Prevalence,molecular characterization and risk factor analysis of Ehrlichia canis and Anaplasma platys in domestic dogs from Paraguay

This is the first study to investigate the prevalence and risk factors associated with Ehrlichia canis and Anaplasma platys positivity in dogs from Paraguay. Conventional PCR assays for the E. canis 16SrRNA gene and A. platys p44 gene were carried out in blood samples from 384 dogs from Asunción city, Paraguay. Sequencing and phylogenetic analysis were performed in selected positive E. canis and (16SrRNA gene) and A. platys (16S and p44 genes) samples. The overall prevalence of E. canis and A. platys in dogs in Paraguay was 10.41% (40/384) and 10.67% (41/384), respectively. Older dogs without veterinary care had higher odds for E. canis positivity and a higher number of dogs in the same household, as well as absence of anti-tick treatment were considered risk factors for A. platys. Ehrlichia canis and A. platys circulate in the dog population from Asunción, and are described for the first time in Paraguay.     

食源性单核细胞增生李斯特菌lmoF2365_0037基因克隆及序列分析

为了对食源性单核细胞增生李斯特菌LM5567的lmoF23650037基因进行克隆和序列分析。试验利用PCR方法对lmoF23650037基因进行扩增,连接pMD19-T载体进行克隆,筛选阳性菌株进行测序比对分析。结果显示:扩增获得的lmoF23650037基因序列长为548 bp,克隆得到lmoF23650037基因的阳性转化子;生物信息学分析表明:lmoF23650037蛋白属于跨膜蛋白,无信号肽序列;二级结构中无规则卷曲和延伸链比例较大,分别是46.31%和32.89%;序列比对结果表明,LM5567株lmoF23650037基因核苷酸序列与02-6680株(4b,奶酪,加拿大)、10-0811株(1/2b,螃蟹,加拿大)、10-0809株(4b,粪便,加拿大)、81-0558株(4b,脑脊髓液、加拿大)、02-1103株、02-1792(4b,奶酪,加拿大)、81-0861株(4b,卷心菜,加拿大)相似性均为100%;LM5567 lmoF23650037基因编码的氨基酸序列与上述菌株相似性均为93.3%。本试验成功克隆了lmoF23650037基因,为进一步探究lmoF23650037基因的功能奠定了基础。     

川西地区鸡滑液囊支原体分离株的部分生物学特性及其VlhA基因遗传进化分析

为了解鸡滑液囊支原体(MS)在川西地区的感染情况,本研究采集15个肉鸡场疑似MS感染的病鸡咽拭子、跗关节和胸部滑液囊样本共75份,对经PCR检测为阳性的样本进行MS分离,并对分离株的主要生物学特性进行研究,以及对分离株的VlhA基因进行遗传进化分析。结果显示,75份样本MS PCR阳性检出率为41.33%(31/75),11个鸡场感染该病,场阳性率为73.33%(11/15);但仅从其中3个鸡场分离到9株MS,分离株菌落与菌体形态与已知MS培养特性相符,各分离株培养浓度为10~4CCU/mL^10~6CCU/mL,分离株以5×10~5CCU接种7日龄SPF鸡胚,鸡胚于接种后7 d^9 d死亡,且分离回收到接种菌株;9株MS分离株VlhA基因的同源性为86.1%~99.9%,与参考株同源性为86.2%~95.4%,其中分离自同一鸡场的7株MS VlhA基因同源性为96.4%~99.9%;分离株VlhA基因遗传进化分析显示,其中分离自两个不同鸡场的两株MS与国内流行株的亲缘关系最近,而分离自另一鸡场的7株MS与中东地区分离的3株MS亲缘关系较近,表明MS在川西地区肉鸡场呈高感染率,MS VlhA基因变异较大。本研究为川西地区MS的进一步研究提供了基础材料和科学依据。     

水稻复合农艺性状QTL剖析

为了解水稻复合性状的数量性状基因座(QTL),在利用单片段代换系进行QTL鉴定的基础上,剖析了水稻株高QTL与主茎高和穗长QTL,主茎高与倒一节间长、倒二节间长、倒三节间长和倒四及以下节间长QTL,谷粒长宽比QTL与粒长和粒宽QTL,每穗粒数QTL与一次枝梗数和二次枝梗数QTL的关系。结果表明:鉴定出株高QTL的6个单片段代换系中有4个只检测出了主茎高QTL,其加性效应百分率为86.00%～99.55%,有1个只检测出了穗长QTL,其加性效应百分率为48.31%,有1个同时检测出了主茎高QTL和穗长QTL,其中主茎高QTL的加性效应百分率为81.72%,穗长QTL加性效应百分率为18.28%;在检测出主茎高QTL的7个单片段代换系中,有1个只检测出倒一节间长QTL,有2个只检测出倒二节间长QTL,有2个检测出倒一节间长QTL和倒二节间长QTL,有2个只检测出倒三节间长QTL;不同的单片段代换系中检测出的节间长的QTL加性效应百分率变化范围为-128.62%～172.07%;7个检测出谷粒长宽比QTL的单片段代换系中,有5个只检测出粒长QTL,1个只检测出粒宽QTL,1个同时检测出了粒长QTL和粒宽QTL;检测出每穗粒数QTL的3个单片段代换系中,有2个只检测出二次枝梗数的QTL,有1个同时检测出一次枝梗数QTL和二次枝梗数的QTL。这些结果表明,代换片段中如能检出复合性状QTL,也可以检出其构成性状QTL;复合性状QTL的加性效应的大部分可由其构成性状QTL的综合效应来解析,但相同的复合性状,不同代换片段检出的构成性状QTL不同。     

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.     

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。