Transcriptome analysis and development of EST-SSR markers in the mushroom Auricularia heimuer.

Auricularia heimuer, the third most frequently cultivated edible mushroom species worldwide, has high medicinal value. However, a shortage of molecular marker hinders the efficiency and accuracy of genetic breeding efforts for A. heimuer. High-throughput transcriptome sequencing data are essential for gene discovery and molecular markers development. This study aimed to clarify the distribution of SSR loci across the A. heimuer transcriptome and to develop highly informative EST-SSR markers. These tools can be used for phylogenetic analysis, functional gene mining, and molecular marker-assisted breeding of A. heimuer. This study used Illumina high-throughput sequencing technology to obtain A. heimuer transcriptome data. The results revealed 37,538 unigenes in the A. heimuer transcriptome. Of these unigenes, 24,777 (66.01%) were annotated via comparison with the COG, Pfam, and NR databases. Overall, 2510 SSRs were identified from the unigenes, including 6 types of SSRs. The most abundant type of repeats were trinucleotides (1425, 56.77%), followed by mononucleotides (391, 15.58%) and dinucleotides (456, 18.17%). Primer pairs for 102 SSR loci were randomly designed for validity confirmation and polymorphism identification; this process yielded 53 polymorphic EST-SSR markers. Finally, 13 pairs of highly polymorphic EST-SSR primers were used to analyze the genetic diversity and population structure of 52 wild A. heimuer germplasms, revealing that the 52 germplasms could be divided into three categories. These results indicated that SSR loci were abundant in types, numbers, and frequencies, providing a potential basis for germplasm resource identification, genetic diversity analysis, and molecular marker-assisted breeding of A. heimuer.

Development of EST-SSR markers in Bergenia ciliata using de novo transcriptome sequencing.

Bergenia ciliata (Haw.) Sternb. is an important herb predominantly found in the Indian Himalayan Region. It is widely used in medicines, healthcare systems, cosmetics, fodder, and ornamental purposes. The Illumina sequencing and de novo transcriptome assembly were carried out in B. ciliata to develop and identify simple sequence repeat markers. A total of 18 226 simple sequence repeats (SSRs) were identified wherein di-nucleotides were found to be abundant (47.88%), followed by mono-nucleotide (35.03%) and tri-nucleotide (15.88%) repeats. A total of 11 839 EST-SSR primers were designed, of which 96 primer pairs were commercially synthesized. Finally, 17 primer pairs revealed clear, distinct polymorphic bands, and these primers were validated with 40 diverse B. ciliata accessions. The present study revealed moderate level of genetic diversity (Ho = 0.389, He = 0.542, and PIC = 0.513). Furthermore, the transcriptome data and EST-SSR markers generated during the present investigation could be an important genetic resource for functional genomics, population studies, and conservation genetics of the genus Bergenia.

Characterization and development of transcriptome-derived novel EST-SSR markers to assess genetic diversity in Chaetomium globosum.

Chaetomium globosum Kunze, an internationally recognized biocontrol fungus. It mycoparasitizes various plant pathogens and produce antifungal metabolites to suppress the growth of pathogenic fungi. Lack of detailed genome level diversity studies has delimited the development and utilization of potential C. globosum strains. The present study was taken to reveal the distribution, identification, and characterization of expressed sequence tag-simple sequence repeats (EST-SSRs) in C. globosum. RNA-Seq experiment was performed for C. globosum potential isolate Cg2 (AY429049) using Illumina HiSeq 2500. Reference-guided de novo assembly yielded 45,582 transcripts containing 27,957 unigenes. We generated a new set of 8485 EST-SSR markers distributed in 5908 unigene sequences with one SSR locus distribution density per 6.1 kb. Six distinct classes of SSR repeat motifs were identified. The most abundant were mononucleotide repeats (51.67%), followed by tri-nucleotides (36.61%). Out of 5034 EST-SSR primers, 50 primer pairs were selected and validated for the polymorphic study of 15 C. globosum isolates. Twenty-two SSR markers showed average genetic polymorphism among C. globosum isolates. The number of alleles (Na) per marker ranges from 2 to 4, with a total of 74 alleles detected for 22 markers with a mean polymorphism information content (PIC) value of 0.4. UPGMA hierarchical clustering analysis generated three main clusters of C. globosum isolates and exhibited a lower similarity index range from 0.59 to 0.85. Thus, the newly developed EST-SSR markers could replace traditional methods for determining diversity. The study will also enhance the genomic research in C. globosum to explore its biocontrol potential against phytopathogens. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03794-7.

