Development Of Simple Sequence Repeats Research Articles

Development of simple sequence repeat markers for sugarcane from data mining of expressed sequence tags.

Sugarcane (Saccharum spp. hybrids) is a worldwide acclaimed important agricultural crop used primarily for sugar production and biofuel. Sugarcane's genetic complexity, aneuploidy, and extreme heterozygosity make it a challenging crop in developing improved varieties. The molecular breeding programs promise to develop nutritionally improved varieties for both direct consumption and commercial application. Therefore, to address these challenges, the development of simple sequence repeats (SSRs) has been proven to be a powerful molecular tool in sugarcane. This study involved the collection of 285216 expressed sequence tags (ESTs) from sugarcane, resulting in 23666 unigenes, including 4547 contigs. Our analysis identified 4120 unigenes containing a total of 4960 SSRs, with the most abundant repeat types being monomeric (44.33%), dimeric (13.10%), and trimeric (39.68%). We further chose 173 primers to analyze the banding pattern in 10 sugarcane accessions by PAGE analysis. Additionally, functional annotation analysis showed that 71.07%, 53.6%, and 10.3% unigenes were annotated by Uniport, GO, and KEGG, respectively. GO annotations and KEGG pathways were distributed across three functional categories: molecular (46.46%), cellular (33.94%), and biological pathways (19.6%). The cluster analysis indicated the formation of four distinct clusters among selected sugarcane accessions, with maximum genetic distance observed among the varieties. We believe that these EST-SSR markers will serve as valuable references for future genetic characterization, species identification, and breeding efforts in sugarcane.

Multiple-Genome-Based Simple Sequence Repeat Is an Efficient and Successful Method in Genotyping and Classifying Different Jujube Germplasm Resources.

Jujube (Ziziphus jujuba Mill.) is a commercially important tree native to China, known for its high nutritional value and widespread distribution, as well as its diverse germplasm resources. Being resilient to harsh climatic conditions, the cultivation of jujube could provide a solution to food insecurity and income for people of arid and semi-arid regions in and outside of China. The evaluation of germplasm resources and genetic diversity in jujube necessitates the use of Simple Sequence Repeat (SSR) markers. SSR markers are highly polymorphic and can be used to evaluate the genetic diversity within and between cultivars of Chinese jujube, and are important for conservation biology, breeding programs, and the discovery of important traits for Chinese jujube improvement in China and abroad. However, traditional methods of SSR development are time-consuming and inadequate to meet the growing research demands. To address this issue, we developed a novel approach called Multiple-Genome-Based SSR identification (MGB-SSR), which utilizes the genomes of three jujube cultivars to rapidly screen for polymorphic SSRs in the jujube genome. Through the screening process, we identified 12 pairs of SSR primers, which were then used to successfully classify 249 jujube genotypes. Based on the genotyping results, a digital ID card was established, enabling the complete identification of all 249 jujube plants. The MGB-SSR approach proved efficient in rapidly detecting polymorphic SSRs within the jujube genome. Notably, this study represents the first successful differentiation of jujube germplasm resources using 12 SSR markers, with 4 markers successfully identifying triploid jujube genotypes. These findings offer valuable information for the classification of Chinese jujube germplasm, thereby providing significant assistance to jujube researchers and breeders in identifying unknown jujube germplasm.

Characterization of Simple Sequence Repeat (SSR) Markers Mined in Whole Grape Genomes.

