茶树CsPLK基因的鉴定与表达分析

茶树中富含茶氨酸、儿茶素和咖啡碱等重要功能成分,具有较高的价值功效,茶树在生命周期中经常遭受逆境胁迫,维生素B6(VB6)在植物体内参与逆境应答,吡哆醛激酶(pyridoxal kinase,PLK)是VB6补救途径中的关键酶。为进一步了解PLK在茶树生物合成中的功能和作用机理,该研究基于茶树基因组数据库,以龙井43为材料,采用逆转录PCR(RT-PCR)的方法从茶树中克隆出CsPLK的基因。结果表明:该基因序列长为1 179 bp,编码393个氨基酸; CsPLK蛋白和已知物种中PLK蛋白具有较高的同源性,都是核糖激酶超家族成员;通过构建pET-CsPLK载体进行原核表达,并鉴定出重组蛋白有很强的催化活性;组织表达特异性分析表明,叶中的表达量比茎、根的高,在根中最低;荧光定量PCR表示,低温诱导CsPLK上调表达,干旱诱导CsPLK下调表达,发现该基因在茶树中有明显的逆境应答,推测CsPLK在茶树的生长发育、逆境胁迫发挥重要作用。     

Construction and characterization of a bacterial artificial chromosome library for <Emphasis Type="Italic">Camellia sinensis</Emphasis>

Tea is a popular and natural non-alcoholic beverage, and is produced from fresh leaves of Camellia sinensis. Tea leaves contain many bioactive compounds that have significant health benefits. We constructed a high quality bacterial artificial chromosome (BAC) library by using the fresh petals of C. sinensis “Shuchazao” for genome sequencing and improvement of genomic assembly. BAC library is still a significant tool for studies of functional genomes and preservation of precious genetic resources. The BAC library contains 161,280 clones with an average insert size of 113 kb, which represents approximately 6.2-fold coverage of haploid genome equivalents of C. sinensis. We characterized 20 complete BAC clones and 738 BAC end sequences (BESs) ranging from 105 to 917 bp. In addition, we predicted cis-regulatory elements of LAR (leucoanthocyanidin reductase), TCS (caffeine synthase), and TS (theanine synthetase) involved in tea characteristic metabolite synthesis and identified a larger number of light-responsive cis-acting elements in these three genes. Meanwhile, we analyzed alternative splicing of these three genes. Furthermore, 12 pairs of SSR primers were successfully amplified in tea plant DNA. The tea BAC library was a critical resource to accomplish de novo whole-genome sequencing, accelerate gene discovery and enhance molecular breeding of C. sinensis.     

Complete Genome Sequence of a Novel Wild tomato mosaic virus Isolate Infecting Nicotiana tabacum in China

Virus particles of approximately 740–760 nm in length and 13 nm in diameter were observed from a diseased Nicotiana tabacum (tobacco) plant in Sichuan Province, China. The complete genomic sequence of the virus isolate XC1 was determined to contain 9659 nucleotides without 3′ terminal poly(A) tail. XC1 has a genome typical of members of the genus Potyvirus, encoding a large polyprotein of 3075 amino acids. Putative proteolytic cleavage sites and a number of well characterized functional motifs were identified by sequence comparisons with those of known potyviruses. Sequence comparison revealed that XC1 shared the highest level of nucleotide sequence identity (76.5%) with Wild tomato mosaic virus (WTMV). Phylogenetic analysis showed that XC1 was closely related to the WTMV Guangdong isolate with an identity of 94.3% between CP gene sequence of the two viruses. We thus named XC1 WTMV‐XC‐1 as a novel isolate of WTMV. The full sequence of WTMV‐XC‐1 may serve as a basis for future investigations on the gene diversity of WTMV.     

The bottle gourd genome provides insights into Cucurbitaceae evolution and facilitates mapping of a Papaya ring‐spot virus resistance locus