De Novo Transcriptome Profiling for the Generation and Validation of Microsatellite Markers, Transcription Factors, and Database Development for Andrographis paniculata.

Andrographis paniculata belongs to the family Acanthaceae and is known for its medicinal properties owing to the presence of unique constituents belonging to the lactones, diterpenoids, diterpene glycosides, flavonoids, and flavonoid glycosides groups of chemicals. Andrographolide, a major therapeutic constituent of A. paniculata, is extracted primarily from the leaves of this plant and exhibits antimicrobial and anti-inflammatory activities. Using 454 GS-FLX pyrosequencing, we have generated a whole transcriptome profile of entire leaves of A. paniculata. A total of 22,402 high-quality transcripts were generated, with an average transcript length and N50 of 884 bp and 1007 bp, respectively. Functional annotation revealed that 19,264 (86%) of the total transcripts showed significant similarity with the NCBI-Nr database and were successfully annotated. Out of the 19,264 BLAST hits, 17,623 transcripts were assigned GO terms and distributed into three major functional categories: molecular function (44.62%), biological processes (29.19%), and cellular component (26.18%) based on BLAST2GO. Transcription factor analysis showed 6669 transcripts, belonging to 57 different transcription factor families. Fifteen TF genes that belong to the NAC, MYB, and bHLH TF categories were validated by RT PCR amplification. In silico analysis of gene families involved in the synthesis of biochemical compounds having medicinal values, such as cytochrome p450, protein kinases, heat shock proteins, and transporters, was completed and a total of 102 different transcripts encoding enzymes involved in the biosynthesis of terpenoids were predicted. Out of these, 33 transcripts belonged to terpenoid backbone biosynthesis. This study also identified 4254 EST-SSRs from 3661 transcripts, representing 16.34% of the total transcripts. Fifty-three novel EST-SSR markers generated from our EST dataset were used to assess the genetic diversity among eighteen A. paniculata accessions. The genetic diversity analysis revealed two distinct sub-clusters and all accessions based on the genetic similarity index were distinct from each other. A database based on EST transcripts, EST-SSR markers, and transcription factors has been developed using data generated from the present study combined with available transcriptomic resources from a public database using Meta transcriptome analysis to make genomic resources available in one place to the researchers working on this medicinal plant.

De novo full-length transcriptome analysis of two ecotypes of Phragmites australis (swamp reed and dune reed) provides new insights into the transcriptomic complexity of dune reed and its long-term adaptation to desert environments.

BACKGROUND: The extremely harsh environment of the desert is changing dramatically every moment, and the rapid adaptive stress response in the short term requires enormous energy expenditure to mobilize widespread regulatory networks, which is all the more detrimental to the survival of the desert plants themselves. The dune reed, which has adapted to desert environments with complex and variable ecological factors, is an ideal type of plant for studying the molecular mechanisms by which Gramineae plants respond to combinatorial stress of the desert in their natural state. But so far, the data on the genetic resources of reeds is still scarce, therefore most of their research has focused on ecological and physiological studies. RESULTS: In this study, we obtained the first De novo non-redundant Full-Length Non-Chimeric (FLNC) transcriptome databases for swamp reeds (SR), dune reeds (DR) and the All of Phragmites australis (merged of iso-seq data from SR and DR), using PacBio Iso-Seq technology and combining tools such as Iso-Seq3 and Cogent. We then identified and described long non-coding RNAs (LncRNA), transcription factor (TF) and alternative splicing (AS) events in reeds based on a transcriptome database. Meanwhile, we have identified and developed for the first time a large number of candidates expressed sequence tag-SSR (EST-SSRs) markers in reeds based on UniTransModels. In addition, through differential gene expression analysis of wild-type and homogenous cultures, we found a large number of transcription factors that may be associated with desert stress tolerance in the dune reed, and revealed that members of the Lhc family have an important role in the long-term adaptation of dune reeds to desert environments. CONCLUSIONS: Our results provide a positive and usable genetic resource for Phragmites australis with a widespread adaptability and resistance, and provide a genetic database for subsequent reeds genome annotation and functional genomic studies.