SSR (simple sequence repeat) DNA markers are widely used for genotype DNA identification, QTL mapping, and analyzing genetic biodiversity. However, SSRs in grapes are still in their early stages, with a few primer pairs accessible. With the whole-genome sequencing (WGS) of several grape varieties, characterization of grape SSR changed to be necessary not only to genomics but to also help SSR development and utility. Based on this, we identified the whole-genome SSR of nine grape cultivars ('PN40024', 'Cabernet Sauvignon', 'Carménère', 'Chardonnay', 'Merlot', 'Riesling', 'Zinfandel', 'Shine Muscat', and 'Muscat Hamburg') with whole-genome sequences released publicly and found that there are great differences in the distribution of SSR loci in different varieties. According to the difference in genome size, the number of SSRs ranged from 267,385 (Cabernet Sauvignon) to 627,429 (Carménère), the density of the SSR locus in the genome of nine cultivars was generally 1 per Kb. SSR motif distribution characteristic analysis of these grape cultivars showed that the distribution patterns among grape cultivars were conservative, mainly enriched in A/T. However, there are some differences in motif types (especially tetranucleotides, pentanucleotides, and hexanucleotides), quantity, total length, and average length in different varieties, which might be related to the size of the assembled genome or the specificity of variety domestication. The distribution characteristics of SSRs were revealed by whole-genome analysis of simple repeats of grape varieties. In this study, 32 pairs of primers with lower polymorphism have been screened, which provided an important research foundation for the development of molecular markers of grape variety identification and the construction of linkage maps of important agronomic traits for crop improvement.

Development of SSR and SNP markers for identifying opium poppy.

Papaver somniferum L. (opium poppy) is the original plant of heroin, which is a major narcotic drug, and this plant has brought great harm to human health. However, the ban on opium poppy cultivation and trafficking is facing great challenges because of abnormal profits. Therefore, rapid and accurate identification is important to address the abovementioned problems. In this study, eleven simple sequence repeats (SSR) markers and two single nucleotide polymorphism (SNP) markers were mined to distinguish opium poppy from other six Papaver species. These molecular markers were further verified through a large number of plant materials of these seven Papaver species. An excellent multiplex polymerase chain reaction (PCR) system that simultaneously amplifies the three of eleven SSR markers was developed, which effectively improves the efficiency and speed of identification. The present research is of great implication for identifying and investigating the illegal cultivation and trafficking of opium poppy.

A pipeline for effectively developing highly polymorphic simple sequence repeats markers based on multi-sample genomic data.

Simple sequence repeats (SSRs) are widely used genetic markers in ecology, evolution, and conservation even in the genomics era, while a general limitation to their application is the difficulty of developing polymorphic SSR markers. Next‐generation sequencing (NGS) offers the opportunity for the rapid development of SSRs; however, previous studies developing SSRs using genomic data from only one individual need redundant experiments to test the polymorphisms of SSRs. In this study, we designed a pipeline for the rapid development of polymorphic SSR markers from multi‐sample genomic data. We used bioinformatic software to genotype multiple individuals using resequencing data, detected highly polymorphic SSRs prior to experimental validation, significantly improved the efficiency and reduced the experimental effort. The pipeline was successfully applied to a globally threatened species, the brown eared‐pheasant (Crossoptilon mantchuricum), which showed very low genomic diversity. The 20 newly developed SSR markers were highly polymorphic, the average number of alleles was much higher than the genomic average. We also evaluated the effect of the number of individuals and sequencing depth on the SSR mining results, and we found that 10 individuals and ~10X sequencing data were enough to obtain a sufficient number of polymorphic SSRs, even for species with low genetic diversity. Furthermore, the genome assembly of NGS data from the optimal number of individuals and sequencing depth can be used as an alternative reference genome if a high‐quality genome is not available. Our pipeline provided a paradigm for the application of NGS technology to mining and developing molecular markers for ecological and evolutionary studies.

Genetic variation and association mapping in the F2 population of the Perilla crop (Perilla frutescens L.) using new developed Perilla SSR markers

