建立一种同时鉴别诊断猪瘟病毒（classical swine fever virus, CSFV)、非洲猪瘟病毒（African swine fever virus, ASF）和猪非典型瘟病毒（atypical porcine pestivirus, APPV）的快速、灵敏、有效的检测方法。依据GenBank中登录的CSFV (5¢ UTR)、ASFV (B646L) 和 APPV (5¢ UTR) 的高度保守基因序列分别设计和优化了多对特异性引物和Taq-man探针，以保守区基因序列分别制备三种阳性质粒，用矩阵法优化单重/多重荧光PCR的反应体系和条件，为避免荧光通道的交叉干扰多重荧光PCR扩增，结合所标记的荧光报告基团做颜色补偿试验，构建标准曲线的扩增图和对应的线性方程，并进行特异性、敏感性、重复性、符合性以及临床样本的检测等试验。三种病毒的标准曲线相关系数均达到0.995以上，具有良好的线性关系；与其它常见猪病无交叉扩增反应，具有很好的特异性；多重荧光PCR的最低检测量均为1 copy/mL，具有较高的敏感性；组内和组间的变异系数均小于1%，具有很好的重复性。该方法与CSF的国标(GB/T 27540-2011), ASF的国标 (GB/T 18648-2020)，APPV的发明专利 (CN108611442A)检测样本盘的22个毒株样本符合率为100%。本研究建立的多重荧光PCR检测方法具有快速、高效、通量高、特异性好、灵敏度高等特点，可以对CSFV、ASFV和APPV病毒进行鉴别检测，为动物疫病的流行病学调查、疫情的检测提供一种新型的检测手段。本研究结合荧光PCR仪不同荧光通道设计CSFV、ASFV和APPV探针荧光信号强度较高且干扰较小的FAM、CY5和HEX报告基团，建立多重荧光PCR检测方法，用于同时鉴别诊断3种主要猪病毒的检测方法，在临床诊断中具有重要的应用价值。

Skin grafting has been used as one of the most reliable tests to determine the genetic stability of laboratory animal such as mice and rats inbred line, but no identification of swine inbred lines by skin grafting has been reported. At present, Wuzhishan miniature pig (WZSP) inbred line has acquired the F24 individuals in China. In order to verify whether WZSP inbred line had been cultivated successfully, allogeneic skin grafts and related research were performed on F20 individuals of WZSP inbreeding population, compared with a control group of autologous transplantation. We observed the transplant recipients’ wounds, detected peripheral blood-related indicators interleukin-2, 4 and 10, CD4+ and CD8+ lymphocytes, and conducted hematoxylin-eosin (HE) and Masson’s staining of skin to judge whether the immune rejection reactions occurred within 28 days after transplantation. Chr. 7 genomic heterozygosity of 48 WZSP individuals from F20 to F22 was analyzed by high-density single nucleotide polymorphism (SNP) chips (60 000 SNPs). The result showed that there were no significant differences in graft skin, the plasma interleukin-2, 4, 10, CD4+ and CD8+, HE and Masson’s staining results between the allograft and autograft groups, and no immune rejection occurred on the allograft group. We found that 11 genes in Chr. 7 of major histocompatibility complex (MHC) I and MHC II were homozygous which confirmed that immune antibody of the allograft and autograft groups were highly identical and also provided a theoretical basis to no immune rejection occurred on the allograft in the inbred WZSP. The result proved that the WZSP inbred line had been cultivated successfully for the first time in the world. The test methods also provide a scientific basis for the identification of swine and mammal inbred lines.

indica and japonica are the two most important subspecies of Asian cultivated rice. Identifying mechanisms responsible for population differentiation in these subspecies is important for indica-japonica hybridization breeding. In this study, subspecies and economic trait differentiation patterns were analyzed using morphological and molecular (InDel and Intron Length Polymorphism) data in F2 and F3 populations derived from indica-japonica hybridization. Populations were grown in Liaoning and Guangdong provinces, China, with F3 populations generated from F2 populations using bulk harvesting (BM) and single-seed descent methods (SSD). Segregation distortion was detected in F3-BM populations, but not in F3- SSD or in F2 populations. Superior performance was observed with respect to economic traits in Liaoning compared with that in Guangdong and 1 000-grain weight (KW), seed setting rate (SSR) and grain yield per plant (GYP) were significantly correlated with indica and japonica subspecies types. Analysis of molecular and morphological data demonstrated that the environment is the main factor giving rise to population differentiation in indica-japonica hybridization. In addition, we also found that KW, SSR and GYP are related to subspecies characteristics and kinship, which is possibly a significant factor resulting in economic trait differentiation and determining environmental adaptability. Our study has provided new insights into the process of population differentiation in these subspecies to inform indica-japonica hybridization breeding.