EST-SSR Primer Pairs Research Articles

OPTIMASI SUHU ANNEALING UNTUK PRIMER g-SSR DAN EST-SSR PADA KACANG HIJAU (Vigna radiata L.)

Mungbean (Vigna radiata L.) is one of important legume in Indonesia. G-SSR and EST-SSR markers had been widely used in mungbean genetic diversity research. DNA isolation and DNA amplification are required to obtain genetic information about mungbean to obtain accurate data on the genetic diversity of mungbean. This study aims to determine the Temperature of annealing (Ta) of the g-SSR and EST-SSR primer pairs. The total DNA was isolated from young leaves mungbean origin Pelalawan and the eight primer pairs of the g-SSR and EST-SSR were optimized. The optimal Ta for G2436, G3598, G2516, G7472, G0483. G1671, G3302, and G3427 were 50,55ºC, 51,15ºC,51,25ºC, 51,2ºC, 51,6ºC, 49,0ºC, 49,8ºC, and 52,8ºC respectively. Meanwhile, the optimal Ta for E51985, E19823, E24080, E22860, E26637, E16266, E11659, and E10675 were 52,2ºC, 54,4ºC, 52,5ºC,51,25ºC, 53,25ºC, 54ºC, 54,35ºC, and 53,6ºC respectively.

EST-SSR marker development based on RNA-sequencing of E. sibiricus and its application for phylogenetic relationships analysis of seventeen Elymus species

BackgroundElymus L. is the largest genus in the tribe Triticeae Dumort., encompassing approximately 150 polyploid perennial species widely distributed in the temperate regions of the world. It is considered to be an important gene pool for improving cereal crops. However, a shortage of molecular marker limits the efficiency and accuracy of genetic breeding for Elymus species. High-throughput transcriptome sequencing data is essential for gene discovery and molecular marker development.ResultsWe obtained the transcriptome dataset of E. sibiricus, the type species of the genus Elymus, and identified a total of 8871 putative EST-SSRs from 6685 unigenes. Trinucleotides were the dominant repeat motif (4760, 53.66%), followed by dinucleotides (1993, 22.47%) and mononucleotides (1876, 21.15%). The most dominant trinucleotide repeat motif was CCG/CGG (1119, 23.5%). Sequencing of PCR products showed that the sequenced alleles from different Elymus species were homologous to the original SSR locus from which the primer was designed. Different types of tri-repeats as abundant SSR motifs were observed in repeat regions. Two hundred EST-SSR primer pairs were designed and selected to amplify ten DNA samples of Elymus species. Eighty-seven pairs of primer (43.5%) generated clear and reproducible bands with expected size, and showed good transferability across different Elymus species. Finally, thirty primer pairs successfully amplified ninety-five accessions of seventeen Elymus species, and detected significant amounts of polymorphism. In general, hexaploid Elymus species with genomes StStHHYY had a relatively higher level of genetic diversity (H = 0.219, I = 0.330, %P = 63.7), while tetraploid Elymus species with genomes StStYY had low level of genetic diversity (H = 0.182, I = 0.272, %P = 50.4) in the study. The cluster analysis showed that all ninety-five accessions were clustered into three major clusters. The accessions were grouped mainly according to their genomic components and origins.ConclusionsThis study demonstrated that transcriptome sequencing is a fast and cost-effective approach to molecular marker development. These EST-SSR markers developed in this study are valuable tools for genetic diversity, evolutionary, and molecular breeding in E. sibiricus, and other Elymus species.

Development and application of EST–SSRs markers for analysis of genetic diversity in erect milkvetch (Astragalus adsurgens Pall.)

Erect milkvetch (Astragalus adsurgens Pall.) is a major legume forage plant widely grown in Northern China. However, the lack of molecular markers has limited its research into its genetic diversity and work on germplasm improvement. In this study, a total of 39,163 EST-SSR loci were identified from 30,262 unigene sequences in the erect milkvetch transcriptome using Illumina sequencing. Moreover, 22,367 EST-SSR primer pairs (PPs) were successfully designed. In addition, 100 PPs were synthesized and preliminarily screened in two accessions; of these, 90 were determined to be clear and stable EST-SSR markers. Fifty-one PPs were randomly selected in order to assess the genetic diversity of 27 erect milkvetch accessions. The average polymorphism information content of the 51 PPs was 0.682. Greater genetic diversity was detected in accessions from Inner Mongolia and in the group of landrace and wild erect milkvetch accessions. This study provides an important resource for germplasm improvement and genetic diversity analysis in erect milkvetch.

