RFLP和AFLP分析白菜型油菜和甘蓝型油菜遗传多样性及其在油菜改良中的应用价值

采用RFLP和AFLP标记对来自中国和欧美的7份甘蓝型油菜和22份白菜型油菜进行了遗传多样性分析。在这29份材料中,166个酶-探针组合和2对AFLP引物共检测到1477个RFLP标记和183个AFLP标记。RFLP数据显示以拟南芥EST克隆作探针比用油菜基因组克隆做探针能检测到更多的多态性位点,且采用EcoR Ⅰ或BamH Ⅰ酶切比HindⅢ酶切多态性好,白菜型油菜和甘蓝型油菜中基因的拷贝数平均都为3个左右。UPGMA聚类分析表明中国白菜型油菜的遗传多样性比甘蓝型油菜和欧美白菜型油菜丰富,欧美甘蓝型油菜与欧美白菜型油菜聚为一类,而与中国甘蓝型油菜差异更大。中国白菜型油菜丰富的遗传多样性为中国甘蓝型油菜的改良提供了宝贵的资源,揭示了利用白菜型油菜A基因组和甘蓝型油菜A基因组间亚基因组杂种优势的可能性。     

微测序技术分析人类单核苷酸多态性

单核苷酸多态性 (singlenucleotidepolymorphism)是指染色体基因组水平上单个核苷酸变异引起的DNA序列多态性 ,即一种二等位基因标记。目前推断在基因组中至少有 30万个SNP[1 ] 。随着分子遗传学的发展 ,疾病研究从对单基因疾病的研究转向探讨多基因疾病 (如心血管疾病、神经系统疾病、各种肿瘤等 )的相关因素。因此 ,需要在人类基因组中找到一种数目众多、分布广泛且相对稳定的遗传标记 ,单核苷酸多态性正代表了这样一种标记。因此SNPs已成为继第一代限制性片段多态性标记 ,第二代微卫星多态性标记后具有重要研究价值的第三代基因遗传…     

油菜简单重复序列SSR(simple sequence repeat)研究进展

简单重复序列(simple sequence repeat,SSR)是重复单元少于6个核苷酸重复序列,广泛分布于动植物基因组中,呈孟德尔遗传,已被作为一种理想的共显性标记应用于动植物遗传研究中。本文重点介绍了SSR分类、特点,及近几年来油菜SSR标记的开发和SSR技术在油菜基因定位、品种鉴定中的应用,并对SSR标记在油菜中的应用进行了探讨。     

人类基因组中单核苷酸多态性的检测技术

随着人类基因组测序工作的完成,单核苷酸多态性作为继限制性片段长度多态性和微卫星多态性这2种遗传标记之后的第3代遗传标记,已成为人类后基因组时代的主要研究内容之一。系统地介绍了人类基因组中单核苷酸多态性的传统检测方法、新的方法以及基于生物信息学的方法,并对SNP检测技术的发展进行了展望。     

谷子SSR标记研究进展

谷子是一种重要的杂粮作物.谷子是二倍体自花授粉作物,基因组较小,与水稻基因组存在明显的共线性,是开展基因组研究的理想模式植物.SSR标记具有共显性、多态性高、操作简单等优点,广泛应用于遗传多样性研究、品种鉴定、遗传图谱构建、功能基因研究、分子标记辅助选择育种等领域.本文对谷子SSR标记的开发和应用进行总结,提出了谷子分子遗传研究中存在的问题,并对今后的发展趋势作了初步探讨,旨在为谷子分子遗传研究提供参考.     

