单核苷酸多态性与甜瓜抗枯萎病分子育种研究

目的:结合单核苷酸多态性标记技术,利用甜瓜本身的抗病性以解决新疆甜瓜病害问题。方法:对新疆甜瓜抗枯萎病基因Fom-2基因进行克隆分析,并根据Fom-2基因在不同抗性甜瓜亲本的单核苷酸多态性,设计检测SNP标记的PCR扩增引物,验证其多态性;并利用F2代分析该标记与筛选获得的甜瓜抗枯萎病基因连锁的SSR标记的遗传关系。结果:在抗病与感病甜瓜品种中均扩增获得PCR条带,试验中设计单核苷酸多态性分子标记在抗病品种为显性,与筛选的和抗枯萎病基因紧密连锁的共显性标记SSR430共分离。结论:不同抗性甜瓜品种均含有Fom-2基因或其高度同源序列,SNP显性标记和共显性标记SSR430均可用于甜瓜抗枯萎病分子标记辅助育种。     

小麦品系5R618抗叶锈基因的初步定位

5R618是高抗叶锈病小麦品系。为了确定该品系所携带的抗叶锈基因,以5R618与感病小麦品种郑州5389杂交获得F1,自交获得F2分离群体以及F2∶3家系,用叶锈菌生理小种THJP对亲本、F2分离群体以及F2∶3家系进行叶锈抗性鉴定,然后进行分子标记分析。结果显示,5R618对生理小种THJP的抗病性由1对显性基因控制,该基因暂命名为Lr5R。经过亲本和抗感池间分子标记筛选以及F2∶3家系的标记检测,Lr5R定位于染色体3DL上,barc71和STS24-16是Lr5R最近的2个标记,遗传距离分别为0.9 c M和2.1 c M。     

新疆野生油菜胞质雄性不育系1193A对应恢复基因的SSR标记分析

以油菜细胞质雄性不育系1193A和恢复系1193R2为亲本构建F2分离群体,并运用BSA法构建了可育和不育基因池。利用1521对SSR引物进行了多态性分析,结果表明有36对引物在亲本和基因池间都表现多态性,用F2单株验证表明有11对引物与恢复基因连锁,离恢复基因较近的2个标记CB10316和Bn GMS171分布在恢复基因Rf的两侧,遗传距离分别为3.9 c M和5.7 c M,可作为恢复系标记辅助育种的候选标记。     

利用SSR标记定位油菜细胞质雄性不育系1193A的恢复基因

以油菜细胞质雄性不育系1193A和恢复系1193R2为亲本构建F2分离群体,并运用BSA法构建了可育和不育基因池。利用1521对SSR引物进行了多态性分析,结果表明有36对引物在亲本和基因池间都表现多态性,用F2单株验证表明有11对引物与恢复基因连锁,离恢复基因较近的2个标记CB10316和Bn GMS171分布在恢复基因Rf的两侧,遗传距离分别为3.9 c M和5.7 c M,可作为恢复系标记辅助育种的候选标记。     

黄瓜霜霉病抗病基因的RAPD及SCAR标记

以感霜霉病黄瓜L18-10-2和抗霜霉病黄瓜129为亲本构建F2代分离群体,以F3代植株霜霉病抗性鉴定表示F2代各单株抗病性并得以区分各单株杂合或纯合感病性,采用RAPD技术和转SCAR的方法筛选黄瓜抗霜霉病基因分子标记.结果显示,在318条RAPD引物中有18条引物表现出两亲本间多态性,其中引物P18的SB-SP18561扩增片段与霜霉病抗病基因之间紧密连锁,根据交换率和Kosambi函数公式计算其遗传距离为7.85 cM.回收SBSP18561片段并克隆和测序,其准确长度为561 bp.将该RAPD标记转换为SCAR标记,长度为494 bp,命名为SSBSP18494.     

小麦品系19HRWSN-76的抗叶锈性研究

小麦叶锈病是影响小麦产量的最主要病害之一,CIMMYT品系19HRWSN-76高抗小麦叶锈病,以该品系与感病品系郑州5389杂交得到F2群体,利用叶锈菌生理小种FHJP对F2群体接菌鉴定,结果显示群体的抗感比例符合3∶1的理论比值,推测19HR WSN-76的抗叶锈性由一对显型基因控制,暂命名为Lr HR76。利用分子标记技术和分离群体分组分析法对F2群体进行分子标记检测,位于3DL的SSR标记barc71与该抗病基因连锁,遗传距离为3.0 c M。     