The transcriptome sequencing approach RNA-seq represents a powerful tool for transcriptional analysis and development of simple sequence repeat (SSR) markers for nonmodel crop. In the Perilla crop, analysis of the distribution of different repeat motifs showed that the most abundant type was dinucleotide repeats (62.0%), followed by trinucleotide repeats (35.3%), with the two together comprising 97.3% of the eSSR repeats. In this study, we developed 39 new SSR primer sets by the transcriptome sequencing approach RNA-sEq. In total, 130 alleles were detected segregating in nine Perilla accessions with an average of 3.3 alleles per locus, ranging from 125 to 360 bp. The number of alleles per locus ranged from two to six. To detect SSR markers associated with morphological characteristics of Perilla crop, 40 individuals from an F2 population of Perilla were selected for association analysis based on their leaf- and plant-related characteristics. An association analysis of 37 SSR markers and 9 leaf- and plant-related traits in the 40 individuals of the F2 population was conducted. From the analysis, we identified 12 SSR markers associated with leaf-related traits and 11 SSR markers associated with plant-related traits. Therefore, the new Perilla SSR primers described in this study could be helpful in identifying genetic diversity and genetic mapping, designating important genes/QTLs for Perilla crop breeding programs, and allowing Perilla breeders to improve leaf and plant quality through marker-assisted selection (MAS) breeding programs.

Genome-wide simple sequence repeats (SSR) markers discovered from whole-genome sequence comparisons of multiple spinach accessions

The availability of well-assembled genome sequences and reduced sequencing costs have enabled the resequencing of many additional accessions in several crops, thus facilitating the rapid discovery and development of simple sequence repeat (SSR) markers. Although the genome sequence of inbred spinach line Sp75 is available, previous efforts have resulted in a limited number of useful SSR markers. Identification of additional polymorphic SSR markers will support genetics and breeding research in spinach. This study aimed to use the available genomic resources to mine and catalog a large number of polymorphic SSR markers. A search for SSR loci on six chromosome sequences of spinach line Sp75 using GMATA identified a total of 42,155 loci with repeat motifs of two to six nucleotides in the Sp75 reference genome. Whole-genome sequences (30x) of additional 21 accessions were aligned against the chromosome sequences of the reference genome and in silico genotyped using the HipSTR program by comparing and counting repeat numbers variation across the SSR loci among the accessions. The HipSTR program generated SSR genotype data were filtered for monomorphic and high missing loci, and a final set of the 5986 polymorphic SSR loci were identified. The polymorphic SSR loci were present at a density of 12.9 SSRs/Mb and were physically mapped. Out of 36 randomly selected SSR loci for validation, two failed to amplify, while the remaining were all polymorphic in a set of 48 spinach accessions from 34 countries. Genetic diversity analysis performed using the SSRs allele score data on the 48 spinach accessions showed three main population groups. This strategy to mine and develop polymorphic SSR markers by a comparative analysis of the genome sequences of multiple accessions and computational genotyping of the candidate SSR loci eliminates the need for laborious experimental screening. Our approach increased the efficiency of discovering a large set of novel polymorphic SSR markers, as demonstrated in this report.

Genome-wide identification of simple sequence repeats and assessment of genetic diversity in Hedychium

Hedychium is an important industrial crop, which is widely cultivated for its medicinal and horticultural significance. However, the development of simple sequence repeat (SSR) for Hedychium has not yet been reported. In this study, 266,825 SSR markers were identified in H. coronarium ‘ZS’ genome. The relationship between the type, repeat times, content, and content of SSR markers and chromosome length was specifically analyzed. The results revealed that the mononucleotides (59.17 %) were the most abundant repeat motifs, followed by dinucleotides (27.97 %) and trinucleotides (10.85 %). To verify the availability of SSR and its polymorphism among different species, 120 primer pairs were randomly selected. Finally, 22 markers were screened to genotype 44 accessions collected from different geographic locations of China, Thailand, Japan, and USA. A number of 224 alleles were identified, with an average of 10.18 alleles and polymorphism information content ranged from 0.17 to 0.96 with a mean of 0.88. Genetic structure analysis indicated that 44 accessions had strong regional characteristics and a clear distinction between cultivated and wild genotypes. Population structure and PCoA analysis revealed that from 44 accessions, 7 cultivated accession were distinctly grouped into cluster A, whilst other wild accessions belong to cluster B. These new SSR markers will provide important references for genetic diversity analysis, identification of germplasm resources, and breeding of Hedychium.