MITE转座子在不同甘蓝型油菜品种中的多态性研究

微型反向重复转座因子(MITE)在真核生物基因组中普遍存在,其活动能够在物种内形成丰富的多态性,在基因组进化和基因调控中均发挥重要作用。该研究利用286对不同MITE家族侧翼序列筛选的特异引物,对101个中国油菜、27个加拿大油菜和29个合成油菜品系(共157个品系)的基因组DNA进行多态性研究,以明确MITE家族的插入/缺失多态性以及在不同油菜品种之间插入的遗传多样性,进而探讨他们之间的亲缘关系。结果显示：(1)286对引物中筛选出60对条带清晰重复性高的多态性引物,多态性比例21.0%;其中Stowaway like家族和Tourist like家族特异引物的多态性比例分别为24.6%和20.0%。(2)PCR扩增结果显示,60对多态性引物对中国、加拿大、合成油菜品系的基因组DNA分别扩增出4 029、1 044、1 087条清晰可辨的条带。(3)UPGMA聚类显示,中国油菜品种间遗传相似系数在0.59~0.95,加拿大油菜品种间遗传相似系数在0.73~0.95;合成油菜品种间遗传相似系数值在0.64~0.93。研究表明,MITE在基因组中大量随机插入,形成种内丰富的多态性,利用MITE家族引物检测不同地区的油菜种质资源的多态性,可为油菜的育种找到许多尚未被开发的遗传变异。     

通过白菜型油菜和埃塞俄比亚芥的Ar和Cc基因组导入创造甘蓝型油菜新材料

油菜是目前我国主要种植的油料作物之一,但现有的种质资源限制了产量的进一步提高。本研究采取了一种新的育种方式来增加甘蓝型油菜的种质资源,即通过远缘杂交结合分子标记辅助选择的方式将白菜型油菜的Ar基因组和埃塞俄比亚芥的Cc对现有的甘蓝型油菜品种的基因组(AnAnCnCn)进行部分替换。通过对五倍体杂交后代(ArAnBcCcCn)进行染色体选择,找到了染色体数目为38的材料。为了和现有的甘蓝型油菜进行区分,得到的新材料被认定为甘蓝型油菜新材料。实验结果表明,得到的部分甘蓝型油菜新材料具有基本正常的减数分裂过程、正常的花粉萌发以及胚囊发育过程,这说明甘蓝型油菜新材料达到了遗传平衡。分子标记分析表明:甘蓝型油菜新材料的约50%的基因组被白菜型油菜的Ar基因组和埃塞俄比亚芥的Cc替换,并且这些甘蓝型油菜新材料之间具有丰富的遗传多样性。因此,白菜型油菜的Ar基因组和埃塞俄比亚芥的Cc基因组导入对于丰富现有的甘蓝型油菜种质资源具有明显的效果。     

油菜硫甙性状QTL 区间的加密和整合

目的:加密油菜控制硫甙性状QTL区间,并进行QTL整合预测候选基因。方法:利用生物信息学方法根据已知测序的白菜BAC序列信息设计引物,在油菜TN DH群体中进行多态性扩增和定位,并根据加密后构建的遗传连锁图重新检测QTL,进行QTL整合。结果:将根据白菜BAC设计的3对多态性标记成功定位到油菜控制硫甙性状QTL区间,进行QTL整合后将QTL置信区间进一步缩小,并判定了初步的候选基因。结论:充分利用白菜已测序的BAC或者基因组信息,将能加快油菜基础研究的步伐。     

不同DNA分子标记技术信息量比较

本对RFLP、RAPD、SSR和AFLP四种主要的分子标记在研究多态性时所揭示的信息量进行比较研究,结果表明：(1)SSR标记的多态性最高,多态性信息含量PIC值最大,适于品种指纹图谱绘制、基因型鉴定和核心种质构建。另外,由于SSR标记主要检测基因组的重复区,因此用SSR标记研究功能基因区的差异效果较差;(2)AFLP标记评价效率最高,评价效率指数Ai值最大,在一次扩增中能有上百条带,不但能比较不同样品的相异的带,而且可以从相同的带型及带的多少来比较遗传背景的相似程度,适于研究不同种质问的遗传关系、亲子间的系谱分析等。     

