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Effective Procedure for Development of EST-SSR Markers Using cDNA Library  [PDF]
Kyung A Kim, Hee-Cheon Park, Jae-Keun Sohn, Kyung-Min Kim
American Journal of Plant Sciences (AJPS) , 2012, DOI: 10.4236/ajps.2012.39159
Abstract: The present study was conducted to develop EST-SSR markers using the cDNA library from rice plant. Total RNA extracted from the leaves of brown plant hopper resistance gene originated from a rice cultivar “Cheongcheong” and sensitive rice cultivar “Nakdong” were used to synthesize a cDNA library. As a result of analyzing the cDNA library, the 17 EST-SSR primer sets were developed. This study enables to provide effective marker assisted selection (MAS) methods on the selection of white-backed planthopper resistance gene originated from a rice plant more simply, quickly and precisely. Furthermore, using this marker’s advantage of deriving from cDNA, it is possible to identify the white-backed planthopper resistance gene. In addition, this study introduces a technique for construction of a cDNA library safely without using radioactivity.
Development and Molecular Characterization of 55 Novel Polymorphic cDNA-SSR Markers in Faba Bean (Vicia faba L.) Using 454 Pyrosequencing  [PDF]
Sundan Suresh,Jong-Hyun Park,Gyu-Taek Cho,Ho-Sun Lee,Hyung-Jin Baek,Sok-Young Lee,Jong-Wook Chung
Molecules , 2013, DOI: 10.3390/molecules18021844
Abstract: Faba bean ( Vicia faba L.) is a major food source and fodder legume, popularly known for its high content of seed-protein. Its role is critical in crop rotation, and for fixing nitrogen effectively. Polymorphic simple sequence repeat markers from transcript sequences (cDNA; simple sequence repeat [SSR]) were developed for faba bean ( Vicia faba). We found that 1,729 SSR loci from 81,333 individual sequence reads and 240 primer pairs were designed and synthesized. In total, 55 primer pairs were found to be polymorphic and scorable consistently when screened in 32 accessions. The number of alleles ranged from 2 to 15, frequency of major alleles per locus varied from 0.17 to 0.91, the genotypes number ranged from 2 to 17, observed and expected heterozycosity values ranged from 0.00 to 0.44 and 0.17 to 0.89 and overall PIC values ranged from 0.16 to 0.88 respectively. These markers will be a useful tool for assessing the genetic diversity, understanding the population structure, and breeding patterns of faba bean.
Genetic Diversity among 18 Accessions of African Rice (Oryza glaberrima Steud.) Using Simple Sequence Repeat (SSR) Markers
H.A. Doku,E.Y. Danquah,A.N. Amoah,K. Nyalemegbe,H.M. Amoatey
Agricultural Journal , 2013, DOI: 10.3923/aj.2013.106.112
Abstract: Studies were carried out on 18 accessions of African rice (Oryza glaberrima Steud.) collected from four geographical regions of Ghana to assess genetic diversity and potential of these accessions, towards a thorough exploitation of the species, in a region-wide breeding programme to obtain new rice varieties better adapted to the harsh growing conditions of West and Central Africa (WCA). Simple Sequence Repeat (SSR) markers were used to estimate diversity among the accessions. Out of 24 SSR primers used, 23 (i.e., 95.83%) showed allelic polymorphism in the accessions studied. Overall genetic diversity was high (I = 1.178, He = 0.625 and Nei s He = 0.608) and the Fixation index statistics (Fst) revealed that 51.5% of the total variation exists among populations collected from the four geographical regions. All accessions were identified as separate entries with no duplications.
