Abundance Of Simple Sequence Repeats Research Articles

The complete chloroplast genome sequences of three Pedicularis species (Orobanchaceae).

Pedicularis L., a generally bothersome genus of hemiparasitic plants, is primarily native to southwestern China. The phylogenetic relationship and evolutionary history of this genus have not yet been fully resolved. In this study, we sequenced and assembled chloroplast genomes of three Pedicularis species, P. chinensis, P. melampyriflora, and P. striata using high-throughput Illumina sequencing. The assembled plastomes were 142,059 bp (P. chinensis) to 152,146 bp (P. striata) in size, containing 110 (P. chinensis) to 117 (P. striata) genes. Moreover, we identified 13-15 pseudogenes within the three plastomes, nine of which were pseudogenized in all three species. The three plastomes exhibited a similar codon usage pattern. Moreover, the plastomes contained abundant simple sequence repeats and long repeats, which showed slight variations between the three species. A maximum likelihood analysis was performed to elucidate the phylogenetic positions of the three species within the Pedicularis genus. The plastomes presented in our study can be used as valuable genomic resources for further genetic and genomic studies of the Pedicularis genus.

Chloroplast Genomes of Genus Tilia: Comparative Genomics and Molecular Evolution.

Tilia is a complex genus in the family Malvaceae that has high ecological and economical values. Owing to the lack of sufficient distinguishable morphological and molecular characteristics, interspecific relationships in this genus are not clear. Chloroplast (cp) genomes are small, meanwhile most angiosperms usually undergo matrilineal inheritance. Consequently, they can be used in molecular systematics and phylogenetic analyses. Here, we sequenced and assembled cp genomes from T. endochrysea, T. tomentosa, T. miqueliana, T. americana and T. cordata, and compared them with those of seven previously reported Tilia species. Similar gene contents, gene orders and GC contents existed among the 12 cp genomes, which ranged from 162,564 to 162,855 bp and encoded 113 unique genes. Abundant simple sequence repeats (119–127) and dispersed repeats (97–135) were detected in Tilia cp genomes. In total, 11 hypervariable regions were identified that could be suitable for species identification and phylogenetic studies. A phylogenetic analysis of Malvaceae based on 5 hypervariable genes (matK + ndhF + rpoB + rpoC2+ycf1) revealed that all eight subfamilies were monophyletic groups. Additionally, the genus Tilia was divided into three groups on the basis of all 521 molecular variation loci. The current study provides valuable insights into the genomic evolution of the genus Tilia.

Genome survey sequencing of common vetch (Vicia sativa L.) and genetic diversity analysis of Chinese germplasm with genomic SSR markers

BackgroundCommon vetch (Vicia sativa L.) is an annual legume with excellent suitability in cold and dry regions. Despite its great applied potential, the genomic information regarding common vetch currently remains unavailable.Methods and resultsIn the present study, the whole genome survey of common vetch was performed using the next-generation sequencing (NGS). A total of 79.84 Gbp high quality sequence data were obtained and assembled into 3,754,145 scaffolds with an N50 length of 3556 bp. According to the K-mer analyses, the genome size, heterozygosity rate and GC content of common vetch genome were estimated to be 1568 Mbp, 0.4345 and 35%, respectively. In addition, a total of 76,810 putative simple sequence repeats (SSRs) were identified. Among them, dinucleotide was the most abundant SSR type (44.94%), followed by Tri- (35.82%), Tetra- (13.22%), Penta- (4.47%) and Hexanucleotide (1.54%). Furthermore, a total of 58,175 SSR primer pairs were designed and ten of them were validated in Chinese common vetch. Further analysis showed that Chinese common vetch harbored high genetic diversity and could be clustered into two main subgroups.ConclusionThis is the first report about the genome features of common vetch, and the information will help to design whole genome sequencing strategies. The newly identified SSRs in this study provide basic molecular markers for germplasm characterization, genetic diversity and QTL mapping studies for common vetch.

Characterization and Comparative Analysis of Complete Chloroplast Genomes of Three Species From the Genus Astragalus (Leguminosae).

