Simple Sequence Repeats Repeat Research Articles

MegaSSR: a web server for large scale microsatellite identification, classification, and marker development.

Next-generation sequencing technologies have opened new avenues for using genomic data to study and develop molecular markers and improve genetic resources. Simple Sequence Repeats (SSRs) as genetic markers are increasingly used in molecular diversity and molecular breeding programs that require bioinformatics pipelines to analyze the large amounts of data. Therefore, there is an ongoing need for online tools that provide computational resources with minimal effort and maximum efficiency, including automated development of SSR markers. These tools should be flexible, customizable, and able to handle the ever-increasing amount of genomic data. Here we introduce MegaSSR (https://bioinformatics.um6p.ma/MegaSSR), a web server and a standalone pipeline that enables the design of SSR markers in any target genome. MegaSSR allows users to design targeted PCR-based primers for their selected SSR repeats and includes multiple tools that initiate computational pipelines for SSR mining, classification, comparisons, PCR primer design, in silico PCR validation, and statistical visualization. MegaSSR results can be accessed, searched, downloaded, and visualized with user-friendly web-based tools. These tools provide graphs and tables showing various aspects of SSR markers and corresponding PCR primers. MegaSSR will accelerate ongoing research in plant species and assist breeding programs in their efforts to improve current genomic resources.

Development of polymorphic simple sequence repeat markers in Agastache rugosa and their application in genetic evaluation and cross-taxon transferability of Agastache species

Agastache is a genus of perennial herbaceous plants belonging to the mint family, Lamiaceae. Several Agastache species are commercially cultivated and used as medicinal, culinary, and ornamental plants. However, information on the genetic diversity and population structure of the species remains unclear. In the present study, genetic diversity within Agastache species was analyzed using simple sequence repeat (SSR) markers. In this study, 249,746 SSRs were identified in the A. rugosa genome and primer pairs were designed for 56,675 SSRs. The majority of SSR repeat types were dinucleotides (60.65%), followed by trinucleotides (12.38%), and pentanucleotides (12.10%). PCR conditions were established for 250 primer pairs, 111 of which were found to be polymorphic in A. rugosa germplasm. The number of alleles (NA) ranged from 2 to 19, major allele frequency (MAF) ranged from 0.11 to 0.95, observed heterozygosity (HO) ranged from 0 to 0.89, and polymorphic information content (PIC) ranged from 0.09 to 0.92. Cross-species amplification of SSRs markers in other Agastache species showed amplification rates of 82.6% for A. foeniculum and 78.1% in A. urticifolia, with an average of 80.37%. Cluster analysis of the 19 A. rugosa accessions using SSRs markers revealed four major clusters, and population STRUCTURE analysis using 79 SSRs markers revealed three groups and three subgroups among the A. rugosa populations. The SSRs markers developed can contribute to applications such as varietal identification, genetic diversity analysis, and population structure analysis of A. rugosa germplasm.

Development, Identification and Validation of a Novel SSR Molecular Marker for Heat Resistance of Grapes Based on miRNA

The adverse effects of high temperatures on fruit quality and yield have been reported in many studies of grapevines. MiRNAs play crucial roles in plant growth and development and also fulfill functions in regulating the high-temperature response. In this research, miRNA-based SSR (simple sequence repeat) polymorphisms were analyzed according to the grape genome sequence and then combined with polymerase chain reaction (PCR) amplification and Sanger sequencing methods to analyze the miRNA-SSR diversity of different heat-resistant grape varieties. A statistical analysis of SSR sequences showed that 391 and 12 SSRs were specific to pri-miRNA and pre-miRNA, respectively. In pri-miRNA containing SSR, hexa-nucleotide repeats were the most abundant (52.69%), followed by tetra-nucleotide (13.04%) and minimum penta-nucleotide (4.09%), which were also observed in pre-miRNA sequences. On the other hand, differences in heat resistance among grape varieties were observed. Based on the results of leaf Fv/Fm images, values and phenotypic changes under high-temperature stress, 20 heat-resistant (e.g., Niagara Rosada and Grand Noir) and 20 heat-sensitive (e.g., Shine Muscat and Jumeigui) grape varieties were identified. Further, PCR-Sanger sequencing was used to screen SSR differences in four thermos-tolerant and four thermos-sensitive grape varieties, and finally, eight SSR differential primers were found to be able to distinguish these varieties. Of these, four pairs of primers were selected for validation in 40 grape germplasm resources (20 thermo-tolerance and 20 thermo-sensitivity). The VMIRSSR167c3 highlights that the ratio of SSR (CT) repeat values greater than 17 in heat-resistant varieties was 90%. In conclusion, the VMIRSSR167c3 marker can accurately distinguish between heat-resistant and heat-sensitive grape varieties. The results provide a novel molecular marker for the genetic improvement of grape germplasm resources and will be beneficial to the breeding of heat-resistant varieties in the future.