Comparison of Population Genetic Structures between Asian and American Mungbean Accessions Using SSR Markers  [cached]
Xiao-Qiang Wang,Soon-Wook Kwon,Yong-Jin Park
Journal of Agricultural Science , 2012, DOI: 10.5539/jas.v4n9p150
Abstract: The purpose of this study was to evaluate the genetic diversity and population structure of 65 mungbean accessions collected from East and Southeast Asia, the United States and Guatemala using 15 simple sequence repeat (SSR) markers. In total, 47 alleles were detected, the number of the alleles per locus range from two to six, with an average of 3.13. The mean major allele frequency (MAF), expected heterozygosity (HE), and polymorphic information content (PIC) of the 15 SSR loci were 0.76, 0.05, and 0.28, respectively. Of the 47 alleles, 17 (36.2%) were common, with a frequency of 0.05– 0.5; 16 (34.0%) were rare (frequency < 0.05) and 14 (29.8%) were abundant (frequency > 0.5). On the basis of the UPGMA dendrogram, most of the accessions were clustered into two main groups. The first group (Group I) included seven accessions and the second comprised 58 accessions, which were further divided into four subgroups. Four subpopulations were detected by model-based structure analysis. Fifty-five accessions (84.6%) showed a clear relation to each cluster based on their inferred ancestry value (>75%), while the remaining 10 accessions (15.4%) were categorized as admixtures. Mungbean accessions from US distributed to almost all clusters and 2 accessions shared genetic constituents showing it derived from mixed ancestry with Asean accessions. These results could be useful in identifying mungbean germplasms and facilitating their improvement programs.
Genetic variability among cassava accessions based on SSR markers
Ribeiro, Márcia de Nazaré Oliveira;Carvalho, Samuel Pereira de;Santos, Jo?o Bosco dos;Antonio, Rafaela Priscila;
Crop Breeding and Applied Biotechnology , 2011, DOI: 10.1590/S1984-70332011000300009
Abstract: the aim of this study was to characterize and estimate the genetic similarity among 93 cassava accessions. the dna amplification was performed with 14 microsatellite primers. the amplification products were separated by a polyacrylamide gel electrophoresis, showing a polymorphism formation, through which the accessions were discriminated against. the genetic similarity among accessions of cassava was estimated by the dice coefficient. cluster analysis was carried out using the upgma method. the polymorphic primers amplified a total of 26 alleles with 2-4 alleles per loci. the genetic similarity ranged from 0.16 to 0.96. the average values for observed and expected heterozygosity were 0.18 and 0.46, respectively. twenty genetic similarity clusters were determined, demonstrating diversity among accessions, suggesting the possibility of heterotic hybrid generation.
NOTE - Genetic variability among cassava accessions based on SSR markers  [PDF]
Márcia de Nazaré Oliveira Ribeiro,Samuel Pereira de Carvalho,Jo?o Bosco dos Santos,Rafaela Priscila Antonio
Crop Breeding and Applied Biotechnology , 2011,
Abstract: The aim of this study was to characterize and estimate the genetic similarity among 93 cassava accessions. The DNAamplification was performed with 14 microsatellite primers. The amplification products were separated by a polyacrylamide gelelectrophoresis, showing a polymorphism formation, through which the accessions were discriminated against. The genetic similarityamong accessions of cassava was estimated by the Dice coefficient. Cluster analysis was carried out using the UPGMA method. Thepolymorphic primers amplified a total of 26 alleles with 2-4 alleles per loci. The genetic similarity ranged from 0.16 to 0.96. Theaverage values for observed and expected heterozygosity were 0.18 and 0.46, respectively. Twenty genetic similarity clusters weredetermined, demonstrating diversity among accessions, suggesting the possibility of heterotic hybrid generation.
