Expressed Sequence Tag-simple Sequence Repeat Markers Research Articles

Development and characterization of novel EST-SSR markers for Gentiana straminea Maxim., a traditional Tibetan herb in China and cross-amplification in related species

Abstract Gentiana straminea Maxim. (Gentianaceae) is an important traditional Tibetan herb that is mainly distributed on the Qinghai-Tibetan Plateau. Despite its agricultural and pharmacological importance, there remains a paucity of microsatellite markers, particularly expressed sequence tag-simple sequence repeat (EST-SSR) markers, available for this local endemic species. In this study, based on previous Illumina transcriptome data of G. straminea, a total of 96 EST-SSR markers were initially designed and tested. Thirty-two of 96 loci (33.33%) were successfully amplified and verified for validation. Among them, 10 were polymorphic and had clear bands. The polymorphism information content values were 0.09–0.799, the number of alleles per locus ranged from 3 to 14, and the levels of observed and expected heterozygosity were 0.078–0.722 and 0.238–0.884, respectively, which suggested a high level of information. Moreover, cross-amplification was successful for 10 loci in two other related species, Gentiana macrophylla Pallas and Gentiana dahurica Fischer. These EST-SSR markers provide a valuable tool for investigating the genetic diversity related to quantitative traits and population genetic studies on G. straminea and related species in sect. Cruciata Gaudin.

Cross species transferability of G-SSR and EST-SSR markers to Neltuma affinis Spreng.

Aim of study: To examine the transferability of G-SSR (genomic simple sequence repeats) and EST-SSR (expressed sequence tag simple sequence repeats) markers developed for several Neltuma species to N. affinis, a species with no genomic data. Area of study: West-Center of Entre Ríos province, Argentina. The set of molecular markers here proposed can be used to analyze samples from the entire species’ distribution range. Material and methods: Twenty-five genomic G-SSRs and eleven EST-SSRs from multiple species were amplified in thirty N. affinis genotypes. Polymorphism, discrimination power and possible deviations from Hardy-Weinberg equilibrium were assessed. Main results: Seventeen highly polymorphic G-SSRs were successfully transferred to N. affinis, with a PIC (polymorphic information content) average value of 0.811 and a He (expected heterozygosity) average value of 0.694; thirteen were validated, showing very low frequencies of null alleles and no linkage disequilibrium. Additionally, seven polymorphic EST-SSRs were transferred. As expected, PIC and He average values were low. Six out of seven markers were validated, and very low frequencies of null alleles and no linkage disequilibrium were observed. Research highlights: This work provides information on the levels of microsatellites’ cross transferability to N. affinis, and its polymorphism degree. Two sets of polymorphic SSRs (genomic and expressed) to study the genetic status of the species are proposed.

Exploring the efficacy of targeted fingerprinting marker techniques in genetic studies of the Indian poppy (Papaver somniferum L.): A comparative analysis

Targeted fingerprinting marker techniques have emerged as promising tools in plant sciences, particularly in plant breeding. To initiate an effective breeding program for a licit but industrially potential plant, the opium poppy, a comparative assessment of these markers was conducted. In this study, we compared the Start Codon Targeted polymorphism (SCoT), CAAT box-derived polymorphism (CBDP), and Expressed sequence tag-simple sequence repeats (EST-SSRs) markers for their competence and informativeness in revealing genetic identity, relatedness, and diversity among ten released varieties of the Indian opium poppy. Among the markers studied, EST-SSR markers demonstrated higher discriminating power, attributed to their ability to unveil comparatively higher polymorphic information content, marker index, and effective marker index. While all three marker systems effectively discriminated the poppy varieties, EST-SSR markers showed greater genetic diversity and a more informative ancestral history-based dendrogram topology. Significant correlation coefficients were observed between EST-SSR and CBDP markers, particularly in the combined marker dataset. Furthermore, the co-phenetic correlation was significant for three marker systems, with EST-SSR markers showing maximum correlation. Notably, the dendrograms constructed using EST-SSR markers and combined marker data displayed a significant and positive correlation with phenotypic traits assessed for the ten poppy varieties. Overall, the findings highlight the superiority of EST-SSR markers in deciphering genetic relationships among Indian opium poppy varieties and suggest their potential as valuable tools in breeding and genetic diversity studies.

