Insights into the genome of the 'Loco' Concholepas concholepas (Gastropoda: Muricidae) from low-coverage short-read sequencing: genome size, ploidy, transposable elements, nuclear RNA gene operon, mitochondrial genome, and phylogenetic placement in the family Muricidae.

BACKGROUND: The Peruvian 'chanque' or Chilean 'loco' Concholepas concholepas is an economically, ecologically, and culturally important muricid gastropod heavily exploited by artisanal fisheries in the temperate southeastern Pacific Ocean. In this study, we have profited from a set of bioinformatics tools to recover important biological information of C. concholepas from low-coverage short-read NGS datasets. Specifically, we calculated the size of the nuclear genome, ploidy, and estimated transposable elements content using an in silico k-mer approach, we discovered, annotated, and quantified those transposable elements, we assembled and annotated the 45S rDNA RNA operon and mitochondrial genome, and we confirmed the phylogenetic position of C. concholepas within the muricid subfamily Rapaninae based on translated protein coding genes. RESULTS: Using a k-mer approach, the haploid genome size estimated for the predicted diploid genome of C. concholepas varied between 1.83 Gbp (with kmer = 24) and 2.32 Gbp (with kmer = 36). Between half and two thirds of the nuclear genome of C. concholepas was composed of transposable elements. The most common transposable elements were classified as Long Interspersed Nuclear Elements and Short Interspersed Nuclear Elements, which were more abundant than DNA transposons, simple repeats, and Long Terminal Repeats. Less abundant repeat elements included Helitron mobile elements, 45S rRNA DNA, and Satellite DNA, among a few others.The 45S rRNA DNA operon of C. concholepas that encodes for the ssrRNA, 5.8S rRNA, and lsrRNA genes was assembled into a single contig 8,090 bp long. The assembled mitochondrial genome of C. concholepas is 15,449 bp long and encodes 13 protein coding genes, two ribosomal genes, and 22 transfer RNAs. CONCLUSION: The information gained by this study will inform the assembly of a high quality nuclear genome for C. concholepas and will support bioprospecting and biomonitoring using environmental DNA to advance development of conservation and management plans in this overexploited marine snail.

Whole-Genome Survey Analyses of Five Goby Species Provide Insights into Their Genetic Evolution and Invasion-Related Genes.

As one of the most abundant groups in marine fish families, Gobiidae fish are important fishery resources in China, and some are also invasive species in certain regions worldwide. However, the phylogenetic relationships of Gobiidae fish remain ambiguous, and the study of their invasion-related genes is still scarce. This study used high-throughput sequencing technology to conduct a whole-genome survey of five Gobiidae fish species: Acanthogobius flavimanus, Acanthogobius stigmothonus, Favonigobius gymnauchen, Ctenotrypauchen microcephalus, and Tridentiger barbatus. De novo assembly of five fish genomes was performed, and genomic traits were compared through K-mer analysis. Among the five Gobiidae fish genomes, F. gymnauchen had the largest genome size (1601.98 Mb) and the highest heterozygosity (1.56%) and repeat rates (59.83%). Phylogenetic studies showed that A. flavimanus was most closely linked to A. stigmothonus, while Apogonidae and Gobiidae were closely related families. PSMC analysis revealed that C. microcephalus experienced a notable population expansion than the other four fish species in the Early Holocene. By using the KOG, GO, and KEGG databases to annotate single-copy genes, the annotated genes of the five fish were mainly classified as "signal transduction mechanisms", "cellular process", "cellular anatomical entity", and "translation". Acanthogobius flavimanus, A. stigmothonus, and T. barbatus had more genes classified as "response to stimulus" and "localization", which may have played an important role in their invasive processes. Our study also provides valuable material about Gobiidae fish genomics and genetic evolution.

A first insight into the genomic background of Ilex pubescens (Aquifoliaceae) by flow cytometry and genome survey sequencing.