Identification and validation of the first EST-SSR markers based on transcriptome of Anopheles darlingi, the primary transmitter of malaria in Brazil.

Anopheles darlingi is a monotypic species in terms of its morphological, genetic, and behavioral aspects and is the primary transmitter of human malaria (99%) in Brazil, especially in the Brazilian Amazon. In this pioneering study, 15 expressed sequence tag (EST)-simple sequence repeat (SSR) markers were obtained and characterized in samples from the municipality of São Gabriel da Cachoeira, Amazonas state, Brazil, with polymorphisms that can be used for further genetic research. The specimens (from egg to larval stage) collected were bred in the insectary at INPA (National Institute for Amazonian Research). The SSR repeats within the contigs of the A. darlingi EST banks were confirmed on the Vector Base site. DNA was extracted and amplified using polymerase chain reaction and then genotyped. Fifteen polymorphic SSR loci were identified and characterized. The number of alleles totaled 76 and ranged from 2 to 9. The observed heterozygosity varied between 0.026 and 0.769, the expected heterozygosity between 0.025 and 0.776, and the mean polymorphism information content was 0.468. Eight loci showed Hardy-Weinberg equilibrium (HWE) after Bonferroni correction (P: (5%) ≤ 0.0033). No linkage disequilibrium was found among the loci. The polymorphic SSRs of the loci have been shown to be efficient for investigation of the variability and genetic population structure of A. darlingi.

Chloroplast genomic characterization and phylogenetic analysis of Castanopsis hystrix

The structure and size of the chloroplast genome of Castanopsis hystrix was determined by Illumina HiSeq 2500 sequencing platform to understand the difference between C. hystrix and the chloroplast genome of the same genus, and the evolutionary position of C. hystrix in the genus, so as to facilitate species identification, genetic diversity analysis and resource conservation of the genus. Bioinformatics analysis was used to perform sequence assembly, annotation and characteristic analysis. R, Python, MISA, CodonW and MEGA 6 bioinformatics software were used to analyze the genome structure and number, codon bias, sequence repeats, simple sequence repeat (SSR) loci and phylogeny. The genome size of C. hystrix chloroplast was 153 754 bp, showing tetrad structure. A total of 130 genes were identified, including 85 coding genes, 37 tRNA genes and 8 rRNA genes. According to codon bias analysis, the average number of effective codons was 55.5, indicating that the codons were highly random and low in bias. Forty-five repeats and 111 SSR loci were detected by SSR and long repeat fragment analysis. Compared with the related species, chloroplast genome sequences were highly conserved, especially the protein coding sequences. Phylogenetic analysis showed that C. hystrix is closely related to the Hainanese cone. In summary, we obtained the basic information and phylogenetic position of the chloroplast genome of red cone, which will provide a preliminary basis for species identification, genetic diversity of natural populations and functional genomics research of C. hystrix.

Occurrence of simple sequence repeats in cDNA sequences of safflower (Carthamus tinctorius) reveals the importance of SSR-containing genes for cell biology and dynamic response to environmental cues.

Safflower (Carthamus tinctorius) is a diploid crop plant belonging to the family Asteraceae and is well known as one of important oilseed crops due to edible oil containing unsaturated fatty acids. In recent years it is gaining increased attention for food, pharmaceutical and industrial uses, and hence the updating its breeding methods is necessary. Genic simple sequence repeats (SSRs) in addition of being desire molecular markers, are supposed to influence gene function and the respective phenotype. This study aimed to identify SSRs in cDNA sequences and further analysis of the functional features of the SSR-containing genes to elucidate their role in biological and cellular processes. We identified 1,841 SSR regions in 1,667 cDNA sequences. Among all types of repeats, trinucleotide repeats were the most abundant (35.7%), followed by hexanucleotide (29.6%) and dinucleotide repeats (22.0%). Thirty five SSR primer pairs were validated by PCR reaction, detected a high rate of polymorphism (>57%) among safflower accessions, physically mapped on safflower genome and could clearly discriminate the cultivated accessions from wild relatives. The cDNA-derived SSR markers are suitable for evaluation of genetic diversity, linkage and association mapping studies and genome-based breeding programmes. Occurrence of SSR repeats in biologically-important classes of proteins such as kinases, transferases and transcription factors was inferred from functional analyses, which along with variability of their repeat copies, can endow the cell and whole organism the flexibility of facing with continuously changing environment, and indicate a structure-based evolution mechanism of the genome which acts as an up-to-dating tool for the cell and whole origanism, which is realized in GO terms such as involvement of most SSR-containing genes in biological, cellular and metabolic processes, especially in response to stimulus, response to stress, interaction to other organisms and defense responses.