SSR fingerprinting of 203 sweetpotato (Ipomoea batatas (L.) Lam.) varieties

Simple sequence repeat (SSR) markers have been shown to be a powerful tool for varieties identification in plants. However, SSR fingerprinting of sweetpotato varieties has been a little reported. In this study, a total of 1294 SSR primer pairs, including 1215 genomic-SSR and 79 expressed sequence tag (EST)-SSR primer pairs, were screened with sweetpotato varieties Zhengshu 20 and Luoxushu 8 and their 2 F1 individuals randomly sampled, and 273 and 38 of them generated polymorphic bands, respectively. Four genomic-SSR and 3 EST-SSR primer pairs, which showed good polymorphism, were selected to amplify 203 sweetpotato varieties and gave a total of 172 bands, 85 (49.42%) of which were polymorphic. All of the 203 sweetpotato varieties showed unique fingerprint patterns, indicating the utility of SSR markers in variety identification of this crop. Polymorphism information content (PIC) ranged from 0.5824 to 0.9322 with an average of 0.8176. SSR-based genetic distances varied from 0.0118 to 0.6353 with an average of 0.3100 among these varieties. Thus, these sweetpotato varieties exhibited high levels of genetic similarity and had distinct fingerprint profiles. The SSR fingerprints of the 203 sweetpotato varieties have been successfully constructed. The highly polymorphic SSR primer pairs developed in this study have the potential to be used as core primer pairs for variety identification, genetic diversity assessment and linkage map construction in sweetpotato and other plants.

Characterization of the Lycium barbarum fruit transcriptome and development of EST-SSR markers.

Lycium barbarum, commonly known as goji, is important in Chinese herbal medicine and its fruit is a very important agricultural and biological product. However, the molecular mechanism of formation of its fruit and associated medicinal and nutritional components is unexplored. Moreover, this species lacks SSR markers due to lack of genomic and transcriptomic information. In this study, a total of 139,333 unigenes with average length of 1049 bp and N50 of 1579 bp are obtained by trinity assembly from Illumina sequencing reads. A total of 92,498 (66.38%) unigenes showed similarities in at least one database including Nr (46.15%), Nt (56.56%), KO (15.56%), Swiss-prot (33.34%), Pfam (33.43%), GO (33.62%) and KOG/COG (17.55%). Genes in flavonoid and taurine biosynthesis pathways were found and validated by RT-qPCR. A total of 50,093 EST-SSRs were identified from 38,922 unigenes, and 22,537 EST-SSR primer pairs were designed. Four hundred pairs of SSR markers were randomly selected to validate assembly quality, of which 352 (88%) were successful in PCR amplification of genomic DNA from 11 Lycium accessions and 210 produced polymorphisms. The polymorphic loci showed that the genetic similarity of the 11 Lycium accessions ranged from 0.50 to 0.99 and the accessions could be divided into 4 groups. These results will facilitate investigations of the molecular mechanism of formation of L. barbarum fruit and associated medicinal and nutritional components, and will be of value to novel gene discovery and functional genomic studies. The EST-SSR markers will be useful for genetic diversity evaluation, genetic mapping and marker-assisted breeding.

New Sugarcane Microsatellites and Target Region Amplification Polymorphism Primers Designed from Candidate Genes Related to Disease Resistance

Many types of molecular marker systems have been developed to detect and map genomic regions related to disease resistance in many major agronomic crops. However, in sugarcane, molecular marker systems for genetic mapping of genomic regions associated to disease resistance is not yet established mainly due to its complex polyploid genome. In addition, there is a lack of information to develop molecular marker systems (e.g., TRAP and EST–SSR) targeting specific genic regions related to disease resistance. In the present work, a set of 10 EST–SSR primer pairs and 16 fixed TRAP primers designed from sugarcane-derived sequence related to disease resistance are reported. Most of the primers were amplifiable and polymorphic in the sugarcane genotypes tested. In addition, these set of primers were also tested in sorghum, Miscanthus and a cross between sugarcane commercial varieties showing good cross-transferability. This new set of primers, in particular the TRAP ones, will be useful in genetic mapping of genome regions related to disease resistance in sugarcane and related genera.