BACKGROUND: Ilex pubescens is an important traditional Chinese medicinal plant with many naturally occurring compounds and multiple pharmacological effects. However, the lack of reference genomic information has led to tardiness in molecular biology research and breeding programs of this plant. RESULTS: To obtain knowledge on the genomic information of I. pubescens, a genome survey was performed for the first time by next generation sequencing (NGS) together with genome size estimation using flow cytometry. The whole genome survey of I. pubescens generated 46.472 Gb of sequence data with approximately 82.2 × coverage. K-mer analysis indicated that I. pubescens has a small genome of approximately 553 Mb with 1.93% heterozygosity rate and 39.1% repeat rate. Meanwhile, the genome size was estimated to be 722 Mb using flow cytometry, which was possibly more precise for assessment of genome size than k-mer analysis. A total of 45.842 Gb clean reads were assembled into 808,938 scaffolds with a relatively short N50 of 760 bp. The average guanine and cytosine (GC) content was 37.52%. In total, 197,429 microsatellite motifs were detected with a frequency of 2.8 kb, among which mononucleotide motifs were the most abundant (up to 62.47% of the total microsatellite motifs), followed by dinucleotide and trinucleotide motifs. CONCLUSION: In summary, the genome of I. pubescens is small but complex with a high level of heterozygosity. Even though not successfully applied for estimation of genome size due to its complex genome, the survey sequences will help to design whole genome sequencing strategies and provide genetic information support for resource protection, genetic diversity analysis, genetic improvement and artificial breeding of I. pubescens.

The First Genome-Wide Survey of Shortbelly Eel (Dysomma anguillare Barnard, 1923) to Provide Genomic Characteristics, Microsatellite Markers and Complete Mitogenome Information.

Dysomma anguillare is a demersal eel widespread distributing in tropical waters of the Indo-West Pacific and Atlantic. As an important component of the coastal fishery and marine ecosystem, the lack of genomic information for this species severely restricts the progress of relevant researches. In this study, the abecedarian genome-wide characteristics and phylogenetic relationships analyses were carried out based on next-generation sequencing (NGS) platform. The revised genome size was approximately 1 310 Mb, with the largest scaffold length reaching 23 878 bp through K-mer (K = 17) method. The heterozygosity, repetitive rate and average GC content were about 0.94%, 51.93% and 42.23%, respectively. A total of 1 160 104 microsatellite motifs were identified from the de novo assembled genome of D. anguillare, in which dinucleotide repeats accounted for the largest proportion (592 234, 51.05%), the highest occurrence frequency (14.58%) as well as the largest relative abundance (379.27/Mb). The high-polymorphic and moderate-polymorphic loci composed around 73% of the total single sequence repeats (SSRs), showing a latent capacity for subsequent population genetic structure and genetic diversity appraisal researches. Another byproduct of whole-genome sequencing, the double-stranded and circular mitogenome (16 690 bp) was assembled to investigate the evolutionary relationships of D. anguillare. The phylogenic tree constructed with maximum likelihood (ML) method showed that D. anguillare was closely related to Synaphobranchidae species, and the molecular systematic results further supported classical taxonomy status of D. anguillare.

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

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

Genome-Wide Survey Reveals the Microsatellite Characteristics and Phylogenetic Relationships of Harpadon nehereus.

Harpadon nehereus forms one of the most important commercial fisheries along the Bay of Bengal and the southeast coast of China. In this study, the genome-wide survey dataset first produced using next-generation sequencing (NGS) was used to provide general information on the genome size, heterozygosity and repeat sequence ratio of H. nehereus. About 68.74 GB of high-quality sequence data were obtained in total and the genome size was estimated to be 1315 Mb with the 17-mer frequency distribution. The sequence repeat ratio and heterozygosity were calculated to be 52.49% and 0.67%, respectively. A total of 1,027,651 microsatellite motifs were identified and dinucleotide repeat was the most dominant simple sequence repeat (SSR) motif with a frequency of 54.35%. As a by-product of whole genome sequencing, the mitochondrial genome is a powerful tool to investigate the evolutionary relationships between H. nehereus and its relatives. The maximum likelihood (ML) phylogenetic tree was constructed according to the concatenated matrix of amino acids translated from the 13 protein-coding genes (PCGs). Monophyly of two species of the genus Harpadon was revealed in the present study and they formed a monophyletic clade with Saurida with a high bootstrap value of 100%. The results would help to push back the frontiers of genomics and open the doors of molecular diversity as well as conservation genetics studies on this species.

