Genome-Wide Identification of Seven Polyamine Oxidase Genes in Camellia sinensis (L.) and Their Expression Patterns Under Various Abiotic Stresses.

Polyamines (PAs) in plant play a critical role in growth and development and in response to environmental stress. Polyamine oxidase (PAO) is a flavin adenine dinucleotide dependent enzyme that plays a major role in PA catabolism. For the first time, PAO genes in tea plant were screened for the whole genome-wide and seven CsPAO genes were identified, which were named CsPAO1-7. Phylogenetic tree analysis revealed seven CsPAO protein sequences classed into three groups, including clade I, III, and IV. Compared with other plants, the tea plant lacked clade II members. Genetic structure and tissue specific expression analysis showed that there were significant differences among members of the CsPAO gene family. Among members of the CsPAOs family, CsPAO4 and CsPAO5 contain more introns and are highly expressed in various organizations. CsPAO1, CsPAO4, and CsPAO5 genes were cloned and expressed heterologously to verify theirs function. Heat map showed high response of CsPAO5 to drought stress, while CsPAO1 and CsPAO2 were sensitive to changes in nitrogen nutrition. Furthermore, exogenous abscisic acid (ABA) treatment indicated that the expression of most CsPAO genes in roots and leaves was significantly induced. In the root, Spm content increased significantly, while Put and Spd content decreased, suggesting that ABA has great influence on the biosynthesis of PAs. Anaerobic treatment of picked tea leaves showed that the decomposition of PAs was promoted to a certain extent. The above data help to clarify the role of CsPAO in response abiotic and nitrogen nutritional stresses in tea plants, and provide a reference perspective for the potential influence of PAs on the tea processing quality.

Improved hybrid de novo genome assembly and annotation of African wild rice, Oryza longistaminata, from Illumina and PacBio sequencing reads.

African wild rice Oryza longistaminata, one of the eight AA- genome species in the genus Oryza, possesses highly valued traits, such as the rhizomatousness for perennial rice breeding, strong tolerance to biotic and abiotic stresses, and high biomass production on poor soils. To obtain the high-quality reference genome for O. longistaminata we employed a hybrid assembly approach through incorporating Illumina and PacBio sequencing datasets. The final genome assembly comprised only 107 scaffolds and was approximately ∼363.5 Mb, representing ∼92.7% of the estimated African wild rice genome (∼392 Mb). The N50 lengths of the assembled contigs and scaffolds were ∼46.49 Kb and ∼6.83 Mb, indicating ∼3.72-fold and ∼18.8-fold improvement in length compared to the earlier released assembly (∼12.5 Kb and 364 Kb, respectively). Aided with Hi-C data and syntenic relationship with O. sativa, these assembled scaffolds were anchored into 12 pseudo-chromosomes. Genome annotation and comparative genomic analysis reveal that lineage-specific expansion of gene families that respond to biotic- and abiotic stresses are of great potential for mining novel alleles to overcome major diseases and abiotic adaptation in rice breeding programs. This reference genome of African wild rice will greatly enlarge the existing database of rice genome resources and unquestionably form a solid base to understand genomic basis underlying highly valued phenotypic traits and search for novel gene sources in O. longistaminata for the future rice breeding programs.

Transcriptome data reveal conserved patterns of fruiting body development and response to heat stress in the mushroom-forming fungus Flammulina filiformis.

Mushroom-forming fungi are complex multicellular organisms that form the basis of a large industry, yet, our understanding of the mechanisms of mushroom development and its responses to various stresses remains limited. The winter mushroom (Flammulina filiformis) is cultivated at a large commercial scale in East Asia and is a species with a preference for low temperatures. This study investigated fruiting body development in F. filiformis by comparing transcriptomes of 4 developmental stages, and compared the developmental genes to a 200-genome dataset to identify conserved genes involved in fruiting body development, and examined the response of heat sensitive and -resistant strains to heat stress. Our data revealed widely conserved genes involved in primordium development of F. filiformis, many of which originated before the emergence of the Agaricomycetes, indicating co-option for complex multicellularity during evolution. We also revealed several notable fruiting-specific genes, including the genes with conserved stipe-specific expression patterns and the others which related to sexual development, water absorption, basidium formation and sporulation, among others. Comparative analysis revealed that heat stress induced more genes in the heat resistant strain (M1) than in the heat sensitive one (XR). Of particular importance are the hsp70, hsp90 and fes1 genes, which may facilitate the adjustment to heat stress in the early stages of fruiting body development. These data highlighted novel genes involved in complex multicellular development in fungi and aid further studies on gene function and efforts to improve the productivity and heat tolerance in mushroom-forming fungi.

