多堆柄锈菌潜育期侵染量实时荧光定量PCR检测体系的建立

为快速及时诊断并定量监测玉米内多堆柄锈菌Puccinia polysora潜育期的侵染量,根据多堆柄锈菌的ITS序列和玉米Actin2基因序列,分别设计多堆柄锈菌特异性引物PpoF/PpoR和Taq Man探针PpoP以及玉米特异性引物ZmF/ZmR和Taq Man探针ZmP,对引物和探针的特异性和灵敏度进行测定,并用这2对引物和探针建立多堆柄锈菌潜育期的实时荧光定量PCR检测体系,定量监测接种后玉米叶片中多堆柄锈菌DNA量随时间的变化。结果表明,多堆柄锈菌和玉米的特异性引物和探针对各自靶标片段均具有良好的特异性,灵敏度分别为10^-3ng/μL和10^-2ng/μL;在接种后24 h,利用所建实时荧光定量PCR检测体系即可在玉米叶片中检测到多堆柄锈菌,且潜育期内多堆柄锈菌的侵染量随时间呈指数增长。表明所建实时荧光定量PCR检测体系可用于多堆柄锈菌潜育期侵染量的定量检测和监测。     

中国鲎幼鲎形态性状与体重的通径分析及异速生长研究

为了探究不同龄期中国鲎幼鲎生长特性,实验测定了2～4龄幼鲎头胸部长(X₁)、头胸部宽(X₂)、腹部长(X₃)、腹部宽(X₄)、剑尾长(X₅)、全长(X₆)和体重(Y)等参数,运用相关分析和通径分析方法,探讨形态性状对体重的作用效果。结果显示,2～4龄幼鲎体重相关系数最高的形态性状均为头胸部宽(X₂);通径分析发现,头胸部宽(X₂)对2龄和3龄幼鲎体重直接影响最大,腹部宽(X₄)对4龄幼鲎体重直接影响最大;各形态性状对体重决定程度分析与通径分析变化趋势一致。对2～4龄幼鲎头胸部宽和体重进行对数处理,获得异速生长方程回归系数(b)为2.746,表明此阶段幼鲎体重增长率大于头胸部宽增长率,处于正生长期。实验表明,不同龄期阶段中国鲎苗种筛选所依据的形态性状有所差异,中国鲎幼鲎阶段种质评价应考虑其异速生长规律,同时也为探究鲎的生长与性状、结构与生理之间的关系奠定基础。     

高产粳稻沈农265产量相关性状的QTL分析

利用沈农265/丽江新团黑谷的176个F2 3家系群体构建包含90个SSR标记的连锁图谱,并对穗长、每穗颖花数、每穗实粒数、着粒密度、结实率、有效穗数和千粒重等7个产量相关性状进行QTL分析。结果表明,共检测到15个控制产量性状的QTL,分布在第3、4、6、8、9以及第12染色体上。包括3个控制穗长的QTL;3个控制每穗颖花数的QTL;1个控制每穗实粒数的QTL;2个控制着粒密度的QTL;2个控制结实率的QTL;3个控制千粒重的QTL以及1个控制有效穗数的QTL。其中,主效QTL有4个,分别是qPL9,qSPP4-1,qSD9和qRG12。这些QTL将为水稻分子设计育种提供"元件",同时为阐明水稻产量相关性状分子机制提供基础信息。     

荧光定量PCR法检测副溶血弧菌tdh基因的表达差异

以pvuA为内标基因,运用实时荧光定量PCR检测不同来源以及不同应激条件下副溶血弧菌热稳定直接溶血素基因tdh的表达量。pvuA和tdh基因的荧光定量PCR融解曲线分析表明,两者均为特异性扩增。尽管相同来源的不同菌株间tdh表达量存在显著差异,副溶血弧菌临床分离株的tdh mRNA平均表达量显著高于海产品分离株((57.2比13.8)。在pH4.0、0.5%和8%NaCl应激条件下,临床株ZJ2和海产品分离株FJ14A的tdh mRNA表达量显著高于对照组;另一海产品分离株KP34在8%NaCl条件下的表达量显著提高,而低pH应激时tdh mRNA的表达量显著降低。结果表明,不同副溶血弧菌分离株的tdh mRNA表达差异显著,临床分离株的tdh mRNA表达量总体上高于海产品分离株,副溶血弧菌在不同应激条件下主要表现为tdh mRNA表达上调。     

鼠源细胞TaqMan实时荧光定量PCR检测方法的建立

为对鼠源细胞进行鉴别检测,以鼠线粒体16S rRNA基因序列为靶位点设计特异性引物及探针,建立实时荧光定量PCR检测方法,并评价该方法的特异性及敏感性。结果显示,所建立的检测方法特异性好,针对鼠源细胞基因组荧光定量PCR扩增曲线良好,其他物种来源细胞基因组及生物制品原辅材料未出现特异性扩增曲线;敏感性高,基因拷贝数检出限度为45.3拷贝。本试验建立的荧光定量PCR检测方法能够有效地对鼠源细胞进行快速检测,为细胞质量控制提供了有效方法。     