Comprehensive Draft Genome Analyses of Three Rockfishes (Scorpaeniformes, Sebastiscus) via Genome Survey Sequencing.

Sebastiscus species, marine rockfishes, are of essential economic value. However, the genomic data of this genus is lacking and incomplete. Here, whole genome sequencing of all species of Sebastiscus was conducted to provide fundamental genomic information. The genome sizes were estimated to be 802.49 Mb (S. albofasciatus), 786.79 Mb (S. tertius), and 776.00 Mb (S. marmoratus) by using k-mer analyses. The draft genome sequences were initially assembled, and genome-wide microsatellite motifs were identified. The heterozygosity, repeat ratios, and numbers of microsatellite motifs all suggested possibly that S. tertius is more closely related to S. albofasciatus than S. marmoratus at the genetic level. Moreover, the complete mitochondrial genome sequences were assembled from the whole genome data and the phylogenetic analyses genetically supported the validation of Sebastiscus species. This study provides an important genome resource for further studies of Sebastiscus species.

The karyotype, genome survey, and assembly of Mud artemisia (Artemisia selengensis).

BACKGROUND: Artemisia selengensis is traditional Chinese medicine and phytochemical analysis indicated that A. selengensis contains essential oils, fatty acids and phenolic acids. The lack of reference genomic information may lead to tardiness in molecular biology research of A. selengensis. METHOD AND RESULTS: Karyotype analysis, genome survey, and genome assembly was employed to acquire information on the genome structure of A. selengensis. The chromosome number is 2n = 2x = 36, karyotype formula is 28 m + 8Sm, karyotype asymmetry coefficient is 58.8%, and karyotypes were symmetric to Stebbins' type 2A. Besides, the flow cytometry findings reported that the mean peak value of fluorescent intensity is 1,170,677, 2C DNA content is 12 pg and the genome size was estimated to be approximately 5.87 Gb. Furthermore, the genome survey generates 341,478,078 clean reads, unfortunately, after K-mer analysis, no significant peak can be observed, the heterozygosity, repetitive rate and genome size was unable to estimated. It is speculated that this phenomenon might be due to the complexity of genome structure. 37,266 contigs are preliminary assembled with Oxford Nanopore Technology (ONT) sequencing, totaling 804 Mb and GC content was 34.08%. The total length is 804,475,881 bp, N50 is 29,624 bp, and the largest contig length is 239,792 bp. CONCLUSION: This study reveals the preliminary information of genome size of A. selengensis. These findings may provide supportive information for sequencing and assembly of whole-genome sequencing and encourage the progress of functional gene discovery, genetic improvement, evolutionary study, and structural studies of A. selengensis.

Genome survey sequencing and genetic diversity of cultivated Akebia trifoliata assessed via phenotypes and SSR markers.

Akebia trifoliata (Lardizabalaceae) is an important medicinal plant with multiple pharmacological effects. However, the lack of genomic information had limited the further excavation and utilization of this plant. An initial survey of the genome A. trifoliata was performed by next-generation sequencing, and then the genome size was inferred by flow cytometry. The whole genome survey of A. trifoliata generated 61.90 Gb of sequence data with approximately 95.51 × coverage. The genome size, heterozygosity and GC content obtained by k-mer analysis were almost 648.07 Mb, 0.72% and 36.11%, respectively. The genome size calculated by flow cytometry was 685.77 Mb, which was consistent with the results of genome survey. A total of 851,957 simple sequence repeats (SSR) were identified in the A. trifoliata genome. Twenty-eight phenotypic traits and thirty pairs of SSR primers were selected for the analysis of the genetic diversity of 43 accessions of cultivated A. trifoliata. The results showed that 216 bands were generated by 30 pairs of SSR primers, of which 189 (87.5%) were polymorphic. In addition, the phenotypes and SSR markers were used for cluster analysis of 43 cultivated accessions. The results of the two clustering methods were partially consistent. The genome survey of A. trifoliata demonstrated that the genome size of this plant was about 648.07 Mb. In the present study, the size and characteristics of the genome of A. trifoliata were reported for the first time, which greatly enriched the genomic resources of A. trifoliata for the further research and utilization.