Astragalus is the largest genus in Leguminosae. Several molecular studies have investigated the potential adulterants of the species within this genus; nonetheless, the evolutionary relationships among these species remain unclear. Herein, we sequenced and annotated the complete chloroplast genomes of three Astragalus species—Astragalus adsurgens, Astragalus mongholicus var. dahuricus, and Astragalus melilotoides using next-generation sequencing technology and plastid genome annotator (PGA) tool. All species belonged to the inverted repeat lacking clade (IRLC) and had similar sequences concerning gene contents and characteristics. Abundant simple sequence repeat (SSR) loci were detected, with single-nucleotide repeats accounting for the highest proportion of SSRs, most of which were A/T homopolymers. Using Astragalus membranaceus var. membranaceus as reference, the divergence was evident in most non-coding regions of the complete chloroplast genomes of these species. Seven genes (atpB, psbD, rpoB, rpoC1, trnV, rrn16, and rrn23) showed high nucleotide variability (Pi), and could be used as DNA barcodes for Astragalus sp. cemA and rpl33 were found undergoing positive selection by the section patterns in the coded protein. Phylogenetic analysis showed that Astragalus is a monophyletic group closely related to the genus Oxytropis within the tribe Galegeae. The newly sequenced chloroplast genomes provide insight into the unresolved evolutionary relationships within Astragalus spp. and are expected to contribute to species identification.

Genome-wide identification of microsatellite and development of polymorphic SSR markers for spotted sea bass (Lateolabrax maculatus)

The spotted sea bass (Lateolabrax maculatus) is a valuable commercial mariculture fish in China and has huge potential for future genetic improvement. However, only limited number of SSR (simple sequence repeat) markers have been developed for L. maculatus. In this study, a total of 220,709 SSRs were identified from the L. maculatus genome. The total length, density and frequency of SSRs were 5,119,809 bp, 9898.28 bp/Mb and 425.06 loci/Mb, respectively. Dinucleotide repeats were predominant among SSRs (85.00 %) and AC was the most abundant SSR motifs. Both the number and length of SSRs in each chromosome were positively associated with the chromosome length. A total of 155,905 SSR markers were successfully designed (with an average of 6496 SSRs per chromosome). The frequency of SSR markers varied from 245.63 per Mb to 346.60 per Mb. 14 polymorphic SSR primers were used to evaluate the genetic diversity of L. maculatus wild population from Zhuhai. A total of 74 alleles were detected and most SSR markers showed high polymorphism information content (PIC). The cross-species transferability of 11 out of 67 tested loci were proven successful in largemouth bass Micropterus salmoides. The genome-wide SSRs and newly developed SSR markers made available in this study will facilitate future genetic breeding and genomic studies of spotted sea bass.

Complete Plastid Genome Sequencing of Eight Species from Hansenia, Haplosphaera and Sinodielsia (Apiaceae): Comparative Analyses and Phylogenetic Implications.

Hansenia Turcz., Haplosphaera Hand.-Mazz. and Sinodielsia H.Wolff are three Apiaceae genera endemic to the Hengduan Mountains and the Himalayas, which usually inhabit elevations greater than 2000 m. The phylogenetic relationships between and within the genera were uncertain, especially the placement of Hap. himalayensis and S. microloba. Therefore, we aimed to conduct comparative (simple sequence repeat (SSR) structure, codon usage bias, nucleotide diversity (Pi) and inverted repeat (IR) boundaries) and phylogenetic analyses of Hansenia, Haplosphaera and Sinodielsia (also compared with Chamaesium and Bupleurum) to reduce uncertainties in intergeneric and interspecific relationships. We newly assembled eight plastid genomes from Hansenia, Haplosphaera and Sinodielsia species, and analyzed them with two plastid genomes from GenBank of Hap. phaea, S. yunnanensis. Phylogenetic analyses used these ten genomes and another 22 plastid genome sequences of Apiaceae. We found that the newly assembled eight genomes ranged from 155,435 bp to 157,797 bp in length and all had a typical quadripartite structure. Fifty-five to 75 SSRs were found in Hansenia, Haplosphaera and Sinodielsia species, and the most abundant SSR was mononucleotide, which accounted for 58.47% of Hansenia, 60.21% of Haplosphaera and 48.01% of Sinodielsia. There was no evident divergence of codon usage frequency between the three genera, where codons ranged from 21,134 to 21,254. The Pi analysis showed that trnE(UUC)-trnT(GGU), trnH(GUG)-psbA and trnE(UUC)-trnT(GGU) spacer regions had the highest Pi values in the plastid genomes of Hansenia (0.01889), Haplosphaera (0.04333) and Sinodielsia (0.01222), respectively. The ndhG-ndhI spacer regions were found in all three genera to have higher diversity values (Pi values: 0.01028–0.2), and thus may provide potential DNA barcodes in phylogenetic analysis. IR boundary analysis showed that the length of rps19 and ycf1 genes entering IRs were usually stable in the same genus. Our phylogenetic tree demonstrated that Hap. himalayensis is sister to Han. weberbaueriana; meanwhile, Haplosphaera and Hansenia are nested together in the East Asia clade, and S. microloba is nested within individuals of S. yunnanensis in the Acronema clade. This study will enrich the complete plastid genome dataset of the Apiaceae genera and has provided a new insight into phylogeny reconstruction using complete plastid genomes of Hansenia, Haplosphaera and Sinodielsia.