Draft genomes of two outcrossing wild rice, Oryza rufipogon and O. longistaminata, reveal genomic features associated with mating-system evolution.

Oryza rufipogon and O. longistaminata are important wild relatives of cultivated rice, harboring a promising source of novel genes for rice breeding programs. Here, we present de novo assembled draft genomes and annotation of O. rufipogon and O. longistaminata. Our analysis reveals a considerable number of lineage-specific gene families associated with the self-incompatibility (SI) and formation of reproductive separation. We show how lineage-specific expansion or contraction of gene families with functional enrichment of the recognition of pollen, thus enlightening their reproductive diversification. We documented a large number of lineage-specific gene families enriched in salt stress, antifungal response, and disease resistance. Our comparative analysis further shows a genome-wide expansion of genes encoding NBS-LRR proteins in these two outcrossing wild species in contrast to six other selfing rice species. Conserved noncoding sequences (CNSs) in the two wild rice genomes rapidly evolve relative to selfing rice species, resulting in the reduction of genomic variation owing to shifts of mating systems. We find that numerous genes related to these rapidly evolving CNSs are enriched in reproductive structure development, flower development, and postembryonic development, which may associate with SI in O. rufipogon and O. longistaminata.

SMRT sequencing of the Oryza rufipogon genome reveals the genomic basis of rice adaptation.

Asian cultivated rice is believed to have been domesticated from a wild progenitor, Oryza rufipogon, offering promising sources of alleles for world rice improvement. Here we first present a high-quality chromosome-scale genome of the typical O. rufipogon. Comparative genomic analyses of O. sativa and its two wild progenitors, O. nivara and O. rufipogon, identified many dispensable genes functionally enriched in the reproductive process. We detected millions of genomic variants, of which large-effect mutations could affect agronomically relevant traits. We demonstrate how lineage-specific expansion of gene families may have contributed to the formation of reproduction isolation. We document thousands of genes with signatures of positive selection that are mainly involved in the reproduction and response to biotic- and abiotic stresses. We show that selection pressures may serve as forces to govern substantial genomic alterations that form the genetic basis of rapid evolution of mating and reproductive systems under diverse habitats.

Tea plant genomics: achievements, challenges and perspectives.

Tea is among the world's most widely consumed non-alcoholic beverages and possesses enormous economic, health, and cultural values. It is produced from the cured leaves of tea plants, which are important evergreen crops globally cultivated in over 50 countries. Along with recent innovations and advances in biotechnologies, great progress in tea plant genomics and genetics has been achieved, which has facilitated our understanding of the molecular mechanisms of tea quality and the evolution of the tea plant genome. In this review, we briefly summarize the achievements of the past two decades, which primarily include diverse genome and transcriptome sequencing projects, gene discovery and regulation studies, investigation of the epigenetics and noncoding RNAs, origin and domestication, phylogenetics and germplasm utilization of tea plant as well as newly developed tools/platforms. We also present perspectives and possible challenges for future functional genomic studies that will contribute to the acceleration of breeding programs in tea plants.

Optimized sequencing depth and de novo assembler for deeply reconstructing the transcriptome of the tea plant, an economically important plant species.

BACKGROUND: Tea is the oldest and among the world's most popular non-alcoholic beverages, which has important economic, health and cultural values. Tea is commonly produced from the leaves of tea plants (Camellia sinensis), which belong to the genus Camellia of family Theaceae. In the last decade, many studies have generated the transcriptomes of tea plants at different developmental stages or under abiotic and/or biotic stresses to investigate the genetic basis of secondary metabolites that determine tea quality. However, these results exhibited large differences, particularly in the total number of reconstructed transcripts and the quality of the assembled transcriptomes. These differences largely result from limited knowledge regarding the optimized sequencing depth and assembler for transcriptome assembly of structurally complex plant species genomes. RESULTS: We employed different amounts of RNA-sequencing data, ranging from 4 to 84 Gb, to assemble the tea plant transcriptome using five well-known and representative transcript assemblers. Although the total number of assembled transcripts increased with increasing sequencing data, the proportion of unassembled transcripts became saturated as revealed by plant BUSCO datasets. Among the five representative assemblers, the Bridger package shows the best performance in both assembly completeness and accuracy as evaluated by the BUSCO datasets and genome alignment. In addition, we showed that Bridger and BinPacker harbored the shortest runtimes followed by SOAPdenovo and Trans-ABySS. CONCLUSIONS: The present study compares the performance of five representative transcript assemblers and investigates the key factors that affect the assembly quality of the transcriptome of the tea plants. This study will be of significance in helping the tea research community obtain better sequencing and assembly of tea plant transcriptomes under conditions of interest and may thus help to answer major biological questions currently facing the tea industry.