节瓜果皮颜色相关基因的ISSR标记分析

本研究选用节瓜F2代分离群体作为研究材料,采用分离体分组混合(bulked segregation analysis,BSA)分析法,结合ISSR标记技术,分别构建节瓜的淡绿色果皮基因池和墨绿色果皮基因池并对100条ISSR引物进行筛选扩增,进而对与节瓜果皮颜色性状控制基因连锁的分子标记进行分析研究,最终得到了在两基因池间具有多态性的标记G855-6。通过F2代群体验证,最终确定了ISSR标记G855-6与节瓜墨绿色果皮控制基因连锁,其交换值为8.95%,遗传距离为9.04 c M。     

黄瓜白色果皮基因遗传规律及定位研究

以黄瓜嫩果深绿色果皮自交系1507(P1)和白色果皮自交系1508(P2)为亲本,构建6世代遗传群体(P1、P2、F1、F2、BC1P1、BC1P2),对黄瓜嫩果白色果皮基因(w)进行遗传规律分析和基因定位研究。结果表明,黄瓜白色果皮性状由隐性单基因(w)控制,深绿色对白色为显性。利用F2群体,结合分离群体分组分析法筛选得到了14个与w基因相关的SSR标记,构建了该基因的SSR连锁群,将其定位到黄瓜3号染色体上,两侧的标记为SSR23517和SSR23141,遗传距离分别为4.9cM和1.9cM。侧翼标记之间的物理距离为1 150kb,在该区域中共预测了500个候选基因。该研究对w基因的初步定位,为该基因精细定位及分子标记辅助选择育种奠定了良好的基础。     

黄瓜枯萎病病原菌拮抗菌的筛选及其生物防治效应

分离筛选到一株对黄瓜枯萎病病原菌——尖孢镰刀菌具有显著拮抗作用的菌株YHJ15,并根据其生理生化特征和16SrRNA基因序列将其初步鉴定为短小芽孢杆菌;研究了其最适的生长条件。为黄瓜枯萎病的生物防治提供了种质资源。     

大白菜—结球甘蓝1号二体异附加系分子标记鉴定及其自交后代的性状分析

利用与抗霜霉病、抗芜菁花叶病毒基因连锁的SCAR标记和能够区分大白菜和结球甘蓝FLCs基因的特异引物对大白菜—结球甘蓝1号二体异附加系(AC1d)及其亲本进行分析,结果表明:AC1d及其双亲中均含有与抗霜霉病基因连锁的标记;AC1d和亲本结球甘蓝中含有与抗芜菁花叶病毒基因连锁的标记,亲本大白菜中无该标记;AC1d除具有4个大白菜Br FLCs基因外,同时还添加了结球甘蓝Bo FLC3基因。AC1d自交后代的株高、株展、球高、维生素C含量、可溶性蛋白质及其7种硫苷组分含量超出了亲本大白菜和结球甘蓝;叶形指数、可溶性糖含量和对小菜蛾的抗性等超出了亲本大白菜。为利用AC1d后代选育携有目标性状的易位系提供了依据。     

利用cDNA - AFLP技术分离棉花抗枯萎病相关基因

目的:分离出棉花抗枯萎病基因片段,并Blast分析其差异片段.方法:以抗枯萎病的棉花品种中棉12号为材料,采用苗期水培方法于三叶期进行枯萎病菌诱导处理,以不接菌为对照.用64对选择性扩增引物对诱导处理和对照的cDNA进行AFLP分析.结果:得到25个阳性差异片段,在GenBank中进行同源性序列比对,24个片段在GenBank数据库中发现同源序列,有1个功能未知.结论:作物的抗病机理复杂,涉及物质和能量代谢,信号传导,逆境响应及蛋白功能调控等方面.     

Interaction of Fusarium oxysporum f. sp. ciceri and Meloidogyne javanica on Cicer arietinum

Interaction of Meloidogyne javanica and Fusarium oxysporum f. sp. ciceri was studied on Fusarium wilt-susceptible (JG 62 and K 850) and resistant (JG 74 and Avrodhi) chickpea cultivars. In greenhouse experiments, inoculation of M. javanica juveniles prior to F. oxysporum f. sp. ciceri caused greater wilt incidence in susceptible cultivars and induced vascular discoloration in roots of resistant cultivars. Nematode reproduction was greatest (P = 0.05) at 25 °C. Number of galls and percentage of root area galled increased when the temperature was increased from 15 °C to 25 °C. Wilt incidence was greater at 20 °C than at 25 °C. Chlorosis of leaves and vascular discoloration of plants did not occur at 15 °C. The nematode enhanced the wilt incidence in wilt-susceptible cultivars only at 25 °C. Interaction between the two pathogens on shoot and root weights was significant only at 20 °C, and F. o. ciceri suppressed the nematode density at this temperature. Wilt incidence was greater in clayey (48% clay) than in loamy sand (85% sand) soils. The nematode caused greater plant damage on loamy sand than on clayey soil. Fusarium wilt resistance in Avrodhi and JG 74 was stable in the presence of M. javanica across temperatures and soil types.     