Tolerance evaluation and genetic relationship analysis among some economically important chestnut cultivars in Türkiye using drought-associated SSR and EST-SSR markers.

The aim of this study was to evaluate drought tolerance and genetic relationships among some important chestnut cultivars for Türkiye by using drought-related genomic simple sequence repeat (SSR) markers and genic expressed sequence tag-simple sequence repeat (EST-SSR) markers. Using five SSR markers, the average number of alleles (avNa), mean heterozygosity (Havp) and polymorphism information content (PIC) were determined to be 9.22, 0.395 and 0.375, respectively. In addition, using eight EST-SSR markers, the values of avNa, Havp and PIC were determined to be 7.75, 0.309 and 0.262, respectively. All microsatellite markers used in this study showed 100% polymorphism among chestnut cultivars. In UPGMA dendrograms obtained with both SSR and EST-SSR markers, the Erfelek and Hacıömer chestnut cultivars were determined to be the most similar cultivars. Some assessments are discussed regarding drought tolerance for specific alleles obtained from the EST-SSR markers GOT045, GOT021, GOT004, FIR094 and VIT033 in chestnut cultivars. Some preliminary results regarding drought tolerance in chestnut cultivars were obtained in our study with the help of these markers. Our study also characterized the genetic relationships among chestnut cultivars of great importance using drought-related character-specific markers.

De Novo Transcriptome Assembly and EST-SSR Marker Development and Application in Chrysosplenium macrophyllum.

Chrysosplenium macrophyllum Oliv., belonging to the family Saxifragaceae, is a traditional and unique Chinese herbal medicine. However, the lack of adequate molecular markers has hampered the progress regarding population genetics and evolution within this species. In this research, we used the DNBSEQ-T7 Sequencer (MGI) sequencing assay to analyze the transcriptome profiles of C. macrophyllum. SSR markers were developed on the basis of transcriptomic sequences and further validated on C. macrophyllum and other Chrysosplenium species. The genetic diversity and structure of the 12 populations were analyzed by using polymorphic expressed sequence tag simple sequence repeat (EST-SSR) markers. A potential pool of 3127 non-redundant EST-SSR markers were identified for C. macrophyllum in this study. The developed EST-SSR markers had high amplification rates and cross-species transferability in Chrysosplenium. Our results also showed that the natural populations of C. macrophyllum had a high level of genetic diversity. Genetic distance, principal component analysis, and popular structure analysis revealed that all 60 samples clustered into two major groups that were consistent with their geographical origins. This study provided a batch of highly polymorphic EST-SSR molecular markers that were developed via transcriptome sequencing. These markers will be of great significance for the study of the genetic diversity and evolutionary history of C. macrophyllum and other Chrysosplenium species.

EST-SSR Markers' Development Based on RNA-Sequencing and Their Application in Population Genetic Structure and Diversity Analysis of Eleusine indica in China.

Goosegrass (Eleusine indica) is one of the worst agricultural weeds in China. Molecular markers were developed for genetic diversity and population structure analyses. In this study, we identified 8391 expressed sequence tag-simple sequence repeat (EST-SSR) markers from the de novo assembled unigenes of E. indica. Mononucleotides were the most abundant type of repeats (3591, 42.79%), followed by trinucleotides (3162, 37.68%). The most dominant mononucleotide and trinucleotide repeat motifs were A/T (3406, 40.59%) and AAT/ATT (103, 1.5%), respectively. Fourteen pairs of EST-SSR primers were verified and used to analyze the genetic diversity and population structure of 59 goosegrass populations. A total of 49 alleles were amplified, with the number of alleles (Na) ranging from two to eleven per locus, and the effective number of alleles (Ne) ranged from 1.07 to 4.53. The average polymorphic information content (PIC) was 0.36. Genetic structure analysis (K = 2) and principal coordinate analysis divided 59 E. indica populations into two groups in a manner similar to the unweighted pair-group method (Dice genetic similarity coefficient = 0.700). This study developed a set of EST-SSR markers in E. indica and successfully analyzed the diversity and population genetic structures of 59 E. indica populations in China.

