用生物信息学挖掘玉米中的microRNAs及其靶基因

microRNA (miRNA)是一类内源性的、19~24碱基长度的小分子非编码RNA,通过碱基互补调控靶基因的表达,在多细胞生物的基因表达调控过程中扮演着十分重要的角色。植物中的miRNAs具有高度的保守性,这为通过同源比对发现保守的miRNAs提供了思路和途径。本研究通过对拟南芥、水稻等植物已知的miRNAs与玉米EST和GSS数据库的比对,并设置一系列严格的筛选标准,共筛选到23条新的玉米miRNAs;利用WMD 3在线植物miRNAs靶基因预测软件,对新发现的玉米miRNAs进行靶基因预测,总共预测到89个靶基因,进一步功能分析发现这些靶基因参与玉米的生长发育、信号转导、转录调节、新陈代谢及逆境胁迫响应等调控过程。     

野生小豆和栽培小豆microRNA全基因组鉴定与比较分析

microRNA(miRNA)是一种非编码的小RNA分子, 对真核生物基因表达起着非常重要的调控作用,miRNA系统鉴定对于研究其功能及作用机制具有重要意义。本研究分别以栽培小豆京农6号(JN6)和野生小豆CWA108为试验材料,进行了深度miRNA测序。系统鉴定和注释这2个物种的miRNA,并分析了其miRNA种类的异同、表达量的差异和靶基因在功能富集上的差别。鉴定出了JN6和CWA108共有和特有的miRNA,明确了它们之间差异表达的miRNA,以及这些特有和差异miRNA的靶基因在功能富集上的差异,发现可能和抗病与抗逆途径相关。     

植物关联分析的研究进展及其在茶树分子标记辅助育种上的应用前景

茶树是我国重要的经济和饮料作物,由于自交不亲和和长期的异花授粉使其高度异质杂合,选育一个良种耗时长,提高早期选育效率和准确鉴定目标性状成为茶树育种家关心的重大问题之一。关联分析是研究分子标记或候选基因与目标性状关系的一种新方法,现已广泛应用于植物等位基因发掘、功能基因验证以及功能性标记的开发和应用等各方面的研究中,为茶树遗传育种研究提供了新思路。本文主要介绍了关联分析的基本原理、策略和及其在植物中的应用现状,并且探讨了其在茶树分子标记辅助育种研究上的应用前景。     

连续回交对消除农杆菌介导转化引起水稻体细胞变异的影响

农杆菌介导的转化引起许多体细胞变异, 影响了转基因植物的农艺性状。因此, 转基因作物的培育需要大量的T₀代再生植株。在本研究中, 我们将转基因水稻株系与原始品种连续回交, 然后评价其回交后代的表现, 消除体细胞变异, 恢复转基因亲本的农艺性状。回交的供体亲本是3个转基因水稻株系, 分别带有来自于苏云金芽胞杆菌(Bt)的抗虫基因。与原始品种连续回交至BC₃F₁代, 每代BC_nF₁单株再自交两代, 同时对各个世代进行抗虫性选择。通过发芽试验获得转基因纯合的BC_nF₃株系, 在室内抗性试验中, 所有的BC_nF₃纯合株系都能杀死100%的幼虫。在田间试验中, 这些株系的单株产量明显高于供体亲本, 大部分农艺性状与原品种没有显著的差异。这些结果说明连续回交能够在很大程度上恢复转基因水稻株系的农艺性状, 从而减少转基因育种过程中所需的工作量。     

Analysis of genomic and functional RFLP derived markers associated with sucrose content,fiber and yield QTLs in a sugarcane (<Emphasis Type="Italic">Saccharum</Emphasis> spp.) commercial cross

Expressed sequence tags derived markers have a great potential to be used in functional map construction and QTL tagging. In the present work, sugarcane genomic probes and expressed sequence tags having homology to genes, mostly involved in carbohydrate metabolism were used in RFLP assays to identify putative QTLs as well as their epistatic interactions for fiber content, cane yield, pol and tones of sugar per hectare, at two crop cycles in a progeny derived from a bi-parental cross of sugarcane elite materials. A hundred and twenty marker trait associations were found, of which 26 at both crop cycle and 32 only at first ratoon cane. A sucrose synthase derived marker was associated with a putative QTL having a high negative effect on cane yield and also with a QTL having a positive effect on Pol at both crop cycles. Fifty digenic epistatic marker interactions were identified for the four traits evaluated. Of these, only two were observed at both crop cycles.     