Suppression of Fusarium oxysporum and induced resistance of plants involved in the biocontrol of Cucumber Fusarium Wilt by Streptomyces bikiniensis HD-087

Cucumber Fusarium Wilt, caused by Fusarium oxysporum f. sp. cucumerinum, which usually leads to severe economic damage, is a common destructive disease worldwide. To date, no effective method has yet been found to counteract this disease. A fungal isolate, designated HD-087, which was identified as Streptomyces bikiniensis using physiological-biochemical identification and 16S rRNA sequence analysis, is shown to possess distinctive inhibitory activity against F. oxysporum. The fermentation broth of HD-087 leads to certain abnormalities in pathogen hyphae. It peroxidizes cell membrane lipids, which leads to membrane destruction along with cytoplasm leakage. This broth also restrains germination of the conidia. The activities of the enzymes peroxidase, phenylalanine ammonia-lyase, and β-1,3-glucanase in cucumber leaves were dramatically increased after treated with fermentation broth of HD-087. The levels of chlorophyll and soluble sugars were also found to be increased, with the relative conductivity of leaves being reduced. In short, the metabolites of strain HD-087 can effectively suppress F. oxysporum and trigger induced resistance in cucumber.     

Identification of quantitative trait loci contributing to Fusarium wilt resistance on an AFLP linkage map of flax (Linum usitatissimum)

 An AFLP genetic linkage map of flax (Linum usitatissimum) was used to identify two quantitative trait loci (QTLs) on independent linkage groups with a major effect on resistance to Fusarium wilt, a serious disease caused by the soil pathogen Fusarium oxysporum (lini). The linkage map was constructed using a mapping population from doubled-haploid (DH) lines. The DH lines were derived from the haploid component of F₂ haploid-diploid twin seed originating from a cross between a polyembryonic, low-linolenic-acid genotype (CRZY8/RA91) and the Australian cultivar ‘Glenelg’. The AFLP technique was employed to generate 213 marker loci covering approximately 1400 cM of the flax genome (n=15) with an average spacing of 10 cM and comprising 18 linkage groups. Sixty AFLP markers (28%) deviated significantly (P<0.05) from the expected segregation ratio. The map incorporated RFLP markers tightly linked to flax rust (Melamspora lini) resistance genes and markers detected by disease resistance gene-like sequences. The study illustrates the potential of the AFLP technique as a robust and rapid method to generate moderately saturated linkage maps, thereby allowing the molecular analysis of traits, such as resistance to Fusarium wilt, that show oligogenic patterns of inheritance. Received: 8 December 1997 / Accepted: 7 April 1998     

AFLP and STS tagging of a major QTL for Fusarium head blight resistance in wheat

Large-scale field screening for Fusarium head blight (FHB) resistance in wheat is difficult because environmental factors strongly influences the expression of resistance genes. Marker-assisted selection (MAS) may provide a powerful alternative. Conversion of amplified fragment length polymorphism (AFLP) markers into sequence-tagged site (STS) markers can generate breeder-friendly markers for MAS. In a previous study, one major quantitative trait locus (QTL) on chromosome 3BS was identified by using EcoRI-AFLP and a recombinant inbred population derived from the cross Ning 7840/Clark. Further mapping with PstI-AFLPs identified five markers that were significantly associated with the QTL. Three of them individually explained 38% to 50% of the phenotypic variation for FHB resistance. Two of them (pAGT/mCTG57, pACT/mCTG136) were linked to the QTL in coupling, and another (pAG/mCAA244) was linked to the QTL in repulsion. Successful conversion of one AFLP marker (pAG/mCAA244) yielded a co-dominant STS marker that explains about 50% of the phenotypic variation for FHB resistance in the population. The STS was validated in 14 other cultivars and is the first STS marker for a FHB resistance QTL converted from an AFLP marker.     

黄瓜霜霉病抗性相关基因的AFLP标记

运用AFLP技术,采用集群分析法研究与黄瓜霜霉病抗性基因相关的分子标记.结果表明,在F2群体中E25M63-103标记与霜霉病病情指数的相关系数(0.337)和回归分析的F值(20.98)都达到了极显著水平.用E25M63-103标记对国内外的其它27份黄瓜材料进行检测,该标记与霜霉病病情指数的相关系数为0.555,也达到极显著相关水平,进一步证明该标记与控制黄瓜霜霉病感病的相关基因是连锁的.E25M63-103片段长度为103 bp,通过BLAST查询,该片段的同源性较小,表明E25M63-103标记可能是黄瓜基因组特有的一段DNA序列.     