Bottle gourd (Lagenaria siceraria) is an important vegetable crop as well as a rootstock for other cucurbit crops. In this study, we report a high‐quality 313.4‐Mb genome sequence of a bottle gourd inbred line, USVL1VR‐Ls, with a scaffold N50 of 8.7 Mb and the longest of 19.0 Mb. About 98.3% of the assembled scaffolds are anchored to the 11 pseudomolecules. Our comparative genomic analysis identifies chromosome‐level syntenic relationships between bottle gourd and other cucurbits, as well as lineage‐specific gene family expansions in bottle gourd. We reconstructed the genome of the most recent common ancestor of Cucurbitaceae, which revealed that the ancestral Cucurbitaceae karyotypes consisted of 12 protochromosomes with 18 534 protogenes. The 12 protochromosomes are largely retained in the modern melon genome, while have undergone different degrees of shuffling events in other investigated cucurbit genomes. The 11 bottle gourd chromosomes derive from the ancestral Cucurbitaceae karyotypes followed by 19 chromosomal fissions and 20 fusions. The bottle gourd genome sequence has facilitated the mapping of a dominant monogenic locus, Prs, conferring Papaya ring‐spot virus (PRSV) resistance in bottle gourd, to a 317.8‐kb region on chromosome 1. We have developed a cleaved amplified polymorphic sequence (CAPS) marker tightly linked to the Prs locus and demonstrated its potential application in marker‐assisted selection of PRSV resistance in bottle gourd. This study provides insights into the paleohistory of Cucurbitaceae genome evolution, and the high‐quality genome sequence of bottle gourd provides a useful resource for plant comparative genomics studies and cucurbit improvement.     

SNP genotyping and population genomics from expressed sequences – current advances and future possibilities

With the rapid increase in production of genetic data from new sequencing technologies, a myriad of new ways to study genomic patterns in nonmodel organisms are currently possible. Because genome assembly still remains a complicated procedure, and because the functional role of much of the genome is unclear, focusing on SNP genotyping from expressed sequences provides a cost‐effective way to reduce complexity while still retaining functionally relevant information. This review summarizes current methods, identifies ways that using expressed sequence data benefits population genomic inference and explores how current practitioners evaluate and overcome challenges that are commonly encountered. We focus particularly on the additional power of functional analysis provided by expressed sequence data and how these analyses push beyond allele pattern data available from nonfunction genomic approaches. The massive data sets generated by these approaches create opportunities and problems as well – especially false positives. We discuss methods available to validate results from expressed SNP genotyping assays, new approaches that sidestep use of mRNA and review follow‐up experiments that can focus on evolutionary mechanisms acting across the genome.     

Divergent DNA methylation contributes to duplicated gene evolution and chilling response in tea plants

The tea plant (Camellia sinensis) is a thermophilic cash crop and contains a highly duplicated and repeat-rich genome. It is still unclear how DNA methylation regulates the evolution of duplicated genes and chilling stress in tea plants. We therefore generated a single-base-resolution DNA methylation map of tea plants under chilling stress. We found that, compared with other plants, the tea plant genome is highly methylated in all three sequence contexts, including CG, CHG and CHH (where H = A, T, or C), which is further proven to be correlated with its repeat content and genome size. We show that DNA methylation in the gene body negatively regulates the gene expression of tea plants, whereas non-CG methylation in the flanking region enables a positive regulation of gene expression. We demonstrate that transposable element-mediated methylation dynamics significantly drives the expression divergence of duplicated genes in tea plants. The DNA methylation and expression divergence of duplicated genes in the tea plant increases with evolutionary age and selective pressure. Moreover, we detect thousands of differentially methylated genes, some of which are functionally associated with chilling stress. We also experimentally reveal that DNA methyltransferase genes of tea plants are significantly downregulated, whereas demethylase genes are upregulated at the initial stage of chilling stress, which is in line with the significant loss of DNA methylation of three well-known cold-responsive genes at their promoter and gene body regions. Overall, our findings underscore the importance of DNA methylation regulation and offer new insights into duplicated gene evolution and chilling tolerance in tea plants.     

Comprehensive definition of genome features in Spirodela polyrhiza by high‐depth physical mapping and short‐read DNA sequencing strategies

Spirodela polyrhiza is a fast‐growing aquatic monocot with highly reduced morphology, genome size and number of protein‐coding genes. Considering these biological features of Spirodela and its basal position in the monocot lineage, understanding its genome architecture could shed light on plant adaptation and genome evolution. Like many draft genomes, however, the 158‐Mb Spirodela genome sequence has not been resolved to chromosomes, and important genome characteristics have not been defined. Here we deployed rapid genome‐wide physical maps combined with high‐coverage short‐read sequencing to resolve the 20 chromosomes of Spirodela and to empirically delineate its genome features. Our data revealed a dramatic reduction in the number of the rDNA repeat units in Spirodela to fewer than 100, which is even fewer than that reported for yeast. Consistent with its unique phylogenetic position, small RNA sequencing revealed 29 Spirodela‐specific microRNA, with only two being shared with Elaeis guineensis (oil palm) and Musa balbisiana (banana). Combining DNA methylation data and small RNA sequencing enabled the accurate prediction of 20.5% long terminal repeats (LTRs) that doubled the previous estimate, and revealed a high Solo:Intact LTR ratio of 8.2. Interestingly, we found that Spirodela has the lowest global DNA methylation levels (9%) of any plant species tested. Taken together our results reveal a genome that has undergone reduction, likely through eliminating non‐essential protein coding genes, rDNA and LTRs. In addition to delineating the genome features of this unique plant, the methodologies described and large‐scale genome resources from this work will enable future evolutionary and functional studies of this basal monocot family.     