Characterization and Application of EST-SSR Markers Developed from Transcriptome Sequences in Elymus breviaristatus (Poaceae: Triticeae).

BACKGROUND: Elymus L. is the largest genus in the Triticeae tribe. Most species in this genus are highly stress resistant, with excellent forage value. Elymus breviaristatus, a rare species endemic to the Qinghai-Tibet Plateau (QTP), is declining due to habitat fragmentation. However, genetic data for E. breviaristatus are limited, with expressed sequence tag (EST) markers being particularly rare, hampering genetic studies and protection measures. RESULTS: We obtained 9.06 Gb clean sequences from the transcriptome of E. breviaristatus, generating 171,522 unigenes, which were assembled and functionally annotated against five public databases. We identified 30,668 SSRs in the E. breviaristatus transcriptome, from which 103 EST-SSR primer pairs were randomly selected. Of these, 58 pairs of amplified products of the expected size, and 18 of the amplified products were polymorphic. Model-based Bayesian clustering, the unweighted pair group method with arithmetic average (UPGMA), and principal coordinate analysis (PCoA) of 179 wild E. breviaristatus in 12 populations using these EST-SSRs were generally consistent, grouping the 12 populations into two major clades. Analysis of molecular variance (AMOVA) found 70% of the genetic variation among the 12 populations and 30% within the populations, indicating a high level of genetic differentiation (or low gene exchange) among the 12 populations. The transferability of the 58 successful EST-SSR primers to 22 related hexaploid species was 86.2-98.3%. UPGMA analysis generally grouped species with similar genome types together. CONCLUSIONS: Here, we developed EST-SSR markers from the transcriptome of E. breviaristatus. The transferability of these markers was evaluated, and the genetic structure and diversity of E. breviaristatus were explored. Our results provide a basis for the conservation and management of this endangered species, and the obtained molecular markers represent valuable resources for the exploration of genetic relationships among species in the Elymus genus.

Develop a preliminary core germplasm with the novel polymorphism EST-SSRs derived from three transcriptomes of colored calla lily (Zantedeschia hybrida).

The development of high-throughput sequencing technology has made it possible to develop molecular markers such as EST-SSR from transcriptome sequences in non-model plants such as bulbous flowers. However, the EST-SSR markers that have been developed are weakly validated and low polymorphic due to the short read size and poor quality of the assembled sequences. This study therefore used the CandiSSR pipeline to identify 550 potential polymorphic SSR loci among 487 homologous unigenes based on the transcriptomic sequences of three varieties of colored calla lily, and 460 of these loci with appropriate flanking sequences were suitable for primer pairs design. A further validation with 200 randomly selected EST-SSRs demonstrated an increase of more than 30% and 100% in amplification validity and polymorphism, respectively, in comparison with our previous study. In addition, since most of the current varieties of colored calla lily are hybridized from a few species, which have low genetic diversity, we subsequently identified primary core germplasm for 160 colored calla lily accessions using the aforementioned 40 polymorphic EST-SSRs. It was concluded that the core germplasm containing 42 accessions derived from the M strategy incorporated into the software Power Core was the most representative of all 160 original germplasm, as evidenced by the preservation of 100% of the EST-SSR variation, with a higher level of genetic diversity and heterogeneity (Nei = 0.40, I = 0.66, PIC = 0.43). This study provides a practical example of polymorphism EST-SSR markers developed from multiple transcriptomes for non-model plants. A future breeding program for colored calla lily will also benefit from the core germplasm defined by those molecular markers.