Female flowers first: QTL mapping in eastern black walnut (Juglans nigra L.) identifies a dominant locus for heterodichogamy syntenic with that in Persian walnut (J. regia L.)

Eastern black walnut (Juglans nigra L.), one of the most valuable timber and veneer trees in North America, provides nut shells with unique industrial uses and nut kernels with distinctive culinary attributes. A mature F1 full-sib progeny orchard of 248 individuals from the cross of two eastern black walnut cultivars provides a long-term resource for discovering genetic mechanisms controlling life history, quality traits, and stress resistance. The genetic linkage map, constructed with 356 single nucleotide polymorphism (SNP) markers and 62 expressed sequence tag simple sequence repeats (EST-SSRs), is 1645.7 cM in length, distributed across the expected 16 linkage groups. In this first application of QTL mapping in J. nigra, we report QTL for budbreak, peak pistillate bloom, peak staminate bloom, and heterodichogamy. A dominant major QTL for heterodichogamy is reported, the sequence for which is syntenic with the heterodichogamy QTL on chromosome 11 of Persian walnut (J. regia L.). The mapping population parents are both protogynous, and segregation suggests a Mendelian component, with a 3:1-like inheritance pattern from heterozygous parents. Mapping the sequenced EST-SSR markers to the J. regia “Chandler” V2.0 genome sequence revealed evidence for collinearity and structural changes on two of the sixteen chromosomes. The inclusion of sequenced EST-SSR markers enables the direct comparison of this and subsequent J. nigra maps and other Juglandaceae genetic maps. This investigation initiates long-term QTL detection studies for quality and stress resistance traits in black walnut.

Maximizing genetic gain through unlocking genetic variation in different ecotypes of kalmegh (Andrographis paniculata (Burm. f.) Nee).

Andrographis paniculata, commonly known as kalmegh is among the most popular medicinal herbs in Southeast Asia. It is widely cultivated for medicinal purposes. The bioactive molecule, Andrographolide accumulated in herb leaves has immense therapeutic and economic potential. However, comprehensive information regarding genetic diversity is very limited in this species. The present study assessed genetic diversity between and within the six populations (ecotypes) of twenty-four kalmegh accessions using multiple datasets (agro-morphological traits, phytochemical traits, and genic markers). This is the established report where EST-SSR (Expressed sequence tags-Simple Sequence Repeat) markers have been used to unlock genetic variation in kalmegh. Here, we identified and developed ninety-one metabolic pathway-specific EST-SSR markers. Finally, 32 random EST-SSR primer pairs were selected for genetic diversity assessment. Multivariate analysis to unveil the agro-morphological, phytochemical and genotypic variability was helpful in discriminating various germplasms studied in the present study. Among all the morphological discriptors used in present study, days to fifty percent flowering and dry herb yield were found as potential selection index for AP genetic improvement. Hierarchical cluster analysis built with agro-morphological data identified three major groups. However, corresponding analysis with phytochemical and molecular data generated two clear-cut groups among the studied individuals. Moreover, the grouping of individuals into different clusters using multiple datasets was geographically independent, and also showed inconsistency in grouping among agromorphological, phytochemical and molecular dataset based clusters. However, joint analysis using agro-morphological, phytochemical and genotypic information generated two genetic groups, which could be a valuable resource for identifying complementary crossing panels in the kalmegh breeding program. The accessions AP7, AP13, AP5, AP3 belong to cluster I and accessions AP17, AP18 belong to cluster II could be utilized as potential donors for high dry herb yield and andrographolide content, respectively in different selective breeding programs of AP. Thus, our results provided useful information about the overall genetic diversity and variation in economic traits useful for initiating selective breeding programs for contrasting traits of interest and maximizing genetic gain in kalmegh.