Genome Sequences Provide Insights into the Reticulate Origin and Unique Traits of Woody Bamboos.

Polyploidization is a major driver of speciation and its importance to plant evolution has been well recognized. Bamboos comprise one diploid herbaceous and three polyploid woody lineages, and are members of the only major subfamily in grasses that diversified in forests, with the woody members having a tree-like lignified culm. In this study, we generated four draft genome assemblies of major bamboo lineages with three different ploidy levels (diploid, tetraploid, and hexaploid). We also constructed a high-density genetic linkage map for a hexaploid species of bamboo, and used a linkage-map-based strategy for genome assembly and identification of subgenomes in polyploids. Further phylogenomic analyses using a large dataset of syntenic genes with expected copies based on ploidy levels revealed that woody bamboos originated subsequent to the divergence of the herbaceous bamboo lineage, and experienced complex reticulate evolution through three independent allopolyploid events involving four extinct diploid ancestors. A shared but distinct subgenome was identified in all polyploid forms, and the progenitor of this subgenome could have been critical in ancient polyploidizations and the origin of woody bamboos. Important genetic clues to the unique flowering behavior and woody trait in bamboos were also found. Taken together, our study provides significant insights into ancient reticulate evolution at the subgenome level in the absence of extant donor species, and offers a potential model scenario for broad-scale study of angiosperm origination by allopolyploidization.

Tea Plant Information Archive: a comprehensive genomics and bioinformatics platform for tea plant.

Tea is the world's widely consumed nonalcohol beverage with essential economic and health benefits. Confronted with the increasing large-scale omics-data set particularly the genome sequence released in tea plant, the construction of a comprehensive knowledgebase is urgently needed to facilitate the utilization of these data sets towards molecular breeding. We hereby present the first integrative and specially designed web-accessible database, Tea Plant Information Archive (TPIA; http://tpia.teaplant.org). The current release of TPIA employs the comprehensively annotated tea plant genome as framework and incorporates with abundant well-organized transcriptomes, gene expressions (across species, tissues and stresses), orthologs and characteristic metabolites determining tea quality. It also hosts massive transcription factors, polymorphic simple sequence repeats, single nucleotide polymorphisms, correlations, manually curated functional genes and globally collected germplasm information. A variety of versatile analytic tools (e.g. JBrowse, blast, enrichment analysis, etc.) are established helping users to perform further comparative, evolutionary and functional analysis. We show a case application of TPIA that provides novel and interesting insights into the phytochemical content variation of section Thea of genus Camellia under a well-resolved phylogenetic framework. The constructed knowledgebase of tea plant will serve as a central gateway for global tea community to better understand the tea plant biology that largely benefits the whole tea industry.

De novo transcriptome sequencing of Camellia sasanqua and the analysis of major candidate genes related to floral traits.

Camellia sasanqua is one of the most famous horticultural plants in Camellia (Theaceae) due to its aesthetic appeal as landscape plant. Knowledge regarding the genetic basis of flowering time, floral aroma and color in C. sasanqua is limited, but is essential to breed new varieties with desired floral traits. Here, we described the de novo transcriptome of young leaves, flower buds and flowers of C. sasanqua. A total of 60,127 unigenes were functionally annotated based on the sequence similarity. After analysis, we found that two floral integrator genes, SOC1 and AP1, in flowering time pathway showed evidence of gene family expansion. Compared with 1-deoxy-D-xylulose-5-phosphate pathway, some genes in the mevalonate pathway were most highly expressed, suggesting that this might represent the major pathway for terpenoid biosynthesis related to floral aroma in C. sasanqua. In flavonoid biosynthesis pathway, PAL, CHI, DFR and ANS showing significantly higher expression levels in flowers and flower buds might have important role in regulation of floral color. The top five most transcription factors (TFs) families in C. sasanqua transcriptome were MYB, MIKC, C3H, FAR1 and HD-ZIP, many of which have a direct relationship with floral traits. In addition, we also identified 33,540 simple sequence repeats (SSRs) in the C. sasanqua transcriptome. Collectively, the C. sasanqua transcriptome dataset generated from this study along with the SSR markers provide a new resource for the identification of novel regulatory transcripts and will accelerate the genetic improvement of C. sasanqua breeding programs.