Genetic Diversity and Population Structure of Cannabis Based on the Genome-Wide Development of Simple Sequence Repeat Markers.

Cannabis has been used as a source of nutrition, medicine, and fiber. However, lack of genomic simple sequence repeat (SSR) markers had limited the genetic research on Cannabis species. In the present study, 92,409 motifs were identified, and 63,707 complementary SSR primer pairs were developed. The most abundant SSR motifs had six repeat units (36.60%). The most abundant type of motif was dinucleotides (70.90%), followed by trinucleotides, tetranucleotides, and pentanucleotides. We randomly selected 80 pairs of genomic SSR markers, of which 69 (86.25%) were amplified successfully; 59 (73.75%) of these were polymorphic. Genetic diversity and population structure were estimated using the 59 (72 loci) validated polymorphic SSRs and three phenotypic markers. Three hundred ten alleles were identified, and the major allele frequency ranged from 0.26 to 0.85 (average: 0.56), Nei’s genetic diversity ranged from 0.28 to 0.82 (average: 0.56), and the expected heterozygosity ranged from 0.28 to 0.81 (average: 0.56). The polymorphism information content ranged from 0.25 to 0.79 (average: 0.50), the observed number of alleles ranged from 2 to 8 (average: 4.13), and the effective number of alleles ranged from 0.28 to 0.81 (average: 0.5). The Cannabis population did not show mutation-drift equilibrium following analysis via the infinite allele model. A cluster analysis was performed using the unweighted pair group method using arithmetic means based on genetic distances. Population structure analysis was used to divide the germplasms into two subgroups. These results provide guidance for the molecular breeding and further investigation of Cannabis.

Low Base-Substitution Mutation Rate but High Rate of Slippage Mutations in the Sequence Repeat-Rich Genome of Dictyostelium discoideum.

We describe the rate and spectrum of spontaneous mutations for the social amoeba Dictyostelium discoideum, a key model organism in molecular, cellular, evolutionary and developmental biology. Whole-genome sequencing of 37 mutation accumulation lines of D. discoideum after an average of 1,500 cell divisions yields a base-substitution mutation rate of 2.47 × 10−11 per site per generation, substantially lower than that of most eukaryotic and prokaryotic organisms, and of the same order of magnitude as in the ciliates Paramecium tetraurelia and Tetrahymena thermophila. Known for its high genomic AT content and abundance of simple sequence repeats, we observe that base-substitution mutations in D. discoideum are highly A/T biased. This bias likely contributes both to the high genomic AT content and to the formation of simple sequence repeats in the AT-rich genome of Dictyostelium discoideum. In contrast to the situation in other surveyed unicellular eukaryotes, indel rates far exceed the base-substitution mutation rate in this organism with a high proportion of 3n indels, particularly in regions without simple sequence repeats. Like ciliates, D. discoideum has a large effective population size, reducing the power of random genetic drift, magnifying the effect of selection on replication fidelity, in principle allowing D. discoideum to evolve an extremely low base-substitution mutation rate.

Comparison of microsatellite distribution patterns in twenty-nine beetle genomes.