Development and Application of EST-SSR Markers in Cephalotaxus oliveri From Transcriptome Sequences.

Cephalotaxus oliveri is an endemic conifer of China, which has medicinal and ornamental value. However, the limited molecular markers and genetic information are insufficient for further genetic studies of this species. In this study, we characterized and developed the EST-SSRs from transcriptome sequences for the first time. The results showed that a total of 5089 SSRs were identified from 36446 unigenes with a density of one SSR per 11.1 kb. The most common type was trinucleotide repeats, excluding mononucleotide repeats, followed by dinucleotide repeats. AAG/CTT and AT/AT exhibited the highest frequency in the trinucleotide and dinucleotide repeats, respectively. Of the identified SSRs, 671, 1125, and 1958 SSRs were located in CDS, 3'UTR, and 5'UTR, respectively. Functional annotation showed that the SSR-containing unigenes were involved in growth and development with various biological functions. Among successfully designed primer pairs, 238 primer pairs were randomly selected for amplification and validation of EST-SSR markers and 47 primer pairs were identified as polymorphic. Finally, 28 high-polymorphic primers were used for genetic analysis and revealed a moderate level of genetic diversity. Seven natural C. oliveri sampling sites were divided into two genetic groups. Furthermore, the 28 EST-SSRs had 96.43, 71.43, and 78.57% of transferability rate in Cephalotaxus fortune, Ametotaxus argotaenia, and Pseudotaxus chienii, respectively. These markers developed in this study lay the foundation for further genetic and adaptive evolution studies in C. oliveri and related species.

Development of a new set of genic SSR markers in the genus Gentiana: in silico mining, characterization and validation.

Gentiana is an important genus of around 360 medicinally important species, majority of which are not well characterized. Despite its importance, very few genomic resources are available for Gentiana L. Till date, the number of informative and robust simple sequence repeat (SSR)-based markers is limited and very few efforts have been made for their development. A set of robust, freely accessible and informative SSR markers for Gentiana is a pre-requisite for any molecular systematic as well as improvement studies in this group of pharmacologically valuable plants. In view of the importance of these plants, Expressed Sequence Tag (EST) sequences of 18 Gentiana species were surveyed for the development of a large set of non-redundant SSR markers. A total of 5808 perfect SSR with an average length of 17 bp and relative abundance of 214 loci/Mb were identified in the analysed 47,487 EST sequences using Krait software. Mapping of the ESTs resulted in gene ontology annotations of 49.14% of the sequences. Based on these perfect SSRs, 2902 primer pairs were designed, and 60 markers were randomly selected and validated on a set of Gentiana kurroo Royle accessions. Among the screened markers, 39 (65%) were found to be cross-species transferable. This is the first report of the largest set of functional, novel genic SSR markers in Gentiana, which will be a valuable resource for future characterization, genotype identification, conservation and genomic studies in the various species of this group of important medicinal plants. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02969-4.

Large-scale advances in SSR markers with high-throughput sequencing in Euphorbia fischeriana Steud

BACKGROUND: Euphorbia fischeriana Steud is a very important medicinal herb and has significant medical value for healing cancer, edema and tuberculosis in China. The lack of molecular markers for Euphorbia fischeriana Steud is a dominant barrier to genetic research. For the purpose of developing many simple sequence repeat (SSR) molecular markers, we completed transcriptome analysis with the Illumina HiSeq 2000 platform. RESULTS: Approximately 9.1 million clean reads were acquired and then assembled into approximately 186.3 thousand nonredundant unigenes, 53,146 of which were SSR-containing unigenes. A total of 76,193 SSR loci were identified. Of these SSR loci, 28,491 were detected at the terminal position of ESTs, which made it difficult to design SSR primers for these SSR-containing sequences, and the residual SSRs were thus used to design primer pairs. Analyzing the results of these markers revealed that the mononucleotide motif A/T (44,067, 57.83% of all SSRs) was the most abundant, followed by the dinucleotide type AG/CT (9430, 12.38%). Using 100 randomly selected primer pairs, 77 primers were successfully amplified in Euphorbia fischeriana Steud, and 79 were successfully amplified in three other related species. The markers developed displayed relatively high quality and cross-species transferability. CONCLUSIONS: The large number of EST-SSRs exploited successfully in Euphorbia fischeriana Steud for the first time could provide genetic information for research on linkage maps, variety identification, genetic diversity analysis, and molecular marker-assisted breeding.