Mixed-reproductive strategies, competitive mating-type distribution and life cycle of fourteen black morel species.

Morchella species are well known world-round as popular and prized edible fungi due to their unique culinary flavor. Recently, several species have been successfully cultivated in China. However, their reproductive modes are still unknown, and their basic biology needs to be elucidated. Here, we use the morel genome information to investigate mating systems and life cycles of fourteen black morel species. Mating type-specific primers were developed to screen and genotype ascospores, hymenia and stipes from 223 ascocarps of the 14 species from Asia and Europe. Our data indicated that they are all heterothallic and their life cycles are predominantly haploid, but sterile haploid fruiting also exists. Ascospores in all species are mostly haploid, homokaryotic, and multinuclear, whereas aborted ascospores without any nuclei were also detected. Interestingly, we monitored divergent spatial distribution of both mating types in natural morel populations and cultivated sites, where the fertile tissue of fruiting bodies usually harbored both mating types, whereas sterile tissue of wild morels constantly had one MAT allele, while the sterile tissue of cultivated strains always exhibited both MAT alleles. Furthermore, MAT1-1-1 was detected significantly more commonly than MAT1-2-1 in natural populations, which strongly suggested a competitive advantage for MAT1-1 strains.

The Tea Tree Genome Provides Insights into Tea Flavor and Independent Evolution of Caffeine Biosynthesis.

Tea is the world's oldest and most popular caffeine-containing beverage with immense economic, medicinal, and cultural importance. Here, we present the first high-quality nucleotide sequence of the repeat-rich (80.9%), 3.02-Gb genome of the cultivated tea tree Camellia sinensis. We show that an extraordinarily large genome size of tea tree is resulted from the slow, steady, and long-term amplification of a few LTR retrotransposon families. In addition to a recent whole-genome duplication event, lineage-specific expansions of genes associated with flavonoid metabolic biosynthesis were discovered, which enhance catechin production, terpene enzyme activation, and stress tolerance, important features for tea flavor and adaptation. We demonstrate an independent and rapid evolution of the tea caffeine synthesis pathway relative to cacao and coffee. A comparative study among 25 Camellia species revealed that higher expression levels of most flavonoid- and caffeine- but not theanine-related genes contribute to the increased production of catechins and caffeine and thus enhance tea-processing suitability and tea quality. These novel findings pave the way for further metabolomic and functional genomic refinement of characteristic biosynthesis pathways and will help develop a more diversified set of tea flavors that would eventually satisfy and attract more tea drinkers worldwide.

The caterpillar fungus, Ophiocordyceps sinensis, genome provides insights into highland adaptation of fungal pathogenicity.

To understand the potential genetic basis of highland adaptation of fungal pathogenicity, we present here the ~116 Mb de novo assembled high-quality genome of Ophiocordyceps sinensis endemic to the Qinghai-Tibetan Plateau. Compared with other plain-dwelling fungi, we find about 3.4-fold inflation of the O. sinensis genome due to a rapid amplification of long terminal repeat retrotransposons that occurred ~38 million years ago in concert with the uplift of the plateau. We also observe massive removal of thousands of genes related to the transport process and energy metabolism. O. sinensis displays considerable lineage-specific expansion of gene families functionally enriched in the adaptability of low-temperature of cold tolerance, fungal pathogenicity and specialized host infection. We detect signals of positive selection for genes involved in peroxidase and hypoxia to enable its highland adaptation. Resequencing and analyzing 31 whole genomes of O. sinensis, representing nearly all of its geographic range, exhibits latitude-based population divergence and nature selection for population inhabitation towards higher altitudes on the Qinghai-Tibetan Plateau.

Full transcription of the chloroplast genome in photosynthetic eukaryotes.

Prokaryotes possess a simple genome transcription system that is different from that of eukaryotes. In chloroplasts (plastids), it is believed that the prokaryotic gene transcription features govern genome transcription. However, the polycistronic operon transcription model cannot account for all the chloroplast genome (plastome) transcription products at whole-genome level, especially regarding various RNA isoforms. By systematically analyzing transcriptomes of plastids of algae and higher plants, and cyanobacteria, we find that the entire plastome is transcribed in photosynthetic green plants, and that this pattern originated from prokaryotic cyanobacteria - ancestor of the chloroplast genomes that diverged about 1 billion years ago. We propose a multiple arrangement transcription model that multiple transcription initiations and terminations combine haphazardly to accomplish the genome transcription followed by subsequent RNA processing events, which explains the full chloroplast genome transcription phenomenon and numerous functional and/or aberrant pre-RNAs. Our findings indicate a complex prokaryotic genome regulation when processing primary transcripts.