Evaluation of Perfect Microsatellites in Nile Tilapia (Oreochromis niloticus) Genome

Microsatellites or simple sequence repeats (SSRs) consist of a sizable part of genomes and play a crucial role in the function of genes and the organization of the genome. The complete availability of a genome sequence for Nile tilapia (Oreochromis niloticus) provides the possibility of accomplishing a genome-wide analysis of SSRs in this species. I analyzed the abundance and density of perfect SSRs in the Nile tilapia genome and observed a sum of 252,047 microsatellites with 1–6 bp nucleotide motifs. This indicates that about 2.7 % of the Nile tilapia whole genome sequence (927.77Mb) is made up of perfect SSRs, with an average length of 135.68bp/Mb. The average density and frequency of perfect SSRs were 271.69 loci/Mb and 5834.46 bp/Mb, respectively. The six classes of perfect SSRs proportional distribution within the Nile tilapia genome were not even. Dinucleotide repeats (40.13 %) with a total count of 101145 of an average length of 26.11 bp happen to be the most abundant class of SSRs, while the percentages of mononucleotide, trinucleotide, tetranucleotide, pentanucleotide, and hexanucleotide repeats was 31.88 %, 11.98 %, 11.52 %, 4.22 %, and 0.26 %, accordingly. The various classes of SSRs repeat differ in their number of repeats with the highest being 95. My results indicate that 21 motifs contain the prevalent categories with a frequency above 1 locus/Mb: A, AAC, AAG, AATAG, AATTC, AC, AG, AGAT, AT, ATCT, ATG, ATGG, ATT, ATTT, C, CCT, CTG, CTTT, GT, GTTT.

Genome-Wide Analysis and Polymorphism Evaluation of Microsatellites Involved in Photoperiodic Flowering-time Genes in Kenaf (Hibiscus Cannabinus L.)

ABSTRACT Kenaf (Hibiscus cannabinus L.) is one of the most important crops, which is grown for natural fiber production. However, the deficiency of SSR markers limits the genetic improvement of kenaf. In this study, SSRs (simple sequence repeats) were identified from the reference genome and coding sequence (CDS) of Fuhong952, a sequenced variety of kenaf. SSR loci were mined using the Seiners 2.0 software and the characteristics of SSR loci were analyzed. A total of 301, 505 and 14, 269 SSRs with an overall density of 279.70 SSRs/Mb and 182.70 SSRs/Mb were identified from whole-genome and CDS, respectively. 12bp was the predominant repeat in the entire genome and CDS, trinucleotide repeats were the most abundant among both types of sequences. For different SSR repeat types, the SSR frequency decreased dramatically as repeat times increased. AT was the most frequent single motif across the entire genome, while AG was the most abundant motif in CDS. The homologous genes in kenaf were obtained from the 71 genes associated with photoperiodic flowering-time. As such, 115 primer pairs were designed and synthesized using these homologous genes in kenaf. The amplification of 108 pairs revealed stable and clear bands, and two pair of primers located in HcFCA and HcVIN3 were significantly associated with photoperiodic flowering-time using one-way ANOVA and regression analysis. The detailed results of the SSR markers in this study provides valuable information about the frequency distribution of SSRs in the whole genome and will expedite genomic research for improvement in kenaf.