Development of 65 Novel Polymorphic cDNA-SSR Markers in Common Vetch (Vicia sativa subsp. sativa) Using Next Generation Sequencing  [PDF]
Jong-Wook Chung,Tae-Sung Kim,Sundan Suresh,Sok-Young Lee,Gyu-Taek Cho
Molecules , 2013, DOI: 10.3390/molecules18078376
Abstract: Vetch ( Vicia sativa L.) is one of the most important annual forage legumes in the World due to its multiple uses ( i.e., hay, grain, silage and green manure) and high nutritional value. However, detrimental cyanoalanine toxins in its plant parts including seeds and its vulnerability to hard winter conditions are currently reducing the agronomic values of vetch varieties. Moreover, the existence in the public domain of very few genomic resources, especially molecular markers, has further hampered breeding efforts. Polymorphic simple sequence repeat markers from transcript sequences (cDNA; simple sequence repeat [SSR]) were developed for Vicia sativa subsp. sativa. We found 3,811 SSR loci from 31,504 individual sequence reads, and 300 primer pairs were designed and synthesized. In total, 65 primer pairs were found to be consistently scorable when 32 accessions were tested. The numbers of alleles ranged from 2 to 19, frequency of major alleles per locus were 0.27–0.87, the genotype number was 2–19, the overall polymorphism information content ( PIC) values were 0.20–0.86, and the observed and expected heterozygosity values were 0.00–0.41 and 0.264–0.852, respectively. These markers provide a useful tool for assessing genetic diversity, population structure, and positional cloning, facilitating vetch breeding programs.
Assessment of EST-SSR Markers for Evaluating Genetic Diversity in Watermelon Accessions from Zimbabwe  [PDF]
Claid Mujaju, Jasna Sehic, Hilde Nybom
American Journal of Plant Sciences (AJPS) , 2013, DOI: 10.4236/ajps.2013.47177
Abstract:

Fifteen expressed sequence tag (EST)-derived simple sequence repeats (EST-SSRs) were used to investigate genetic diversity in 139 plants obtained from seeds of 35 watermelon accessions collected from all the geographical provinces of Zimbabwe. In addition, 15 plants representing three commercial varieties developed in the United States (USA) were analyzed for comparison. A total of 65 alleles were detected among all the watermelon accessions. For the 13 polymorphic EST-SSR loci, number of alleles per locus varied from 2 to 13, with an average of 5 alleles per locus. Values for the polymorphic information content increased as the number of alleles increased, and varied from 0.15 to 0.77 with an average of 0.54 suggesting sufficient discriminatory power. Both cluster analysis and principal coordinate analysis (PCA) produced two major clusters; one with the 22 cow-melon accessions and the other with the 16 sweet watermelon accessions. Within the sweet watermelon group, two distinct sub-clusters formed, one of which contained only two of the commercial varieties from USA. Partitioning of genetic variation in the Zimbabwean material using analysis of molecular variation (AMOVA) revealed that 64% of the total variation resides between the two major forms, i.e. sweet watermelons and cow-melons, 28% between accessions within forms and 8% within accessions. The EST-SSR markers revealed a somewhat higher diversity in sweet watermelon accessions compared to that of cow-melons. This finding is contrary to previous reports using other markers (genomic SSR loci or RAPD) and/or a plant material that is likely to have experienced more stringent selection procedures compared to the landraces analyzed in our study.

Development and validation of genic-SSR markers in sesame by RNA-seq
Haiyang Zhang, Libin Wei, Hongmei Miao, Tide Zhang, Cuiying Wang
BMC Genomics , 2012, DOI: 10.1186/1471-2164-13-316
Abstract: In this study, 75?bp and 100?bp paired-end RNA-seq was used to sequence 24 cDNA libraries, and 42,566 uni-transcripts were assembled from more than 260 million filtered reads. The total length of uni-transcript sequences was 47.99?Mb, and 7,324 SSRs (SSRs ≥15?bp) and 4,440 SSRs (SSRs ≥18?bp) were identified. On average, there was one genic-SSR per 6.55?kb (SSRs ≥15?bp) or 10.81?kb (SSRs ≥18?bp). Among perfect SSRs (≥18?bp), di-nucleotide motifs (48.01%) were the most abundant, followed by tri- (20.96%), hexa- (25.37%), penta- (2.97%), tetra- (2.12%), and mono-nucleotides (0.57%). The top four motif repeats were (AG/CT)n [1,268 (34.51%)], (CA/TG)n [281 (7.65%)], (AT/AT)n [215 (5.85%)], and (GAA/TTC)n [131 (3.57%)]. A total of 2,164 SSR primer pairs were identified in the 4,440 SSR-containing sequences (≥18?bp), and 300 SSR primer pairs were randomly chosen for validation. These SSR markers were amplified and validated in 25 sesame accessions (24 cultivated accessions, one wild species). 