Whole genome survey and genetic markers development of crocodile flathead Cociella crocodilus.

Flatheads in family Platycephalidae are ecologically and commercially important marine fish species in the Indo-West Pacific. Due to similar morphological characters, the taxonomy and phylogenetics of flatheads are in confusion. Studies on phylogenetics and molecular marker development are required to discriminate congeners of flatheads. In the present study, we performed whole genome survey sequencing of crocodile flathead Cociella crocodilus to provide genomic information and genetic markers of this species. In total, 54.03 Gb of clean genomic data were generated. The genome size was estimated to be 732.99 Mb with the heterozygosity ratio of 0.73% and the repeat sequence ratio of 33.48%. The preliminary assembled genome sequences were 794.07 Mb with contig N50 of 1504 bp. We detected 2 624 875 genome-wide SNPs with transition/transversion ratio of 1.422. A total of 313 842 microsatellite motifs were identified, most of which were dinucleotide motifs with a frequency of 74.89%. In addition, we assembled the complete mitogenome of C. crocodilus and subsequent phylogenetic analysis were performed. Phylogenetic analyses revealed numbers of polyphyletic groups in family Platycephalidae. The reported genomic data and genetic markers in our study should be useful in further phylogeny and phylogenomics studies of flathead species.

Comprehensive whole genome survey analyses of male and female brown-spotted flathead fish Platycephalus sp.1.

The flathead fish Platycephalus sp.1 is an ecologically and commercially important marine fish in the northwestern Pacific with notable sexual differences in growth and development. Yet the genomic data of this species is lacking. In the present study, whole genome sequencing of two individuals (one male and one female) of Platycephalus sp.1 were conducted to provide fundamental genomic information. The genome sizes were estimated to be 674.96 Mb (male) and 684.15 Mb (female) by using k-mer analyses. The heterozygosity and repeat ratios suggested possible male heterogamety of this species. The draft genome sequences were initially assembled and genome-wide microsatellite motifs were identified. Besides, the complete mitochondrial genome sequences were assembled and the phylogenetic analyses genetically supported the validation of Platycephalus sp.1. The reported genomic data and genetic markers in this study could be useful in future comparative genomics and evolutionary biology studies.

Genome survey and development of polymorphic microsatellite loci for Sillago sihama based on Illumina sequencing technology.

In this study, we first conducted a genome survey assay for Sillago sihama by Illumina sequencing platform, and then developed 15 polymorphic microsatellite loci in a wild population. A total of 129.46 Gb raw data were obtained, of which 115.07 Gb were clean data, with a sequencing depth of 179.3-folds. This genome was estimated to be 522.6 Mb in size, with the heterozygosity, repeat content and GC content being 0.63%, 21% and 44%. A total of 630,028 microsatellites were identified from the genome, of which, dinucleotide repeat was the most abundant (56.80%), followed by mononucleotide repeat (30.23%). Furthermore, 60 pairs of primers were designed and synthesized based on microsatellite sequences, of which 15 were polymorphic in a wild population. A total of 91 alleles were found, with an average of 6.07 per locus. Number of alleles, observed and expected heterozygosity per locus ranged from two to 13, from 0.250 to 0.862, and from 0.396 to 0.901, respectively. Twelve loci were highly informative (PIC > 0.5), and the others were medium informative (0.25 < PIC < 0.5). Seven loci deviated from Hardy-Weinberg equilibrium after Bonferroni correction (P < 0.0033). No significant linkage disequilibrium was detected between loci pairs. This study provided a large number of genomic resources and 15 polymorphic microsatellite loci that should be helpful for the further genetic studies in S. sihama.

The First Genome Survey of the Antarctic Krill (Euphausia superba) Provides a Valuable Genetic Resource for Polar Biomedical Research.