Development of novel genome‑wide simple sequence repeats (SSR) markers in Bunium persicum

Bunium persicum (Boiss.) Fedtsch or Kala jeera is an economically important medicinal spice that grows in cold temperate regions of Central Asia and Northern India. It is mainly used as spice and condiment in various food preparations. However, its high aroma and richness in essential oils make it industrially important. The bioactivity and pharmacological activities of its phytochemicals have been well elucidated. Due to limited cultivation and huge harvest pressure on natural resources, this species is under severe threat of diversity loss. Further, genetics and molecular breeding studies are lagging for this important crop plant due to a dearth of efficient genome wide molecular markers. In the present study, genomic resources for B. persicum have been generated for the first time through de novo sequencing. In addition, novel genomic SSRs from these resources are identified for their use in future diversity analysis and breeding programs. A total of 177029 perfect and 5915 compound SSR motifs were identified in 212585 assembled sequences. Mononucleotide repeats were the most abundant (55.18%) followed by di- (32.35%), tri- (7.21%), tetra- (3.15%) and hexa- nucleotide (0.76%) repeats. Total 101519 and 2649 primers were successfully designed for sequences containing perfect and compound SSR repeat motifs, respectively. For validation, 88 primer pairs were synthesized for perfect SSR motifs, from which 68 pairs gave scorable amplification. From these, 16 primer pairs were found monomorphic and 52 were polymorphic. These 68 primer pairs altogether yielded 101 alleles with an average of 1.71 alleles per loci. Overall average allele frequency (0.7908), PIC value (0.213), heterozygosity value (0.2995), and gene diversity (0.2680) was recorded for the evaluated Bunium accessions. UPGMA tree constructed based on Nei genetic distances successfully distinguished the evaluated B. persicum accessions. Low genetic diversity recorded in the present study emphasizes the need for restoration and maintenance of effective B. persicum population sizes in situ for its future sustenance.

Development of Genome-wide Simple Sequence Repeat Markers from Whole-genome Sequence of Mungbean (Vigna radiata)

Background: Mungbean is an important pulse crop and it is mainly cultivated in Asia for human consumption. Its small genome and diploid nature make it a well-suited model organism among legume crops. Thus, cost-effective, reliable and highly polymorphic molecular markers distributing the whole genome are needed for diversity, mapping and functional genomics studies in this model species. Methods: The whole-genome sequence of mungbean was obtained and used as a source of identification of simple sequence repeats (SSR). The sequence reads were aligned and SSRs detection was performed using the Phobos plugin tandem repeat finder in the Geneious software program. A total of 12 mungbean genotypes were selected to validate the newly developed SSR markers. Result: In the present study, a total of 12, 49,774 and 11, 86, 386 perfect and imperfect SSR repeats were identified from the mungbean genome. The tri-repeats were the most abundant (26.10%), followed by hexa (20.82%), penta (20.45%), tetra (17.65%) and di-repeats (14.95%). We designed 1330 SSR primers based on the genomic sequence of flanking perfect SSRs (Di and tri-repeats). Among them, 50 SSR primers uniformly distributed across the 11 mungbean chromosomes were selected and used to validate 12 mungbean genotypes. The newly developed genomic SSR markers generated in the present study are a valuable genomic resource for the mungbean breeding programs.

Genome-wide in silico identification and characterization of Simple Sequence Repeats in diverse completed SARS-CoV-2 genomes

Simple sequence repeats (SSRs) or, Microsatellites are short repeat sequences that have been extensively studied in eukaryotic (plants) and prokaryotic (bacteria) organisms. Compared to other organisms, the presence and incidence of SSR on viral genomes are less studied. With the emergence of novel infectious viruses over the past few decades, it is imperative to study the genetic diversity in such viruses to predict their evolutionary and functional changes over time. Following the emergence of SARS-CoV-2, we have assembled 121 complete genomes reported from 31 countries across the six continents for the identification and characterization of SSR repeats. Using two independent SSR identification tools, we have found remarkable consistency in the diversity of microsatellites pattern (38–42 per genome) found in the 121 analyzed SARS-CoV-2 genomes indication their important role for genome stability. Among the identified motifs, trinucleotide and hexanucleotide repeats were found to be the most abundant form followed by mono- and di-nucleotide. There were no tetra- or penta-nucleotide repeats in the analyzed SARS-CoV-2 genomes. The discovery of microsatellites in SARS-CoV-2 genomes may become useful for the population genetics, evolutionary analysis, strain identification and genetic variation.

Fungus-specific SSR markers in the Antarctic lichens Usnea antarctica and U. aurantiacoatra (Parmeliaceae, Ascomycota).

