Development of a core set of KASP markers for assaying genetic diversity in Brassica rapa subsp. chinensis Makino

Single‐nucleotide polymorphisms (SNPs) are rapid, economical and reliable genotyping tools. Non‐heading Chinese cabbage (Brassica rapa L. subsp. chinensis Makino) is now an economically important vegetable crop worldwide. In this study, 1,167 SNPs were evaluated for 7polymorphism among 70 representative non‐heading Chinese cabbage inbred lines using a Kompetitive Allele Specific PCR (KASP) genotyping assay. On the basis of identified polymorphisms and the results of a principal component analysis, we selected 50 core SNPs that were balanced sufficiently to provide adequate information for genetic identification. The core SNPs were used for construction of a neighbour‐joining dendrogram that separated the 70 inbred lines into four main groups and several subgroups corresponding to Caixin, Heiyebaicai, Huangxinwu, Naibaicai, Taitsai, Pak‐choi, and Wutatsai. This categorization was superior to that achieved using a dataset of 479 polymorphic SNPs. To confirm the utility of the core SNP markers in genetic identification, we tested their stability and resolution using 162 commercial hybrid cultivars. The SNPs, which represent a cost‐effective, accurate marker set for germplasm analysis and cultivar identification, are suitable for molecular marker‐assisted breeding in non‐heading Chinese cabbage.     

Association of TNF-α, IL-10 and IL-6 promoter polymorphisms in pulmonary tuberculosis patients and their household contacts of younger age group

Single nucleotide polymorphisms (SNPs) of cytokine genes have been found to be involved in the clinical outcome of Tuberculosis. The present study was aimed to identify the high risk genotypes in Tuberculosis patients and their household contacts. A total of 490 subjects were studied which includes 150 active pulmonary tuberculosis patients (APTB), 190 household contacts (HHC) and 150 healthy controls (HC). The SNPs of TNF-α (-308A/G), IL-10(-1082G/A) and IL-6(-174G/C) were performed by ARMs PCR. The IL-10 GA genotype showed significant association in APTB and HHC and was 2.3 times higher risk in APTB and 3.7 times in HHC compared to HCs. The A allele was found to be significantly associated with the risk of disease. The CC genotype of IL-6 was found to be significantly associated in APTB and an insignificant positive association in HHCs. The multifactor dimensionality reduction (MDR) analysis indicated that the genotypes of IL-6 were showing high risk with GA genotype of IL-10. In conclusion the gene interaction may be useful for identification of genotypes as biomarkers to distinguish high risk individuals.     

BmNPV半胱氨酸蛋白酶基因的核苷酸序列研究

对家蚕核型多角体病毒苏州株（BmNPVsu）光胱氨酸蛋白酶基因（CP）的序列分析表明,该基因读码框为972个核苷酸,编码323个氨基酸。同源性分析表明,BmNPVsu的CP与首蓿银纹夜蛾核型多角体病毒（AcNPV）、美国白蛾核型多角体病毒（HcNPV）、云杉卷叶蛾核型多角体病毒（CfNPV）、黄杉毒蛾核型多角体病毒（OpNPV）、舞毒蛾核型多角体病毒（Ld-NPV）在DNA水平上的同源性分别为96.5％、76.2％、74.9％、72.7％、62.9％;在氨基酸水平上的同源性分别为96.9％、77.1％、79.3％、77.1％、65.6％。BmNPV CP的氨基酸序列与不同来源的木瓜蛋白酶超家族的CP也具有较高的同源性,特别与 Trpanosoma brucei的CP具有较高的一致性,达32％。在组织蛋白酶B、H、L、S以及木瓜蛋白酶的36个保守氨基酸残基中有31种出现在BmNPVsu的CP中,BmNPVsu CP同其它杆状病毒CP一样,可看作木瓜蛋白酶超家族成员。     

A streak disease of pearl millet caused by a leafhopper-transmitted geminivirus

The cause of a streak disease of pearl millet (Pennisetum glaucum), originating from Nigeria, has been attributed to a geminivirus belonging to the African streak virus cluster. A full-length, infectious clone of the virus was obtained which was transmissible by the vectorCicadulina mbila (Naudé). Analysis of the complete nucleotide sequence of the coat protein gene of this virus shows it to be most closely related to sugarcane streak virus. The possible evolutionary implications of this finding are discussed.     

Characterisation of a maize-infecting potyvirus from Spain

In order to characterise and classify an unknown maize-infecting potyvirus isolated from fields in northeast Spain, the entire coat protein gene and the C-terminal twothirds of the large nuclear inclusion protein (NIb) gene were cloned and sequenced. Protein sequencing enabled the cleavage site between the two proteins to be deduced and also revealed that on storage the viral coat protein undergoes a specific degradation in which the N-terminal 39 amino acids are removed. Comparison of the nucleotide sequence of the 3 non-coding region of the viral RNA and the predicted amino acid sequence of the coat protein with the equivalent regions of other members of the potyvirus group revealed that the Spanish virus is closely related to maize dwarf mosaic virus strain A.     