Microsatellite markers: an overview of the recent progress in plants

In recent years, molecular markers have been utilized for a variety of applications including examination of genetic relationships between individuals, mapping of useful genes, construction of linkage maps, marker assisted selections and backcrosses, population genetics and phylogenetic studies. Among the available molecular markers, microsatellites or simple sequence repeats (SSRs) which are tandem repeats of one to six nucleotide long DNA motifs, have gained considerable importance in plant genetics and breeding owing to many desirable genetic attributes including hypervariability, multiallelic nature, codominant inheritance, reproducibility, relative abundance, extensive genome coverage including organellar genomes, chromosome specific location and amenability to automation and high throughput genotyping. High degree of allelic variation revealed by microsatellite markers results from variation in number of repeat-motifs at a locus caused by replication slippage and/or unequal crossing-over during meiosis. In spite of limited understanding of the functions of the SSR motifs within the plant genes, SSRs are being widely utilized in plant genome analysis. Microsatellites can be developed directly from genomic DNA libraries or from libraries enriched for specific microsatellites. Alternatively, microsatellites can also be found by searching public databases such as GenBank and EMBL or through cross-species transferability. At present, EST databases are an important source of candidate genes, as these can generate markers directly associated with a trait of interest and may be transferable in close relative genera. A large number of SSR based techniques have been developed and a quantum of literature has accumulated regarding the applicability of SSRs in plant genetics and genomics. In this review we discuss the recent developments (last 4–5 years) made in plant genetics using SSR markers.     

苹果炭疽病抗性miRNA的筛选

为筛选炭疽病菌侵染苹果属植物后相关miRNA的响应情况,以抗病品种‘八棱脆’海棠、感病品种‘嘎啦’苹果2个为试验材料,分别用胶胞炭疽孢子悬浮液侵染组培苗,4天后发病,提取植物总RNA并反转miRNA,用qRT-PCR分析2种植物中抗病相关miRNA相对表达量的差异,并对其靶基因进行预测。在抗性品种中miR390a和miR396b/c/f表达量下调,而在感病品种中表达量上调,且其靶基因均有LRR受体样丝氨酸/苏氨酸蛋白,miR482b靶基因为抗性蛋白。miR390a、miR482b和miR396b/c/f可能在苹果炭疽病的侵染过程中起重要作用。     

利用SSCP技术分析甘蓝型油菜10个功能基因序列差异

以甘蓝型油菜SI-1300和Eagle为材料,利用DNA单链构象多态性(single-strand conformation polymorphism, SSCP)技术,对10对功能基因特异性引物进行多态性分析,每对引物均检测到1个多态性位点。随后随机挑选10个多态性片段进行测序,并利用bl2seq软件比较测序序列与基因原始序列。结果显示测序序列与所对应的基因原始序列之间相似程度平均高达98%,差异碱基数平均仅为2.3个。进一步选取5对引物比较分析两个材料间的差异扩增片段序列,发现差异扩增片段在2个材料中高度保守,平均相似度达97%;在所测序的5对引物扩增序列中,共存在39个单核苷酸多态性(single-nucleotide polymorphisms, SNPs)和5个插入/缺失突变(insertion-deletions, INDELs),SNP和INDEL的发生频率分别为1 SNP/30 bp和1 INDEL /233 bp。结果表明,SSCP标记能够真实代表原始功能基因,甘蓝型油菜功能基因序列在不同材料间高度保守,其遗传变异类型主要来源于SNP。     

作物数量性状(QTL)克隆研究进展

此文首先简述了数量性状基因（QTL）克隆研究方法,然后较全面的总结了作物QTL克隆的研究进展,最后讨论了QTL调控的分子机理。对37个已成功克隆的QTL进行汇总分析表明：控制作物数量性状的有转录因子、蛋白激酶、细胞分裂素氧化酶类等基因;其中12个已成功克隆的QTL编码转录因子,说明转录调控因子是新的植株形态进化和变异的重要原因。随着测序技术的发展,海量的基因组数据将为QTL的遗传定位和分子剖析提供更多便利,越来越多的QTL将会被克隆出来。     

Simple sequence repeat markers for botanical varieties of cultivated peanut (Arachis hypogaea L.)