Premise of the study:Usnea antarctica and U. aurantiacoatra (Parmeliaceae) are common lichens in the maritime Antarctic. These species share the same habitats on King George Island (South Shetland Islands, Antarctica) and are distinguishable based on reproductive strategies.Methods and Results:We developed 23 fungus-specific simple sequence repeat (SSR) markers that cross-amplify between the two species. We used a low-coverage genome-skimming approach on one sample of each species to identify SSR repeats in the two species. Primers were designed for 3–4-bp repeats, and only the loci common to both species were selected for further analyses. Seventy-seven samples of the two species were selected to assess fungal specificity, genetic variability, and linkage of the markers. In addition, we tested cross-amplification in other Usnea species.Conclusions:The 23 newly designed SSR markers are suitable for population genetic and phylogeographic studies of Usnea species.

Genome-Wide Discovery of Microsatellite Markers from Diploid Progenitor Species, Arachis duranensis and A. ipaensis, and Their Application in Cultivated Peanut (A. hypogaea).

Despite several efforts in the last decade toward development of simple sequence repeat (SSR) markers in peanut, there is still a need for more markers for conducting different genetic and breeding studies. With the effort of the International Peanut Genome Initiative, the availability of reference genome for both the diploid progenitors of cultivated peanut allowed us to identify 135,529 and 199,957 SSRs from the A (Arachis duranensis) and B genomes (Arachis ipaensis), respectively. Genome sequence analysis showed uneven distribution of the SSR motifs across genomes with variation in parameters such as SSR type, repeat number, and SSR length. Using the flanking sequences of identified SSRs, primers were designed for 51,354 and 60,893 SSRs with densities of 49 and 45 SSRs per Mb in A. duranensis and A. ipaensis, respectively. In silico PCR analysis of these SSR markers showed high transferability between wild and cultivated Arachis species. Two physical maps were developed for the A genome and the B genome using these SSR markers, and two reported disease resistance quantitative trait loci (QTLs), qF2TSWV5 for tomato spotted wilt virus (TSWV) and qF2LS6 for leaf spot (LS), were mapped in the 8.135 Mb region of chromosome A04 of A. duranensis. From this genomic region, 719 novel SSR markers were developed, which provide the possibility for fine mapping of these QTLs. In addition, this region also harbors 652 genes and 49 of these are defense related genes, including two NB-ARC genes, three LRR receptor-like genes and three WRKY transcription factors. These disease resistance related genes could contribute to resistance to viral (such as TSWV) and fungal (such as LS) diseases in peanut. In summary, this study not only provides a large number of molecular markers for potential use in peanut genetic map development and QTL mapping but also for map-based gene cloning and molecular breeding.

Development of genomic simple sequence repeat markers in faba bean by next-generation sequencing

Faba bean (Vicia faba L.) is an important food legume crop with a huge genome. Development of genetic markers for faba bean is important to study diversity and for molecular breeding. In this study, we used Next Generation Sequencing (NGS) technology for the development of genomic simple sequence repeat (SSR) markers. A total of 14,027,500 sequence reads were obtained comprising 4,208 Mb. From these reads, 56,063 contigs were assembled (16,367 Mb) and 2138 SSRs were identified. Mono and dinucleotides were the most abundant, accounting for 57.5 % and 20.9 % of all SSR repeats, respectively. A total of 430 primer pairs were designed from contigs larger than 350 nucleotides and 50 primers pairs were tested for validation of SSR locus amplification. Nearly all (96 %) of the markers were found to produce clear amplicons and to be reproducible. Thirty-nine SSR markers were then applied to 46 faba bean accessions from worldwide origins, resulting in 161 alleles with 87.5 % polymorphism, and an average of 4.1 alleles per marker. Gene diversity (GD) of the markers ranged from 0 to 0.48 with an average of 0.27. Testing of the markers showed that they were useful in determining genetic relationships and population structure in faba bean accessions.

Cytomolecular discrimination of the Am chromosomes of Triticum monococcum and the A chromosomes of Triticum aestivum using microsatellite DNA repeats.