The world-famous Antarctic krill (Euphausia superba) plays a fundamental role in the Antarctic food chain. It resides in cold environments with the most abundant biomass to support the Antarctic ecology and fisheries. Here, we performed the first genome survey of the Antarctic krill, with genomic evidence for its estimated genome size of 42.1 gigabases (Gb). Such a large genome, however, is beyond our present capability to obtain a good assembly, although our sequencing data are a valuable genetic resource for subsequent polar biomedical research. We extracted 13 typical protein-coding gene sequences of the mitochondrial genome and analyzed simple sequence repeats (SSRs), which are useful for species identification and origin determination. Meanwhile, we conducted a high-throughput comparative identification of putative antimicrobial peptides (AMPs) and antihypertensive peptides (AHTPs) from whole-body transcriptomes of the Antarctic krill and its well-known counterpart, the whiteleg shrimp (Penaeus vannamei; resident in warm waters). Related data revealed that AMPs/AMP precursors and AHTPs were generally conserved, with interesting variations between the two crustacean species. In summary, as the first report of estimated genome size of the Antarctic krill, our present genome survey data provide a foundation for further biological research into this polar species. Our preliminary investigations on bioactive peptides will bring a new perspective for the in-depth development of novel marine drugs.

Genome survey sequencing and genetic background characterization of yellow horn based on next-generation sequencing.

Yellowhorn (Xanthoceras sorbifolium Bunge) is an important wood oil tree species, with high ornamental and medicinal value. Nevertheless, genomic information of yellowhorn is currently unavailable. Here, for the first time, we conducted a genome survey of two yellowhorn cultivars, Zhongshi 4 and Zhongshi 9, which had distinct differences on the phenotype and drought resistance, to obtain knowledge on the genomic information by next generation sequencing (NGS). Meanwhile, its genome size was estimated using flow cytometry. As a result, the whole genome survey of Zhongshi 4 and Zhongshi 9 generated 34.40 and 39.55 GB sequence data. The genome size of Zhongshi 4 and Zhongshi 9 estimated were about 536.58 Mb and 569.52 Mb, which were closed to results of flow cytometry. The heterozygosity rates were calculated to be 0.75% and 0.89%, and the repeat rates were 60.08% and 62.00%. These reads were assembled into 1024,373 and 885,404 contigs with a N50 length of 1005 bp and 1219 bp, respectively, which were further assembled into 714,369 and 686,128 scaffolds with scaffold N50 length of ~ 1963 bp and ~ 1938 bp, total length of 386,915 Kb and 391,904 Kb. These results indicated that there was little difference in genome size and complexity among different cultivars. In addition, 63137 and 65271 high-quality genomic simple sequence repeat (SSR) markers in Zhongshi 4 and Zhongshi 9 were generated. We suggest that the technologies combining Illumina and PacBio, assisted by Hi-C and matching assemble software should be used to one of two yellowhorn cultivars genome sequencing. The result will help to design whole genome sequencing strategies for yellowhorn, and provided a large amount of gene resources for further excavation and utilization of yellowhorn.

[Genome survey analysis and SSR loci mining of Bupleurum falcatum].

Buplewrum falcatum is a traditional Chinese medicine,which is mainly used for the treatment of cold and liver protection. B. falcatum is dominantly cultivated in Japan as well as planted in China,Korea and other countries and regions. In order to determine the appropriate sequencing strategy,the genome survey before large-scale genome sequencing is needed. This survey can provide information about the size and complexity of the whole genome of the target species. In the present study,the next generation sequencing technology( Illumina Hiseq 2000) was used to analyze the genome size and complexity of B. falcatum. In addition,SSR loci were analyzed from the sequenced data. Primer 3 was used to design specific primers and 33 pairs of primers were randomly selected for PCR with template DNA of B. falcatum,and the PCR system and optimal annealing temperature were screened. A total of 288. 64 G genome sequence data was obtained,and the estimated genome size of B. falcatum was 2 119. 58 Mb. The measured genome data depth was138×; the rate of heterozygosity was 1. 84%; and the ratio of repeat sequence was 83. 89%. It is speculated that the genome of B. falcatum is complex. The preliminary assembly was performed with K-mer = 41,and the contig N50 was 224 bp,the total length 896. 97 Mb,the scaffold N50 313 bp,and the total length was 922. 67 Mb. A total of 91 377 SSR sequences were detected in the sequenced genome data which were distributed in 70 809 unigenes.The main type is dinucleotide repeats,with 49 680 sequences,accounting for70. 16%. Among the 33 pairs of primers randomly synthesized according to the obtained SSR sequences,21 pairs were successfully amplifying the target sequences. The results will be helpful for later large scale genome sequencing and SSR molecular markers development for germplasm identification and trait mapping.