鲤鱼多态性EST标记的筛选与特性分析

表达序列标签(Expressed sequence tag,EST)标记在基因组作图和分子标记辅助育种研究中具有重要价值.筛选和开发可用于遗传作图的鲤鱼多态性EST标记,对研究鲤鱼的基因组结构、遗传多样性研究和遗传育种有重要意义.本研究根据GenBank数据库中鲤鱼EST序列设计了67对EST引物,有47对在鲤鱼基因组DNA中成功扩增得到稳定的特异性条带,经单链构象多态性(SSCP)分析,12对(25.5%)引物扩增的EST在1个鲤鱼回交家系中具有多态性,其中6个为鲤鱼功能基因,5个与斑马鱼(Danio rerio)功能基因具较高相似性,1个与斑点叉尾(Ictalurus punctatus)功能基因具较高相似性.这些多态性的EST在鲤鱼二倍体家系和单倍体群体中的等位基因分离均符合孟德尔规律(1∶1或3∶1),单倍体中的SSCP条带数目为二倍体的二分之一.选用其中4对多态性的EST引物对洞庭湖鲤鱼进行初步群体遗传分析,结果显示基因多样性(H)为0.437,远低于微卫星标记所揭示的该群体基因多样性(接近于1).结果表明,多态性的EST-SSCP标记尽管遗传变异性较低,但是作为来源于编码区的Ⅰ型遗传标记,在遗传作图和种群遗传适应性等研究中有较好的应用潜力.     

一种快速生长的甘蓝型油菜DH系的获得和鉴定

从快速生长的甘蓝型油菜的小孢子培养中共获得23个再生植株。经倍性鉴定,其中自发加倍成二倍体的有10株,单倍体13株。单倍体再用秋水仙碱处理后获得DH系,所得材料对油菜功能基因组学的研究可能有一定的价值。     

整株转化法及其在油菜上的应用与展望

近年来, 在植物遗传转化和功能基因组学研究的迫切需求下, 一些不依赖于受体细胞脱分化、再分化过程的转化技术(即所谓的整株转化法)相继出现, 它们大多以分生组织或生殖细胞作为受体, 不受基因型限制, 具有操作简单、转化效率高、无体细胞变异和后代遗传稳定等优点。该文对整株转化法的最新研究进展进行综述, 并重点讨论了其在油菜(Brassica napus) 上的应用现状与前景。     

Yield reduction in Brassica napus, B. rapa, B. juncea, and Sinapis alba caused by flea beetle (Phyllotreta cruciferae (Goeze) (Coleoptera: Chrysomelidae)) infestation in northern Idaho

Phyllotreta cruciferae is an important insect pest of spring-planted Brassica crops, especially during the seedling stage. To determine the effect of early season P. cruciferae infestation on seed yield, 10 genotypes from each of two canola species (Brassica napus L. and Brassica rapa L.) and two mustard species (Brassica juncea L. and Sinapis alba L.) were grown in 2 yr under three different P. cruciferae treatments: (1) no insecticide control; (2) foliar applications of endosulfan; and (3) carbofuran with seed at planting plus foliar application of carbaryl. Averaged over 10 genotypes, B. rapa showed most visible P. cruciferae injury and showed greatest yield reduction without insecticide application. Mustard species (S. alba and B. juncea) showed least visible injury and higher yield without insecticide compared with canola species (B. napus and B. rapa). Indeed, average seed yield of S. alba without insecticide was higher than either B. napus or B. rapa with most effective P. cruciferae control. Significant variation occurred within each species. A number of lines from B. napus, B. juncea, anid S. alba showed less feeding injury and yield reduction as a result of P. cruciferae infestation compared with other lines from the same species examined, thus having potential genetic background for developing resistant cultivars.     

Diversity array technology markers: genetic diversity analyses and linkage map construction in rapeseed (Brassica napus L.)