The cytomolecular discrimination of the Am- and A-genome chromosomes facilitates the selection of wheat-Triticum monococcum introgression lines. Fluorescence in situ hybridisation (FISH) with the commonly used DNA probes Afa family, 18S rDNA and pSc119.2 showed that the more complex hybridisation pattern obtained in T. monococcum relative to bread wheat made it possible to differentiate the Am and A chromosomes within homoeologous groups 1, 4 and 5. In order to provide additional chromosomal landmarks to discriminate the Am and A chromosomes, the microsatellite repeats (GAA)n, (CAG)n, (CAC)n, (AAC)n, (AGG)n and (ACT)n were tested as FISH probes. These showed that T. monococcum chromosomes have fewer, generally weaker, simple sequence repeat (SSR) signals than the A-genome chromosomes of hexaploid wheat. A differential hybridisation pattern was observed on 6Am and 6A chromosomes with all the SSR probes tested except for the (ACT)n probe. The 2Am and 2A chromosomes were differentiated by the signals given by the (GAA)n, (CAG)n and (AAC)n repeats, while only (GAA)n discriminated the chromosomes 3Am and 3A. Chromosomes 7Am and 7A could be differentiated by the lack of (GAA)n and (AGG)n signals on 7A. As potential landmarks for identifying the Am chromosomes, SSR repeats will facilitate the introgression of T. monococcum chromatin into wheat.

Cytogenetic Diversity of Simple Sequences Repeats in Morphotypes of Brassica rapa ssp. chinensis.

A significant fraction of the nuclear DNA of all eukaryotes is comprised of simple sequence repeats (SSRs). Although these sequences are widely used for studying genetic variation, linkage mapping and evolution, little attention had been paid to the chromosomal distribution and cytogenetic diversity of these sequences. In this paper, we report the distribution characterization of mono-, di-, and tri-nucleotide SSRs in Brassica rapa ssp. chinensis. Fluorescence in situ hybridization was used to characterize the cytogenetic diversity of SSRs among morphotypes of B. rapa ssp. chinensis. The proportion of different SSR motifs varied among morphotypes of B. rapa ssp. chinensis, with tri-nucleotide SSRs being more prevalent in the genome of B. rapa ssp. chinensis. We determined the chromosomal locations of mono-, di-, and tri-nucleotide repeat loci. The results showed that the chromosomal distribution of SSRs in the different morphotypes is non-random and motif-dependent, and allowed us to characterize the relative variability in terms of SSR numbers and similar chromosomal distributions in centromeric/peri-centromeric heterochromatin. The differences between SSR repeats with respect to abundance and distribution indicate that SSRs are a driving force in the genomic evolution of B. rapa species. Our results provide a comprehensive view of the SSR sequence distribution and evolution for comparison among morphotypes B. rapa ssp. chinensis.

Genome-wide identification and validation of simple sequence repeats (SSRs) from Asparagus officinalis

Garden asparagus (Asparagus officinalis), an important vegetable cultivated worldwide, can also serve as a model dioecious plant species in the study of sex determination and sex chromosome evolution. However, limited DNA marker resources have been developed and used for this species. To expand these resources, we examined the DNA sequences for simple sequence repeats (SSRs) in 163,406 scaffolds representing approximately 400 Mbp of the A. officinalis genome. A total of 87,576 SSRs were identified in 59,565 scaffolds. The most abundant SSR repeats were trinucleotide and tetranucleotide, accounting for 29.2 and 29.1% of the total SSRs, respectively, followed by di-, penta-, hexa-, hepta-, and octanucleotides. The AG motif was most common among dinucleotides and was also the most frequent motif in the entire A. officinalis genome, representing 14.7% of all SSRs. A total of 41,917 SSR primers pairs were designed to amplify SSRs. Twenty-two genomic SSR markers were tested in 39 asparagus accessions belonging to ten cultivars and one accession of Asparagus setaceus for determination of genetic diversity. The intra-species polymorphism information content (PIC) values of the 22 genomic SSR markers were intermediate, with an average of 0.41. The genetic diversity between the ten A. officinalis cultivars was low, and the UPGMA dendrogram was largely unrelated to cultivars. It is here suggested that the sex of individuals is an important factor influencing the clustering results. The information reported here provides new information about the organization of the microsatellites in A. officinalis genome and lays a foundation for further genetic studies and breeding applications of A. officinalis and related species.