The Complete Nucleotide Sequence of a Japanese Isolate of White clover mosaic virus Strain RC

The complete nucleotide sequence was determined for genomic RNA of White clover mosaic virus (WClMV-RC) isolated from red clover (Trifolium pratense) in Japan, It is 5843 nucleotides in length, excluding the poly(A) tail at the 3' terminus. Similar to other potexviruses, it contains five open reading frames (ORFs 1 through 5), which putatively encode an RNA-dependent RNA polymerase (RdRp) (147 kDa), a triple gene block (TGB) (26 kDa/13 kDa/7 kDa), and a coat protein (CP) (22 kDa), respectively. The deduced amino acid sequence of the WClMV-RC CP was identical to that of WClMV-O, one of two New Zealand isolates, but only 85% identical to that of WClMV-M, the other New Zealand isolate, because of heterogeneity in the C-termini of CP amino acid sequences. The implication of this CP heterogeneity is discussed. Received 30 August 2001/ Accepted in revised form 11 January 2002     

Identification of ALS inhibitor‐resistant Amaranthus biotypes using polymerase chain reaction amplification of specific alleles

Summary Polymerase chain reaction (PCR) amplification of specific alleles (PASA) was adapted as a molecular marker‐based method for the rapid detection of point mutations in Amaranthus retroflexus and Amaranthus rudis leading to ALS inhibitor resistance. Two pairs of primers were designed for the specific amplification of alleles of the ALS gene of susceptible and resistant biotypes. The allele‐specific primer matched the desired allele, but mismatched the different allele at its 3′ end. Differentiation was carried out by comparison of the amplified DNA fragments in gel electrophoresis after PASA‐PCR. In A. rudis, differentiation was possible with one PCR and genomic DNA as probe. A ‘nested’ PCR was necessary for the differentiation of sensitive and resistant A. retroflexus. PASA is useful for the identification of resistant weed biotypes and also as a monitoring tool to map resistance occurrence and distribution. Advantages include the fast and clear separation of those plants with and without mutations at an early stage of development, its easy and consistent performance and quick results compared with existing resistance detection tests. These advantages, when combined with management strategies, enable further activities to reduce herbicide resistance.     

A new approach to evaluate relative resistance and tolerance of tomato cultivars to begomoviruses causing the tomato yellow leaf curl disease in Spain

A simple procedure to evaluate relative resistance and tolerance of tomato cultivars to the begomoviruses causing tomato yellow leaf curl (TYLC) disease in Spain was developed. To estimate the resistance and tolerance levels of a cultivar, several formulae were developed based on the ratio of infected plants, virus titre (estimated by tissue–print hybridization) and symptom intensity. The formulae were applied to five commercial tomato cultivars (Amoretto, Birloque, Royesta, Tovigreen and Ulises) naturally infected by TYLC viruses. The analyses showed that Ulises, Birloque and Tovigreen exhibited a moderate resistance, and Ulises was also highly tolerant. There was a positive correlation between symptom intensity and virus titre in infected plants, suggesting that the hybridization technique could also be used as an early estimator of tolerance. Finally, molecular hybridization and nucleotide sequence analyses of the begomovirus intergenic region showed that the local TYLC virus population consisted of a single species, Tomato yellow leaf curl virus (TYLCV, formerly TYLCV-Israel), with low genetic variation (nucleotide identity between isolates higher than 97%).     

鸡新城疫云南分离株HN基因序列测定和分析

用RT—PCR法对分离自云南省的2株鸡新城疫病毒(编号YN—C1、YN—C2)进行HN基因的扩增和克隆．并对其进行核苷酸序列测定和分析。结果表明：2毒株HN基因开放性阅读框架(ORF)均为l716nt；二者之间的核苷酸同源性为95．3％，氨基酸同源性为95．8％；YN—C1毒株与参考毒株GD／1／98／Go核苷酸、氨基酸同源性均最高，分别为97．9％和97．6％；YN—C2毒株与参考毒株JS／5／01／Go核苷酸同源性最高为98．5％，而与参考毒株JS／3／98／Go氨基酸同源性最高为98．1%。     

猪2型圆环病毒核酸疫苗免疫效应研究

PCR扩增PCV2 ORF2和BVP22基因。将ORF2基因克隆入真核表达载体pCDNA3．1（＋），构建了pCORF2作为核酸疫苗免疫小白鼠。同时将具有蛋白转导功能的BVP22基因分别克隆到ORF2基因的上游和下游，使二者融合表达，命名为pCORF2BVP22和pCBVP220RF2。分4组肌肉免疫BALB/c小白鼠，分别注射pCDNA3．1（＋）、pCORF2、pCORF2BVP22和pCBVP220RF2，共免疫2次，间隔2周，分别于首免后2周和二免后4周采血，ELISA法检测体液抗体。同时为在体外验证ORF2和BVP22基因的真核表达，将ORF2和BVP22基因分别克隆入pEGFP-N1载体，构建了pNORF2和pNBVP22，转染Hela细胞后在荧光显微镜下观察到了ORF2和BVP22在体外的瞬时高效表达。结果显示，通过两次免疫后，各疫苗组均产生了针对ORF2的体液抗体，同时证明BVP22可增强核酸疫苗的免疫效果，其中以BVP22在ORF2上游效果更好。本研究为防制猪2型圆环病毒感染提供了较为理想的侯选疫苗。