Development of EST-SSR markers for genetic diversity analysis in coconut (Cocos nucifera L.).

Genetic improvement in coconut relies on exploiting the vast existing diversity among coconut accessions. Robust molecular markers are a pre-requisite for efficient characterization of genetic diversity. Microsatellites or simple sequence repeats (SSRs), mined from expressed sequence tags (ESTs), constitute an important resource for analysis of genetic diversity as they are abundant, polymorphic and represent function regions of the genome. We have identified a total of 318,528 putative EST-SSRs from 130,942 unigenes utilizing a leaf transcriptome dataset of coconut. Among the EST-SSRs, dinucleotide repeats were abundant (219,912; 69.04%) followed by trinucleotide (70,722; 22.2%) and tetra-nucleotide repeats (6281; 1.9%). Among the dinucleotide repeat motifs, the dominant repeat was AG/CT (35.87%), followed by AT/AT (18.59%), while the dominant trinucleotide repeat was AAG/CTT (4.59%). One hundred and twenty EST-SSR primer pairs were designed and utilized to amplify six DNA samples of coconut accessions. Fifty primers (41.7%) produced reproducible polymorphic fragments of expected sizes, from which a total of 10 primers were selected for the diversity assessment in 186 palms of 50 coconut accessions, comprising of 25 each of tall and dwarf accessions. A total of 137 alleles were detected with an average of 13.7 alleles per SSR locus. The number of alleles observed at each locus in the data set ranged from 7 to 22. All the loci showed 100% polymorphism with respect to the samples screened. The average observed heterozygosity was 0.46. The PIC values ranged from 0.79 (CnKGDEST129 and CnKGDEST100) to 0.91 (CnKGDEST117 and CnKGDEST122) with a mean value of 0.85, indicating the capacity of the EST-SSR markers to detect high levels of polymorphism. The cluster analysis revealed that accessions were generally clustered based on their relative similarity and irrespective of their geographic origins. The present study demonstrates the usefulness of transcriptome sequencing as a rapid and cost-effective methodology for the development of molecular markers. The EST-SSR markers generated through this study constitute useful and reliable tools for assessment of genetic diversity and marker-assisted selection in coconut.

De novo assembly and Transcriptome characterization of an endemic species of Vietnam, Panax vietnamensis Ha et Grushv., including the development of EST-SSR markers for population genetics.

BACKGROUND: Understanding the genetic diversity in endangered species that occur inforest remnants is necessary to establish efficient strategies for the species conservation, restoration and management. Panax vietnamensis Ha et Grushv. is medicinally important, endemic and endangered species of Vietnam. However, genetic diversity and structure of population are unknown due to lack of efficient molecular markers. RESULTS: In this study, we employed Illumina HiSeq™ 4000 sequencing to analyze the transcriptomes of P. vietnamensis (roots, leaves and stems). Raw reads total of 23,741,783 was obtained and then assembled, from which the generated unigenes were 89,271 (average length = 598.3191 nt). The 31,686 unigenes were annotated in different databases i.e. Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, Nucleotide Collection (NR/NT) and Swiss-Prot for functional annotation. Further, 11,343 EST-SSRs were detected. From 7774 primer pairs, 101 were selected for polymorphism validation, in which; 20 primer pairs were successfully amplified to DNA fragments and significant amounts of polymorphism was observed within population. The nine polymorphic microsatellite loci were used for population structure and diversity analyses. The obtained results revealed high levels of genetic diversity in populations, the average observed and expected heterozygosity were HO = 0.422 and HE = 0.479, respectively. During the Bottleneck analysis using TPM and SMM models (p < 0.01) shows that targeted population is significantly heterozygote deficient. This suggests sign of the bottleneck in all populations. Genetic differentiation between populations was moderate (FST = 0.133) and indicating slightly high level of gene flow (Nm = 1.63). Analysis of molecular variance (AMOVA) showed 63.17% of variation within individuals and 12.45% among populations. Our results shows two genetic clusters related to geographical distances. CONCLUSION: Our study will assist conservators in future conservation management, breeding, production and habitats restoration of the species.