Microsatellites-based population analysis revealed micro-diversity in two major genepools of Brassica juncea

Microsatellites or simple sequence repeats (SSRs) prove to be an important marker system for genetic analysis of a diverse population. The study presents the development of SSRs from A-genome of Brassica followed by genotyping with diverse genotypes from two major gene pools of Brassica juncea. A total of 61,918 SSRs were identified, of which dinucleotide repeats were most common. SSR markers were able to amplify 269 alleles among 85 genotypes. The average number of alleles per locus and PIC value was 3.24 and 0.41, respectively. Gene diversity and heterozygosity value was also able to identify a huge variability among the genotypes from two gene pools. Dendrogram based on Euclidean index was able to group genotypes into three clusters and identified micro-diversity within European gene pool. The present study will serve as a tool in the selection of contrast genotypes for Brassica juncea breeding programs.

De novo assembly, annotation, marker discovery, and genetic diversity of the Stipa breviflora Griseb. (Poaceae) response to grazing.

Grassland is one of the most widely-distributed ecosystems on Earth and provides a variety of ecosystem services. Grasslands, however, currently suffer from severe degradation induced by human activities, overgrazing pressure and climate change. In the present study, we explored the transcriptome response of Stipa breviflora, a dominant species in the desert steppe, to grazing through transcriptome sequencing, the development of simple sequence repeat (SSR) markers, and analysis of genetic diversity. De novo assembly produced 111,018 unigenes, of which 88,164 (79.41%) unigenes were annotated. A total of 686 unigenes showed significantly different expression under grazing, including 304 and 382 that were upregulated and downregulated, respectively. These differentially expressed genes (DEGs) were significantly enriched in the "alpha-linolenic acid metabolism" and "plant-pathogen interaction" pathways. Based on transcriptome sequencing data, we developed eight SSR molecular markers and investigated the genetic diversity of S. breviflora in grazed and ungrazed sites. We found that a relatively high level of S. breviflora genetic diversity occurred under grazing. The findings of genes that improve resistance to grazing are helpful for the restoration, conservation, and management of desert steppe.

Transferability, development of simple sequence repeat (SSR) markers and application to the analysis of genetic diversity and population structure of the African fan palm (Borassus aethiopum Mart.) in Benin

BackgroundIn Sub-Saharan Africa, Borassus aethiopum Mart. (African fan palm) is an important non-timber forest product-providing palm that faces multiple anthropogenic threats to its genetic diversity. However, this species is so far under-studied, which prevents its sustainable development as a resource. The present work is a first attempt at characterizing the genetic diversity and population structure of B. aethiopum across nine collection sites spanning the three climatic regions of Benin, West Africa, through the use of microsatellite markers.ResultsDuring a first phase we relied on the reported transferability of primers developed in other palm species. We find that, in disagreement with previously published results, only 22.5% of the markers tested enable amplification of B. aethiopum DNA and polymorphism detection is very low.In a second phase, we generated a B. aethiopum-specific genomic dataset through high-throughput sequencing and used it for the de novo detection of microsatellite loci. Among the primer pairs targeting these, 11 detected polymorphisms and were further used for analyzing genetic diversity. Across the nine sites, expected heterozygosity (He) ranges from 0.263 to 0.451 with an overall average of 0.354, showing a low genetic diversity. Analysis of molecular variance (AMOVA) shows that within-site variation accounts for 53% of the genetic variation. Accordingly, the low number of migrants and positive values of the fixation index (F) in sites from both the Central (Sudano-Guinean) and the Southern (Guinean) climatic regions suggest limited gene flow between sites. The global correlation between genetic and geographic distances is weak; however, our clustering analyses indicate that B. aethiopum palms from Savè (Center) are genetically more similar to those from the North than to samples from other Central sites.ConclusionsIn the light of our results, we discuss the use of inter-species transfer vs. de novo development of microsatellite markers in genetic diversity analyses targeting under-studied species, and suggest future applications for our molecular resources. We propose that, while prominent short-range pollen and seed dispersal in Benin explain most of our results, gene flux between the Central and Northern regions, as a result of animal and/or human migrations, might underlie the Savè discrepancy.

