分子标记辅助快速回交选育抗草铵膦油菜不育系及恢复系

为快速培育抗草铵膦除草剂的油菜不育系和恢复系,以含bar、barnase、barstar基因的抗草铵膦材料C5-1及含bar、barnase基因的不育材料C5-2为供体亲本,以甘蓝型冬油菜Gr-1、Gr-2为轮回亲本,以回交转育为基本方法,利用bar基因的PCR检测、育性鉴定和喷除草剂方法对回交后代进行前景选择,并利用SSR分子标记进行背景辅助选择,将bar、barnase和bartar基因导入Gr-1、Gr-2中,在回交第3代BC3中选育得到遗传背景回复率大于96%的抗草铵膦油菜不育系和恢复系。     

油菜抗除草剂性状的遗传及利用

以抗草甘膦油菜“Q3”和MICMS保持系“宁B6”、“宁B7”及恢复系“宁R1”、“宁R3”为亲本材料,配制杂交组合,研究抗性遗传。结果表明,油菜对草甘膦的抗性为显性性状,由1对基因控制,抗草甘膦基因在F_2和BC_1群体遵循孟德尔分离规律。因此,应用杂交、回交育种方法将CP_4 gox双价基因转育到油菜推广品种中是可行的。     

转基因抗除草剂油菜相关基因的PCR检测及其在育种中的应用

以抗草甘膦杂交油菜三系和抗草丁膦杂交油菜二系亲本及其F1杂种为材料,探索建立和优化抗草甘膦的CP4、gox基因和抗草丁膦的bar基因及其Barnase、Barstar基因的PCR检测方法,为转基因抗除草剂油菜的品种选育、F1种子纯度鉴定和转基因油菜的生态安全性研究提供可靠、实用的检测方法。     

转基因抗除草剂油菜的分子鉴定

根据抗草甘膦基因gox、cp4-EPSPS、aroA及抗草丁膦基因PAT的基因序列设计特异引物，对抗草甘膦油菜Q3、抗草丁膦油菜HCN-19及其后代材料进行检测，结果表明，在Q3及其后代中检测到gox及cp4-EPSPS双价抗草甘膦基因，在HCN-19及其后代中检测到PAT基因特异片段。利用该方法对F1(NA6／HCN-19)纯度进行了鉴定。     

转基因抗草甘膦油菜的实用PCR检测方法

 利用定性PCR技术 ,建立了检测孟山都公司培育的转基因抗草甘膦油菜中的epsps、gox和fmv35s的方法 ,同时设立了阳性内对照napin。该方法的检测灵敏度为 0 .0 5 %。利用复合PCR可以在一个PCR反应中同时检测出epsps、gox、fmv35s和napin基因。这种方法可以有效地用于转基因抗草甘膦油菜中的外源基因的检测。     

利用除草剂快速回交选育抗除草剂谷子新品种

为快速培育抗除草剂谷子新品种,以抗除草剂材料"冀谷19×SK492"为供体亲本,以当地优良品种豫谷18为轮回亲本,以回交转育为基本方法,利用喷除草剂方法对回交后代进行选择,将抗除草剂基因导入豫谷18中,选育出抗除草剂谷子新品种。     

野芥菜向抗除草剂转基因油菜基因流动的研究

采用人工授粉的方法,分别以转基因抗草丁膦和抗草甘膦油菜为母本,野芥菜为父本,研究野芥菜的基因流动到转基因油菜中的可能性。结果表明,野芥菜和抗草丁膦及抗草甘膦油菜的亲和性指数明显低于其自交授粉的亲和性指数,只有0.44和0.54。经除草剂筛选和PCR检测证明,所有F1都携带了相应的抗性基因并表现出对相应除草剂的抗性。对F1的适合度研究表明,两种F1种子萌发率及营养生长情况和野芥菜没有明显差异,但结实明显下降,携带抗草丁膦基因的F1和携带抗草甘膦基因的F1每个角果饱满种子粒数分别是0.50和0.61。分别以两种F1为母本,野芥菜为父本进行回交,结实仍然很低,携带抗草丁膦基因的F1和携带抗草甘膦基因的F1与野芥菜回交每个角果饱满种子粒数分别只有0.35和0.33。上述结果表明,野芥菜基因流动到两种抗性油菜的可能性均比较小。     

