Genetic diversity, population genetic structure and gene flow in the rare and endangered wild plant Cypripedium macranthos revealed by genotyping-by-sequencing.

BACKGROUND: Genetic diversity, genetic structure, and gene flow in plant populations and their influencing factors are important in conservation biology. Cypripedium macranthos is one of the few wild orchids with high ornamental value in northern China. However, over the past decade, excessive collection, trading, tourism development, habitat fragmentation, deceptive pollination, and seed germination difficulties have all caused a sharp decline in the number of C. macranthos individuals and its population. In order to propose a scientific and effective conservation strategy, the genetic diversity, genetic structure and gene flow of the current CM population are urgent scientific issues to be clarified. RESULTS: Here, 99 individuals of C. macranthos from north and northeast China were analyzed to evaluate the genetic diversity, gene flow among populations, and genetic structure by genotyping-by-sequencing. More than 68.44 Gb high-quality clean reads and 41,154 SNPs were obtained. Our data based on bioinformatics methods revealed that C. macranthos has lower genetic diversity, high levels of historical gene flow, and moderate-to-high genetic differentiation between populations. The gene migration model revealed that the direction of gene flow was mainly from northeast populations to north populations in China. The results of genetic structure analysis showed that 11 C. macranthos populations can be considered as two groups, and further divided into four subgroups. Moreover, the Mantel test detected no significant "Isolation by Distance" between populations. CONCLUSIONS: Our study demonstrates that the present genetic diversity and genetic structure of C. macranthos populations were mainly caused by biological characteristics, human interference, habitat fragmentation, and restricted gene flow. Finally, constructive measures, which can provide a basis for the proposal of conservation strategies, have been suggested.

Genome of Lindera glauca provides insights into the evolution of biosynthesis genes for aromatic compounds.

Lindera glauca is a crucial source of diverse industrial oil and medicines. The spicy aroma of tender leaves is caused by the presence of abundant aromatic compounds. Here, we present its chromosome-level genome assembly comprising 12 pseudochromosomes (2,092.2 Mb; scaffold N50: 186.5 Mb), which was predicted to have 65,145 protein-coding genes. Comparative genomic analyses indicated two whole-genome duplication (WGD) events in the Lauraceae family, contributing to the production of numerous terpene synthase (TPS) genes. We identified 138 TPS genes in L. glauca. Comparative transcriptomic analyses revealed high expression of genes Lg03G2346 and Lg08G140 in TPS-a and Lg07G2961 and Lg12G971 in TPS-b subfamilies, which regulated the biosynthesis of the monoterpenoid ß-ocimene and sesquiterpenoid D-germacrene in L. glauca. The results suggested a molecular basis for species-specific terpenoid biosynthesis and provided a foundation for molecular breeding to produce desired characteristics and a valuable reference genome.

The complete chloroplast genome sequence of the endemic and rare orchid Nothodoritis zhejiangensis (Orchidaceae) in China.

Nothodoritis zhejiangensis (Orchidaceae) is a rare orchid unique to China of great horticultural value. The complete chloroplast (cp) genome of this species is 143,522 bp in length and the total GC content is 36.8%. It includes a large single copy (LSC) region of 83,830 bp in length, a small single copy (SSC) region of 10,764 bp, and a pair of inverted repeat regions (IRs) with 24,464 bp. The cp genome contains 120 functional genes, including 74 protein-coding genes, 38 tRNA genes, and eight rRNA genes. The phylogenetic tree constructed by the maximum-likelihood method showed that the taxonomic status of N. zhejiangensis was clustered with the species in Phalaenopsis.

Population genetic structure and gene flow of rare and endangered Tetraena mongolica Maxim. revealed by reduced representation sequencing.

BACKGROUND: Studying population genetic structure and gene flow of plant populations and their influencing factors is of particular significance in the field of conservation biology, especially important for species such as rare and endangered plants. Tetraena mongolica Maxim. (TM), belongs to Zygophyllaceae family, a rare and endangered plant with narrow distribution. However, for the last decade, due to excessive logging, urban expansion, industrial and tourism development, habitat fragmentation and loss of natural habitats have become major threats to the population of endangered plants. RESULTS: In this study, genetic diversity, population genetic structure and gene flow of TM populations were evaluated by reduced representation sequencing technology, and a total of more than 133.45 GB high-quality clean reads and 38,097 high-quality SNPs were generated. Analysis based on multiple methods, we found that the existing TM populations have moderate levels of genetic diversity, and very low genetic differentiation as well as high levels of gene flow between populations. Population structure and principal coordinates analysis showed that 8 TM populations can be divided into two groups. The Mantel test detected no significant correlation between geographical distances and genetic distance for the whole sampling. Moreover, the migration model indicated that the gene flow is more of a north to south migration pattern in history. CONCLUSIONS: This study demonstrates that the present genetic structure is mainly due to habitat fragmentation caused by urban sprawl, industrial development and coal mining. Our recommendation with respect to conservation management is that, all 8 populations should be preserved as a whole population, rather than just those in the core area of TM nature reserve. In particular, the populations near the edge of TM distribution in cities and industrial areas deserve our special protection.