基于植物rbcL基因测序对茶叶进行掺杂检验

茶叶是世界上最受人们欢迎的饮品之一,但茶叶中掺杂其他植物成分的现象时有发生。依靠传统的感官和理化检验方法难以准确判断茶叶中掺杂的植物种类。报道一种基于植物核酮糖-1,5-二磷酸羧化酶/加氧酶大亚基(rbc L)基因序列进行茶叶掺杂定性检测的方法,包括rbc L基因片段的扩增、测序和序列分析等步骤。利用所建立的方法对7份茶叶样品进行分析,发现岳阳黄茶(黄茶)和信阳毛尖(绿茶)未掺杂其他植物成分,而正山小种(红茶)、铁观音(乌龙茶)、太姥银针(白茶)、六堡茶和普洱茶(黑茶)均一定程度上混杂有其他植物成分。所建立的检测方法对样品的需求量小,操作简便,检测结果可靠性高,能定性检测各类茶叶中是否掺杂及掺杂了何种植物成分。     

Theanine transporters identified in tea plants (Camellia sinensis L.)

Theanine, a unique non‐proteinogenic amino acid, is an important component of tea, as it confers the umami taste and relaxation effect of tea as a beverage. Theanine is primarily synthesized in tea roots and is subsequently transported to young shoots, which are harvested for tea production. Currently, the mechanism for theanine transport in the tea plant remains unknown. Here, by screening a yeast mutant library, followed by functional analyses, we identified the glutamine permease, GNP1 as a specific transporter for theanine in yeast. Although there is no GNP1 homolog in the tea plant, we assessed the theanine transport ability of nine tea plant amino acid permease (AAP) family members, with six exhibiting transport activity. We further determined that CsAAP1, CsAAP2, CsAAP4, CsAAP5, CsAAP6, and CsAAP8 exhibited moderate theanine affinities and transport was H⁺‐dependent. The tissue‐specific expression of these six CsAAPs in leaves, vascular tissues, and the root suggested their broad roles in theanine loading and unloading from the vascular system, and in targeting to sink tissues. Furthermore, expression of these CsAAPs was shown to be seasonally regulated, coincident with theanine transport within the tea plant. Finally, CsAAP1 expression in the root was highly correlated with root‐to‐bud transport of theanine, in seven tea plant cultivars. Taken together, these findings support the hypothesis that members of the CsAAP family transport theanine and participate in its root‐to‐shoot delivery in the tea plant.     

贵州苦丁茶植物资源及化学成分分析

苦丁茶是我国南方各民族长期饮用的一类植物代用茶。在贵州,苦丁茶原植物有６科６属９种,主流品种为木樨科女贞属植物粗壮女贞〔Ｌｉｇｕｓｔｒｕｍｒｏｂｕｓｔｕｍ（Ｒｏｘｂ．）Ｂｌ．〕。分析了贵州５种苦丁茶植物叶的主要化学成分并和绿茶进行了对比,分析结果表明,苦丁茶类植物是一种营养成分含量丰富,不含咖啡因的营养保健的功能性饮料新资源     

Likelihood‐based inference of population history from low‐coverage de novo genome assemblies

Short‐read sequencing technologies have in principle made it feasible to draw detailed inferences about the recent history of any organism. In practice, however, this remains challenging due to the difficulty of genome assembly in most organisms and the lack of statistical methods powerful enough to discriminate between recent, nonequilibrium histories. We address both the assembly and inference challenges. We develop a bioinformatic pipeline for generating outgroup‐rooted alignments of orthologous sequence blocks from de novo low‐coverage short‐read data for a small number of genomes, and show how such sequence blocks can be used to fit explicit models of population divergence and admixture in a likelihood framework. To illustrate our approach, we reconstruct the Pleistocene history of an oak‐feeding insect (the oak gallwasp Biorhiza pallida), which, in common with many other taxa, was restricted during Pleistocene ice ages to a longitudinal series of southern refugia spanning the Western Palaearctic. Our analysis of sequence blocks sampled from a single genome from each of three major glacial refugia reveals support for an unexpected history dominated by recent admixture. Despite the fact that 80% of the genome is affected by admixture during the last glacial cycle, we are able to infer the deeper divergence history of these populations. These inferences are robust to variation in block length, mutation model and the sampling location of individual genomes within refugia. This combination of de novo assembly and numerical likelihood calculation provides a powerful framework for estimating recent population history that can be applied to any organism without the need for prior genetic resources.     