抗除草剂油菜雄性不育系选育及利用研究

对抗草甘膦油菜“Quest”的抗性遗传研究结果表明,油菜对草甘膦的抗性为显性性状,由1对基因控制,抗草甘麟基因在F2和BC1群体遵循孟德尔分离规律。以抗草甘膦油菜“Quest”和双低雄性不育系“G851A”、保持系“G851B”为亲本材料,经5个轮回世代的回交、自交和测交,育成了抗除草剂油菜雄性不育系“K851A”和保持系“K851B”。“K851A”不育系株型紧凑,不育性彻底,双低品质稳定,且具有除草剂抗性,组配的杂交组合K851A/C-1平均产量达3426.0 kg/hm2,较对照增产12.84%。     

大豆高代新品系草甘膦抗性的检测与筛选

[目的]对大豆杂交高代品系草甘膦抗性进行检测,并从中筛选综合性状优良的抗草甘膦大豆新品系。[方法]以普通大豆晋大73为母本,抗草甘膦转基因大豆RR1为父本进行杂交,采用常规育种方法,早代以农艺性状为标记进行选择,F7代对34个后代品系进行蛋白脂肪含量测定和大田草甘膦抗性测定,并以大豆凝集素基因(lectin)为内置标准,对大豆外源CaMV35S启动子、NOS终止子、抗草甘膦基因(CP4-EPSPS)进行PCR检测。[结果]27个品系对草甘膦具有完全抗性,且均检测到CaMV35S启动子、NOS终止子和CP4-EPSPS基因;5个品系未检测到CP4-EPSPS基因,田间测定也均为完全不抗;另有2个品系PCR检测结果一致,但田间抗性测定1个为完全抗性、1个为完全不抗。[结论]田间测定结果与PCR检测结果基本一致,符合率达到了94.1%。筛选出27个具备抗草甘膦特性的新品系,其中4个品质优良,表现为超亲优势。     

双抗棉花新型核不育系Yu98-8A2的选育及鉴定

以抗除草剂核单基因隐性不育系Yu98-8A1不育株为母本,转Bt基因抗虫棉中棉所41为父本,通过连续回交,选育出双抗(抗虫和抗除草剂)棉花核不育系Yu98-8A2.花冠、花药表型及花粉发育显微观察表明,新型不育系保持了原不育系转育受体Yu98-8A1的败育特性.棉铃虫抗性及草甘膦抗性试验暗示,Yu98-8A2既保持了原不育系Yu98-8A1的除草剂抗性,又聚合了棉铃虫抗性;PCR分子鉴定表明,Yu98-8A2聚合了Bt及EPSPS-G6基因.     

油菜中转基因成分的筛查检测策略

旨在建立油菜中转基因成分的快速筛查方法,服务于农业转基因生物安全监管。根据转基因油菜常用转化遗传元件信息建立转基因油菜的筛查策略,并使用定性PCR法检测。结果表明,利用CaMV35S启动子、NOS终止子、bar基因、pat基因、CP4-epsps基因和NPT II基因6个靶标元件的组合,理论上可筛查92%的已知商业化转基因油菜转化事件。该方法的检测灵敏度达到1g/kg,可对油菜中的大多数转化事件进行快速、高灵敏度的筛查。     

Structural Changes of 2V Chromosome of Haynaldia villosa Induced by Gametocidal Chromosome 3C of Aegilops triuncialis