Transcriptome wide SSR discovery cross-taxa transferability and development of marker database for studying genetic diversity population structure of Lilium species

Lily belongs to family liliaceae, which mainly propagates vegetatively. Therefore, sufficient number of polymorphic, informative, and functional molecular markers are essential for studying a wide range of genetic parameters in Lilium species. We attempted to develop, characterize and design SSR (simple sequence repeat) markers using online genetic resources for analyzing genetic diversity and population structure of Lilium species. We found di-nucleotide repeat motif were more frequent (4684) within 0.14 gb (giga bases) transcriptome than other repeats, of which was two times higher than tetra-repeat motifs. Frequency of di-(AG/CT), tri-(AGG/CTT), tetra-(AAAT), penta-(AGAGG), and hexa-(AGAGGG) repeats was 34.9%, 7.0%, 0.4%, 0.3%, and 0.2%, respectively. A total of 3607 non-redundant SSR primer pairs was designed based on the sequences of CDS, 5′-UTR and 3′-UTR region covering 34%, 14%, 23%, respectively. Among them, a sub set of primers (245 SSR) was validated using polymerase chain reaction (PCR) amplification, of which 167 primers gave expected PCR amplicon and 101 primers showed polymorphism. Each locus contained 2 to 12 alleles on average 0.82 PIC (polymorphic information content) value. A total of 87 lily accessions was subjected to genetic diversity analysis using polymorphic SSRs and found to separate into seven groups with 0.73 to 0.79 heterozygosity. Our data on large scale SSR based genetic diversity and population structure analysis may help to accelerate the breeding programs of lily through utilizing different genomes, understanding genetics and characterizing germplasm with efficient manner.

Development of nuclear microsatellite markers to facilitate germplasm conservation and population genetics studies of five groups of tropical perennial plants with edible fruits and shoots: rambutan (Nephelium lappaceum L.), sapodilla (Manilkara zapota (L.) P. Royen), lychee (Litchi chinensis Sonn.), mangosteen (Garcinia mangostana Linn. and Garcinia cochinchinensis (Lour.) Choisy) and bamboo (Bambusa vulgaris Schrad. ex J.C. Wendl and Guadua angustifolia Kunth)

Simple sequence repeat (SSR) enriched libraries for five groups of tropical perennial plants with edible fruits and shoots were prepared and sequenced in a GS-FLX Roche 454: sapodilla (Manilkara zapota (L.) P. Royen), lychee (Litchi chinensis Sonn.), mangosteen (Garcinia mangostana Linn. and G. cochinchinensis (Lour.) Choisy), rambutan (Nephelium lappaceum L.), and bamboo (Bambusa vulgaris Schrad. ex J.C. Wendl and Guadua angustifolia Kunth). For SSR development, these species were organized by their common names in five groups. A total of 3870 SSR primer sets were designed, using capillary electrophoresis 1872 nuclear SSRs were tested on 4 to 10 DNA samples within each plant group, that is 384 loci for each of the four groups of fruit trees and 336 loci for the bamboo group. Only 7.9% of the primers tested did not result in amplification. All 1872 SSRs are provided, we highlight 178 SSRs (between 26 and 47 per group) considered top-quality polymorphic SSRs that amplified all the samples, had strong fluorescence signal, presented no stutters and showed minimum non-specific amplification or background fluorescence. A total of 66,057 contig sequences were submitted to GenBank Database. Markers presented here will be useful not only for conservation efforts in banks of germplasm, but also for in-depth analysis of population genetics which usually requires evaluation of large number of loci.