De novo transcriptome assembly, gene annotation, and EST-SSR marker development of an important medicinal and edible crop, Amomum tsaoko (Zingiberaceae)

BackgroundAmomum tsaoko is a medicinal and food dual-use crop that belongs to the Zingiberaceae family. However, the lack of transcriptomic and genomic information has limited the understanding of the genetic basis of this species. Here, we performed transcriptome sequencing of samples from different A. tsaoko tissues, and identified and characterized the expressed sequence tag-simple sequence repeat (EST-SSR) markers.ResultsA total of 58,278,226 high-quality clean reads were obtained and de novo assembled to generate 146,911 unigenes with an N50 length of 2002 bp. A total of 128,174 unigenes were successfully annotated by searching seven protein databases, and 496 unigenes were identified as annotated as putative terpenoid biosynthesis-related genes. Furthermore, a total of 55,590 EST-SSR loci were detected, and 42,333 primer pairs were successfully designed. We randomly selected 80 primer pairs to validate their polymorphism in A. tsaoko; 18 of these primer pairs produced distinct, clear, and reproducible polymorphisms. A total of 98 bands and 96 polymorphic bands were amplified by 18 pairs of EST-SSR primers for the 72 A. tsaoko accessions. The Shannon's information index (I) ranged from 0.477 (AM208) to 1.701 (AM242) with an average of 1.183, and the polymorphism information content (PIC) ranged from 0.223 (AM208) to 0.779 (AM247) with an average of 0.580, indicating that these markers had a high level of polymorphism. Analysis of molecular variance (AMOVA) indicated relatively low genetic differentiation among the six A. tsaoko populations. Cross-species amplification showed that 14 of the 18 EST-SSR primer pairs have transferability between 11 Zingiberaceae species.ConclusionsOur study is the first to provide transcriptome data of this important medicinal and edible crop, and these newly developed EST-SSR markers are a very efficient tool for germplasm evaluation, genetic diversity, and molecular marker-assisted selection in A. tsaoko.

Analysis of the genetic diversity and population structure of Monochasma savatieri Franch. ex Maxim using novel EST-SSR markers

BackgroundMonochasma savatieri Franch. ex Maxim is a medicinally valuable herb. However, the collection and protection of the wild germplasm resources of M. savatieri are still insufficient, and their genetic diversity and population structure have been poorly studied.ResultsWe collected and examined 46 M. savatieri individuals from Fujian, Hunan, Jiangxi, and Zhejiang provinces for genetic diversity and population structure, using 33 newly developed expressed sequence tag-simple sequence repeat (EST-SSR) markers. Applying these markers, we detected a total of 208 alleles, with an average of 6.303 alleles per locus. The polymorphic information content varied from 0.138 to 0.884 (average: 0.668), indicating a high level of polymorphism. At the population level, there was a low degree of genetic diversity among populations (I = 0.535, He = 0.342), with Zhejiang individuals showing the highest genetic diversity among the four populations (Fst = 0.497), which indicated little gene flow within the M. savatieri populations (Nm = 0.253). Mantel test analysis revealed a significant positive correlation between geographical and genetic distance among populations (R2 = 0.3304, p < 0.05), and structure and principal coordinate analyses supported classification of populations into three clusters, which was consistent with the findings of cluster analysis.ConclusionsAs a rare medicinal plants, the protection of M. savatieri does not look optimistic, and accordingly, protective efforts should be beefed up on the natural wild populations. This study provided novel tools and insights for designing effective collection and conservation strategies for M. savatieri.

Development of EST-SSR Markers Related to Polyphyllin Biosynthesis Reveals Genetic Diversity and Population Structure in Paris polyphylla

Paris polyphylla is an important medicinal plant that can biosynthesize polyphyllins with multiple effective therapies, ranging from anti-inflammation to antitumor; however, the genetic diversity of Paris polyphylla is still unclear. To explore the genetic characteristics of cultivation populations in primary planting areas, we developed 10 expressed sequence tag simple sequence repeat (EST-SSR) markers related to polyphyllin backbone biosynthesis and utilized them in 136 individuals from 10 cultivated populations of P. polyphylla var. yunnanensis. The genetic diversity index showed that ten loci had relatively high genetic polymorphism levels. Shannon information of loci suggested that more information occurred within population and less information occurred among population. In addition, the overall populations exhibited a low degree of differentiation among populations, but maintained a high degree of genetic diversity among individuals, resulting in high gene flow and general hybridization. The genetic structure analysis revealed that 10 populations possibly derived from two ancestral groups and all individuals were found with different levels of admixture. The two groups were different from the cultivation groups at population level, suggesting the cross-pollination among cultivars. These findings will provide insights into the genetic diversity of the germplasm resources and facilitate marker-assisted breeding for this medicinal herb.