RNA sequencing, de novo assembly, and functional annotation of an endangered Nymphalid butterfly, Fabriciana nerippe Felder, 1862

AbstractGrassland butterflies are considered representative indicators of biodiversity and ecosystem health. Their dramatic decline caused by habitat destruction, intensifying agriculture and global warming has prompted concern for conservation. While ecological indices of butterflies have been documented, there is a lack of genomic resources relative to the biological importance of this group. Here, we report, first whole‐transcriptomic resource for Fabriciana nerippe, a brush‐footed butterfly member that is endangered in Korea. Approximately, 241.3 million clean reads were obtained from paired‐end Illumina sequencing of adult whole‐body RNA. The de novo assembly resulted in 114 405 unigenes with length ranging from 133 to 33 218 bp. We found 41 868 assembled unigenes homologous to sequences in locally curated PANM‐DB (Protostome DB). We assigned gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) orthology terms to 18 085 and 5779 unigenes, respectively. InterProScan predicted 6691 protein domains for the assembled unigene sequences, with the reverse transcriptase and zinc finger domains found to be the most predominant. A total of 315 282 simple sequence repeats (SSRs) were identified from the assembled unigenes, when considering dinucleotide to octanucleotide repeats with a minimum of three repeat units. AT/AT, AAT/ATT, and AAAT/ATTT represented the most prominent SSR repeat types in the transcriptome. The unigene annotation profile and microsatellites generated in the study can serve as a reference resource for closely related species and gene functional analysis studies. Our data can be utilized to study ecological drift and loss of the species, consequently leading to better protection of the population.

Molecular basis of off-type microsatellite markers in papaya

Papaya (Carica papaya L.) is a major fruit crop in the tropics and is a model system for studying sex chromosome evolution in plants. From a simple sequence repeat (SSR) mapping project in papaya, 37 SSRs displayed non-parental alleles in the F2 population, known as off-type markers. They all belong to Class I (≥20 bp, hypervariable markers). To investigate the molecular basis of these non-parental alleles, seven SSRs with off-type alleles were cloned and sequenced from two parents and selected F2 individuals. SSR repeat motif variation was the major cause for the generation of off-type alleles and single nucleotide polymorphisms (SNPs) and Insertion/deletion also contributed to off-type polymorphism. Off-type SSRs arose after one meiosis at 3 % frequency. Two of the seven markers had no polymorphisms between the parents but non-parental alleles segregated among the F2 individuals. Four of the seven markers exhibited SNPs between parents with transition and transversion at 2:1 ratio. Six markers showed additional SNPs between F2 offspring and parents with transition and transversion at about 1:1 ratio, suggesting higher than expected level of transversion mutations at the F2 population after one meiosis event between the parental genomes. These off-type SSRs were analyzed in a second F2 population and 43 % of them were conserved. The major cause of off-type SSRs in papaya is the variation of repeat length in Type I SSRs, and the underlining mechanism might be DNA replication slippage of repeat motifs. The presence and high conservation of these off-type markers demonstrated that hypervariable class I SSRs could generate genetic variation in one generation and increase genetic diversity for natural selection to enhance fitness.

Evolution Analysis of Simple Sequence Repeats in Plant Genome.

Simple sequence repeats (SSRs) are widespread units on genome sequences, and play many important roles in plants. In order to reveal the evolution of plant genomes, we investigated the evolutionary regularities of SSRs during the evolution of plant species and the plant kingdom by analysis of twelve sequenced plant genome sequences. First, in the twelve studied plant genomes, the main SSRs were those which contain repeats of 1–3 nucleotides combination. Second, in mononucleotide SSRs, the A/T percentage gradually increased along with the evolution of plants (except for P. patens). With the increase of SSRs repeat number the percentage of A/T in C. reinhardtii had no significant change, while the percentage of A/T in terrestrial plants species gradually declined. Third, in dinucleotide SSRs, the percentage of AT/TA increased along with the evolution of plant kingdom and the repeat number increased in terrestrial plants species. This trend was more obvious in dicotyledon than monocotyledon. The percentage of CG/GC showed the opposite pattern to the AT/TA. Forth, in trinucleotide SSRs, the percentages of combinations including two or three A/T were in a rising trend along with the evolution of plant kingdom; meanwhile with the increase of SSRs repeat number in plants species, different species chose different combinations as dominant SSRs. SSRs in C. reinhardtii, P. patens, Z. mays and A. thaliana showed their specific patterns related to evolutionary position or specific changes of genome sequences. The results showed that, SSRs not only had the general pattern in the evolution of plant kingdom, but also were associated with the evolution of the specific genome sequence. The study of the evolutionary regularities of SSRs provided new insights for the analysis of the plant genome evolution.