玉米大斑病抗性基因Ht1定位区域内候选序列的生物信息学分析

为获得玉米大斑病抗性基因Ht1候选序列, 文章采用生物信息学方法对与玉米大斑病抗性基因Ht1紧密连锁的分子标记umc22和umc122定位区域内候选序列进行了分析, 其中得到的63条ORF序列中有14条序列可编码蛋白质结构域。将14条核苷酸酸序列预测出的氨基酸序列与已克隆的24条抗性基因编码氨基酸序列进行Blast比对及进化树构建。结果发现, 候选序列gpm565a具有植物抗性基因编码产物的高度保守结构域, 而且与抗性基因Xal相似性高、亲缘关系近, 推测可能与抗性基因Ht1有关。其他候选序列由于不具有植物抗性基因编码产物的高度保守结构域或者相似性低、亲缘关系远等原因, 不能确定与抗性基因Ht1有关。通过对候选序列gpm565a进行二级结构及三维结构分析, 发现有大量构成蛋白质特异功能结构组件的无规则卷曲存在, 推测gpm565a可能是Ht1功能域的一部分。     

用微卫星标记定位太空诱变玉米核不育基因

用姊妹交多代的太空诱变玉米雄性不育材料RP3195(A)×S37(自交系)的两个不同果穗的F2代群体作为育性调查和基因定位群体,这两个果穗的F2代群体分别为138株和247株。用326对微卫星引物进行差异筛选,其中有56对引物出现多态性,然后用56对引物对F2代群体进行分析,结果表明引物bnlg197和umc1012与不育基因连锁,其中在F2代群体的不同果穗中引物bnlg197与不育基因之间的遗传距离分别为7cM和14.5cM,标记umc1012在F2代群体(138株)中与不育基因之间的遗传距离为28.5cM,据此将该核不育基因定位在3L染色体上。     

小麦品种Triticum spelta album中抗条锈病基因Yr5的RAPD标记

共用520个10碱基随机引物对小麦抗条锈基因Yr5的近等基因系进行了RAPD分析,发现了3个特异性DNA片段S1496、S14181950与Yr5基因连锁,其中S1496761与Yr5基因紧密连锁,遗传距离为2．7cM。经对特异性DNA片段S1496 761进行克隆,测序,设计了PCR扩增用专化引物SC－S1496 761a和SC－S149676b,用该引物可扩增出与原RAPD引物扩增出的相似的特异DNA片段,由于该引物还可扩增出迁移率极为相近的另1条非特异带,在琼脂糖凝胶上难以分辨,需用聚丙烯酰胺凝胶电泳结合银染进行检测,经用F2分离群体及部分相关品种材料检测,已证明该标记的可靠性。     

抗茎腐病分子标记在159份玉米自交系中的验证及实用性评价

为了评价抗茎腐病基因分子标记在辅助育种中的实用性,本研究对159份玉米自交系进行了茎腐病田间抗性鉴定,并检测了与4个茎腐病抗性QTL(qRfg1、qRfg2、Rpi QI319-1和Rpi QI319-2)紧密连锁的11个分子标记在上述材料中的扩增情况。结果表明:供试玉米自交系的平均发病率为26.30%,发病率低于30.0%的材料占67.92%,抗病资源丰富。来源于国外、东北、西南和黄淮海地区的材料平均发病率分别为27.67%、17.92%、15.12%和36.80%,与东北和西南地区种质相比,黄淮海地区抗性种质相对缺乏。通过比较分子标记扩增带型与田间茎腐病表型,发现与同一QTL连锁的不同分子标记的检测结果存在较大差异,其中分子标记STS01(qRfg1)、STSZ479(qRfg2)、bnlg1866(Rpi QI319-1)和bnlg1716(Rpi QI319-2)的阳性检测结果与田间表型符合度较高,分别为76.79%、78.95%、91.67%、73.33%,具有上述特异扩增多态性的材料平均发病率分别为22.06%、19.01%、10.65%、19.63%,可作为抗茎腐病分子检测的有效标记。本研究为开展玉米抗茎腐病分子育种提供了重要参考。     