Genetic diversity of Ethiopian potato (Plectranthus edulis (Vatke) Agnew) genotypes using simple sequence repeat markers

ABSTRACT Plectranthus edulis is among the most ancient and native tuber crops in Ethiopia, which is mainly cultivated by smallholder farmers. Being an orphan crop, it has not received due research attention. To tackle major crop-production constraints through breeding, knowledge of its genetic diversity is a prerequisite. Thus, the current study was carried out to assess the nature and extent of genetic diversity in 130 genotypes using 20 cross-transferable expressed sequence tag-simple sequence repeat (EST-SSR) markers developed from a related species, Plectranthus barbatus. Among the 20 SSR markers, 15 were transferable to Plectranthus edulis, from which 14 polymorphic markers were used for diversity analysis. A total of 101 alleles were detected, with a mean of 7.2 alleles per locus. Allele frequency ranged from 0.23 to 0.54, with mean value of 0.40. Polymorphic information content (PIC) ranged from 0.54 to 0.84, with a mean of 0.70, indicating a high level of informativeness. The Nei’s genetic distance ranged from 0.026 to 0.247, with the highest similarity being between Wolaita and Gamo Gofa populations. About 97% of the genetic variation was attributable to differences within populations; only 3% was attributable to variation among populations. The Neighbor-Joining cluster analysis grouped the 130 genotypes into three clusters. In general, the EST-SSR markers were effective in identifying genetic diversity in Ethiopian potato. This information can help to identify parental lines for crossing and exploiting the existing variability through selection.

Genetic diversity and inferred ancestry of Asian lotus (Nelumbo nucifera) germplasms in Thailand and Vietnam

Tropical lotus (Nelumbo) is an important and unique ecological type of lotus germplasm. Understanding the genetic relationship and diversity of the tropical lotus is necessary for its sustainable conservation and utilization. Using 42 EST-SSR (expressed sequence tag-simple sequence repeats) and 30 SRAP (sequence-related amplified polymorphism) markers, we assessed the genetic diversity and inferred the ancestry of representative tropical lotus from Thailand and Vietnam. In total, 164 and 41 polymorphic bands were detected in 69 accessions by 36 EST-SSR and seven SRAP makers, respectively. Higher genetic diversity was revealed in Thai lotus than in Vietnamese lotus. A Neighbor-Joining tree of five main clusters was constructed using combined EST-SSR and SRAP markers. Cluster I included 17 accessions of Thai lotus; cluster II contained three Thai accessions and 11 accessions from southern Vietnam; and cluster III was constituted by 13 accessions of seed lotus. Consistent with the results from the Neighbor-Joining tree, the genetic structure analysis showed that the genetic background of most Thai and Vietnamese lotus was pure, as artificial breeding has been rare in both countries. Furthermore, these analyses indicate that Thai and Vietnamese lotus germplasms belong to two different gene pools or populations. Most lotus accessions are genetically related to geographical distribution patterns in Thailand or Vietnam. Our findings showed that the origin or genetic relationships of some unidentified lotus sources can be evaluated by comparing morphological characteristics and the data of molecular markers. In addition, these findings provide reliable information for the targeted conservation of tropical lotus and parent selection in breeding novel cultivars of lotus.

Genetic Diversity Analysis of Sapindus in China and Extraction of a Core Germplasm Collection Using EST-SSR Markers.

Sapindus is an important forest tree genus with utilization in biodiesel, biomedicine, and it harbors great potential for biochemical engineering applications. For advanced breeding of Sapindus, it is necessary to evaluate the genetic diversity and construct a rationally designed core germplasm collection. In this study, the genetic diversity and population structure of Sapindus were conducted with 18 expressed sequence tag-simple sequence repeat (EST-SSR) markers in order to establish a core germplasm collection from 161 Sapindus accessions. The population of Sapindus showed high genetic diversity and significant population structure. Interspecific genetic variation was significantly higher than intraspecific variation in the Sapindus mukorossi, Sapindus delavayi, and combined Sapindus rarak plus Sapindus rarak var. velutinus populations. S. mukorossi had abundant genetic variation and showed a specific pattern of geographical variation, whereas S. delavayi, S. rarak, and S. rarak var. velutinus showed less intraspecific variation. A core germplasm collection was created that contained 40% of genetic variation in the initial population, comprising 53 S. mukorossi and nine S. delavayi lineages, as well as single representatives of S. rarak and S. rarak var. velutinus. These results provide a germplasm basis and theoretical rationale for the efficient management, conservation, and utilization of Sapindus, as well as genetic resources for joint genomics research in the future.