Simple sequence repeats (SSRs) represent an important source of genetic variation that provides a basis for adaptation to different environments in organisms. In this study, we examined the distribution patterns of SSRs in twenty-nine beetle genomes and carried out Gene Ontology (GO) analysis of CDSs embedded with perfect SSRs (P-SSRs). The results demonstrated that imperfect SSRs (I-SSRs) represented the most abundant SSR category in beetle genomes and in different genomic regions (CDS, exon, and intron regions). The numbers of P-SSRs, I-SSRs, compound SSRs, and variable number tandem repeats were positively correlated with beetle genome size, whereas neither the frequency nor the density of the SSRs was correlated with genome size. Moreover, our results demonstrated that common genomic features of P-SSRs within the same suborder or family of Coleoptera were rare. Mono-, di-, tri-, or tetranucleotide SSRs were the most abundant P-SSR categories in beetle genomes. The preferred predominant repeat motif among the mononucleotide P-SSRs was (A)n, but the most frequent repeat motifs for other length classes varied differentially among these genomes. Furthermore, the P-SSR type with the highest GC content differed in the beetle genomes and in different genomic regions. CV (coefficient of variability) analysis demonstrated that the repeat copy numbers of P-SSRs presented relatively higher variation in introns than in CDSs and exons. The GO terms of CDSs containing P-SSRs for molecular functions were mainly enriched in "binding" and "transcription". Our findings will be useful for studying the functional roles of microsatellite heterogeneity in beetle adaptation.

Comparative Analyses of Five Complete Chloroplast Genomes from the Genus Pterocarpus (Fabacaeae).

Pterocarpus is a genus of trees mainly distributed in tropical Asia, Africa, and South America. Some species of Pterocarpus are rosewood tree species, having important economic value for timber, and for some species, medicinal value as well. Up to now, information about this genus with regard to the genomic characteristics of the chloroplasts has been limited. Based on a combination of next-generation sequencing (Illumina Hiseq) and long-read sequencing (PacBio), the whole chloroplast genomes (cp genomes) of five species (rosewoods) in Pterocarpus (Pterocarpus macrocarpus, P. santalinus, P. indicus, P. pedatus, P. marsupium) have been assembled. The cp genomes of five species in Pterocarpus have similar structural characteristics, gene content, and sequence to other flowering plants. The cp genomes have a typical four-part structure, containing 110 unique genes (77 protein coding genes, 4 rRNAs, 29 tRNAs). Through comparative genomic analysis, abundant simple sequence repeat (SSR)loci (333–349) were detected in Pterocarpus, among which A /T single nucleotide repeats accounted for the highest proportion (72.8–76.4%). In the five cp genomes of Pterocarpus, eight hypervariable regions, including trnH-GUG_psbA, trnS-UGA_psbC, accD-psaI, ndhI-exon2_ndhI-exon1, ndhG_ndhi-exon2, rpoC2-exon2, ccsA, and trnfM-CAU, are proposed for use as DNA barcode regions. In the comparison of gene selection pressures (P. santalinus as the reference genome), purifying selection was inferred as the primary mode of selection in maintaining important biological functions. Phylogenetic analysis shows that Pterocarpus is a monophyletic group. The species P. tinctorius is resolved as early diverging in the genus. Pterocarpus was resolved as sister to the genus Tipuana.

Genome-wide distribution of simple sequence repeats in pomegranate and their application to the analysis of genetic diversity

Pomegranate (Punica granatum L.) germplasm collections are increasingly appreciated as a repository for the genetic improvement of species, and their genetic analysis is an essential prerequisite for their utilization in pomegranate breeding. Sufficient markers are a prerequisite for genetic analysis. In this study, we identified 36,792 simple sequence repeat (SSR) markers in the pomegranate genome. Dinucleotide repeats are the most abundant SSRs, and of the mono-, di-, tri-, and tetranucleotide repeats, A/T, AT/AT, AAT/ATT, and AAAT/ATTT were the most dominant motif type. There were only 1.80% SSR motifs located in the coding regions, while most (98.20%) SSR motifs were located in noncoding regions. We confirmed the reliability of 50 SSR markers by PCR amplification, from which 11 highly polymorphic SSR markers were selected to study the genetic diversity of 218 pomegranate accessions. A total of 63 alleles was detected for the 11 SSR primers, ranging from 2 (PG152) to 9 (PG093, PG080) across the pomegranate accessions. The polymorphism information content values of each SSR primer pair ranged from 0.22 to 0.70, with a mean value of 0.45. Based on population structure analysis, these accessions were clustered into three main populations and 42 accessions were chosen as the core set. The availability of these SSR markers and the clustering result for the pomegranate germplasm provide new important information for pomegranate genetics and breeding programs.