Haynaldia villosa （2n=2X= 14, VV）, a relative of wheat, plays important roles in wheat improvement mainly owing to its disease resistance. Powdery mildew resistance gene Pm21 has been successfully transferred into wheat by Cytogenetic Institute, Nanjing Agricultural University, China, and is widely used in the current wheat breeding programs. In this research, our objective is to further transfer and utilize the beneficial genes such as eye-spot resistance, yellow rust resistance, and gene of the tufted bristles on the glume ridge （a remarkable morphology） mapped on 2V of Haynaldia villosa. A disomic addition line with gametocidal chromosome 3C ofAegilops triuncialis added in Norin-26 was crossed to the wheat-H, villosa disomic substitution 2V（2D） and the hybrid F1 was then self-crossed. Chromosome C-banding, genomic in situ hybridization （GISH）, and meiotic analysis in combination with molecular markers were applied to detect the chromosome variations derived from hybrids Fz and F3. To date, four translocations including one small segmental translocation T6BS·6BL-2VS, two whole arm translocations （preliminarily designed as T3DS·2VL and T2VS.7DL） and one intercalary translocation T2VS·2VL-W-2VL, one deletion Del. 2VS·2VL-, one monotelosomic Mt2VS, and one isochromosome 2VS·2VS line have been developed and characterized. One wheat SSR marker Xwmc25.120 tagging 2VS and one wheat STS marker NAU/STSBCD135-1 （2BL） tagging 2VL were successfully used to confirm the alien chromosome segments involved in the seven lines. The tufted bristles on the glume ridge appeared in lines T2VS-7DL, Mt2VS, 2VS-2VS as well as the parent DS2V（2D）, whereas in T3DS·2VL, this trait did not appear. The gene controlling the tufted bristles was located on 2VS. Gametocidal chromosome 3C ofAegilops triuncialis could successfully induce chromosome 2V structural changes.     

多效唑处理后油菜苗在低温胁迫下的光合及生理特性

为探讨多效唑提高油菜抗寒性的生理机制,以2个甘蓝型油菜品种秦优7号和秦优33为材料,研究多效唑处理油菜苗在低温胁迫下的光合生理特性。结果表明：油菜5~6叶期时在叶面喷施150 mg·L^-1多效唑可以显著减缓受低温胁迫的油菜叶片叶绿素含量、净光合速率（P_n）、气孔导度(G_s)、蒸腾速率（T_r）、气孔限制值（L_s）的下降幅度;多效唑处理的秦优7号和秦优33的叶绿素含量、P_n、G_s、T_r、L_s分别比对照高22.90%和24.20%、18.13%和20.71%、29.16%和50.00%、13.00%和29.19%、48.00%和27.59%。多效唑还可以降低油菜在低温胁迫下叶片的相对电导率,并提高叶片可溶性糖含量,从而维持叶片质膜的稳定性,增强油菜的抗寒性。总之,在油菜苗期喷施多效唑能缓解低温胁迫对油菜的抑制影响,促进低温胁迫下油菜幼苗的生长,减缓低温对其伤害。     

除草剂对茶园土壤尿素态氮转化和温室气体排放的影响

通过室内培养试验,研究了几种常用茶园除草剂对土壤尿素态氮转化和温室气体排放的影响。设置CK(未施尿素和除草剂)、单施尿素、尿素+草甘膦钾盐、尿素+草甘膦异丙胺盐、尿素+草铵膦、尿素+百草枯共6个处理,尿素用量为200 mg N·kg~(-1)干土,除草剂用量为10 mg有效成分·kg~(-1)干土。结果表明:4种除草剂均在前期(5 d)显著抑制了尿素的水解作用(P0.05),此后促进尿素水解,并延长了尿素水解时间,百草枯施用效果较为显著,其他3种除草剂施用效果之间差异不显著。与单施尿素处理相比,施用除草剂均显著抑制土壤硝化作用(P0.05),硝化抑制率为38.42%~58.08%。茶园土壤单施尿素显著促进温室气体CO_2和N_2O的排放(P0.05)。与单施尿素处理相比,4种除草剂均促进了茶园土壤CO_2的排放,累积排放量增幅为6.05%~65.46%,其中草甘膦异丙胺和百草枯处理CO_2排放量显著增加(P0.05);4种除草剂显著抑制N_2O排放(P0.05),累积排放量降幅为46.21%~63.58%,百草枯处理抑制效果最显著(P0.05);4种除草剂不同程度抑制了CH_4的排放,累积排放量降幅为13.71%~32.09%,草甘膦异丙胺和百草枯处理CH_4累积排放量显著降低(P0.05),但4种除草剂之间差异不显著(P0.05)。由于不同除草剂对不同微生物生理代谢影响的复杂性,其对氮素转化和温室气体的影响还需长期田间试验研究。     