Genetic Diversity Analysis of Sugarcane Germplasm Based on Fluorescence-Labeled Simple Sequence Repeat Markers and a Capillary Electrophoresis-based Genotyping Platform

Genetic diversity analysis, which refers to the elaboration of total extent of genetic characteristics in the genetic makeup of a certain species, constitutes a classical strategy for the study of diversity, population genetic structure, and breeding practices. In this study, fluorescence-labeled seven gSSR and eight EST-SSR primer pairs and a capillary electrophoresis genotyping platform were used to assess the genetic diversity among 181 sugarcane clones. The clones were sorted into 14 series based on their origin. A total of 205 polymorphic SSR alleles were identified. The mean polymorphic information content (PIC) value was 0.94 for gSSRs and 0.93 for EST-SSRs, respectively. Gene differentiation coefficient (Gst) of inter-series variation (13.71 %) was much lower than intra-series variation (86.29 %). Gene flow value (Nm = 3.15) suggested that there was no significant genetic differentiation or population structure variations among the 14 series. The 181 clones could be clustered into seven groups based on neighbor-joining cluster analysis. Three major groups, namely the USA Group, the Guangxi-Hainan-Fujian Group, and the Guangdong Group, consisted of 36, 64, and 39 clones, respectively. The genotyping data provide valuable information for selecting cross parents, designing cross combinations, and future hybrid breeding strategies.

Constructing DNA fingerprinting of Hemarthria cultivars using EST-SSR and SCoT markers

DNA fingerprinting of known cultivars may provide much-needed data to assist with the identification of such cultivars. From 86 pairs of expressed sequence tag SSRs (EST-SSR) and 45 start-codon targeted polymorphism (SCoT) primers, eight pairs of EST-SSR primers and seven SCoT primers were chosen to construct the DNA fingerprinting of six Hemarthria cultivars in this study. Using genomic DNA from six cultivars, a total of eight pairs of EST-SSR primers were able to amplify 193 bands, producing an average of 24.1 bands per primer pair. The percentage of polymorphic bands (PPB) was 83.4 %, and the polymorphism information content (PIC) ranged from 0.480 to 0.695, with an average of 0.602. A total of seven SCoT primers amplified 105 bands with an average of 15 bands per sample. The PPB was 84.8 %, and the PIC ranged from 0.471 to 0.758, with an average of 0.612. The unweighted pair-group method with arithmetic mean dendrogram from EST-SSR and SCoT markers grouped the six Hemarthria cultivars into two major groups of the same. These clusters are in accordance with their known species and origin. Our results indicate a rich genetic diversity in these six Hemarthria cultivars. The six cultivars we assayed could be easily identified using these eight pairs of EST-SSR and seven pairs of SCoT primers.

Development and Characterization of Simple Sequence Repeat (SSR) Markers Based on RNA-Sequencing of Medicago sativa and In silico Mapping onto the M. truncatula Genome

Sufficient codominant genetic markers are needed for various genetic investigations in alfalfa since the species is an outcrossing autotetraploid. With the newly developed next generation sequencing technology, a large amount of transcribed sequences of alfalfa have been generated and are available for identifying SSR markers by data mining. A total of 54,278 alfalfa non-redundant unigenes were assembled through the Illumina HiSeqTM 2000 sequencing technology. Based on 3,903 unigene sequences, 4,493 SSRs were identified. Tri-nucleotide repeats (56.71%) were the most abundant motif class while AG/CT (21.7%), AGG/CCT (19.8%), AAC/GTT (10.3%), ATC/ATG (8.8%), and ACC/GGT (6.3%) were the subsequent top five nucleotide repeat motifs. Eight hundred and thirty- seven EST-SSR primer pairs were successfully designed. Of these, 527 (63%) primer pairs yielded clear and scored PCR products and 372 (70.6%) exhibited polymorphisms. High transferability was observed for ssp falcata at 99.2% (523) and 71.7% (378) in M. truncatula. In addition, 313 of 527 SSR marker sequences were in silico mapped onto the eight M. truncatula chromosomes. Thirty-six polymorphic SSR primer pairs were used in the genetic relatedness analysis of 30 Chinese alfalfa cultivated accessions generating a total of 199 scored alleles. The mean observed heterozygosity and polymorphic information content were 0.767 and 0.635, respectively. The codominant markers not only enriched the current resources of molecular markers in alfalfa, but also would facilitate targeted investigations in marker-trait association, QTL mapping, and genetic diversity analysis in alfalfa.