Cultivated peanut (Arachis hypogaea L.) consists of six botanical varieties. Identification of DNA markers associated with botanical varieties would be useful in plant genotyping, germplasm management, and evolutionary studies. We have developed 130 simple sequence repeat (SSR) markers in peanut, 38 of which were used in this study because of their ability in detecting genetic polymorphism among 24 peanut accessions. Eight SSR markers were found useful to classify botanical varieties. Among them, six SSR markers were specific to botanical varieties fastigiata and vulgaris, one to botanical varieties hypogaea and hirsuta, and one to botanical varieties peruviana, and aequatoriana. Also, three of them derived from peanut expressed sequence tags (ESTs) were associated with putative genes. As botanical varieties have different morphological traits and belong to different subspecies in A. hypogaea, these markers might be associated with genes involved in the expression of morphological traits. The results also suggested that SSRs (also called microsatellites) might play a role in shaping evolution of cultivated peanut. Multiplex PCR of botanical variety-specific markers could be applied to facilitate efficient genotyping of the peanut lines.     

Detection of linkage between RFLP markers and genes affecting anthocyanin pigmentation in maize (Zea mays L.)

Summary Linkages between molecular markers and genes involved in the expression of agronomical traits have already been described in all of the major crops. In most cases, the genetic model underlying the Quantitative Traits Loci (QTL) is discussed. Here, Restriction Fragment Length Polymorphisms (RFLPs) and Mapmaker-QTL have been used to pinpoint seven regions of the genome significantly correlated with four pigmentation qualitative traits of maize (Zea mays L.). Two of these, located on chromosomes 2 and 10, explain most of the variation of these traits. The R and B gene loci known to be involved in the regulation of the anthocyanin pathway map to the same regions and we suggest that these loci could be the candidate genes involved in the correlations detected with RFLPs. This type of result is in accordance with the hypothesis of the candidate gene which supposes that, if we have a very high density map of randomly-selected cDNA clones, it should theoretically be possible to associate a cloned genic sequence with a phenotypic trait where correlations are found.     

棉花盐胁迫下叶片miRNAs的鉴定与分析

microRNAs(miRNAs)是一类21~24个核酸长度的非编码小分子RNA(sRNA,small RNA),通过介导目标mRNA的切割或者抑制翻译来调节植物基因的表达。本文利用200 mmol·L-1NaCl分别处理3叶期耐盐品系早熟长绒7号和盐敏感品系南丹巴地大花4 h,构建了4个小RNA文库并测序。以雷蒙德氏棉(Gossypium raimondii L.)D5基因组和本实验室陆地棉(Gossypium hirsutum L.)盐胁迫转录组测序拼接得到的序列作为参考,共发现360个miRNA,包括308个保守的miRNA和52个可信度高的新miRNA。2个品系共出现94个差异表达的miRNA,表达模式可分为2大类,筛选出20个候选miRNA。KEGG(Kyoto encyclopedia of genes and genomes)分析表明,耐盐品系显著富集的信号通路是抗原加工过程,而盐敏感品系显著富集的是生物碱合成途径。     

The use of general and specific combining abilities in a context of gene expression relevant to plant breeding

Many common traits are believed to be a composite reflection of multiple genetic and environmental factors. Recent advances suggest that subtle variations in the regulation of gene expression may contribute to quantitative traits. The nature of sequence variation affecting the regulation of gene expression either in cis (that is, affecting the expression of only one of the two alleles in a heterozygous diploid) or in trans (that is, affecting the expression of both alleles in a heterozygous diploid) is a key and usually unknown feature for the breeders. If the change in expression acts entirely in cis, then the structural gene can be treated as a candidate gene and a potential target for marker-assisted selection. Therefore, gene surveys for cis-regulatory variation are a first step in identifying potential targets for marker-assisted breeding. Here, we discuss in detail the “genome-wide analysis of allele-specific expression differences” (GASED) approach. The GASED approach was developed to screen for cis-regulatory variation on a genome-wide scale. In GASED, mRNA abundance is treated as if it were a quantitative phenotypic response variable, whose genetic between-F₁ hybrid variance is partitioned into additive and non-additive components. In plant breeding, this partitioning of the genetic variance is well known in the context of estimation of general and specific combining abilities for diallel crossing schemes. We demonstrate the GASED method using Arabidopsis thaliana data. The method can be used to screen for cis-regulatory variation in any crop species for which diallel crossing schemes are appropriate and genomic tools are available.     