Genome Characteristics,Phylogeny and Varying Host Specificity of Polish Kra and Ros Isolates of Tomato torrado virus

The genome sequences of two Polish Kra and Ros isolates of Tomato torrado virus (ToTV) were determined and compared with data of previously described ToTV isolates and other Torradovirus members. Whole‐genome sequence comparisons revealed 97.0–99.6% nucleotide sequence identities and close relatedness, with other known ToTV isolates. The high homology between Kra, Ros and Wal'03 ToTVs is likely responsible for the similar symptoms observed on infected plants. However, the symptoms differed in intensity and various host specificity. We report that Kra ToTV caused a milder expression of symptoms on Solanum tuberosum than Wal'03. We hypothesize this may be a result of the significant variability observed within the 3′‐UTR of RNA1 of Kra as well as of Ros ToTV isolates. In the light of this fact, potato may be considered an indicator plant for distinguishing Kra and Wal'03 ToTV isolates.     

Detection and correction of assembly errors of rice Nipponbare reference sequence

A complete and high‐quality genome reference sequence of an organism provides a solid foundation for a wide research community and determines the outcomes of relevant genomic, genetic, molecular and evolutionary research. Rice is an important food crop and a model plant for grasses, and therefore was the first chosen crop plant for whole genome sequencing. The genome of the japonica representative rice variety, Nipponbare, was sequenced using a gold standard, map‐based clone‐by‐clone strategy. However, although the Nipponbare reference sequence (RefSeq) has the best quality for existing crop genome sequences, it still contains many assembly errors and gaps. To improve the Nipponbare RefSeq, first a robust method is required to detect the hidden assembly errors. Through alignments between BAC‐end sequences (BESs) embedded in the Nipponbare bacterial artificial chromosome (BAC) physical map and the Nipponbare RefSeq, we detected locations on the Nipponbare RefSeq that were inversely matched with BESs and could therefore be candidates for spurious inversions of assembly. We performed further analysis of five potential locations and confirmed assembly errors at those locations; four of them, two on chr4 and two on chr11 of the Nipponbare RefSeq (IRGSP build 5), were found to be caused by reverse repetitive sequences flanking the locations. Our approach is effective in detecting spurious inversions in the Nipponbare RefSeq and can be applied for improving the sequence qualities of other genomes as well.     

Genome‐wide scans detect adaptation to aridity in a widespread forest tree species

Patterns of adaptive variation within plant species are best studied through common garden experiments, but these are costly and time‐consuming, especially for trees that have long generation times. We explored whether genome‐wide scanning technology combined with outlier marker detection could be used to detect adaptation to climate and provide an alternative to common garden experiments. As a case study, we sampled nine provenances of the widespread forest tree species, Eucalyptus tricarpa, across an aridity gradient in southeastern Australia. Using a Bayesian analysis, we identified a suite of 94 putatively adaptive (outlying) sequence‐tagged markers across the genome. Population‐level allele frequencies of these outlier markers were strongly correlated with temperature and moisture availability at the site of origin, and with population differences in functional traits measured in two common gardens. Using the output from a canonical analysis of principal coordinates, we devised a metric that provides a holistic measure of genomic adaptation to aridity that could be used to guide assisted migration or genetic augmentation.     

Towards a whole‐genome sequence for rye (Secale cereale L.)

We report on a whole‐genome draft sequence of rye (Secale cereale L.). Rye is a diploid Triticeae species closely related to wheat and barley, and an important crop for food and feed in Central and Eastern Europe. Through whole‐genome shotgun sequencing of the 7.9‐Gbp genome of the winter rye inbred line Lo7 we obtained a de novo assembly represented by 1.29 million scaffolds covering a total length of 2.8 Gbp. Our reference sequence represents nearly the entire low‐copy portion of the rye genome. This genome assembly was used to predict 27 784 rye gene models based on homology to sequenced grass genomes. Through resequencing of 10 rye inbred lines and one accession of the wild relative S. vavilovii, we discovered more than 90 million single nucleotide variants and short insertions/deletions in the rye genome. From these variants, we developed the high‐density Rye600k genotyping array with 600 843 markers, which enabled anchoring the sequence contigs along a high‐density genetic map and establishing a synteny‐based virtual gene order. Genotyping data were used to characterize the diversity of rye breeding pools and genetic resources, and to obtain a genome‐wide map of selection signals differentiating the divergent gene pools. This rye whole‐genome sequence closes a gap in Triticeae genome research, and will be highly valuable for comparative genomics, functional studies and genome‐based breeding in rye.