276 (92.0%) primer pairs yielded PCR amplification products in 24 cultivars. Thirty two primer pairs (11.59%) exhibited polymorphisms. Moreover, 203 primer pairs (67.67%) yielded PCR amplicons in the wild accession and 167 (60.51%) were polymorphic between species. A UPGMA dendrogram based on genetic similarity coefficients showed that the correlation between genotype and geographical source was low and that the genetic basis of sesame in China is narrow, as previously reported. The 32 polymorphic primer pairs were validated using an F2 mapping population; 18 primer pairs exhibited polymorphisms between the parents, and 14 genic-SSRs could be integrated into 9 main linkage groups.2,164 genic-SSR markers have been developed in sesame using transcriptome sequencing. 276 of 300 validated primer pairs successfully yielded PCR amplicons in 24 cultivated sesame accessions. These markers increase current SSR marker resources and will greatly benefit genetic diversity, qualitative and quantit
Genetic diversity among varieties and wild species accessions of pea (Pisum sativum L.) based on SSR markers
J Nasiri, A Haghnazari, J Saba
African Journal of Biotechnology , 2009,
Abstract: To assess the genetic relations inPisum genus and to examine putative duplicate accessions, 20 pea varieties (Pisum sativum L.) with 57 accessions from wild Pisum species fulvum, subspecies (subsp.) asiaticum, elatius, thebaicum, abyssinicum, transcaucasicum and arvense were analyzed using 10 out of 20 microsatellite primer pairs. We genotyped all accessions. In total, 59 alleles were identified in whole collection. The maximum number of alleles (8 alleles) was obtained from the PEACPLHPP, AF004843, and AA43090 loci. The maximum number of private alleles (4) in the wild collection was detected in AF004843 locus but in the cultivar collection, it was detected in AA430902 and PSBLOX13.2 loci. Cluster analysis and principal coordinate analysis located accessions in 3 groups and cultivated varieties were obviously separated from the wild accessions. Analysis of molecular variance (AMOVA) revealed that the intergroups component of variance (29%) is lower than the intragroups component of variance (71%). The lowest value of genetic differentiation ( Pisum genus and to examine putative duplicate accessions, 20 pea varieties (Pisum sativum L.) with 57 accessions from wild Pisum species fulvum, subspecies (subsp.) asiaticum, elatius, thebaicum, abyssinicum, transcaucasicum and arvense were analyzed using 10 out of 20 microsatellite primer pairs. We genotyped all accessions. In total, 59 alleles were identified in whole collection. The maximum number of alleles (8 alleles) was obtained from the PEACPLHPP, AF004843, and AA43090 loci. The maximum number of private alleles (4) in the wild collection was detected in AF004843 locus but in the cultivar collection, it was detected in AA430902 and PSBLOX13.2 loci. Cluster analysis and principal coordinate analysis located accessions in 3 groups and cultivated varieties were obviously separated from the wild accessions. Analysis of molecular variance (AMOVA) revealed that the intergroups component of variance (29%) is lower than the intragroups component of variance (71%). The lowest value of genetic differentiation (Pisum genus and to examine putative duplicate accessions, 20 pea varieties (Pisum sativum L.) with 57 accessions from wild Pisum species fulvum, subspecies (subsp.) asiaticum, elatius, thebaicum, abyssinicum, transcaucasicum and arvense were analyzed using 10 out of 20 microsatellite primer pairs. We genotyped all accessions. In total, 59 alleles were identified in whole collection. The maximum number of alleles (8 alleles) was obtained from the PEACPLHPP, AF004843, and AA43090 loci. The maximum number of private alleles (4) in the wild collection was detected in AF004843 locus but in the cultivar collection, it was detected in AA430902 and PSBLOX13.2 loci. Cluster analysis and principal coordinate analysis located accessions in 3 groups and cultivated varieties were obviously separated from the wild accessions. Analysis of molecular variance (AMOVA) revealed that the intergroups component of variance
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