基于FRET原理构建AtLHT1转运底物筛选探针

【目的】为提高导向药物筛选通量,加快导向农药分子设计、合成、筛选等研发速度,构建基于拟南芥氨基酸转运蛋白AtLHT1的荧光共振能量转移（Fluorescence resonance energy transfer,FRET）探针作为导向农药筛选平台。【方法】构建CFP-LHT1-YFP的三明治探针,分别在大肠埃希菌Escherichia coli BL21（DE3）原核细胞和BY4741酵母细胞中进行表达、鉴定和纯化。采用荧光酶标仪和激光共聚焦检测FRET效率的变化。【结果】CFPAtLHT1-YFP融合蛋白探针纯化后,分别与供试氨基酸和草甘膦混合,供试氨基酸和草甘膦的加入导致探针蛋白的FRET比率发生明显变化,草甘膦处理使D_535nm/D_480nm升高7%～12%,其变化趋势与阳性配体甘氨酸和谷氨酸接近,而加入阴性配体精氨酸则没有明显变化。同时,FRET比率呈现底物浓度依赖。样品中分别加入1、5和30 mmol/L的草甘膦溶液,20 min后检测到D_535nm/D_480nm分别升高3%、1%和13...     

Genetic analysis and SSR mapping of stem rust resistance gene from wheat mutant D51

Wheat (Triticum aestivum L.) stem rust caused by Puccinia graminis f. sp. tritici is one of the main diseases of wheat worldwide. Wheat mutant line D51, which forms a highly susceptive cultivar ‘L6239’ to the three races notated and cultured with immature embryos, shows resistance to prevailing races 21C3CPH, 21C3CKH, and 21C3CTR of P. graminis f. sp. tritici in China. In this study, the number and the expression stages of the resistance genes in mutant D51 were studied using inoculation identification and microsatellite (SSR) marker analysis. Two F₁ populations from the crosses of D51 × L6239 (60 individuals) and D51 × Chinese Spring (60 individuals), their F₂ populations (185 and 175 individuals respectively) at the seedling stage, and one F₂ population derived from the cross of D51 × L6239 (194 individuals) at the adult stage were inoculated with pathogen race 21C3CPH to test for resistance. All F₁ individuals of the two crosses were immune to stem rust at both seedling and adult stages. The response pattern of the three F₂ populations showed that the R:S segregation ratio was 3:1, suggesting that the stem rust resistance of D51 is controlled by a single dominant gene, and is expressed during the entire growth period. The identification of the stem rust resistance by the F₃ progeny test confirmed the credibility of the F₂ population test. Segregating populations and small population analyses were used to identify chromosomal regions and molecular markers linked to the gene by the SSR marker method. A total of 675 SSR markers and 185 individuals of the D516L6239 F2 population were used to search genetically linked markers to the target gene. Using Mapmaker 3.0 and Map-draw with Kosambi’s function and other options set at default values, molecular mapping revealed that the gene was located on chromosome 5DS, linked with and flanked by two SSR markers, Xgwm190 and Xwmc150, at 18.58 and 21.33 cM, respectively. It has been reported that only one stem rust resistant gene, Sr30, is located on the wheat chromosome 5DL, and that it has no resistance to 34C2MKK and 34C2MFK, while the parent L6239 of mutant D51 has no resistance to 21C3CPH, 21C3CTK and 21C3CTR, but has resistance to 34C2MKK and 34C2MFK. The results above indicate that the gene identified in the study might be a novel resistance gene to stem rust, tentatively designated as SrD51. __________ Translated from Acta Agronomica Sinica, 2007, 33(8): 1262–1266 [译自: 作物学报]     