Genome survey sequencing of Dioscorea zingiberensis.

Dioscorea zingiberensis (Dioscoreceae) is the main plant source of diosgenin (steroidal sapogenins), the precursor for the production of steroid hormones in the pharmaceutical industry. Despite its large economic value, genomic information of the genus Dioscorea is currently unavailable. Here, we present an initial survey of the D. zingiberensis genome performed by next-generation sequencing technology together with a genome size investigation inferred by flow cytometry. The whole genome survey of D. zingiberensis generated 31.48 Gb of sequence data with approximately 78.70× coverage. The estimated genome size is 800 Mb, with a high level of heterozygosity based on K-mer analysis. These reads were assembled into 334 288 contigs with a N50 length of 1079 bp, which were further assembled into 92 163 scaffolds with a total length of 173.46 Mb. A total of 4935 genes, 81 tRNAs, 69 rRNAs, and 661 miRNAs were predicted by the genome analysis, and 263 484 repeated sequences were obtained with 419 372 simple sequence repeats (SSRs). Among these SSRs, the mononucleotide repeat type was the most abundant (up to 54.60% of the total SSRs), followed by the dinucleotide (29.60%), trinucleotide (11.37%), tetranucleotide (3.53%), pentanucleotide (0.65%), and hexanucleotide (0.25%) repeat types. The 1C-value of D. zingiberensis was calibrated against Salvia miltiorrhiza and calculated as 0.87 pg (851 Mb) by flow cytometry, which was very close to the result of the genome survey. This is the first report of genome-wide characterization within this taxon.

Genome survey sequencing of red swamp crayfish Procambarus clarkii.

Red swamp crayfish, Procambarus clarkii, presently is an important aquatic commercial species in China. The crayfish is a hot area of research focus, and its genetic improvement is quite urgent for the crayfish aquaculture in China. However, the knowledge of its genomic landscape is limited. In this study, a survey of P. clarkii genome was investigated based on Illumina's Solexa sequencing platform. Meanwhile, its genome size was estimated using flow cytometry. Interestingly, the genome size estimated is about 8.50 Gb by flow cytometry and 1.86 Gb with genome survey sequencing. Based on the assembled genome sequences, total of 136,962 genes and 152,268 exons were predicted, and the predicted genes ranged from 150 to 12,807 bp in length. The survey sequences could help accelerate the progress of gene discovery involved in genetic diversity and evolutionary analysis, even though it could not successfully applied for estimation of P. clarkii genome size.

A Comprehensive Genome Survey Provides Novel Insights into Bile Salt Hydrolase (BSH) in Lactobacillaceae.

Bile salt hydrolase (BSH) is a well-known enzyme that has been commonly characterized in probiotic bacteria, as it has cholesterol-lowering effects. However, its molecular investigations are scarce. Here, we build a local database of BSH sequences from Lactobacillaceae (BSH⁻SDL), and phylogenetic analysis and homology searches were employed to elucidate their comparability and distinctiveness among species. Evolutionary study demonstrates that BSH sequences in BSH⁻SDL are divided into five groups, named BSH A, B, C, D and E here, which can be the genetic basis for BSH classification and nomenclature. Sequence analysis suggests the differences between BSH-active and BSH-inactive proteins clearly, especially on site 82. In addition, a total of 551 BSHs from 107 species are identified from 451 genomes of 158 Lactobacillaceae species. Interestingly, those bacteria carrying various copies of BSH A or B can be predicted to be potential cholesterol-lowering probiotics, based on the results of phylogenetic analysis and the subtypes that those previously reported BSH-active probiotics possess. In summary, this study elaborates the molecular basis of BSH in Lactobacillaceae systematically, and provides a novel methodology as well as a consistent standard for the identification of the BSH subtype. We believe that high-throughput screening can be efficiently applied to the selection of promising candidate BSH-active probiotics, which will advance the development of healthcare products in cholesterol metabolism.