开花后不同光周期条件下大豆农艺性状和品质性状的QTL分析

以开花期相近的181个大豆重组自交系(RIL)为材料,研究开花后不同光照长度对大豆主要农艺性状的影响,并在利用SSR标记构建大豆遗传图谱的基础上,分别在长日(16 h)和短日(12 h)条件下检测与主要农艺性状及其光周期敏感度(PS)相关的QTL。结果表明,开花后光照处理对大豆农艺性状和品质性状有较大影响,不同性状的光周期敏感度差异明显,株高＞主茎节数＞蛋白质含量、脂肪含量＞百粒重＞单株荚数＞蛋白质和脂肪总量。利用复合区间作图法检测到12个与株高、主茎节数、单株荚数、百粒重、蛋白质和脂肪总量等性状及各性状对开花后光周期处理的敏感度相关的QTL,分别定位于A1、A2、B1、B2、C1、D1a、F、L等8个连锁群上。其中,在短日条件下检测到4个QTL,可解释的遗传变异范围在11.37%~26.63%之间;在长日条件下检测到3个QTL,可解释的遗传变异范围在11.84%~27.85%之间;检测到5个与不同性状光周期敏感度有关的QTL,可解释相对应性状表型变异的范围在6.15%~21.44%之间。针对同一性状,未检测到在长日和短日条件下均起作用的主效QTL, 说明开花后光周期对大豆产量和品质性状相关基因的表达有较大影响。     

Genome‐Wide Identification of MicroRNAs Responsive to High Temperature in Rice (Oryza sativa) by High‐Throughput Deep Sequencing

MicroRNAs (miRNAs) are known to play important roles in plant growth and stress response. Heat stress is a severe abiotic stresses by adversely affecting plant growth and yield. To identify heat‐responsive miRNAs at the genome‐wide level in rice (Oryza sativa), we constructed two small RNA libraries from young panicles treated or not with heat conditions. Ion torrent sequencing of the two libraries identified 294 known miRNAs and 539 novel miRNAs. Differential expression analysis showed that 26 miRNAs were downregulated and 21 miRNAs were upregulated in response to heat stress. Among them, five heat‐responsive miRNAs, including miR162b, miR529a‐p5, PC‐5P‐62245‐9, miR171b and miR169n, were validated by quantitative real‐time polymerase chain reaction. A total of 44 target genes of the differentially expressed miRNAs were predicted. These target genes are most significantly overrepresented in the cell growth process. The results demonstrated that rice miRNAs play critical roles in the heat stress response. This study opens up a new avenue for understanding the regulatory mechanisms of miRNAs involvement in the heat stress response in rice.     

利用高通量测序技术鉴定棉纤维发育相关miRNAs及其靶基因

miRNA (microRNA)是一类21～24个核酸长度的非编码小分子RNA(sRNA),它主要通过抑制或降解靶基因来调控植物生长发育等过程.试验利用在纤维长度上有显著差异的2个回交自交系BILs (Backcross inbred lines)的0DPA (Days post anthesis)、3 DPA的胚珠和10 DPA的纤维构建6个sRNA文库并进行Solexa 测序.以已公布的棉花D5基因组序列和棉属其他序列为参考,经分析共发现561个miRNAs,其中包括254个已知的miRNAs(属于40个miRNA家族),75个候选的miRNAs和232个新的miRNAs,研究结果极大地丰富了棉属miRNAs.通过miRNA靶基因预测分析发现多数miRNAs负调控其对应的靶基因,少数正调控.KEGG(Kyoto encyclopedia of genes and genomes)注释结果表明miRNA的靶基因在植物激素代谢途径中显著富集.     

利用已测序水稻品种分析其农艺性状基因座

水稻重要农艺性状的基因定位研究在育种上具有重要意义。2004年在海南陵水县种植两个完全测序水稻品种日本晴与9311的F₂群体及双亲，分别考察了其单株分蘖数、穗数、有效分蘖数、穗长、主穗长、抽穗期、株高和剑叶8个农艺性状3次重复的平均值。用已构建的连锁遗传图谱（Nipponbare/9311-F₂遗传图谱）及Excel 2000和Mapmaker/QTL 1.1b软件对这8个性状间的相互关系和基因位点进行了分析。结果在LOD>2.0和P＜0.005的条件下共检测到41个QTLs，它们分布在水稻所有染色体上，单个QTL对性状表型贡献率11.0%～46.4%，其中大于20%的有22个。对选用已测序材料为亲本构建图谱来探讨水稻农艺性状的分子基础及其育种意义进行了讨论。     

应用大豆表型设计育种技术选育大豆品种齐黄34

大豆产量由群体结构和个体生产力共同决定。其中,群体结构由个体的结荚习性、株高、主茎节数、分枝数、叶柄和叶片等株型性状决定;个体生产力由单株有效荚数、每荚粒数和粒重等性状决定,这些性状都具有特定的遗传和变异规律。表型设计育种是根据农作物表型性状的遗传和变异规律,设计出理想的表型性状值并培育出理想品种的育种技术。大豆品种齐黄34就是根据生态环境、生产条件和市场需求,采用表型设计育种技术选育成的高产大豆品种,具有株型紧凑、个体生产潜力大等特点。