Population genetic structure and variability in Lindera glauca (Lauraceae) indicates low levels of genetic diversity and skewed sex ratios in natural populations in mainland China.

Lindera glauca (Lauraceae) is a tree of economic and ecological significance that reproduces sexually and asexually via apomictic seeds. It is widely distributed in the low-altitude montane forests of East Asia. Despite the potential implications of a mixed reproductive system in terms of genetic diversity, few studies have focused on this aspect. In this study, the genetic structure of wild populations of L. glauca was investigated via genetic analyses. Overall, 13 nuclear microsatellites (nSSRs) and five chloroplast microsatellites (cpSSRs) were used to genotype 300 individual plants, taken from 20 wild populations (a small sample size in some wild populations is due to the limitation of its specific reproduction, leading to certain limitations in the results of this study) and two cultivated populations ranging across nearly the entire natural distribution of mainland China. The populations exhibited low levels of genetic diversity (nSSR: AR = 1.75, Ho = 0.32, He = 0.36; cpSSR: Nb = 2.01, Hrs = 0.40), and no significant effect of isolation by distance between populations existed, regardless of marker type (nSSR: R2 = 0.0401, P = 0.068; cpSSR: R2 = 0.033, P = 0.091). Haplotype networks showed complex relationships among populations, and the H12 haplotype was predominant in most populations. Analyses of molecular variance obtained with nuclear markers (Fsc = 0.293, F ST = 0.362) and chloroplast markers (Fsc = 0.299, F ST = 0.312) were similar. The migration ratio of pollen flow versus seed flow in this study was negative (r = -1.149). Results suggest that weak barriers of dispersal between populations and/or the similarity of founders shared between neighbors and distant populations are indicative of the gene flow between populations more likely involving seeds. Wild L. glauca in mainland China was inferred to have highly skewed sex ratios with predominant females. In addition, some populations experienced a recent bottleneck effect, especially in Gujianshan, Chongqing, and southwest China (population GJS). It is suggested that few wild male individuals should be conserved in order to maintain overall genetic diversity in the wild populations of this species. These findings provide important information for the sustainable utilization and preservation of the overall genetic diversity of L. glauca.

The complete chloroplast genome sequence of a medicinal orchid species Coelogyne fimbriata (Orchidaceae).

Coelogyne fimbriata is an important orchid species with high medicinal value. Its complete chloroplast genome is 158,935 bp in length, which possesses the typical structure consising of a small single-copy region (SSC) of 18,743 bp, two inverted repeats (IRs) of 26,374 bp, and a large-single copy region (LSC) of 87,444 bp. The genome encodes 137 genes, including 91 protein-coding genes (PCGs), 38 tRNA genes and 8 rRNA genes. And the overall GC content is 37.40%. In addition, our phylogenetic analysis based on cp genome revealed the phylogenetic relationship between C. fimbriata and other 22 species in Orchidaceae.

Transcriptome analysis of Chelidonium majus elaiosomes and seeds provide insights into fatty acid biosynthesis.

BACKGROUND: Elaiosomes are specialized fleshy and edible seed appendages dispersed by ants. Lipids are the primary components of elaiosomes. Chelidonium majus is a well-known plant, the seeds of which are dispersed by ants. Previous studies have identified the presence of primary fatty acids in its elaiosomes and seeds. However, the molecular mechanisms underlying fatty acid biosynthesis in elaiosomes remain unknown. METHODS: In order to gain a comprehensive transcriptional profile of the elaiosomes and seeds of C. majus, and understand the expression patterns of genes associated with fatty acid biosynthesis, four different developmental stages, including the flower-bud (Ch01), flowering (Ch02), young seed (Ch03), and mature seed (Ch04) stages, were chosen to perform whole-transcriptome profiling through the RNA-seq technology (Illumina NGS sequencing). RESULTS: A total of 63,064 unigenes were generated from 12 libraries. Of these, 7,323, 258, and 11,540 unigenes were annotated with 25 Cluster of Orthologous Groups, 43 Gene Ontology terms, and 373 Kyoto Encyclopedia of Genes and Genomes pathways, respectively. In addition, 322 genes were involved in lipid transport and metabolism, and 508 genes were involved in the lipid metabolism pathways. A total of 41 significantly differentially expressed genes (DEGs) involved in the lipid metabolism pathways were identified, most of which were upregulated in Ch03 compared to Ch02, indicating that fatty acid biosynthesis primarily occurs during the flowering to the young seed stages. Of the DEGs, acyl-ACP thioesterases, acyl carrier protein desaturase (DESA1), and malonyl CoA-ACP transacylase were involved in palmitic acid synthesis; stearoyl-CoA desaturase and DESA1 were involved in oleic acid synthesis, and acyl-lipid omega-6 desaturase was involved in linoleic acid synthesis.