Development of simple sequence repeat markers and diversity analysis in alfalfa (Medicago sativa L.)

Efficient and robust molecular markers are essential for molecular breeding in plant. Compared to dominant and bi-allelic markers, multiple alleles of simple sequence repeat (SSR) markers are particularly informative and superior in genetic linkage map and QTL mapping in autotetraploid species like alfalfa. The objective of this study was to enrich SSR markers directly from alfalfa expressed sequence tags (ESTs). A total of 12,371 alfalfa ESTs were retrieved from the National Center for Biotechnology Information. Total 774 SSR-containing ESTs were identified from 716 ESTs. On average, one SSR was found per 7.7 kb of EST sequences. Tri-nucleotide repeats (48.8 %) was the most abundant motif type, followed by di-(26.1 %), tetra-(11.5 %), penta-(9.7 %), and hexanucleotide (3.9 %). One hundred EST-SSR primer pairs were successfully designed and 29 exhibited polymorphism among 28 alfalfa accessions. The allele number per marker ranged from two to 21 with an average of 6.8. The PIC values ranged from 0.195 to 0.896 with an average of 0.608, indicating a high level of polymorphism of the EST-SSR markers. Based on the 29 EST-SSR markers, assessment of genetic diversity was conducted and found that Medicago sativa ssp. sativa was clearly different from the other subspecies. The high transferability of those EST-SSR markers was also found for relative species.

Development of <i>Pinus koraiensis</i> SSR Primers Based on EST-SSR Information Technology

A total 408 SSRs were distributed in 18,181 ESTs sequences in Pinaceae in NCBI (National Center for Biotechnology Information) searched by SSRIT software, accounting for 2.24% of the whole EST sequences. We designed 132 pairs of EST-SSR primers by primer3. Of the designed 132 pairs, 29 pairs were able to produce an amplification product in the 10 Pinus koraiensis DNA samples, but only five primers in the 29 pairs exhibited polymorphism. Dinucleotide repeats were the most common repeat class. The repeated primitives of dinucleotide were 10, accounting for 52.73% of the whole repeated primitives; the repeat numbers were 87. The second most common repeat class was trinucleotide. The repeated primitives of trinucleotide were 27, accounting for 42.27% of the whole repeated primitives, and repeat numbers were 78.

Mining, characterization, and exploitation of EST-derived microsatellites in Gossypium barbadense

Simple sequence repeats (SSRs) have been widely applied as molecular markers in genetic studies. However, the number of expressed sequence tags (ESTs) and SSR markers from Gossypium barbadense is fewer than those from other cotton species. In this study, EST-SSR distribution from G. barbadense was characterized and new G. barbadense-derived EST-SSR markers were determined on the basis of the ESTs obtained by randomly sequencing 2 cDNA libraries associated with fiber development in G. barbadense. By mining 9697 non-redundant ESTs, a total of 638 SSR loci derived from 595 ESTs were observed. In G. barbadense, the frequency of ESTs containing SSRs was 6.13%, with an average of 1 SSR in every 10.4 kb of EST sequence. Furthermore, trinucleotide was found to be the most abundant repeat type among 2–6-nucleotide repeat types. It accounted for 26.6% of the total, followed by the hexanucleotide (26.0%) and pentanucleotide repeats (25.9%). Among all the repeat motifs, (AAG)n accounted for the highest proportion. EST-SSR primer pairs were developed using the Primer3 program, and the redundant primers were removed using the virtual PCR approach. As a result, 380 non-redundant EST-SSR primer pairs were developed and used to detect polymorphisms between the mapping parents G. hirsutum ‘TM-1’ and G. barbadense ‘Hai7124’ for constructing linkage groups in cultivated allotetraploid cotton. Out of these, 98 (25.8%) primer pairs detected polymorphisms. Finally, 95 polymorphic loci from 82 primer pairs were integrated into the backbone genetic map; of these, 42 were mapped into the A subgenome and 53 into the D subgenome. The present work provided the foundation for constructing saturated genetic maps and conducting comparative genomic studies on different cotton species.