甘蓝型油菜polCMS育性恢复位点的全基因组关联分析

【目的】甘蓝型油菜波里马细胞质雄性不育(pol CMS)在中国已被广泛应用于杂交种育种,其育性恢复程度表现出受1对主效基因的控制,并受微效修饰基因的影响。通过全基因组关联分析方法挖掘育性恢复位点,并对候选基因进行比较分析。【方法】通过芸薹属60K SNP芯片对308份甘蓝型油菜自然群体进行基因型分型,并用pol CMS系301A作母本,与上述材料分别进行杂交得到308份F1,每份F-_1分别于2013年和2014年进行种植,每年2次重复,于始花期根据花粉育性和花蕊发育情况调查F1植株的育性等级,同时对测交父本自然群体进行群体结构分析和亲缘关系评估,并结合测交父本的基因型分型结果和F_1的育性等级进行全基因组关联分析(GWAS)。从GWAS分析中显著的SNP左右100 kb区间或与显著SNP处于同一单体型块(R~20.5)的区间内预测候选基因,并对候选基因进行QTL比较分析和单体型或等位基因的效应分析。【结果】方差分析结果显示,两年F_1的育性等级存在显著差异(P0.01),但相关分析发现,两年的育性等级存在显著的正相关(r=0.52,P0.001)。群体结构分析显示,所有测交父本被分为3个亚群(冬性、春性和半冬性),亲缘关系分析发现,任何2个材料之间平均亲缘关系值为0.072,73%的任意材料间亲缘关系值小于0.1,其中,约53%的材料亲缘关系值为0。GWAS分析共检测到13个与育性恢复程度显著关联的SNP,构成了6个候选区间,分别位于A01、A09、C03、C06和C08 5条染色体上,单个SNP解释的表型变异介于2.53%—9.96%。从中共预测到6个与育性恢复位点相关的候选基因,其中4个编码的蛋白含有恢复基因特有的PPR保守基序。共线性分析发现,4个候选基因中的2个(Bna A09g46700D和Bna C08g40710D)位于A09和C08染色体部分同源区间,且与已克隆的pol CMS育性恢复位点ORF2同源。另外2个新鉴定到的候选基因(Bna C03g45840D和Bna C06g13000D)连锁的SNP等位基因或单体型变化都与育性等级显著相关(P0.001)。【结论】通过GWAS分析鉴定到多个与油菜育性恢复有关的候选基因,开发基于与这些基因连锁位点或SNP的功能标记将有助于对该不育系统进行恢复系和保持系的筛选。     

Molecular Mapping of Two Novel Stripe Rust Resistant Genes YrTp1 and YrTp2 in A-3 Derived from Triticum aestivum × Thinopyrum ponticum

Loss of variety resistance to stripe rust (Puccinia striiformis Westend f. sp.tritici) is an important factor causing massive periodical epidemic of rust in wheat production. Creation and development of new races of rust pathogen have led to serious crisis of resistance loss in widely planted varieties. This has quickened the search for new resistance resources. Molecular marker could facilitate the identification of the location of novel genes. A line A-3 with high resistance (immune) to currently epidemic yellow rust races (CY29, 31, 32) was screened out in offspring of Triticum aestivum × Thinopyrum ponticum. Segregation in F₂ and BC₁ populations indicated that the resistance was controlled by two independent genes: one dominant and one recessive. SSR markers were employed to map the two resistant genes in the F₂ and BC₁ populations. A marker WMC477-_167bp located on 2BS was linked to the dominant gene with genetic distance of 0.4 cM. Another marker WMC364-_{208 bp} located on 7BS was linked to the recessive-resistant gene with genetic distance of 5.8 cM. The two genes identified in this paper might be two novel stripe rust resistant genes, which were temporarily designated as YrTp1 and YrTp2, respectively. The tightly linking markers facilitate transfer of the two resistant genes into the new varieties to control epidemic of yellow rust.