陆地棉HB红花性状特异SCAR分子标记的开发

利用Operon系列引物筛选到1个与HB红花性状基因连锁的RAPD标记OPA15^1160,对差异条带进行克隆与核苷酸测序,根据测序结果设计SCAR引物,在HB红花近等基因系及其白花轮回亲本中进行PCR扩增程序优化和鉴定,筛选出一对引物可稳定扩增出与HB红花性状基因连锁的特异片段,获得了与HB红花性状基因紧密连锁的SCAR标记HB^-330。利用具黄色花瓣紫红色基斑的海岛棉与粉红花瓣的红叶棉等种质材料以7LHB红花近等基因系与白花轮回亲本杂交的F1、BC1F1、F2群体,对该SCAR标记的特异性与准确性进行了鉴定与验证,在红花植株中扩增出了330bp大小的片段而在白花植株中未扩增出,证明该标记准确性高、重复性好。HB红花是通过远缘杂交转自野生二倍体比克氏棉的性状,已成功地应用于性状标记杂交棉育种。该SCAR标记不仅为HB红花标记杂交种的纯度鉴定提供了有效技术手段,也为新品种保护提供了技术支持,促进了红花性状杂交种的分子标记辅助育种进程。     

草鱼种质相关SRAP及SCAR的分子标记

采用相关序列扩增多态性(Sequence-realted Amplified Polymorphism, SRAP)技术分析野生草鱼和家养草鱼,筛选与草鱼种质退化相关的分子遗传标记.共进行88对引物组合的检测, 产生标记数目共计905个.依据标记在群体中出现的频率和变化规律,共筛选出2 1个可能与种质相关的特异性标记,对这些特异性标记进行测序并将测序结果进行BLAST分析 .发现测得片段中有8个片段在GenBank中找到同源性较高的序列,而其他片段与数据库中序列的相似性较低.根据序列信息分别设计了3对引物.用这3对引物分别对草鱼三个群体进行 PCR扩增,分别产生了SCAR1(308 bp)、SCAR2(66 bp)、SCAR3(114 bp)3个扩增带.采用大样本对这3个标记进行验证,发现其中SCAR1在家养群体中呈现阳性,在野生群体中为阴性,可区分出这两种群体.以SCAR3为引物在174条家养群体中得到目的片段,在26个家养群体没有扩增出条带,分布频率为87%;在100个野生群体中有6个个体检测到该条带,分布频率为6%.以SCAR2为引物在野生群体中完全扩增出目的条带,淡水中心群体中有7条扩增到条带,前洲群体中没有扩增出条带,标记在家养种群中的分布频率为96.50%.因此SCAR1可作为草鱼家养群体的一个重要的分子遗传特征指标,为进一步进行分子标记辅助育种奠定了基础 [动物学报 54(3):475-481,2008].     

油菜单显性核雄性不育基因的分子标记

以陕西省杂交油菜研究中心选育的单显性核不育油菜分离群体为材料,利用集群分离法（BSA）对该油菜单显性核不育基因进行了RAPD分析。在随机选取的300个10碱基随机引物中,引物S243（5′CTATGCCGAC3′）在可育集团与不育集团间扩增出特异而可重复的1．5kb的多态性片段OPU-031500,而在细胞质雄性不育和其它核不育类型油菜中均未扩增出上述特异性片段,从而确证此RAPD标记OPU-031500。片段是与甘蓝型油菜单显性核不育基因连锁的。将该多态性片段克隆并测序,发现其序列与拟南芥的一段DNA序列高度同源。根据同源序列及测序结果设计两对特异引物（P1／P2和P3／P4）,引物P3／P4在可育系中可扩增到约1．5kb的单一特异片断,而在不育系中无带,从而将RAPD标记转化为稳定可靠的SCAR标记。     

由基因序列开发番茄枯萎病抗性基因Ⅰ-2的共显性分子标记

根据I-2的基因序列设计特异扩增引物对I-2/5F和I-2/5R, 扩增I-2基因3 132～3 765 bp之间片段, 基因型为I-2 / I-2的材料03F-7可扩增出633 bp的条带, 而基因型为i-2/ i-2的材料Moneymaker可扩增出693 bp的条带, 杂合型材料可扩增出以上2个条带。通过这两个特异扩增片段的克隆和测序证明, 抗病材料扩增的633 bp片段为I-2基因的3 132～3 765 bp之间的序列, 而感病等位基因中出现大量的碱基突变和60 bp片段插入。利用引物对I-2/5F和I-2/5R, 可区分纯合抗病材料、杂合抗病材料和纯合感病材料, 从而建立了I-2基因的共显性分子标记。在此基础上, 利用该标记对16个主要番茄品种进行基因型鉴定, 8个品种含有I-2基因, 其中1个品种基因型为I-2 / I-2, 其他品种为I-2 / i-2。通过一次PCR和一次HindⅢ酶切建立了I-2和Tm-22双基因检测体系, 为多基因鉴定及标记辅助选择提供了有力工具。     