Development, chromosome location and genetic mapping of EST-SSR markers in wheat

A number of 151695 wheat expression sequence tags (ESTs) that originated from GenBank/dbEST from July 14, 2003 to August 24, 2004 were used to search for simple sequence repeats (SSRs) with motif 2―5 bp, and 2038 simple sequence repeats (EST-SSRs), which accounted for 1.34% of EST database, were identified. Based on these SSR sequences, 249 EST-SSR primer pairs and 166 amplified clear bands in various wheat cultivars were designed. These EST-SSR markers can be used as new molecular markers in wheat and related species. Using Chinese Spring nulli-tetrasomic lines, 93 EST-SSR primer pairs and 193 EST-SSR loci were located on 19 wheat chromosomes except for 4A and 4B. Forty-three loci were mapped on 11 chromosomes of the genetic frame- work map previously constructed using recombinant inbred lines.

Genetic mapping of EST-derived microsatellites from the diploid Gossypium arboreum in allotetraploid cotton.

To increase the numbers of microsatellites available for use in constructing a genetic map, and facilitate the use of functional genomics to elucidate fiber development and breeding in cotton, we sampled microsatellite sequences from expressed sequence tags (ESTs) transcribed during fiber elongation in the A-genome species Gossypium arboreum to evaluate their frequency of occurrence, level of polymorphism and distribution in the At and Dt subgenomes of tetraploid cotton. From among ESTs derived from G. arboreum fibers at 7-10 days post anthesis (dpa), 931 ESTs were found to contain simple sequence repeats (SSRs); 544 (58.4%) EST-SSR primer pairs were developed, and 468 (86%) amplified PCR products from allotetraploid cotton (G. hirsutumcv. TM-1 and G. barbadense cv. Hai7124). However, only 99 (18.2%) of these were found to be polymorphic and segregating in our interspecific BC1 mapping population [(TM-1xHai7124)xTM-1]. In these amplified and informative EST-SSRs, hexa- and tri-nucleotide repeat motifs were the most frequent, representing 40.1 and 30%, respectively, of the total. A total of 111 loci detected with these 99 EST-SSRs were integrated into our backbone map including 511 SSR loci. The distribution of the EST-SSRs appeared to be non-random, since 72 loci were anchored to the At and 37 to the Dt subgenome of allotetraploid cotton based on linkage tests. Interestingly, out of the 10 pairs of duplicate loci amplified, seven were mapped to the corresponding homologous linkage groups and/or chromosomes. BLASTX analysis revealed that 69 of the 99 ESTs showed significant similarities to known genes. Some genes important for fiber development, such as sucrose synthase, were mapped to corresponding chromosomes. These EST-SSRs provide structural and functional genomic information that will be useful for understanding cotton fiber development.

Analysis of microsatellites in major crops assessed by computational and experimental approaches

Over 85Mb publicly available expressed sequence tags (ESTs) from four agronomically important crops were used to search for the types and frequencies of simple sequence repeats (SSRs) with motif length of 1-6bp. The frequency of EST-SSRs was one in 11.81kb in rice, 17.42kb in wheat, 23.80kb in soybean, and 28.32kb in maize, respectively. Trinucleotide repeats were the most abundant SSR types with up to 47 trinucleotide repeat units in 100kb ESTs. Compared with dicots, monocots contained GC-rich tri- and hexa- motifs, especially rice where 23.6 CCG motif occurred in 100kb ESTs. 597 EST-SSR primer pairs were designed for wheat, of which, 478 primer pairs had successful PCR amplifications. The percentage of polymorphism was up to 41.8% among wheat varieties, that could be used to construct a transcriptional map of wheat. Cross-species amplification was detected. Among the 478 functional primers from wheat, 255 EST-SSRs amplified fragments in rice, maize and soybean way, indicating high degree of transferability between species, that facilitate comparative mapping and homologous gene cloning.