基于VP72基因的非洲猪瘟PCR检测方法的建立与应用

基层兽医快速准确诊断非洲猪瘟,可以加强地方兽医部门的疫情防控能力。笔者以非洲猪瘟结构基因VP72部分序列为检测靶基因,设计特异性引物,优化反应条件,建立常规PCR检测方法。笔者应用新建的检测方法,检测了贵州境内采集的5份病料,其中4份阴性,1份阳性,与非洲猪瘟荧光实时定量PCR复检结果一致。结果表明,笔者新建的PCR检测方法特异性好,与其他病原无交叉性,快捷便利。同时也表明,贵州非洲猪瘟疫情暴发压力较大,应加强疫情防范和扑灭工作。     

Evaluation of a high-throughput nucleic acid extraction method for the detection of Mycobacterium avium subsp. paratuberculosis in bovine fecal samples by PCR

Johne’s disease (paratuberculosis) is an economically important disease of cattle worldwide. The disease is caused by Mycobacterium avium subsp. paratuberculosis (MAP), a fastidious gram-positive bacterium. PCR is increasingly used in diagnostic laboratories for the detection of MAP in fecal samples given the rapid test turnaround time and sensitivity and specificity comparable to fecal culture. However, efficient extraction of DNA for sensitive detection of MAP by PCR is affected by the complex lipid-rich cell wall of MAP and the presence of PCR inhibitors in feces. We evaluated a high-throughput nucleic acid extraction method (MagMAX core nucleic acid purification kit with mechanical lysis module) in conjunction with an hspX gene PCR for the detection of MAP from bovine fecal samples, which resulted in correct identification of all negative (13 of 13) and positive (35 of 35) proficiency test samples obtained from the National Veterinary Services Laboratories. In addition, all 6 negative and 50 of 51 positive diagnostic specimens tested were categorized correctly.     

Quantitative trait loci for fruit size and flowering time-related traits under domestication and diversifying selection in cucumber (Cucumis sativus)

In cucumber, the genetic basis of traits under domestication and/or diversifying selection is not well understood. Here, we reported QTL mapping for flowering time and fruit size-related traits with segregating populations derived from a cultivated × wild cross. Phenotypic data of flowering time (FT), fruit size (FS), fruit number (FN) and fruit weight per plant (FW) were collected in multiple environments. QTL analysis identified 19 QTL for these traits. We found that the major-effect QTL FT1.1 played an important role in regulating flowering time in cultivated cucumber, whereas the minor-effect QTL FT6.3 contributed to photoperiod sensitive flowering time during domestication. Two novel consensus FS QTL, FS1.4 and FS2.3, seem to be the targets of selection during breeding for the US processing cucumber. All other FS QTL were co-localized with previously detected QTL using populations derived from cultivated cucumbers, suggesting that they were under selection during both initial domestication and subsequent improvement. Results from this study also suggested that the wild cucumber is a useful resource for capturing positive transgressive segregation and novel alleles that could be explored in cucumber breeding.     

Variation and heritability of morphological and physiological traits among Leymus chinensis genotypes under different environmental conditions

Intraspecific trait variation and heritability in different environmental conditions not only suggest a potential for an evolutionary response but also have important ecological consequences at the population, community, and ecosystem levels. However, the contribution of quantitative trait variation within a grassland species to evolutionary responses or ecological consequences is seldom documented. Leymus chinensis is an important dominant species in semi-arid grasslands of China, which has seriously suffered from drought and high temperature stresses in recent decades. In the present study, we measured variation and heritability of 10 quantitative traits, namely the number of tillers, maximum shoot height, number of rhizomes, maximum rhizome length, rhizome mass, aboveground mass, root mass, maximum net photosynthetic rate(Pmax), specific leaf area(SLA), and leaf length to leaf width ratio(LL/LW), for 10 genotypes of L. chinensis under one non-stress(Ck) condition and three environmental stress conditions(i.e., drought(Dr), high temperature(Ht), and both drought and high temperature(DrHt)). Result indicated that(1) the interaction of genotype and environmental condition(G×E) was significant for 6 traits but not significant for the other 4 traits as shown by two-way analysis of variance(ANOVA), suggesting that different selection forces were placed for different traits on the factors dominating phenotypic responses to different environmental conditions. Moreover, these significant G×E effects on traits indicated significantly different phenotypic adaptive responses among L. chinensis genotypes to different environmental conditions. Additionally, individuals could be grouped according to environmental condition rather than genotype as shown by canonical discriminant analysis, indicating that environmental condition played a more important role in affecting phenotypic variation than genotype;(2) by one-way ANOVA, significant differences among L. chinensis genotypes were found in all 10 traits under Ck and Dr conditions, in 8 traits under Dr Ht condition and only in 4 traits under Ht condition; and(3) all 10 traits showed relatively low or non-measurable broad-sense heritability(H_2) under stress conditions. However, the lowest H_2 value for most traits did not occur under DrHt condition, which supported the hypothesis of 'unfavorable conditions have unpredictable effects' rather than 'unfavorable conditions decrease heritability'. Results from our experiment might aid to improve predictions on the potential impacts of climate changes on L. chinensis and eventually species conservation and ecosystem restoration.     