Comprehensive evaluation of fuel properties and complex regulation of intracellular transporters for high oil production in developing seeds of Prunus sibirica for woody biodiesel.

BACKGROUND: Based on our previous studies of 17 Prunus sibirica germplasms, one plus tree with high quality and quantity of seed oils has emerged as novel potential source of biodiesel. To better develop P. sibirica seed oils as woody biodiesel, a concurrent exploration of oil content, FA composition, biodiesel yield and fuel properties as well as prediction model construction for fuel properties was conducted on developing seeds to determine the optimal seed harvest time for producing high-quality biodiesel. Oil synthesis required supply of carbon source, energy and FA, but their transport mechanisms still remains enigmatic. Our recent 454 sequencing of P. sibirica could provide long-read sequences to identify membrane transporters for a better understanding of regulatory mechanism for high oil production in developing seeds. RESULTS: To better develop the seed oils of P. sibirica as woody biodiesel, we firstly focused on a temporal and comparative evaluation of growth tendency, oil content, FA composition, biodiesel yield and fuel properties as well as model construction for biodiesel property prediction in different developing seeds from P. sibirica plus tree (accession AS-80), revealing that the oils from developing seeds harvested after 60 days after flowering (DAF) could be as novel potential feedstock for producing biodiesel with ideal fuel property. To gain new insight into membrane transport mechanism for high oil yield in developing seeds of P. sibirica, we presented a global analysis of transporter based on our recent 454 sequencing data of P. sibirica. We annotated a total of 116 genes for membrane-localized transporters at different organelles (plastid, endoplasmatic reticulum, tonoplast, mitochondria and peroxisome), of which some specific transporters were identified to be involved in carbon allocation, metabolite transport and energy supply for oil synthesis by both RT-PCR and qRT-PCR. Importantly, the transporter-mediated model was well established for high oil synthesis in developing P. sibirica seeds. Our findings could help to reveal molecular mechanism of increased oil production and may also present strategies for engineering oil accumulation in oilseed plants. CONCLUSIONS: This study presents a temporal and comparative evaluation of developing P. sibirica seed oils as a potential feedstock for producing high-quality biodiesel and a global identification for membrane transporters was to gain better insights into regulatory mechanism of high oil production in developing seeds of P. sibirica. Our findings may present strategies for developing woody biodiesel resources and engineering oil accumulation.

Development and characterization of microsatellite loci for Lindera glauca (Lauraceae).

PREMISE OF THE STUDY: Microsatellite primers were developed to investigate population genetic structure in Lindera glauca (Lauraceae). METHODS AND RESULTS: Twenty-five microsatellite primers were developed and optimized for L. glauca using Illumina's Solexa sequencing technology. These novel primers were found to be polymorphic in nine wild L. glauca populations with 81 total alleles confirmed and genotyped via capillary gel electrophoresis. The total number of alleles, observed heterozygosity, and expected heterozygosity for each population ranged from one to four, from 0.00 to 0.90, and from 0.00 to 0.79, respectively. In addition, the 25 primers were tested in 10 additional individuals of the related species L. communis, and all but four markers showed good amplification results. CONCLUSIONS: This set of microsatellite primers is the first specifically developed for L. glauca and will facilitate studies of genetic diversity and evolution among populations of this species.

[The expression and clinical significance of Bax and PHF20 in laryngeal squamous cell carcinoma].