普通小麦Qz180中一个抗条锈病基因的分子作图(英文)

普通小麦(Triticum aestivum L.)材料Qz180具有良好的抗条锈病特性,经基因推导发现其含有一个优良的抗条锈病的基因,暂定名为YrQz。用Qz180与感病材料铭贤169和WL1分别杂交构建了两个F_2群体,用条中30号条锈菌小种对这两个群体进行的抗性测验表明,YrQz为显性单基因遗传。通过SSR和AFLP结合BSA的方法对这个基因进行了分子作图,结果鉴定出与YrQz连锁的2个SSR标记和2个AFLP标记。根据SSR标记的染色体位置,该基因被定位在2B染色体的长臂上,位于两个SSR位点Xgwm388和Xgwm526之间;两个AFLP标记P35M48(452)和P36M61(163)分别位于该基因的两侧,遗传距离分别为3.4cM和4.1cM。     

Dissection of the fusarium I2 gene cluster in tomato reveals six homologs and one active gene copy.

The I2 locus in tomato confers resistance to race 2 of the soil-borne fungus Fusarium oxysporum f sp lycopersici. The selective restriction fragment amplification (AFLP) positional cloning strategy was used to identify I2 in the tomato genome. A yeast artificial chromosome (YAC) clone covering approximately 750 kb encompassing the I2 locus was isolated, and the AFLP technique was used to derive tightly linked AFLP markers from this YAC clone. Genetic complementation analysis in transgenic R1 plants using a set of overlapping cosmids covering the I2 locus revealed three cosmids giving full resistance to F. o. lycopersici race 2. These cosmids shared a 7-kb DNA fragment containing an open reading frame encoding a protein with similarity to the nucleotide binding site leucine-rich repeat family of resistance genes. At the I2 locus, we identified six additional homologs that included the recently identified I2C-1 and I2C-2 genes. However, cosmids containing the I2C-1 or I2C-2 gene could not confer resistance to plants, indicating that these members are not the functional resistance genes. Alignments between the various members of the I2 gene family revealed two significant variable regions within the leucine-rich repeat region. They consisted of deletions or duplications of one or more leucine-rich repeats. We propose that one or both of these leucine-rich repeats are involved in Fusarium wilt resistance with I2 specificity.     

普通小麦Qz180中一个抗条锈病基因的分子作图

普通小麦(Triticum aestivum L.)材料Qz180具有良好的抗条锈病特性,经基因推导发现其含有一个优良的抗条锈病的基因,暂定名为YrQz.用Qz180与感病材料铭贤169和WL1分别杂交构建了两个F2群体,用条中30号条锈菌小种对这两个群体进行的抗性测验表明,YrQz为显性单基因遗传.通过SSR和AFLP结合BSA的方法对这个基因进行了分子作图,结果鉴定出与YrQz连锁的2个SSR标记和2个AFLP标记.根据SSR标记的染色体位置,该基因被定位在2B染色体的长臂上,位于两个SSR位点Xgwm388和Xgwm526之间;两个AFLP标记P35M48(452)和P36M61(163)分别位于该基因的两侧,遗传距离分别为3.4 cM和4.1cM.     

Conversion of AFLP markers associated with FHB resistance in wheat into STS markers with an extension-AFLP method.

Amplified fragment length polymorphism (AFLP) has proven a powerful tool for tagging genes or quantitative trait loci (QTLs) of interest in plants. However, conversion of AFLP markers into sequence-tagged site (STS) markers is technically challenging in wheat owing to the complicated nature of its genome. In this study, we developed an "extension-AFLP" method to convert AFLP markers associated with Fusarium head blight (FHB) resistance into STS markers. When an AFLP marker of interest was detected with an EcoRI+3-MseI+4-selective primer combination, the PCR product was used as a template for an additional selective amplification with four primer pairs, in which one additional selective base (either A, C, G, or T) was added to the 3' end of one of the two primers. The extended primer pair that produced the targeted band was further extended by adding each of the four selective nucleotide bases for the next round of selective amplification. Extension selective amplification was performed until the target bands became clear enough for subsequent cloning and sequencing. By using the extension-AFLP method, we successfully converted two AFLP markers located on chromosome 3BS and associated with FHB resistance into STS markers. Our results indicated that the extension-AFLP method is an efficient approach for converting AFLP markers into STS markers in wheat. The developed STS markers might be used for marker-assisted selection (MAS) for FHB resistance in wheat breeding programs.