Identification and verification of quantitative trait loci for eating and cooking quality of rice (Oryza sativa)

This study was conducted to identify new quantitative trait loci (QTLs) that have stable effects for eating and cooking quality (ECQ) of rice. Three recombinant inbred line populations of indica rice were each planted in two years. Three traits for ECQ, amylose content (AC), gel consistency (GC) and alkali spreading value (ASV), were measured for QTL analysis. A total of 13 QTLs were detected, including four for AC, six for ASV and three for GC. Two QTLs, qGC4 in the interval RM16252–RM335 on the short arm of chromosome 4 and qGC6.2 in the Alk region, were validated in a population derived from a residual heterozygote that was homozygous at the major locus Wx. In the absence of segregation at the Wx locus, qGC4 and qGC6.2 had additive effects of 2.46 and 8.18 mm, respectively, offering a potential for improving GC property of rice varieties. Comparison between qGC4 and previous results suggests that qGC4 is likely a new QTL for GC, providing a candidate for gene cloning and functional characterization.     

Frost response in wheat and early detection using proximal sensors

Frost can significantly reduce production of field crops grown in Mediterranean‐type environments, where significant economic losses for Australian wheat occur annually. If non‐destructive sensors could make rapid, spatial assessment of frost damage, this could limit economic losses through timely management decisions. This paper reports on a methodology for imposing frost treatments to wheat under field conditions and the utility of canopy reflectance data for detecting early crop damage. Purpose‐built chambers using stepped additions of dry ice allowed for a range of frost scenarios to be tested when applied at wheat heading and anthesis. For frost treatments applied at anthesis, grain number and yield were reduced by 8.8% and 7.2%, respectively, for every degree Celsius below zero (down to ?4°C). This effect was additive over two consecutive nights. For cold load equivalent, there was a 2.2% and 1.9% reduction in grain number and yield, respectively, per °C hr (below 0°C). For wheat, spectral indices PRI and NDVI (reflectance) and FRF_G and SFR_G (fluorescence) showed significant relationships, with cold load applied for heading treatments. Next steps include targeting frost intensities equivalent to cold loads between 20 and 80°C hr and testing the utility of these proposed indices in a commercial paddock setting.     

长期继代培养的转基因苹果外源基因遗传及表达稳定性分析

为研究长期继代培养的转基因苹果组培苗中外源基因的遗传及表达稳定性，以继代培养9年的7个转GFP(绿色荧光蛋白)基因苹果株系组培苗为试材，分析转基因株系对Kan(卡那霉素)的抗性、DNA水平、转录水平和转录后水平GFP基因的遗传及表达稳定性。结果表明，在7个苹果转基因株系组培苗中均可检测出GFP特异基因片段，并且均可在含有50 mg/L卡那霉素的培养基上正常生长；绝对定量qRT-PCR检测发现，各转化株系GFP基因拷贝数并不相同；利用相对定量qRT-PCR法检测发现7个株系中GFP基因 mRNA表达量有明显差异，同时荧光显微镜下观察各转化株系叶片，绿色荧光强度有明显差异，利用SPSS软件对7个转基因株系中GFP基因拷贝数与GFP mRNA表达量相关性分析结果呈无显著相关性。以上结果表明，外源基因可以在长期继代培养的转基因苹果组培苗中保持其遗传稳定性，但不同转化株系中外源基因的表达量有显著差异，其表达量与拷贝数无显著相关，可能与外源基因在植物基因组中的插入位点有关。     