Development of nuclear SSR and chloroplast genome markers in diverse Liriodendron chinense germplasm based on low-coverage whole genome sequencing

BackgroundLiriodendron chinense ranges widely in subtropical China and northern Vietnam; however, it inhabits several small, isolated populations and is now an endangered species due to its limited seed production. The objective of this study was to develop a set of nuclear SSR (simple sequence repeats) and multiple chloroplast genome markers for genetic studies in L. chinense and their characterization in diverse germplasm.ResultsWe performed low-coverage whole genome sequencing of the L. chinense from four genotypes, assembled the chloroplast genome and identified nuclear SSR loci by searching in contigs for SSR motifs. Comparative analysis of the four chloroplast genomes of L. chinense revealed 45 SNPs, 17 indels, 49 polymorphic SSR loci, and five small inversions. Most chloroplast intraspecific polymorphisms were located in the interspaces of single-copy regions. In total, 6147 SSR markers were isolated from low-coverage whole genome sequences. The most common SSR motifs were dinucleotide (70.09%), followed by trinucleotide motifs (23.10%). The motif AG/TC (33.51%) was the most abundant, followed by TC/AG (25.53%). A set of 13 SSR primer combinations were tested for amplification and their ability to detect polymorphisms in a set of 109 L. chinense individuals, representing distinct varieties or germplasm. The number of alleles per locus ranged from 8 to 28 with an average of 21 alleles. The expected heterozygosity (He) varied from 0.19 to 0.93 and the observed heterozygosity (Ho) ranged from 0.11 to 0.79.ConclusionsThe genetic resources characterized and tested in this study provide a valuable tool to detect polymorphisms in L. chinense for future genetic studies and breeding programs.

Genome-wide development of simple sequence repeats database for flax (Linum usitatissimum L.) and its use for genetic diversity assessment

Flax (Linum usitatissimum L.) is an important industrial crop and is widely cultivated in North America, Europe, and China for its fiber and oil. Identifying flax molecular markers is a vital step towards improving flax yield and quality via marker-assisted breeding. Simple sequence repeat (SSR) markers have proven to be the most valuable type of genetic marker for molecular breeding and genetic diversity studies. However, the development of genome-wide SSRs in flax has not yet been reported, which limits their use in molecular breeding. In this study, we screened 25,871 SSR motifs from L. usitatissimum, mainly consisting of trinucleotide (52.8%) and dinucleotide (36.1%) repeats. Based on the 24,375 SSR motifs, 71,184 primers pairs of SSR markers were designed for the flax genome, covering the whole genome at a density of 225.3/Mb. Furthermore, 96 pairs of SSR markers from different chromosomes were used to assess a panel of 26 flax accessions obtained from different parts of the world. The 21 SSR markers exhibited polymorphisms across the 26 flax accessions, and the flax varieties were accurately clustered into two groups and two subgroups corresponding to their type (wild species or cultivar) and use (fiber or oilseed). This study demonstrates the potential value in analyzing genetic diversity in flax, and the large-scale development of SSR markers developed in this study will facilitate future molecular analyses in flax.

Characterization and Development of Genomic SSRs in Pecan (Carya illinoinensis)

Research Highlights: The distribution of simple sequence repeat (SSR) motifs in two draft genomes of pecan was evaluated. Sixty-six SSR loci were validated by PCR amplification in pecan. Twenty-two new development markers can be used for genetic study in genus Carya. Background and Objectives: Pecan has good nutritional and health benefits and is an important crop worldwide. However, the genetic research in this species is insufficient. One of the main reasons for this is the lack of enough accurate, convenient, and economical molecular markers. Among different marker types, SSR loci are enormously useful in genetic studies. However, the number of SSRs in C. illinoinensis (Wangenh.) K. Koch is limited. Materials and Methods: The distribution of SSR motifs in the pecan genome was analyzed. Then, the primers for each SSR were designed. To evaluate their availability, 74 SSR loci were randomly selected and amplified in pecan. Finally, 22 new SSRs and eight former ones were picked to evaluate the genetic diversity in 60 pecan genotypes and to determine their transferability in other Carya species. Results: 145,714 and 143,041 SSR motifs were obtained from two draft genomes of ‘87MX3-2’ and ‘Pawnee’, respectively. In total, 9145 candidate primers were obtained. Sixty-six (89.19%) primers amplified the target products. Among the 30 SSRs, 29 loci showed polymorphism in 60 pecan genotypes. The polymorphic information content (PIC) values ranged from 0.012 to 0.906. In total, 26, 25, and 22 SSRs can be used in C. cathayensis Sarg., C. dabieshanensis W. C. Cheng & R. H. Chang, and C. hunanensis W.C. Liu, respectively. Finally, the dendrogram of all individuals was constructed. The results agree with the geographic origin of the four species and the pedigree relationships between different pecan cultivars. Conclusions: The characterization of SSRs in the pecan genome and the new SSRs will promote the progress of genetic study and breeding in pecan, as well as other species of genus Carya.