由基因序列开发番茄枯萎病抗性基因I-2的共显性分子标记

根据I-2的基因序列设计特异扩增引物对I-2/5F和I-2/5R, 扩增I-2基因3 132～3 765 bp之间片段, 基因型为I-2 / I-2的材料03F-7可扩增出633 bp的条带, 而基因型为i-2/ i-2的材料Moneymaker可扩增出693 bp的条带, 杂合型材料可扩增出以上2个条带。通过这两个特异扩增片段的克隆和测序证明, 抗病材料扩增的633 bp片段为I-2基因的3 132～3 765 bp之间的序列, 而感病等位基因中出现大量的碱基突变和60 bp片段插入。利用引物对I-2/5F和I-2/5R, 可区分纯合抗病材料、杂合抗病材料和纯合感病材料, 从而建立了I-2基因的共显性分子标记。在此基础上, 利用该标记对16个主要番茄品种进行基因型鉴定, 8个品种含有I-2基因, 其中1个品种基因型为I-2 / I-2, 其他品种为I-2 / i-2。通过一次PCR和一次HindⅢ酶切建立了I-2和Tm-22双基因检测体系, 为多基因鉴定及标记辅助选择提供了有力工具。     

一条卡瓦胡椒特异RAPD 带转化成SCAR 标记的研究

采用27 份不同来源的胡椒属( Piper) 材料和1 份不同属的草胡椒( Peperomia pellucida) 材料用引物OPQ-03 扩增得到一条约400 碱基对( bp) 卡瓦胡椒特异片段。对该片段进行了克隆和序列分析, 并根据序列分析结果将上述RAPD 分子标记转化为重复性和特异性更好的SCAR ( sequence characterized amplified regions, 序列特征化扩增区) 分子标记。本研究设计出了1 对卡瓦胡椒特异SCAR 引物P7. 1 ( 5′-GGT CAC CTC ACC GCA GCA GGA TGA ACG-3′) 和P7 . 2 (5′-GGT CAC CTC AAT GAC ATG GGA TGA ATC-3′) , 用这对特异引物对本次试验的28 份材料进行PCR 扩增, 结果只有不同属的草胡椒材料无任何扩增, 其它材料均扩增出了预期大小440 bp 的特异带。     

Molecular mapping of genes for opposite leafing in maize using simple-sequence repeat markers

Maize with opposite phyllotaxy (OP) and also initiating ears in opposite pairs is an aberrant mutant and also precious material for maize breeding and plant evolution studies. Mapping and identifying the markers closely linked to genes for the OP trait are essential for cloning the gene and marker-assisted selection in breeding. We established H14D, a near-isogenic line of the OP trait with H53 genetic background. We found that the OP trait is regulated by two independent dominant genes with mutually complementary relations, named Opp-1 and Opp-2. Screening of seven simple-sequence repeat (SSR) markers among the 105 pairs of SSR primers showed polymorphism between the inbred lines H14D and H53. The polymorphic SSR markers were then used to determine linkage with the trait in an F(2) population with 441 progeny, suggesting that SSR marker umc2094 in the Bin2.01 region is linked with Opp-1 at 6.7 cM, and bnlg1831 in Bin2.06 is linked with Opp-2 at 6.1 cM. Further investigation showed that bnlg1092 and umc1028 are linked to Opp-1 and Opp-2 genes, with genetic distances of 12.2 and 1.9 cM. It was also found that the four SSR markers flank the two OP genes, respectively. These results will be useful for marker-assisted selection breeding of OP maize and will also strengthen the basis for cloning of the opposite leafing gene.     

The northern corn leaf blight resistance gene Ht1 encodes an nucleotide-binding,leucine-rich repeat immune receptor