基质栽培黄瓜叶片水分状况、光合与荧光参数对不同灌水下限的响应

为探明基质栽培黄瓜适宜的灌水下限,实现农艺节水,以黄瓜品种博特209为试材,采用基质盆栽,共设置4个灌水下限处理,即田间持水量的50%、60%、70%、80%,用A、B、C、D表示,灌水上限统一设定为田间持水量的90%,采用TDR350水分速测仪控制基质的水分含量,研究不同灌水下限对基质栽培黄瓜叶片水分状况、光合日变化及荧光参数的影响。结果表明:随着灌水下限的提高,叶片的相对含水量、自由水含量及自由水/束缚水的比值呈逐渐上升的趋势,水分饱和亏、叶片细胞汁液浓度及束缚水含量则呈下降趋势,处理C、D的相对含水量、自由水含量均显著(P<0.05)高于处理A、B,处理A细胞汁液浓度显著高于处理C、D,处理A、B束缚水含量显著(P<0.05)高于处理C、D。叶绿素a、叶绿素b和叶绿素a+b的含量均随灌水下限的升高呈现为先上升后下降的趋势,而类胡萝卜素含量则表现为先下降后上升的趋势,处理C叶绿素a、叶绿素a+b含量显著(P<0.05)高于处理A、B;处理C、D黄瓜叶片的胞间二氧化碳浓度(C_i)的变化趋势基本呈“V”型,而处理A、B的Ci的变化趋势基本呈“W”型;处理A、B、C、D的蒸腾速率(T_r)日变化均呈先上升后下降的趋势,处理B、C、D的Tr在14:00后呈现逐渐下降的趋势,而处理A在12:00后就呈快速下降的趋势;处理B、C、D的气孔导度(G_s)日变化大致呈“M”型的双峰曲线,处理A则呈单峰曲线;处理A、B、C、D叶片净光合速率(P_n)与G_s日变化趋势基本一致,在整个日变化过程中,处理C、D的P_n始终高于处理A、B。F_v/F_m、Φ_PSⅡ、qP随灌水下限的提高呈逐渐上升的趋势,而NPQ呈逐渐下降的趋势。研究结果表明,灌水下限为田间持水量的70%~80%,基质栽培黄瓜叶片的水分状况良好、光合色素含量高、净光合速率大,有利于黄瓜的光合作用,促进黄瓜的生长发育。     

柑橘黄龙病菌浓度对琼中绿橙果实品质的影响

为了定量衡量黄龙病菌量对琼中绿橙果实品质的影响，本实验以采自海南琼中橘园中感染了柑橘黄龙病的琼中绿橙为实验材料，用实时荧光定量PCR相对定量的方法测定果实中柑橘黄龙病菌的浓度，同时对果实品质的8个外在指标（果实纵经、横径、果形指数、单果重、果皮率、残渣率、种子数量、种子重量）和5个内在指标（果汁率、可食率、可溶性固形物含量、可滴定酸含量、固酸比）进行测定，研究黄龙病菌浓度对琼中绿橙果实品质的影响。结果表明：随着琼中绿橙果实中柑橘黄龙病菌浓度的增大，果实外观品质变化明显的指标包括果实纵经、果形指数、单果重（p<0.05），其中果实纵经呈现明显下降趋势，从64.97mm下降到57.44mm，相对较少11.59%；果形指数呈现明显上升趋势，从0.83上升到0.94，相对增多13.25%；单果重呈现下降趋势，从166.35g下降到109.66g，相对较少34.08%；果实横径、果皮率、残渣率、种子数量、种子重量等指标变化不明显。随着琼中绿橙果实中柑橘黄龙病菌浓度的增大，果实内在品质变化明显的指标为可溶性固形物含量，可溶性固形物含量呈现明显下降趋势（P<0.05），从9.65%下降到8.19%，相对减少15.13%；果汁率、可食率、可滴定酸含量和TSS/TA没有明显变化。结论：在黄龙病菌浓度增加的情况下，琼中绿橙在果实外观、内在品质等多个指标呈现下降趋势。研究结果为定量评价黄龙病对柑橘果实的影响提供参考。     

利用荚果厚度模拟大豆鼓粒进程的研究

大豆籽粒发育可分为鼓粒期和籽粒归圆期,探究无损伤检测鼓粒进程的指标对大豆相关研究具有重要意义。本研究以青鲜819-4、冀豆17、鲁96150和V94-3971为试验材料,通过分期播种、连续测定标记花荚的荚果厚度,分期收获同日标记的大豆籽粒并进行发芽率测定,研究大豆鼓粒期荚果厚度变化。结果表明:1)鼓粒期鲜荚皮厚度基本保持恒定,因此荚果厚度可用于度量籽粒厚度的变化。荚果厚度(y)与开花后日数(x)的关系可以用二次函数(y=ax2+bx+c)进行有效模拟。利用模拟函数可以对大豆鼓粒始期、最大荚果厚度出现期、鼓粒持续期以及平均鼓粒速率等进行有效预测。荚果最大厚度可用作判定大豆籽粒开始具备发芽能力的外观指标。2)荚果厚度变化受材料特性和外界环境的共同影响。同一材料不同时期标记的花荚,荚果厚度增速趋势可能有差异,因此在大豆籽粒灌浆特性研究中,同日标记花荚是籽粒灌浆特性研究中一项必要的控制措施。3)不同材料鼓粒特性存在差异,选择鼓粒启动早、鼓粒速度快的大豆品种是可行的。荚果厚度有望成为研究大豆灌浆特性和适时早收的无损伤选择指标。