黄淮海地区大豆品种主要农艺性状演变分析

对黄淮海地区通过审定的212份大豆品种的主要农艺性状演变进行了分析。结果表明:2000年以后育成品种比70年代育成品种分枝和单株荚数分别降低26.04%和1.66%,百粒重和脂肪含量分别增加了23.17%和7.32%。株高、底荚高度和生育日数90年代育成品种最低;主茎节数80年代育成品种最少,比主茎节数最高的70年代育成品种减少18.37%;在黄淮海育成大豆品种中,亚有限结荚习性大豆所占比例持续增长,但还是以有限结荚习性为主,无限结荚习性大豆所占比例一直在下降;圆形叶的比例逐年增加,而披针叶和椭圆叶比例有所下降。随着年代推进,在分枝数和单株荚数下降的情况下,由于百粒重的增加,单产也随之增加。蛋白质含量以90年代育成品种最高;脂肪含量逐年稳步增长。     

非均质物料链式组合称重定量算法优化与试验

非均质物料质量差异较大且不可分割,组合称重定量过程中组合对象不确定,存在组合称重定量精度与组合速度的矛盾.该研究针对链式组合称重定量系统,提出以定量精度及组合效率为目标,对组合样本数和抽样数进行优化分析,达到保证组合称重定量精度下,减少数据计算量以提高组合定量速度的目的.研究表明,在相同允许组合误差下,增大组合样本数可...     

Real-time detection of <Emphasis Type="Italic">Phytophthora nicotianae</Emphasis> and <Emphasis Type="Italic">P. citrophthora</Emphasis>in citrus roots and soil

Two primers, specific for Phytophthora nicotianae (Pn6) and P. citrophthora (Pc2B), were modified to obtain Scorpion primers for real-time identification and detection of both pathogens in citrus nursery soils and roots. Multiplex PCR with dual-labelled fluorogenic probes allowed concurrent identification of both species ofPhytophthora among 150 fungal isolates, including 14 species of Phytophthora. Using P. nicotianaespecific primers a delayed and lower fluorescence increase was also obtained from P. cactorumDNA. However, in separate real-time amplifications, the aspecific increase of fluorescence from P. cactorum was avoided by increasing the annealing temperature. In multiplex PCR, with a series of 10-fold DNA dilutions, the detection limit was 10 pg l^-1 for P. nicotianaeand 100 pg l^–1 for P. citrophthora, whereas in separate reaction DNA up to 1 pg l^-1 was detected for both pathogens.Simple and rapid procedures for direct DNA extraction from soil and roots were utilised to yield DNA whose purity and quality was suitable for PCR assays. By combining these protocols with a double amplification (nested Scorpion-PCR) using primers Ph2-ITS4 amplifying DNA from the main Phytophthora species (first round) and PnB5-Pn6 Scorpion and Pc2B Scorpion-Pc7 (second round), it was possible to achieve real-time detection of P. nicotianaeand P. citrophthora from roots and soil. The degree of sensitivity was similar to that of traditional detection methods based on the use of selective media. The analyses of artificially and naturally infested soil showed a high and significant correlation between the concentration of pathogen propagules and the real-time PCR cycle threshold.     

葡萄扇叶病毒实时荧光定量RT-PCR检测方法的建立及应用

根据葡萄扇叶病毒（Grapevine fanleaf virus,GFLV）RNA2序列设计4对特异性引物,通过引物筛选、体系优化,建立了以FL-HP1-F/R为引物的GFLV的SYBR GreenⅠ实时荧光定量RT-PCR方法。该方法标准曲线循环阈值与模板浓度呈良好的线性关系,扩增效率为100%,相关性系数为0.998,灵敏性分别是常规RT-PCR和巢式RT-PCR的100倍和10倍。重复性试验表明组内和组间变异系数均小于2.59%,显示该方法具有较好的检测稳定性。内参基因的稳定性测试结果表明葡萄EF1和GAPDH基因具有较好的试验稳定性。利用建立的荧光定量方法测定12个葡萄样品中GFLV含量,并检测58个葡萄样品,结果表明不同葡萄样品中GFLV含量差异较大,最高含量是最低含量的81 245.5倍,与ELISA和巢式RT-PCR相比,实时荧光定量方法能检测出更多的GFLV分离物。     

Development of STS markers and QTL validation for common bacterial blight resistance in common bean

Common bacterial blight (CBB) of common bean ( Phaseolus vulgaris L.), is one of the major diseases that decrease yield and quality. A major quantitative trait locus (QTL) for CBB resistance from line XAN 159 was transferred into two bean lines, HR45 and HR67. Previous studies identified that two markers are linked to this QTL but the chromosome location was not consistent. To identify more tightly linked markers and to verify the chromosome location, 65 additional markers were mapped using 81 recombinant inbred lines (RILs) derived from a cross HR67 × OAC95-4. The QTL was mapped to a 13 cM region on chromosome 1 and defined by eight molecular markers that explained 25–52% of the phenotypic variation. Six tightly linked amplified fragment length polymorphism markers (0.6–9.7 cM from the QTL peak) were converted into seven sequence tagged site markers, three of which were mapped to this QTL. Five tightly linked markers were used to screen 907 F₂ plants derived from a cross HR45 × 'OAC Rex' and four of them were linked to each other within 4.2 cM. These markers may be useful in marker-assisted selection and map-based cloning of this major QTL.