Northern corn leaf blight, caused by the fungal pathogen Exserohilum turcicum, is a major disease of maize. The first major locus conferring resistance to E. turcicum race 0, Ht1, was identified over 50 years ago, but the underlying gene has remained unknown. We employed a map-based cloning strategy to identify the Ht1 causal gene, which was found to be a coiled-coil nucleotide-binding, leucine-rich repeat (NLR) gene, which we named PH4GP-Ht1. Transgenic testing confirmed that introducing the native PH4GP-Ht1 sequence to a susceptible maize variety resulted in resistance to E. turcicum race 0. A survey of the maize nested association mapping genomes revealed that susceptible Ht1 alleles had very low to no expression of the gene. Overexpression of the susceptible B73 allele, however, did not result in resistant plants, indicating that sequence variations may underlie the difference between resistant and susceptible phenotypes. Modelling of the PH4GP-Ht1 protein indicated that it has structural homology to the Arabidopsis NLR resistance gene ZAR1, and probably forms a similar homopentamer structure following activation. RNA sequencing data from an infection time course revealed that 1 week after inoculation there was a threefold reduction in fungal biomass in the PH4GP-Ht1 transgenic plants compared to wild-type plants. Furthermore, PH4GP-Ht1 transgenics had significantly more inoculation-responsive differentially expressed genes than wild-type plants, with enrichment seen in genes associated with both defence and photosynthesis. These results demonstrate that the NLR PH4GP-Ht1 is the causal gene underlying Ht1, which represents a different mode of action compared to the previously reported wall-associated kinase northern corn leaf blight resistance gene Htn1/Ht2/Ht3.     

Construction and characterization of a bacterial artificial chromosome library of maize inbred line 77Ht2

Maize inbred line 77Ht2 contains agriculturally important genes and has been widely used in corn breeding in China. A bacterial artificial chromosome (BAC) library of 77Ht2 has been constructed in order to identify useful genes and to facilitate the study of the maize genome. The library contains 175104 clones with an average insert size of 57 kb and represents about 4 maize haploid genome equivalents. Characterization of the library showed less than 0.5% of clones to not contain large inserts. Significant contamination of chloroplast and mitochondria DNA was not detected. BAC clones (152 arrays) were stored in 96 microtiter plates, with each well containing 12 clones. This is the first maize BAC library constructed in China. It is well suited for map-based cloning of maize genes and genome physical mapping.     

Identification of a RAPD marker linked to the oat stem rust gene Pg3

Summary The feasibility of identifying molecular markers linked to disease resistance genes in oats was investigated utilizing random primers in conjunction with polymerase chain reaction technology. A pair of near-isogenic oat lines were screened for polymorphic DNA fragments linked to the stem rust resistance gene Pg3. Two primers were identified which amplified DNA fragments that were polymorphic between the lines analyzed. One primer (ACOpR-2) was shown to be completely linked to the Pg3 locus; the other primer was not linked to either the ACOpR-2 or the Pg3 loci. This type of analysis, combined with rapid leaf disc DNA extraction techniques, offers an effective means of identifying useful molecular markers and of applying them to plant breeding selection strategies.Plant Research Centre publication number: 1443     

Molecular tagging and genetic mapping of the disease resistance gene<Emphasis Type="Italic"> RppQ</Emphasis> to southern corn rust

Southern corn rust (SCR), Puccinia polysora Underw, is a destructive disease in maize (Zea mays L.). Inbred line Qi319 is highly resistant to SCR. Results from the inoculation test and genetic analysis of SCR in five F₂ populations and five BC₁F₁ populations derived from resistant parent Qi319 clearly indicate that the resistance to SCR in Qi319 is controlled by a single dominant resistant gene, which was named RppQ. Simple sequence repeat (SSR) analysis was carried out in an F₂ population derived from the cross Qi319×340. Twenty SSR primer pairs evenly distributed on chromosome10 were screened at first. Out of them, two primer pairs, phi118 and phi 041, showed linkage with SCR resistance. Based on this result, eight new SSR primer pairs surrounding the region of primers phi118 and phi 041 were selected and further tested regarding their linkage relation with RppQ. Results indicated that SSR markers umc1,318 and umc 2,018 were linked to RppQ with a genetic distance of 4.76 and 14.59 cM, respectively. On the other side of RppQ, beyond SSR markers phi 041 and phi118, another SSR marker umc1,293 was linked to RppQ with a genetic distance of 3.78 cM. Because the five linkage SSR markers (phi118, phi 041, umc1,318, umc 2,018 and umc1,293) are all located on chromosome 10, the RppQ gene should also be located on chromosome 10. In order to fine map the RppQ gene, AFLP (amplified fragment length polymorphism) analysis was carried out. A total 54 AFLP primer combinations were analyzed; one AFLP marker, AF1, from the amplification products of primer combination E-AGC/M-CAA, showed linkage with the RppQ gene in a genetic distance of 3.34 cM. Finally the RppQ gene was mapped on the short arm of chromosome 10 between SSR markers phi 041 and AFLP marker AF1 with a genetic distance of 2.45 and 3.34 cM respectively.Communicated by H. F. Linskens