Genetic Divergence and Relationship Among Opisthopappus Species Identified by Development of EST-SSR Markers.

Opisthopappus Shih is an endemic and endangered genus restricted to the Taihang Mountains that has important ornamental and economic value. According to the Flora Reipublicae Popularis Sinicae (FRPS, Chinese version), this genus contains two species (Opisthopappus longilobus and Opisthopappus taihangensis), whereas in the Flora of China (English version) only one species O. taihangensis is present. The interspecific phylogenetic relationship remains unclear and undefined, which might primarily be due to the lack of specific molecular markers for phylogenetic analysis. For this study, 2644 expressed sequence tag-simple sequence repeats (EST-SSRs) from 33,974 unigenes using a de novo transcript assembly of Opisthopappus were identified with a distribution frequency of 7.78% total unigenes. Thereinto, mononucleotides (1200, 45.39%) were the dominant repeat motif, followed by trinucleotides (992, 37.52%), and dinucleotides (410, 15.51%). The most dominant trinucleotide repeat motif was ACC/GGT (207, 20.87%). Based on the identified EST-SSRs, 245 among 1444 designed EST-SSR primers were selected for the development of potential molecular markers. Among these markers, 63 pairs of primers (25.71%) generated clear and reproducible bands with expected sizes. Eventually, 11 primer pairs successfully amplified all individuals from the studied populations. Through the EST-SSR markers, a high level of genetic diversity was detected between Opisthopappus populations. A significant genetic differentiation between the O. longilobus and O. taihangensis populations was found. All studied populations were divided into two clusters by UPGMA, NJ, STRUCTURE, and PCoA. These results fully supported the view of the FRPS, namely, that O. longilobus and O. taihangensis should be regarded as two distinct species. Our study demonstrated that transcriptome sequences, as a valuable tool for the quick and cost-effective development of molecular markers, was helpful toward obtaining comprehensive EST-SSR markers that could contribute to an in-depth assessment of the genetic and phylogenetic relationships between Opisthopappus species.

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

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

Application of EST-SSR markers developed from the transcriptome of Torreya grandis (Taxaceae), a threatened nut-yielding conifer tree.

Torreya grandis (Taxaceae) is an ancient conifer species endemic to southeast China. Because of its nutrient-rich and delicious seeds, this species has been utilized for centuries by the Chinese. However, transcriptome data and transcriptome-derived microsatellite markers for population genetics studies are still insufficient for understanding of this species' genetic basis. In this study, a transcriptome from T. grandis leaves was generated using Illumina sequencing. A total of 69,920 unigenes were generated after de novo assembly, and annotated by searching against seven protein databases. In addition, 2,065 expressed sequence tag-simple sequence repeats (EST-SSRs) were detected, with the distribution frequency of 2.75% of total unigenes and average number of 0.03 SSRs per unigene. Among these EST-SSRs, 1,339 primer pairs were successfully designed, and 106 primer pairs were randomly selected for the development of potential molecular markers. Among them, 11 EST-SSR markers revealed a moderate level of genetic diversity, and were used to investigate the population structure of T. grandis. Two different genetic groups within this species were revealed using these EST-SSR markers, indicating that these markers developed in this study can be effectively applied to the population genetic analysis of T. grandis.

De novo transcriptome sequencing of Flammulina velutipes uncover candidate genes associated with cold-induced fruiting.