Transcription factors and molecular markers revealed asymmetric contributions between allotetraploid Upland cotton and its two diploid ancestors

ABSTRACT Three Gossypium species have been used to breed cotton as they vary in their fiber production and resistance to stresses. Transcription factors (TFs) mostly are present in different copies or isoforms by which they conduct their regulation. Their copy number can determine organism behavior to a cue. Simple sequence repeats (SSRs) are one of the most informative and versatile molecular markers. Transcription factors of three Gossypium species were compared in silico. Seventy eight percent of TFs were common between the three species. Single copy for each species were 6057 TF. Gossypium hirsutum and G. raimondii shared the most common interspecific TF. Gossypium arboreum species-specific TF were the least. MYB TF family with its subfamilies is the most abundant followed by bHLH and AP2/ERF family. Gossypium hirsutum generally possesses more TF copies compared to other two species. The 2109 single-copy clusters indicate that G. hirsutum has received one copy from only one parent. The five most abundant SSR markers of TF were dinucleotides AT, TA, TC, CT and TG belonging to G. raimondii. For G. arboreum and G. hirsutum they were trinucleotides CAA, CGA, TGA, GAA (CAT: G. hirsutum) and TCA. The findings suggest that there is regulatory difference between the three Gossypium species for fiber production and insect attack response. The differences may be due to some adaptive deletion events during speciation of G. hirsutum from its parents G. arboreum and G. raimondii.

Development of SSR Markers Based on Transcriptome Sequences of the Wolf Spider Pardosa pseudoannulata (Araneae: Lycosidae)1

Pardosa pseudoannulata (Boesenberg et Strand, l906) is one of the most common wandering spiders in agricultural fields, and a potential bioindicator for heavy metal contamination. In this study, a high-throughput method was used for discovering the simple sequence repeat (SSR) genetic markers from this spider. In total, 2805 sequences containing a total of 3450 SSRs were identified from the previously constructed transcriptome database, with 516 of the sequences containing more than one SSR. The most abundant SSRs in P. pseudoannulata are di-nucleotide (42.32%) SSRs, followed by mono-nucleotide (41.83%). A/T (41.36%) repeats were the most abundant, followed by AT/AT (28.32%). A total of 6561 pairs of SSR primers were designed using Primer 5.0 software, and a subset of 26 SSR primer pairs was randomly selected and validated by RT-PCR. The SSR markers identified here will be an important resource for measuring genetic diversity and biological monitoring research in P. pseudoannulata.

De novo assembly, transcriptome characterization, and simple sequence repeat marker development in duckweed Lemna gibba.

Lemna gibba is a species of duckweed showing great potential in bioenergy production and wastewater treatment. However, the relevant transcriptomic and genomic resources are very limited for this species, which dramatically hinders its genetic diversity and genome mapping researches. In this work, ~ 233.5 million clean reads were generated from L. gibba by Illumina paired-end sequencing, and subsequently they were de novo assembled into 131,870 unigenes, of which 61,622 were annotated and 43,319 were expressed with Fragments Per Kilobase of transcript per Million fragments mapped (FPKM) > 5. In total, 19,297 simple sequence repeats (SSRs) were identified from 15,261 SSR-containing unigenes. Dinucleotide (78.4%) were the most abundant SSRs, followed by tri- (14.9%), tetra- (4.1%), and penta-nucleotides (1.5%). The top three motifs were AG/CT (69.9%), AC/GT (6.5%), and ATC/ATG (4.9%). Further analysis revealed that the presence of SSR motif was independent of the expression level for a given gene. Based on the sequence of these SSR-containing unigenes, a total of 10,292 SSR markers were developed, of which only 2671 were further retained after removing those derived from unannotated or extra-low expressed (e.g., FPKM ≤ 5) unigenes. Finally, a subset of 70 SSR markers was randomly selected and examined in nine diverse L. gibba genotypes for the PCR amplification and polymorphism, as well as in other duckweed species for the inter-specifically amplifiability. This work is the first report on the transcriptome-based large-scale SSR markers development and analysis in L. gibba. The transcriptome generated and the SSR markers developed in this work will provide a valuable resource for genetic diversity assessment in L. gibba and also for species relationship investigation in Lemnaceae family.