OBJECTIVE: To investigate the expression of Bax and PHF20 in laryngeal squamous cell carcinoma (LSCC)and to discuss their relevance and the roles in carcinogenesis and development in LSCC. METHOD: The expressions of Bax and PHF20 in the LSCC tissues and normal mucosa tissues adjacent to carcinoma were detected by SP immunohistochemistry assay. The relationship between the expressions of Bax and PHF20 and the clinicopathological characteristics including clinical stage, pathological type, histological grade and lymph node metastasis in LSCC were analyzed according to the clinical data. RESULT: (1) The expressions of Bax and PHF20 were both significantly lower in the LSCC tissue than that in the normal laryngeal tissue (P < 0.01). (2) In clinical stage grouping, there were no statistical differences of the quantity and positive rate of Bax and PHF20 expressions among supraglottic, glottic and subglottic LSCC (P > 0.05). In histological differentiation grouping, the quantity and positive rate of Bax and PHF20 expressions decreased significantly in poorly differentiated LSCC compared with the well and moderately differentiated LSCCs (P < 0.01, P < 0.05, respectively). In T stage grouping, the quantity and positive rate of Bax and PHF20 expressions were both significantly higher in T1 + T2 compared with T3 +T4 (both P < 0.01). In addition, the quantity and positive rate of Bax and PHF20 expressions were both significantly higher in LSCC with lymph node metastasis compared to that without lymph node metastasis (both P < 0.01). CONCLUSION: The lack of Bax and PHF20 might contribute to the carcinogenesis and development in LSCC. The positive expression of Bax and PHF20 maybe relative to T term degree, differentiation degree and lymphamatic metastasis of LSCC.

De novo transcriptome analysis of the Siberian apricot (Prunus sibirica L.) and search for potential SSR markers by 454 pyrosequencing.

The Siberian apricot, an economically and ecologically important plant in China, contains seeds high in oil and can grow on marginal land. Although this species has multiple purposes and may be a feedstock of biofuel in China, transcriptome information and molecular research on this species remain limited. RNA-Seq technology has been widely applied to transcriptomics, genomics and the development of molecular markers, and functional gene studies. In this study, we obtained 1,243,067 high-quality reads with a mean size of 425 bp in a single run, totaling 528.4 Mb of sequence data using 454 GS FLX Titanium sequencing. All reads were assembled de novo into 46,940 unigenes with a mean size of 651 bp (range: 45-5566 bp). Assembled unigenes were annotated in multiple public databases based on similarity alignments to genes and proteins. 191 unigenes involving in lipid biosynthesis and metabolism were found, among them, expression patterns of two desaturase enzymes were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR), based on six tissues from Siberian apricot, the seeds had the highest expression. 7304 simple sequence repeats (SSR) were identified from 6509 unigenes, a total of 9930 primer pairs were designed, 50 primer pairs were randomly selected to validate of the usefulness, and 24 (48%) primer pairs produced bands of the expected size. These data provide a base of sequence information to improve agronomic characters and molecular marker-assisted breeding to alter the composition of fatty acids in seeds from this plant, and hence, facilitate its utilization as a future biodiesel feedstock.

Cloning, characterization, and expression analysis of acyl-acyl carrier protein (ACP)-thioesterase B from seeds of Chinese Spicehush (Lindera communis).

Acyl-acyl carrier protein (ACP) thioesterases (TE EC 3.1.2.14) are fatty acid biosynthesis key enzymes that determine fatty acid carbon chain length in most plant tissues. A full-length cDNA corresponding to one of the fatty acyl-ACP thioesterase (Fat) genes, designated LcFatB, was isolated from developing Lindera communis seeds using PCR and RACE with degenerate primers based on conserved sequences of multiple TE gene sequences obtained from GenBank. The 1788 bp cDNA had an open reading frame (ORF) of 1260 bp encoding a protein of 419 amino acids. The deduced amino acid sequence showed 61-73% identity to proteins in the FatB class of plant thioesterases. Real-time quantitative PCR analysis revealed that LcFatB was expressed in all tissues of L. communis, with the highest expression in the developing seeds 75days after flowering. Recombinant pET-MLcFatB was constructed using the pET-30 a vector and transformed into Escherichia coli BL21(DE3)△FadE, a strain that deleted the acyl-CoA dehydrogenase (FadE). SDS-PAGE analysis of proteins isolated from pET-MLcFatB E. coli cells after induction with IPTG revealed a protein band at ~40.5kDa, corresponding to the predicted size of LcFatB mature protein. The decanoic acid and lauric acid contents of the pET-MLcFatB transformant were increased significantly. These findings suggest that an LcFatB gene from a non-traditional oil-seed tree could be used to function as a saturated acyl-ACP thioesterase and could potentially be used to modify the fatty acid composition of seed oil from L. communis or other species through transgenic approaches.