To understand molecular mechanism of cold-induced fruiting in Flammulina velutipes, which is one of most popular edible fungi in east Asia, de novo assembly of the F. velutipes transcriptome was carried out. There were 26,888,494 and 26,275,146 clean reads obtained from mycelium and primordia of F. velutipes, respectively. A total of 20,157 unigenes were de novo assembled and 15,058 of them were annotated. Moreover, 7935 unigenes were differentially expressed between mycelium and primordia, 4025 of them were up-regulated and 3910 were down-regulated. GO and KEGG pathway analysis of the differentially expressed unigenes indicated that functional groups associated with two-component signaling pathway, calcium signaling, mitogen-actived protein kinase pathway, molecular chaperones, cell wall and membrane system, play an important role in cold-induced fruiting of F. velutipes. In this work 643 EST-SSRs were identified in 20,157 unigenes and 1560 EST-SSRs primers pairs were designed. Moreover, 5548 and 5955 SNPs were detected in mycelium and primordia, respectively. Consequently, results of this work can serve as a valuable resource for functional genomics study of cold-induced fruiting in F. velutipes.

Population structure, genetic diversity and downy mildew resistance among Ocimum species germplasm.

BACKGROUND: The basil (Ocimum spp.) genus maintains a rich diversity of phenotypes and aromatic volatiles through natural and artificial outcrossing. Characterization of population structure and genetic diversity among a representative sample of this genus is severely lacking. Absence of such information has slowed breeding efforts and the development of sweet basil (Ocimum basilicum L.) with resistance to the worldwide downy mildew epidemic, caused by the obligate oomycete Peronospora belbahrii. In an effort to improve classification of relationships 20 EST-SSR markers with species-level transferability were developed and used to resolve relationships among a diverse panel of 180 Ocimum spp. accessions with varying response to downy mildew. RESULTS: Results obtained from nested Bayesian model-based clustering, analysis of molecular variance and unweighted pair group method using arithmetic average (UPGMA) analyses were synergized to provide an updated phylogeny of the Ocimum genus. Three (major) and seven (sub) population (cluster) models were identified and well-supported (P < 0.001) by PhiPT (ΦPT) values of 0.433 and 0.344, respectively. Allelic frequency among clusters supported previously developed hypotheses of allopolyploid genome structure. Evidence of cryptic population structure was demonstrated for the k1 O. basilicum cluster suggesting prevalence of gene flow. UPGMA analysis provided best resolution for the 36-accession, DM resistant k3 cluster with consistently strong bootstrap support. Although the k3 cluster is a rich source of DM resistance introgression of resistance into the commercially important k1 accessions is impeded by reproductive barriers as demonstrated by multiple sterile F1 hybrids. The k2 cluster located between k1 and k3, represents a source of transferrable tolerance evidenced by fertile backcross progeny. The 90-accession k1 cluster was largely susceptible to downy mildew with accession 'MRI' representing the only source of DM resistance. CONCLUSIONS: High levels of genetic diversity support the observed phenotypic diversity among Ocimum spp. accessions. EST-SSRs provided a robust evaluation of molecular diversity and can be used for additional studies to increase resolution of genetic relationships in the Ocimum genus. Elucidation of population structure and genetic relationships among Ocimum spp. germplasm provide the foundation for improved DM resistance breeding strategies and more rapid response to future disease outbreaks.

Generation and classification of transcriptomes in two Croomia species and molecular evolution of CYC/TB1 genes in Stemonaceae.