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.

Distribution, Function and Polymorphism Characteristics of Microsatellites in Pyropia yezoensis Transcriptome

The distribution, putative function and polymorphism characteristics of simple sequence repeats (SSRs) in P. yezoensis transcriptome were analyzed in this study. In tota., 307. SSRs were detected among 2681 unigenes. Trinucleotide types were dominant, constituting 91.87% of all the microsatellites. The most abundant SSR was CCG (71.03%) and the second abundant one was AGC (234, 7.61%). A total of 111 (3.61%) dinucleotide types were found and the most abundant one was AC (51, 1.66%) which was followed by CG (34, 1.11%). SSRs identified showed a GC bases preference; GC bases constituted 89.73% of all the SSR bases. SSRs occurrence number decreased as repetitions increased. Annotation results exhibited that the majority of SSRs-containing unigenes have the functions of ‘metabolic process’, ‘binding’ and ‘catalytic activity’ and existed as the forms of ‘cell’, ‘cell component’ and ‘organelle’. The dominant amino acids that SSRs coded were Ala (28.59%), Arg (26.02%), Gly (14.35%) and Pro (13.65%). Amplification results showed that 3 out 23 SSRs from transcriptome (13.04%) and 8 out 77 SSRs from genome (10.39%) were polymorphic.

An RNA Sequencing Transcriptome Analysis and Development of EST-SSR Markers in Chinese Hawthorn through Illumina Sequencing

Chinese hawthorn (Crataegus pinnatifida) is an important ornamental and economic horticultural plant. However, the lack of molecular markers has limited the development and utilization of hawthorn germplasm resources. Simple sequence repeats (SSRs) derived from expressed sequence tags (ESTs) allow precise and effective cultivar characterization and are routinely used for genetic diversity analysis. Thus, we first reported the development of polymorphic EST-SSR markers in C. pinnatifida with perfect repeats using Illumina RNA-Seq technique. In total, we investigated 14,364 unigenes, from which 5091 EST-SSR loci were mined. Di-nucleotides (2012, 39.52%) were the most abundant SSRs, followed by mono- (1989, 39.07%), and tri-nucleotides (1024, 20.11%). On the basis of these EST-SSRs, a total of 300 primer pairs were designed and used for polymorphism analysis in 70 accessions collected from different geographical regions of China. Of 239 (79.67%) pairs of primer-generated amplification products, 163 (54.33%) pairs of primers showed polymorphism. Finally, 33 primers with high polymorphism were selected for genetic diversity analysis and tested on 70 individuals with low-cost fluorescence-labeled M13 primers using capillary electrophoresis genotyping platform. A total of 108 alleles were amplified by 33 SSR markers, with the number of alleles (Na) ranging from 2 to 14 per locus (mean: 4.939), and the effective number of alleles (Ne) ranging from 1.258 to 3.214 (mean: 2.221). The mean values of gene diversity (He), observed heterozygosity (Ho), and polymorphism information content (PIC) were 0.524 (range 0.205–0.689), 0.709 (range 0.132–1.000), and 0.450 (range 0.184–0.642), respectively. Furthermore, the dendrogram constructed based on the EST-SSR separated the cultivars into two main clusters. In sum, our study was the first comprehensive study on the development and analysis of a large set of SSR markers in hawthorn. The results suggested that the use of NGS techniques for SSR development represented a powerful tool for genetic studies. Additionally, fluorescence-labeled M13 markers proved to be a valuable method for genotyping. All of these EST-SSR markers have agronomic potential and constitute a scientific basis for future studies on the identification, classification, and innovation of hawthorn germplasms.