Development of 16 novel EST-SSR markers for species identification and cross-genus amplification in sambar, sika, and red deer.

Deer genera around the globe are threatened by anthropogenic interference. The translocation of alien species and their subsequent genetic introgression into indigenous deer populations is particularly harmful to the species of greatest conservation concern. Products derived from deer, including venison and antler velvet, are also at risk of fraudulent labeling. The current molecular markers used to genetically identify deer species were developed from genome sequences and have limited applicability for cross-species amplification. The absence of efficacious diagnostic techniques for identifying deer species has hampered conservation and wildlife crime investigation efforts. Expressed sequence tag-simple sequence repeat (EST-SSR) markers are reliable tools for individual and species identification, especially in terms of cross-species genotyping. We conducted transcriptome sequencing of sambar (Rusa unicolor) antler velvet and acquired 11,190 EST-SSRs from 65,074 newly assembled unigenes. We identified a total of 55 unambiguous amplicons in sambar (n = 45), which were selected as markers to evaluate cross-species genotyping in sika deer (Cervus nippon, n = 30) and red deer (Cervus elaphus, n = 46), resulting in cross-species amplification rates of 94.5% and 89.1%, respectively. Based on polymorphic information content (>0.25) and genotyping fidelity, we selected 16 of these EST-SSRs for species identification. This marker set revealed significant genetic differentiation based on the fixation index and genetic distance values. Principal coordinate analysis and STRUCTURE analysis revealed distinct clusters of species and clearly identified red-sika hybrids. These markers showed applicability across different genera and proved suitable for identification and phylogenetic analyses across deer species.

Evidence for hexasomic inheritance in Chrysanthemum morifolium Ramat. based on analysis of EST-SSR markers.

Chrysanthemums (Chrysanthemum morifolium Ramat.) are ornamental flowers, which are famous worldwide. The mode of inheritance has great implications for the genetic analysis of polyploid species. However, genetic analysis of chrysanthemum has been hampered because of its controversial inheritance mode (disomic or hexasomic). To classify the inheritance mode of chrysanthemums, an analysis of three approaches was carried out in an F1 progeny of 192 offspring using 223 expressed sequence tag-simple sequence repeat (EST-SSR) markers. The analysis included segregation analysis, the ratio of simplex marker alleles linked in coupling to repulsion, as well as the transmission and segregation patterns of EST-SSR marker alleles. After segregation analysis, 204 marker alleles fit hexasomic inheritance and 150 marker alleles fit disomic inheritance, showing that marker alleles were inherited predominantly in a hexasomic manner. Furthermore, the results of the analysis of allele configuration and segregation behavior of five EST-SSR markers also suggested random pairing of chromosomes. Additionally, the ratio of simplex marker alleles linked in coupling to repulsion was 1:0, further supporting hexasomic inheritance. Therefore, it could be inferred that chrysanthemum is a complete or near-complete hexasome.

Genetic relationships among Cucurbita pepo ornamental gourds based on EST-SSR markers

The ornamental gourd Cucurbita pepo L. is a ubiquitous crop native to North America, exhibiting highly diverse fruit characteristics. Studying the genetic diversity of ornamental gourds can help identify and evaluate the curated germplasm resources, understand the phylogenetic relationships among them, and highlight ways in which the germplasm resources can be used to address gaps in the understanding. In this study, a set of 85 of 323 previously identified polymorphic expressed sequence tag-simple sequence repeat (EST-SSR) genetic markers were selected to evaluate the genetic relationships among 47 C. pepo accessions and one C. foetidissima accession. This collection consisted of accessions from the subspecies pepo, texana, and the hybrid texana × pepo. Our analyses yielded a total of 271 alleles, with an average of 3.2 alleles per genetic locus. The dendrogram construction, principal coordinate analyses, and genetic value calculation revealed several robust subclusters in the texana subspecies accessions. From these results, we propose five new distinct morphotypes based on our construction of a concise SSR fingerprint. Moreover, our study confirms that the fruit shape similarity among accessions is a fair reflection of genetic relatedness.