Transcriptome Analysis Identifies Candidate Genes Related to Triacylglycerol and Pigment Biosynthesis and Photoperiodic Flowering in the Ornamental and Oil-Producing Plant, Camellia reticulata (Theaceae).

Camellia reticulata, which is native to Southwest China, is famous for its ornamental flowers and high-quality seed oil. However, the lack of genomic information for this species has largely hampered our understanding of its key pathways related to oil production, photoperiodic flowering process and pigment biosynthesis. Here, we first sequenced and characterized the transcriptome of a diploid C. reticulata in an attempt to identify genes potentially involved in triacylglycerol biosynthesis (TAGBS), photoperiodic flowering, flavonoid biosynthesis (FlaBS), carotenoid biosynthesis (CrtBS) pathways. De novo assembly of the transcriptome provided a catalog of 141,460 unigenes with a total length of ~96.1 million nucleotides (Mnt) and an N50 of 1080 nt. Of them, 22,229 unigenes were defined as differentially expressed genes (DEGs) across five sequenced tissues. A large number of annotated genes in C. reticulata were found to have been duplicated, and differential expression patterns of these duplicated genes were commonly observed across tissues, such as the differential expression of SOC1_a, SOC1_b, and SOC1_c in the photoperiodic flowering pathway. Up-regulation of SAD_a and FATA genes and down-regulation of FAD2_a gene in the TAGBS pathway in seeds may be relevant to the ratio of monounsaturated fatty acid (MUFAs) to polyunsaturated fatty acid (PUFAs) in seed oil. MYBF1, a transcription regulator gene of the FlaBS pathway, was found with great sequence variation and alteration of expression patterns, probably resulting in functionally evolutionary differentiation in C. reticulata. MYBA1_a and some anthocyanin-specific biosynthetic genes in the FlaBS pathway were highly expressed in both flower buds and flowers, suggesting important roles of anthocyanin biosynthesis in flower development. Besides, a total of 40,823 expressed sequence tag simple sequence repeats (EST-SSRs) were identified in the C. reticulata transcriptome, providing valuable marker resources for further basic and applied researches on this economically important Camellia plant.

The complete mitochondrial genome of western painted turtle, Chrysemys picta bellii (Chrysemys, Emydidae).

In this study, we report the complete mitochondrial genome sequence of western painted turtle, Chrysemys picta bellii. The genome is found to be 16,875 bp in length and has a base composition of A (34.4%), G (13.0%), C (26.0%), and T (26.6%). Similar to other turtles, it contains a typically conserved structure including 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, and 1 control region (D-loop). Most of the genes are encoded on H-strand, except for the eight tRNA and ND6 genes. All protein-coding genes start with an ATN codon except for COX1 and ND4, which initiate with GTG instead, and terminate with the typical stop codon (TAA/TAG) or a single T (T-) or an unexpected codon of AGG. The complete mitochondrial genome sequence provided here would be useful for further phylogenetic analysis and conservation genetic studies in C. p. bellii.

Autotetraploid rice methylome analysis reveals methylation variation of transposable elements and their effects on gene expression.

Polyploidy, or whole-genome duplication (WGD), serves as a key innovation in plant evolution and is an important genomic feature for all eukaryotes. Neopolyploids have to overcome difficulties in meiosis, genomic alterations, changes of gene expression, and epigenomic reorganization. However, the underlying mechanisms for these processes are poorly understood. One of the most interesting aspects is that genome doubling events increase the dosage of all genes. Unlike allopolyploids entangled by both hybridization and polyploidization, autopolyploids, especially artificial lines, in relatively uniform genetic background offer a model system to understand mechanisms of genome-dosage effects. To investigate DNA methylation effects in response to WGD rather than hybridization, we produced autotetraploid rice with its diploid donor, Oryza sativa ssp. indica cv. Aijiaonante, both of which were independently self-pollinated over 48 generations, and generated and compared their comprehensive transcriptomes, base pair-resolution methylomes, and siRNAomes. DNA methylation variation of transposable elements (TEs) was observed as widespread in autotetraploid rice, in which hypermethylation of class II DNA transposons was predominantly noted in CHG and CHH contexts. This was accompanied by changes of 24-nt siRNA abundance, indicating the role of the RNA-directed DNA methylation pathway. Our results showed that the increased methylation state of class II TEs may suppress the expression of neighboring genes in autotetraploid rice that has obtained double alleles, leading to no significant differences in transcriptome alterations for most genes from its diploid donor. Collectively, our findings suggest that chromosome doubling induces methylation variation in TEs that affect gene expression and may become a "genome shock" response factor to help neoautopolyploids adapt to genome-dosage effects.