Genome-wide identification of conserved and novel microRNAs in one bud and two tender leaves of tea plant (Camellia sinensis) by small RNA sequencing, microarray-based hybridization and genome survey scaffold sequences.

BACKGROUND: MicroRNAs (miRNAs) are important for plant growth and responses to environmental stresses via post-transcriptional regulation of gene expression. Tea, which is primarily produced from one bud and two tender leaves of the tea plant (Camellia sinensis), is one of the most popular non-alcoholic beverages worldwide owing to its abundance of secondary metabolites. A large number of miRNAs have been identified in various plants, including non-model species. However, due to the lack of reference genome sequences and/or information of tea plant genome survey scaffold sequences, discovery of miRNAs has been limited in C. sinensis. RESULTS: Using small RNA sequencing, combined with our recently obtained genome survey data, we have identified and analyzed 175 conserved and 83 novel miRNAs mainly in one bud and two tender leaves of the tea plant. Among these, 93 conserved and 18 novel miRNAs were validated using miRNA microarray hybridization. In addition, the expression pattern of 11 conserved and 8 novel miRNAs were validated by stem-loop-qRT-PCR. A total of 716 potential target genes of identified miRNAs were predicted. Further, Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that most of the target genes were primarily involved in stress response and enzymes related to phenylpropanoid biosynthesis. The predicted targets of 4 conserved miRNAs were further validated by 5'RLM-RACE. A negative correlation between expression profiles of 3 out of 4 conserved miRNAs (csn-miR160a-5p, csn-miR164a, csn-miR828 and csn-miR858a) and their targets (ARF17, NAC100, WER and MYB12 transcription factor) were observed. CONCLUSION: In summary, the present study is one of few such studies on miRNA detection and identification in the tea plant. The predicted target genes of majority of miRNAs encoded enzymes, transcription factors, and functional proteins. The miRNA-target transcription factor gene interactions may provide important clues about the regulatory mechanism of these miRNAs in the tea plant. The data reported in this study will make a huge contribution to knowledge on the potential miRNA regulators of the secondary metabolism pathway and other important biological processes in C. sinensis.

Genome Survey Sequencing of Luffa Cylindrica L. and Microsatellite High Resolution Melting (SSR-HRM) Analysis for Genetic Relationship of Luffa Genotypes.

Luffa cylindrica (L.) Roem. is an economically important vegetable crop in China. However, the genomic information on this species is currently unknown. In this study, for the first time, a genome survey of L. cylindrica was carried out using next-generation sequencing (NGS) technology. In total, 43.40 Gb sequence data of L. cylindrica, about 54.94× coverage of the estimated genome size of 789.97 Mb, were obtained from HiSeq 2500 sequencing, in which the guanine plus cytosine (GC) content was calculated to be 37.90%. The heterozygosity of genome sequences was only 0.24%. In total, 1,913,731 contigs (>200 bp) with 525 bp N50 length and 1,410,117 scaffolds (>200 bp) with 885.01 Mb total length were obtained. From the initial assembled L. cylindrica genome, 431,234 microsatellites (SSRs) (≥5 repeats) were identified. The motif types of SSR repeats included 62.88% di-nucleotide, 31.03% tri-nucleotide, 4.59% tetra-nucleotide, 0.96% penta-nucleotide and 0.54% hexa-nucleotide. Eighty genomic SSR markers were developed, and 51/80 primers could be used in both "Zheda 23" and "Zheda 83". Nineteen SSRs were used to investigate the genetic diversity among 32 accessions through SSR-HRM analysis. The unweighted pair group method analysis (UPGMA) dendrogram tree was built by calculating the SSR-HRM raw data. SSR-HRM could be effectively used for genotype relationship analysis of Luffa species.