Development of simple sequence repeats (SSRs) markers for identification of wild species and somatic hybrids of potato

SSR markers were analysed to reveal allelic variations in wild species and interspecific somatic hybrids of potato. SSR allelic profiles showed high polymorphism and distinctness in the samples. A total of 131 alleles of 13 polymorphic SSR loci were scored in 48 samples of wild species and somatic hybrids of potato. Alleles per locus ranged from 6 (STM5127/STM5114/STM1053/STI0030) to 20 (STM1052) whereas PIC value varied between 0.81 (STM5127/STM5114) to 0.94 (STM1052) in the samples. This study suggests that SSR-based genotyping and development SSR markers dataset would strengthen their utilization in identification and molecular characterization of wild species and somatic hybrids of potato.

Substantial enhancement of high polymorphic SSR marker development using in silico method from 18 available rice blast fungus genome sequences and its application in genetic diversity assessment

Rice blast fungus, Magnaporthe oryzae, causes rice blast disease leading to the reduction of rice yield. Utilization of rice blast resistant cultivar is one of efficient ways to cope with this disease. The genetic diversity of rice blast is of great importance as it indicates the genotype of rice blast isolates in a population of epidemic area and reveals the dynamics of genotype change over time. Simple sequence repeat (SSR) marker is frequently used for indicating the genetic diversity of several organisms including rice blast fungus. The in silico SSR development method provides the enhancement of polymorphic rate and requires less time and labour. This study aimed to develop in silico SSR marker and study genetic diversity of rice blast isolates collected from Thailand. The 11,072 SSR loci were examined in rice blast reference genome 70–15. The most abundant SSR type and pattern were mono-nucleotide repeat and AGC/CTG motif respectively. Fourteen highly polymorphic SSR loci, identified by in silico from 18 rice blast genome sequences, were selected and specific primer were developed. Ten SSR loci were novel and detected the polymorphism in Thai rice blast isolates, which showed 2–7 alleles per locus and PIC values ranged from 0.07 to 0.56. The genetic relationship analysis showed that rice blast isolates were clustered by collection time and site. Our result indicated that in silico SSR development is a powerful technique to enhance the production of highly polymorphic SSR markers and these newly developed SSR markers were useful for genetic diversity assessment.

An efficient strategy for developing genotype identification markers based on simple sequence repeats in grapevine

Microsatellites are valuable resources for breeding and genetic studies of crop species. The development of simple sequence repeat (SSR) markers, which are applied across closely related cultivars, has been slow, labor-intensive, and expensive, with a low success rate. In this study, we demonstrate an efficient strategy for developing cross-species, amplifiable polymorphic SSR markers from grapevine (Vitis vinifera) based on sequence comparison and in silico polymerase chain reaction amplification of the genome sequences of grape cultivars. A total of 21 primer sets targeting unique SSR loci were screened from polymorphism and heterozygosity analyses of 13 grape cultivars and four wild species. The number of alleles varied from 3 to 12, with a mean of 7.52 alleles per locus. The lowest and highest observed heterozygosity values were 0.24 and 0.82, respectively, with an average of 0.54. Polymorphic information content values for the primer sets ranged from 0.32 to 0.86, with an average of 0.70. We successfully identified genetic relationships among 17 grape genotypes using the unweighted pair group method with arithmetic average and principal coordinates analysis of the SSR markers. The results suggest that the strategy developed in this study is applicable to high-throughput cross-amplifiable SSR marker development for the identification of genotype, genetic study, and examination of genetic diversity in grapevines.

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

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.