production and habitats restoration of the species.

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De novo assembly and transcriptome characterization of an endemic species of Vietnam, Panax vietnamensis Ha et Grushv., including the development of EST-SSR markers for population genetics

10.21203/rs.2.18797/v2 ◽

2020 ◽

Author(s):

Duy Dinh Vu ◽

Syed Noor Muhammad Shah ◽

Mai Phuong Pham ◽

Van Thang Bui ◽

Minh Tam Nguyen ◽

...

Keyword(s):

Genetic Diversity ◽

Genetic Structure ◽

Expressed Sequence Tag ◽

De Novo ◽

Average Length ◽

Species Conservation ◽

Group Method ◽

Arithmetic Means ◽

Illumina Hiseq ◽

Genetic Diversity And Structure

Abstract Background: Understanding the genetic diversity in threatened species that occur in forest remnants is necessary to establish efficient strategies for the species conservation, restoration and management. Panax vietnamensis Ha et Grushv. is medicinally important, endemic and endangered species of Vietnam. However, genetic diversity and structure of population is unknown due to lack of efficient molecular markers. Results: In this study, we employed Illumina HiSeq TM 4000 sequencing to analyze the transcriptomes of P. vietnamensis (roots, leaves and stems). A total of 23,741,783 raw reads were obtained and assembled, from which, 89,271 unigenes with an average length of 598.3191 nt were generated. During functional annotation, 31,686 unigenes were annotated in Gene Ontology categories, Kyoto Encyclopedia of Genes and Genomes pathways, Swiss-Prot database, and Nucleotide Collection (NR/NT) database. In addition, 11,343 expressed sequence tag-simple sequence repeat (EST-SSRs) were detected. From 7,774 primer pairs, 101 were selected for polymorphism validation, in which, 20 primer pairs were successfully amplified to DNA fragments and significant amounts of polymorphism was observed within population. The nine polymorphic microsatellite loci were used to analyze genetic diversity and structure of the natural populations. The obtained results revealed that the shows high levels of genetic diversity in populations, the average observed and expected heterozygosity were H O = 0.422 and H E = 0.479. During the Bottleneck analysis using TPM and SMM models (p < 0.01) shows that targeted population is significantly heterozygote deficient. This suggests sign of bottleneck in all populations. Genetic differentiation among populations was moderate (F ST = 0.133) and indicating limited gene flow (Nm = 1.63). Analysis of molecular variance (AMOVA) showed 63.17% of variation within individuals and 12.45% among populations. These results showed a moderate genetic structure of P. vietnamensis. STRUCTURE analysis and the unweighted pair-group method with arithmetic means (UPGMA) tree revealed strong genetic structure and two genetic clusters related to geographical distances, as well. Conclusion: Our study will assist conservators in future conservation management, breeding, production and habitats restoration of the species.

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Transcriptome Sequencing of Different Avocado Ecotypes: de novo Transcriptome Assembly, Annotation, Identification and Validation of EST-SSR Markers

Forests ◽

10.3390/f10050411 ◽

2019 ◽

Vol 10 (5) ◽

pp. 411 ◽

Cited By ~ 9

Author(s):

Yu Ge ◽

Lin Tan ◽

Bin Wu ◽

Tao Wang ◽

Teng Zhang ◽

...

Keyword(s):

Ssr Markers ◽

High Throughput Sequencing ◽

Expressed Sequence Tag ◽

De Novo ◽

Average Length ◽

Transcriptome Assembly ◽

Persea Americana ◽

Illumina Hiseq ◽

Ssr Loci ◽

Ssr Development

Avocado (Persea americana Mill.) could be considered as an important tropical and subtropical woody oil crop with high economic and nutritional value. Despite the importance of this species, genomic information is currently unavailable for avocado and closely related congeners. In this study, we generated more than 216 million clean reads from different avocado ecotypes using Illumina HiSeq high-throughput sequencing technology. The high-quality reads were assembled into 154,310 unigenes with an average length of 922 bp. A total of 55,558 simple sequence repeat (SSR) loci detected among the 43,270 SSR-containing unigene sequences were used to develop 74,580 expressed sequence tag (EST)-SSR markers. From these markers, a subset of 100 EST-SSR markers was randomly chosen to identify polymorphic EST-SSR markers in 28 avocado accessions. Sixteen EST-SSR markers with moderate to high polymorphism levels were detected, with polymorphism information contents ranging from 0.33 to 0.84 and averaging 0.63. These 16 polymorphic EST-SSRs could clearly and effectively distinguish the 28 avocado accessions. In summary, our study is the first presentation of transcriptome data of different avocado ecotypes and comprehensive study on the development and analysis of a set of EST-SSR markers in avocado. The application of next-generation sequencing techniques for SSR development is a potentially powerful tool for genetic studies.