De novo transcriptome assembly of the wild relative of tea tree (Camellia taliensis) and comparative analysis with tea transcriptome identified putative genes associated with tea quality and stress response.

BACKGROUND: Camellia taliensis is one of the most important wild relatives of cultivated tea tree, C. sinensis. The species extensively occupies mountainous habitats representing a wide-range abiotic tolerance and biotic resistance and thus harbors valuable gene resources that may greatly benefit genetic improvement of cultivated tea tree. However, owning to a large genome size of ~3 Gb and structurally complex genome, there are fairly limited genetic information and particularly few genomic resources publicly available for this species. To better understand the key pathways determining tea flavor and enhance tea tree breeding programs, we performed a high-throughput transcriptome sequencing for C. taliensis. RESULTS: In this study, approximate 241.5 million high-quality paired-end reads, accounting for ~24 Gb of sequence data, were generated from tender shoots, young leaves, flower buds and flowers using Illumina HiSeq 2000 platform. De novo assembly with further processing and filtering yielded a set of 67,923 transcripts with an average length of 685 bp and an N50 of 995 bp. Based on sequence similarity searches against public databases, a total of 39,475 transcripts were annotated with gene descriptions, conserved protein domains or gene ontology (GO) terms. Candidate genes for major metabolic pathways involved in tea quality were identified and experimentally validated using RT-qPCR. Further gene expression profiles showed that they are differentially regulated at different developmental stages. To gain insights into the evolution of these genes, we aligned them to the previously cloned orthologous genes in C. sinensis, and found that considerable nucleotide variation within several genes involved in important secondary metabolic biosynthesis pathways, of which flavone synthase II gene (FNSII) is the most variable between these two species. Moreover, comparative analyses revealed that C. taliensis shows a remarkable expansion of LEA genes, compared to C. sinensis, which might contribute to the observed stronger stress resistance of C. taliensis. CONCLUSION: We reported the first large-coverage transcriptome datasets for C. taliensis using the next-generation sequencing technology. Such comprehensive EST datasets provide an unprecedented opportunity for identifying genes involved in several major metabolic pathways and will accelerate functional genomic studies and genetic improvement efforts of tea trees in the future.

CandiSSR: An Efficient Pipeline used for Identifying Candidate Polymorphic SSRs Based on Multiple Assembled Sequences.

Simple sequence repeats (SSRs), also known as microsatellites, are ubiquitous short tandem duplications commonly found in genomes and/or transcriptomes of diverse organisms. They represent one of the most powerful molecular markers for genetic analysis and breeding programs because of their high mutation rate and neutral evolution. However, traditionally experimental screening of the SSR polymorphic status and their subsequent applicability to genetic studies are extremely labor-intensive and time-consuming. Thankfully, the recently decreased costs of next generation sequencing and increasing availability of large genome and/or transcriptome sequences have provided an excellent opportunity and sources for large-scale mining this type of molecular markers. However, current tools are limited. Thus we here developed a new pipeline, CandiSSR, to identify candidate polymorphic SSRs (PolySSRs) based on the multiple assembled sequences. The pipeline allows users to identify putative PolySSRs not only from the transcriptome datasets but also from multiple assembled genome sequences. In addition, two confidence metrics including standard deviation and missing rate of the SSR repetitions are provided to systematically assess the feasibility of the detected PolySSRs for subsequent application to genetic characterization. Meanwhile, primer pairs for each identified PolySSR are also automatically designed and further evaluated by the global sequence similarities of the primer-binding region, ensuring the successful rate of the marker development. Screening rice genomes with CandiSSR and subsequent experimental validation showed an accuracy rate of over 90%. Besides, the application of CandiSSR has successfully identified a large number of PolySSRs in the Arabidopsis genomes and Camellia transcriptomes. CandiSSR and the PolySSR marker sources are publicly available at: http://www.plantkingdomgdb.com/CandiSSR/index.html.