Evaluation of genetic diversity and population structure of Fragaria nilgerrensis using EST-SSR markers.

Fragaria nilgerrensis is a diploid wild strawberry widely distributed in Southwest China. Its white color and "peach-like" fragrance of fruits are valuable characters for the genetic improvement of cultivated strawberry plants. Its strong biotic and abiotic resistance and tolerance also enable it to survive in different habitats in the field. In this study, we evaluated the level of genetic variation within and between 16 populations with 169 individuals of F. nilgerrensis using 16 newly developed EST-SSR (expressed sequence tag-simple sequence repeats) markers. The results show that the genetic diversity of this species was high, based on Nei's genetic diversity (0.26) and polymorphic loci (0.41), although it is self-compatible and has clonal propagation. Significant genetic differentiation among populations was also detected by AMOVA analysis (Fst=0.34), which could be indicative of little gene flow (Nm=0.43) in F. nilgerrensis. The phylogenetic tree indicates that most of individuals from the same population have clustered together. These populations were not grouped based on the geographical distance, consistent with the Mantel test result (R2=0.0063, P>0.05). All the populations were assigned into two ancestral groups, with some individuals admixed, suggesting ancestral gene flow had occurred between these two groups. Our developed EST-SSR markers as well as the genetic diversity and population structure analysis of F. nilgerrensis are important for genetic improvement in the breeding process. Moreover, the populations that contain high genetic diversity would be a priority for collection and conservation.

Analysis of genetic diversity and relationships of Perilla frutescens using novel EST-SSR markers derived from transcriptome between wild-type and mutant Perilla.

Perilla frutescens (Lamiaceae) is distributed in East Asia and is classified into var. frutescens and crispa. P. frutescens is multipurpose crop for human health because of a variety of secondary metabolites such as phenolic compound and essential oil. However, a lack of genetic information has hindered the development and utilization of Perilla genotypes. This study was performed to develop expressed sequence tag-simple sequence repeat (EST-SSR) markers from P. frutescens var. crispa (wild type) and Antisperill (a mutant cultivar) and used them to assess the genetic diversity of, and relationships among, 94 P. frutescens genotypes. We obtained 65Gb of sequence data comprising 632,970 transcripts by de novo RNA-sequencing. Of the 14,780 common SSRs, 102 polymorphic EST-SSRs were selected using in silico polymerase chain reaction (PCR). Overall, successful amplification from 58 EST-SSRs markers revealed remarkable genetic diversity and relationships among 94 P. frutescens genotypes. In total, 268 alleles were identified, with an average of 4.62 alleles per locus (range 2-11 alleles/locus). The average polymorphism information content (PIC) value was 0.50 (range 0.04-0.86). In phylogenetic and population structure analyses, the genotypes formed two major groups: Group I (var. crispa) and Group II (var. frutescens). This results suggest that 58 novel EST-SSR markers derived from wild-type cultivar (var. crispa) and its mutant cultivar (Antisperill) have potential uses for population genetics and recombinant inbred line mapping analyses, which will provide comprehensive insights into the genetic diversity and relationship of P. frutescens.

Development and characterization of EST-SSR markers for Pinus thunbergii

ABSTRACT In the present study, a set of ten Expressed Sequence Tag-Simple Sequence Repeats (EST-SSR) markers have been developed to contribute to the genetic studies of Pinus thunbergii. The number of alleles in the developed EST-SSR markers ranged from one to six. Observed heterozygosity was 0.000 to 0.707; the expected heterozygosity ranged from 0.000 to 0.720. Polymorphism information content (PIC) was ranged from 0.000 to 0.669. The mean of the probability of identity between two unrelated individuals (PI) and two siblings (PI-Sib) were 0.540 and 0.719, respectively. The EST-SSR markers developed in the present study, in combining with the previously developed markers, can help in understanding the genetic diversity and genetic structure of P. thunbergii populations.