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De novo assembly and transcriptome characterization of an endemic species of Vietnam, Panax vietnamensis Ha et Grushv., including the development of EST-SSR markers for population genetics

10.21203/rs.2.18797/v1 ◽

2019 ◽

Author(s):

Duy Dinh Vu ◽

Syed Noor Muhammad Shah ◽

Mai Phuong Pham ◽

Van Thang Bui ◽

Minh Tam Nguyen ◽

...

Keyword(s):

Genetic Diversity ◽

Population Genetics ◽

Genetic Structure ◽

Expressed Sequence Tag ◽

De Novo ◽

Average Length ◽

Group Method ◽

Arithmetic Means ◽

Illumina Hiseq ◽

Genetic Diversity And Structure

Abstract Background: Understanding the genetic diversity in threatened species that occur in forest remnants is necessary to establish efficient strategies for the species conservation, restoration and management. Panax vietnamensis Ha et Grushv. is medicinally important, endemic and endangered species of Vietnam. However, genetic diversity and structure of population is unknown due to lack of efficient molecular markers. Results: In this study, we employed Illumina HiSeq TM 4000 sequencing to analyze the transcriptomes of P. vietnamensis (roots, leaves and stems). A total of 23,741,783 raw reads were obtained and assembled, from which, 89,271 unigenes with an average length of 598.3191 nt were generated. During functional annotation, 31,686 unigenes were annotated in Gene Ontology categories, Kyoto Encyclopedia of Genes and Genomes pathways, Swiss-Prot database, and Nucleotide Collection (NR/NT) database. In addition, 11,343 expressed sequence tag-simple sequence repeat (EST-SSRs) were detected. From 7,774 primer pairs, 101 were selected for polymorphism validation, in which, 20 primer pairs were successfully amplified to DNA fragments and significant amounts of polymorphism was observed within population. The nine polymorphic microsatellite loci were used to analyze genetic diversity and structure of the natural populations. The obtained results revealed that the shows high levels of genetic diversity in populations, the average observed and expected heterozygosity were H O = 0.422 and H E = 0.479. During the Bottleneck analysis using TPM and SMM models (p < 0.01) shows that targeted population is significantly heterozygote deficient. This suggests sign of bottleneck in all populations. Genetic differentiation among populations was moderate (F ST = 0.133) and indicating limited gene flow (Nm = 1.63). Analysis of molecular variance (AMOVA) showed 63.17% of variation within individuals and 12.45% among populations. These results showed a moderate genetic structure of P. vietnamensis. STRUCTURE analysis and the unweighted pair-group method with arithmetic means (UPGMA) tree revealed strong genetic structure and two genetic clusters related to geographical distances, as well. Conclusion: Our study will assist conservators in future conservation management, breeding, production and habitats restoration of the species. Keywords: Conservation, EST-SSRs; Transcriptome; Panax vietnamensis ; Population genetics

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Development of Expressed Sequence Tag-Drived Simple Sequence Repeat Markers and Diversity Analysis of Phytophthora capsici in China

International Journal of Phytopathology ◽

10.33687/phytopath.002.03.0410 ◽

2013 ◽

Vol 2 (3) ◽

pp. 137-146

Author(s):

Liu Pei-Qing ◽

Wu Min-Liang ◽

Li Ben-Jin ◽

Lan Cheng-Zhong ◽

Weng Qi-Yong ◽

...

Keyword(s):

Genetic Diversity ◽

Ssr Markers ◽

Expressed Sequence Tag ◽

Phytophthora Capsici ◽

Management Strategies ◽

Diversity Analysis ◽

Evolutionary Potential ◽

Genetic Clusters ◽

Expressed Sequence ◽

Simple Sequence

Phytophthora capsici is a highly dynamic and destructive pathogen of vegetable and great interests on the genetic structure of P. capsici have grown in the world. However, there is little genetic information about P. capsici based on the EST-SSR markers. In this study, 193 SSR markers were developed and 33 selected markers were successfully detected and they were polymorphic with the number of alleles per locus ranging from 2 to 7. 4 SSR markers were further selected for genetic diversity analysis and Nei’s genetic diversity values of 15 populations ranged from 0.38 to 0.66, with an average of 0.53. The higher polymorphism and greater transport ability of these markers among P. capsici species were proved by the expected heterozygosity (He =0.64) and Shannon’s index of diversity (I =1.14), indicating that they maintained a substantial level of genetic diversity. Additionally, the genetic differentiation among the 4 markers (Fst =0.15) was moderate and the gene flow among groups was consequent (Nm =1.69). Clustering analyses revealed that 15 populations are made of two differentiated genetic clusters and are similar regarding genetic diversity composition. Our results suggest that there are considerable evolutionary potential of P. capsici in China and useful management strategies should be adapt to it.

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