Cytoplasmic male sterility (CMS) has widely been used as an efficient pollination control system in rapeseed hybrid production. Identification of cytoplasm type of rapeseed accessions is becoming the most important basic work for hybrid-rapeseed breeding. In this study, we report a simple multiplex PCR method to distinguish the existing common cytoplasm resources, Pol, Nap, Cam, Ogu and Ogu-NWSUAF cytoplasm, in rapeseed. Cytoplasm type of 35 F(1) hybrids and 140 rapeseed open pollinated varieties or breeding lines in our rapeseed breeding programme were tested by this method. The results indicated that 10 of 35 F(1) hybrids are the Nap, and 25 the Pol cytoplasm type, which is consistent with the information provided by the breeders. Out of 140 accessions tested, 100 (71.4%), 21 (15%) and 19 (13.6%) accessions possess Nap, Cam and Pol cytoplasm, respectively. All 19 accessions with Pol cytoplasm are from China. Pedigree analysis indicated that these accessions with Pol cytoplasm were either restorers for Pol CMS, including Shaan 2C, Huiyehui, 220, etc. or derived from hybrids with Pol CMS as female parent. Our molecular results are consistent with those of the classical testcross, suggesting the reliability of this method. The multiplex PCR assay method can be applied to CMS "three-line" breeding, selection and validation of hybrid rapeseed.

As a zinc-finger protein, PR domain containing 16 (PRDM16) controls brown fat determination by stimulating brown fat-selective genes expression while suppressing the expression of genes selective for white fat cells, whose mutations were associated with myelodysplastic syndrome (MDS) and leukemogenesis in human and murine model of leukemia. To date, no polymorphisms of PRDM16 gene in bovine had been reported. Herein, PCR-SSCP and DNA sequencing methods were employed to screen the genetic variation within PRDM16 gene in 1031 Chinese indigenous bovine. The results revealed two novel silent mutations: XM_001788152: m.1641T>C (547aa), 1881G>A (627aa). Hence, we described the PvuII and HaeIII forced PCR-RFLP methods for detecting these mutations, respectively. In the forced PCR-RFLP analysis with PvuII, the frequencies of bovine PRDM16-C allele varied from 0.044 to 0.506 in four Chinese native breeds. In the forced PCR-RFLP analysis with HaeIII, the frequencies of bovine PRDM16-G allele were 0.474, 0.494, 0.576 and 0.906 for Jiaxian (JX), Nanyang (NY), Qinchuan (QC) and Chinese Holstein (CH) population. Significant statistical differences between genotypic frequencies implied that both of the polymorphic loci were significantly associated with cattle breeds by the chi square test (chi2 = 190.058, P < 0.001 and chi2 = 118.239, P < 0.001 for PvuII; chi2 = 209.842, P < 0.001 and chi2 = 108.711, P < 0.001 for HaeIII). The associations of the PvuII and HaeIII forced PCR-RFLPs of bovine PRDM16 loci with growth traits were analyzed in Nanyang breed. The two SNPs were associated with body weight and average daily gain in Nanyang aged 12 months, individuals with genotype TT and AA showed significantly better body weight (P < 0.05) and average daily gain (P < 0.01) at 12 months, respectively.

The nucleophosmin (nucleolar phosphoprotein B23, numatrin) gene (NPM1, previously known as nucleophosmin/nucleoplasmin family, member 1) encodes a multifunctional nucleolar phosphoprotein that plays a crucial role in cell growth and homeostasis. Seven sequence variants (SVs) were identified in the coding region of bovine NPM1, five of which were in complete linkage disequilibrium. Eight different haplotypes were identified, of which two major haplotypes have a frequency of 23.2% and 20.4%. Three SVs were significantly associated with body weight in the Nanyang population as analysed at different ages. No significant association was detected between 18 combined genotypes and body weight at five different ages. Our results suggest that some polymorphisms in NPM1 are associated with body weight at some ages and may be used as candidates for marker-assisted selection and management in beef cattle breeding programmes.

In this study, polymorphism in the exon 1, 2, 3, 4 and 5 of bovine GAD1 gene was detected by PCR-SSCP and DNA sequencing methods in 726 individuals from three Chinese cattle breeds. Two haplotypes (T and C) and three observed genotypes (TT, TC, and CC) were detected. The result of DNA sequence showed one mutations (T12345C) by comparisons with NC_007300: g.12345T>C. The mutation was at the exon 3, and was identified a missense mutation Phe (TTT)>Leu (CTT) at position 51 (p.F51L). Polymorphism of the GAD1 gene was shown to be associated with growth traits in the Nanyang breed. The SNP in the bovine GAD1 gene had significant effects on body length, body weight, heart girth, and average daily gain at 24 months (P < 0.05). Therefore, these results suggest that the GAD1 gene is a strong candidate gene that affects growth traits in cattle.

In this study, polymorphism in the exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11 of bovine NUCB2 gene was detected by PCR-SSCP and DNA sequencing methods in 686 individuals from three Chinese cattle breeds. Two haplotypes (M and N), three observed genotypes (MM, MN and NN) and two SNPs (NC_007313: g. 27451G>A, NC_007313: g. 27472T>C) were detected. The frequencies of haplotypes M and N in inland Chinese three breeds were 0.531-0.721 and 0.279-0.469 respectively. The studied showed that Nanyang, Jiaxian Red and Qinchuan cattle populations were in Hardy-Weinberg equilibrium at SNPs locus of NUCB2 gene (P > 0.05). Polymorphism of the NUCB2 gene was shown to be associated with growth traits in Qingchuan and Nanyang cattle breed. The linkage of two mutant sites in the bovine NUCB2 gene had significant effects on body length, body weight, heart girth, and average daily gain at 24 months (P < 0.05). Results of this study suggested that the NUCB2-gene-specific SNP may be a useful marker for growth traits in future marker-assisted selection programmes in inland Chinese cattle.