Identification of microsatellites and parentage testing development of bactrian camel (Camelus bactrianus)

This report is aimed to identify polymorphic microsatellites from Bactrian camel transcriptome sequences and apply microsatellite multiplex systems for parentage testing. 1, 820 simple sequence repeats (SSRs) were identified from 23, 624 transcriptome sequences, representing an average density of SSR/26.57 kb. The most abundant SSR, the trinucleotide repeat motif, accounts for 30% of all SSRs. Tetra-, penta-, di-and hexanucleotide repeats account for 29%, 22%, 17% and 2% of SSRs, respectively. Based on sequence redundancy and PCR amplification, we selected 14 polymorphic microsatellites for Bactrian camel parentage testing analysis in a population of 117 unrelated camels from China and Mongolia. We identified a total of 149 alleles, with 5–23 alleles per locus (10.64 ± 5.472) and an average heterozygosity (HE) of 0.676 (range: 0.380–0.888). Based on only the genotype of the offspring, a parentage exclusion probability of 0.9999 was calculated when excluding a candidate parent from parentage of an arbitrary offspring. When excluding a candidate parent from parentage of an arbitrary offspring and based on both the genotype of the offspring and the other parent, an exclusion probability of 0.9999 was calculated. We selected 15 baby camels, 20 sires and 20 dams for parentage testing. According to the parentage assignment, the microsatellite panel assigned all 15 offspring with high confidence. This core set of 14 microsatellites represent a powerful and efficient method for determining parentage in domestic Bactrian camels.

Development of Simple Sequence Repeat Markers in Hazelnut (Corylus avellana L.) by Next-Generation Sequencing and Discrimination of Turkish Hazelnut Cultivars

European hazelnut (Corylus avellana) is a diploid tree species and is widely used in confections. Hazelnuts are, to a large part, produced in Turkey with the cultivar “Tombul” widely grown in the Black Sea region. In this work, the “Tombul” genome was partially sequenced by next-generation sequencing technology yielding 29.2% (111.85 Mb) of the ~ 385 Mb (1C). This sequence information was used to develop genetic markers in order to enable differentiation of material before the long maturation process and to facilitate future breeding strategies. A total of 90,142 simple sequence repeats (SSRs) were identified in the contigs giving a frequency of 1 SSR per 1240 nt in the assembly. Mononucleotides were the most abundant SSR marker type (60.9%) followed by di- and trinucleotides. Primer pairs were designed for 75,139 (83.3%) of the SSRs. Fifty SSR primers were applied to 47 hazelnut accessions from nine countries to test their effectiveness and polymorphism. The markers amplified an average of 3.2 fragments. The highest polymorphism information content value was for cavSSR11062 (0.97) and the lowest (0.04) was for cavSSR13386. Two markers were monomorphic: cavSSR12855 and cavSSR13267. Single-copy SSR primers were also assessed for their ability to discriminate 19 Turkish cultivars, and it was found that seven primer pairs (Cav4217, Cav14875, Cav14418, Cav2704, Cav12862, Cav3909, Cav1361) were sufficient for this task. Thus, this study developed new SSR markers for use in hazelnut breeding and genetic studies and also provide a method to distinguish and identify true-type Turkish cultivars.

Identification and development of microsatellite markers in Hamamelis mollis (Hamamelidaceae).

Premise of the Study Hamamelis mollis (Hamamelidaceae) is a Tertiary relict species endemic to southern China. Polymorphic microsatellite markers were developed to reveal the genetic diversity of this species.Methods and ResultsThe genome of H. mollis was sequenced and de novo assembled into 642,351 contigs. A total of 72,097 paired primers were successfully designed from 80,282 simple sequence repeat (SSR) markers identified in 63,419 contigs. PCR amplification showed that 96 of the 136 synthesized primers could be successfully amplified, and 22 demonstrated polymorphism. The mean number of alleles, levels of observed heterozygosity, and levels of expected heterozygosity were 4.602 ± 0.140, 0.632 ± 0.020, and 0.696 ± 0.010, respectively. The majority of the 96 primer pairs could be amplified in at least one other Hamamelidaceae species, including Distylium myricoides (60), Loropetalum chinense (39), Exbucklandia populnea (24), and E. tonkinensis (24).ConclusionsThese microsatellite loci provide abundant genomic SSR markers to evaluate genetic diversity of this woody ornamental plant.