We developed Diversity Array Technology (DArT) markers for application in genetic studies of Brassica napus and other Brassica species with A or C genomes. Genomic representation from 107 diverse genotypes of B. napus L. var. oleifera (rapeseed, AACC genomes) and B. rapa (AA genome) was used to develop a DArT array comprising 11 520 clones generated using PstI/BanII and PstI/BstN1 complexity reduction methods. In total, 1547 polymorphic DArT markers of high technical quality were identified and used to assess molecular diversity among 89 accessions of B. napus, B. rapa, B. juncea, and B. carinata collected from different parts of the world. Hierarchical cluster and principal component analyses based on genetic distance matrices identified distinct populations clustering mainly according to their origin/pedigrees. DArT markers were also mapped in a new doubled haploid population comprising 131 lines from a cross between spring rapeseed lines 'Lynx-037DH' and 'Monty-028DH'. Linkage groups were assigned on the basis of previously mapped simple sequence repeat (SSRs), intron polymorphism (IP), and gene-based markers. The map consisted of 437 DArT, 135 SSR, 6 IP, and 6 gene-based markers and spanned 2288 cM. Our results demonstrate that DArT markers are suitable for genetic diversity analysis and linkage map construction in rapeseed.     

Exploiting comparative mapping among Brassica species to accelerate the physical delimitation of a genic male-sterile locus (BnRf) in Brassica napus

The recessive genic male sterility (RGMS) line 9012AB has been used as an important pollination control system for rapeseed hybrid production in China. Here, we report our study on physical mapping of one male-sterile locus (BnRf) in 9012AB by exploiting the comparative genomics among Brassica species. The genetic maps around BnRf from previous reports were integrated and enriched with markers from the Brassica A7 chromosome. Subsequent collinearity analysis of these markers contributed to the identification of a novel ancestral karyotype block F that possibly encompasses BnRf. Fourteen insertion/deletion markers were further developed from this conserved block and genotyped in three large backcross populations, leading to the construction of high-resolution local genetic maps where the BnRf locus was restricted to a less than 0.1-cM region. Moreover, it was observed that the target region in Brassica napus shares a high collinearity relationship with a region from the Brassica rapa A7 chromosome. A BnRf-cosegregated marker (AT3G23870) was then used to screen a B. napus bacterial artificial chromosome (BAC) library. From the resulting 16 positive BAC clones, one (JBnB089D05) was identified to most possibly contain the BnRf (c) allele. With the assistance of the genome sequence from the Brassica rapa homolog, the 13.8-kb DNA fragment covering both closest flanking markers from the BAC clone was isolated. Gene annotation based on the comparison of microcollinear regions among Brassica napus, B. rapa and Arabidopsis showed that five potential open reading frames reside in this fragment. These results provide a foundation for the characterization of the BnRf locus and allow a better understanding of the chromosome evolution around BnRf.     

Syntenic quantitative trait loci and genomic divergence for Sclerotinia resistance and flowering time in Brassica napus

Oilseed rape (Brassica napus) is an allotetraploid with two subgenomes descended from a common ancestor. Accordingly, its genome contains syntenic regions with many duplicate genes, some of which may have retained their original functions, whereas others may have diverged. Here, we mapped quantitative trait loci (QTL) for stem rot resistance (SRR), a disease caused by the fungus Sclerotinia sclerotiorum, and flowering time (FT) in a recombinant inbred line population. The population was genotyped using B. napus 60K single nucleotide polymorphism arrays and phenotyped in six (FT) and nine (SSR) experimental conditions or environments. In total, we detected 30 SRR QTL and 22 FT QTL and show that some of the major QTL associated with these two traits were co-localized, suggesting a genetic linkage between them. Two SRR QTL on chromosome A2 and two on chromosome C2 were shown to be syntenic, suggesting the functional conservation of these regions. We used the syntenic properties of the genomic regions to exclude genes for selection candidates responsible for QTL-associated traits. For example, 152 of the 185 genes could be excluded from a syntenic A2-C2 region. These findings will help to elucidate polyploid genomics in future studies, in addition to providing useful information for B. napus breeding programs.     