The genus Croomia (Stemonaceae) is an excellent model for studying the evolution of the Eastern Asia (EA)-Eastern North America (ENA) floristic disjunction and the genetic mechanisms of floral zygomorphy formation. In addition to the presence of both actinomorphic and zygomorphic flowers within the genus, species are disjunctively distributed between EA and ENA. However, due to the limited availability of genomic resources, few studies of Croomia have examined these questions. In this study, we sequenced the floral and leaf transcriptomes of the zygomorphic flowered C roomia heterosepala and the actinomorphic flowered Croomia japonica, and used comparative genomic approaches to investigate the transcriptome evolution of the two closely related species. The sequencing and de novo assembly of transcriptomes from flowers of C. heterosepala (ChFlower), flowers of C. japonica (CjFlower), and leaves of C. japonica (CjLeaf) yielded 57,193, 62,131 and 64,448 unigenes, respectively. In addition, estimation of Ka/Ks ratios for 11,566 potential orthologous groups between ChFlower and CjFlower revealed that only six pairs had Ka/Ks ratios significantly greater than 1 and are likely under positive selection. A total of 429 single copy nuclear genes (SCNGs) and 21,460 expression sequence tags-simple sequence repeats (EST-SSRs) were identified in this study. Specifically, we identified seven CYC/TB1-like genes from Stemonaceae. Phylogenetic and molecular evolution analyses indicated that these CYC/TB1-like genes formed a monophyletic clade (SteTBL1) and were subject to strong purifying selection. The shifts of floral symmetry in Stemonaceae do not appear to be correlated with TBL copy number.

De Novo Assembly of Transcriptome and Development of Novel EST-SSR Markers in Rhododendron rex Lévl. through Illumina Sequencing.

Transcriptome sequences generated by next-generation sequencing (NGS) technologies can be utilized to rapidly detect and characterize a large number of gene-based microsatellites from different plants. Rhododendron rex Lévl. is a perennial woody species from the family Ericaceae and an endangered plant with high ornamental value endemic to Southwestern China. Nevertheless, the genetic and genomic information of R. rex remain unknown. In this study, we performed transcriptome sequencing for R. rex leaf samples, and generated large transcript sequences for functional characterization and development gene-associated SSR markers. A total of 164,242 unigenes were assembled and 115,089 (70.07%) unigenes were successfully annotated in public databases. In addition, a total of 15,314 potential EST-SSRs were identified, and the frequency of SSRs in the R. rex unigenes was 9.32%, with an average of one EST-SSR per 5.65 kb. The most abundant type was repeated di-nucleotide (54.63%), followed by mono- (26.03%) and tri-nucleotide (18.51%) repeats. Based on the SSR-containing sequence, 100 primer pairs were randomly selected and synthesized and used for assessment of the polymorphism. Thirty-six primer pairs were polymorphic and revealed polymorphism among 20 individuals from four R. rex populations. A total of 197 alleles were identified, with an average of 5.472 alleles per locus. The Polymorphism Information Content ranged from 0.154 to 0.870, with a mean of 0.482. The newly developed EST-SSR markers exhibited high transferability (58.33-83.33%) among the six subgenera. Thus, these novel EST-SSR markers developed would provide valuable sequence resources for population structure, genetic diversity analysis, and genetic resource assessments of R. rex and its related species.

Development and Application of Transcriptome-Derived Microsatellites in Actinidia eriantha (Actinidiaceae).

Actinidia eriantha Benth. is a diploid perennial woody vine native to China and is recognized as a valuable species for commercial kiwifruit improvement with high levels of ascorbic acid as well as having been used in traditional Chinese medicine. Due to the lack of genomic resources for the species, microsatellite markers for population genetics studies are scarce. In this study, RNASeq was conducted on fruit tissue of A. eriantha, yielding 5,678,129 reads with a total output of 3.41 Gb. De novo assembly yielded 69,783 non-redundant unigenes (41.3 Mb), of which 21,730 were annotated using protein databases. A total of 8,658 EST-SSR loci were identified in 7,495 unigene sequences, for which primer pairs were successfully designed for 3,842 loci (44.4%). Among these, 183 primer pairs were assayed for PCR amplification, yielding 69 with detectable polymorphism in A. eriantha. Additionally, 61 of the 69 polymorphic loci could be successfully amplified in at least one other Actinidia species. Of these, 14 polymorphic loci (mean NA = 6.07 ± 2.30) were randomly selected for assessing levels of genetic diversity and population structure within A. eriantha. Finally, a neighbor-joining tree and Bayesian clustering analysis showed distinct clustering into two groups (K = 2), agreeing with the geographical distributions of these populations. Overall, our results will facilitate further studies of genetic diversity within A. eriantha and will aid in discriminating outlier loci involved in local adaptation.