Comparative mapping reveals similar linkage of functional genes to QTL of yield-related traits between <Emphasis Type="Italic">Brassica napus</Emphasis> and <Emphasis Type="Italic">Oryza sativa</Emphasis>

Oryza sativa and Brassica napus-two important crops for food and oil, respectively-share high seed yield as a common breeding goal. As a model plant, O. sativa genomics have been intensively investigated and its agronomic traits have been advanced. In the present study, we used the available information on O. sativa to conduct comparative mapping between O. sativa and B. napus, with the aim of advancing research on seed-yield and yield-related traits in B. napus. Firstly, functional markers (from 55 differentially expressed genes between a hybrid and its parents) were used to detect B. napus genes that co-localized with yield-related traits in an F(2:3) population. Referring to publicly available sequences of 55 B. napus genes, 53 homologous O. sativa genes were subsequently detected by screening, and their chromosomal locations were determined using silico mapping. Comparative location of yield-related QTL between the two species showed that a total of 37 O. sativa and B. napus homologues were located in similar yield-related QTL between species. Our results indicate that homologous genes between O. sativa and B. napus may have consistent function and control similar traits, which may be helpful for agronomic gene characterization in B. napus based on what is known in O. sativa.     

模拟氮沉降对青藏高原地区油菜幼苗期生长和生理特性的影响

以甘蓝型油菜(青杂303)为研究材料,通过研究分析油菜幼苗期的生长和生理特性,旨在探讨油菜幼苗对青藏高原地区未来大气氮沉降的响应机制。实验以NH4NO3氮肥模拟大气氮沉降的NH4+、NO3-输入并分别喷施0、2.5、5.0 g/m2.a模拟青藏高原地区大气氮沉降增加的0倍、2倍、4倍背景。结果表明:施氮处理对油菜幼苗生长具有促进作用,油菜幼苗的地上部分干重、叶面积和株高等形态指标都发生了显著变化;随着施氮量增加油菜幼苗叶片的抗氧化酶活性增强,氮肥输入量为5.0 g/m2.a的处理组中,其幼苗叶片超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)活性与对照的相比均具有极显著差异。因此,反映出青藏高原地区生态系统中植物生长受到有效氮的限制,在外界有效氮输入下,对植物生长发育具有促进作用。     

Identification and characterization of candidate Rlm4 blackleg resistance genes in Brassica napus using next-generation sequencing

A thorough understanding of the relationships between plants and pathogens is essential if we are to continue to meet the agricultural needs of the world's growing population. The identification of genes underlying important quantitative trait loci is extremely challenging in complex genomes such as Brassica napus (canola, oilseed rape or rapeseed). However, recent advances in next-generation sequencing (NGS) enable much quicker identification of candidate genes for traits of interest. Here, we demonstrate this with the identification of candidate disease resistance genes from B.?napus for its most devastating fungal pathogen, Leptosphaeria maculans (blackleg fungus). These two species are locked in an evolutionary arms race whereby a gene-for-gene interaction confers either resistance or susceptibility in the plant depending on the genotype of the plant and pathogen. Preliminary analysis of the complete genome sequence of Brassica rapa, the diploid progenitor of B.?napus, identified numerous candidate genes with disease resistance characteristics, several of which were clustered around a region syntenic with a major locus (Rlm4) for blackleg resistance on A7 of B.?napus. Molecular analyses of the candidate genes using B.?napus NGS data are presented, and the difficulties associated with identifying functional gene copies within the highly duplicated Brassica genome are discussed.     

Homoeologous loci control the accumulation of seed glucosinolates in oilseed rape (Brassica napus).

The genetic control of seed glucosinolate content in oilseed rape was investigated using two intervarietal backcross populations. Four QTLs segregating in the population derived from a Brassica napus L. 'Victor' x Brassica napus L. 'Tapidor' cross, together accounting for 76% of the phenotypic variation, were mapped. Three of these loci also appeared to control the accumulation of seed glucosinolates in a Brassica napus L. 'Bienvenu' x 'Tapidor' cross, and accounted for 86% of the phenotypic variation. The three QTLs common to both populations mapped to homoeologous regions of the B. napus genome, suggesting that seed glucosinolate accumulation is controlled by duplicate genes. It was possible to extend the comparative analysis of QTLs controlling seed glucosinolate accumulation by aligning the published genetic maps generated by several research groups. This comparative mapping demonstrated that high-glucosinolate varieties often carry low-glucosinolate alleles at one or more of the loci controlling seed glucosinolate accumulation.