Development of SNP-based dCAPS markers for identifying male sterile gene tms5 in two-line hybrid rice.

Molecular markers can increase both the efficiency and speed of breeding programs. Functional markers that detect the functional mutations causing phenotypic changes offer a precise method for genetic identification. In this study, we used newly derived cleaved amplified polymorphic sequence markers to detect the functional mutations of tms5, which is a male sterile gene that is widely used in rice production in China. In addition, restriction cutting sites were designed to specifically digest amplicons of tms5 but not wild type (TMS5), in order to avoid the risk of false positive results. By optimizing the condition of the polymerase chain reaction amplifications and restriction enzyme digestions, the newly designed markers could accurately distinguish between tms5 and TMS5. These markers can be applied in marker-assisted selection for breeding novel thermo-sensitive genic male sterile (TGMS) lines, as well as to rapidly identify the TGMS hybrid seed purity.

CYFRA21-1 levels could be a biomarker for bladder cancer: a meta-analysis.

The proteolytic region of cytokeratin-19, referred to as CYFRA21-1, is a soluble molecule present in the serum and other body fluids, and is considered a tumor marker in several neoplastic diseases. To examine whether urinary or serum samples containing CYFRA21-1 can be used as biomarkers for bladder cancer, we conducted a comprehensive meta-analysis of 3 case-control studies. In all studies considered, patients with bladder cancer had a higher CYFRA21-1 level than healthy subjects. Subgroup analysis showed that patients with metastatic bladder cancer had a higher CYFRA21-1 level than those with locally invasive disease. However, no significant difference in CYFRA21-1 was observed between patients with stage I and stage II bladder cancer; there was also no difference in patients with stage II local bladder cancer and those with stage III local bladder cancer. Based on our results, CYFRA21-1 level may be a diagnostic biomarker for diagnosing bladder cancer as well as a possible biomarker for differentiation between local and metastatic bladder cancer. However, it cannot be used as a urinary or serum biomarker for differentiating histological stages of local bladder cancer for histological grades I-III.

A novel synthetic Cry1Ab gene resists rice insect pests.

A few insect control genes of Bacillus thuringiensis have been modified successfully to increase the expression in plants by replacing rare codons, increasing GC content, and avoiding the DNA elements that could cause premature transcription termination, mRNA instability, and potential methylation. However, the modification process was intricate and often confused researchers. In this study, we adopted a simple method to modify Cry1Ab only by individually replacing its amino acid sequence with corresponding rice-preferred codons based on analysis of 92,188 coding DNA sequences. Unexpectedly, all elements of A+T richness, which terminate or destabilize transcription in plants, were avoided in the newly designed mCry1Ab. However, mCry1Ab had 2 notable features: less synonymous codons and high GC content. mCry1Ab only employed 22 of the 61 codons to encode protein and had an enhanced GC content of 65%. The increase in GC content caused abundant potential methylation signals to emerge in mCry1Ab. To test whether mCry1Ab could be expressed in rice, we transferred it into Oryza japonica variety Wanjing97. Insect bioassays revealed that transgenic plants harboring this gene driven by 2 promoters, CaMV35S and OsTSP I, were highly resistant to rice leaffolder (Cnaphalocrocis medinalis). Analysis of R0 to R2 generation plants indicated that the mCry1Ab was inherited stably by the progeny. Our study provided a simple modified method for expressing exogenous genes in rice and confirmed that less synonymous codons and high GC content do not affect transgene expression in rice.

Quantitative detection of the rice false smut pathogen Ustilaginoidea virens by real-time PCR.

Rice false smut (RFS) is an important rice disease that is caused by the pathogen Ustilaginoidea virens. In this study, we developed a real-time polymerase chain reaction (PCR) assay to detect U. virens and to estimate the level of disease. The genomic DNAs of U. virens and rice were extracted together from the rice samples. Real-time PCR assays were performed and compared to conventional nested-PCR assays. The real-time PCR assay presented a consistent linearity of the standard curve (R(2) = 0.9999). The detection limit could be as low as 40 fg U. virens DNA with a rice genomic DNA background on using the real-time PCR assay, which showed significantly higher sensitivity than the conventional nested-PCR assay. We conclude that the real-time PCR quantitative assay is a useful tool for detecting U. virens and for early defense and control of RFS.

Association between a single nucleotide polymorphism in the bovine chemerin gene and carcass traits in Qinchuan cattle.

Qinchuan is a red or yellow draft and beef breed in China. In order to identify a predictor of carcass traits on the basis of associations between carcass traits and gene polymorphism, variation in the bovine chemerin gene was investigated using PCR-single-strand conformational polymorphism and DNA sequencing. An SNP of A868G located in exon 2 of the Bos taurus chemerin gene was detected in 716 samples of six breeds (Jiaxian red, Luxi, Nan yang, Qinchuan, Simmental and Luxi crossbred steers, and Xia'nan), all in China, and three genotypes (AA, AG and GG) were found. Based on the χ(2) test, the AA/AG/GG genotype frequencies of all six breeds were found to be in Hardy-Weinberg equilibrium. A possible association of A868G with some carcass traits was investigated in 106 Qinchuan cattle. Animals with the AG genotype were found to have significantly lower mean loin eye area and meat tenderness compared to those with the AA and GG genotypes. However, there was no significant association between any individual haplotype and backfat thickness, water holding capacity or marbling score. We suggest that A868G could be used as a molecular marker in marker-assisted selection for carcass traits.

A novel SNP of the C/EBPα gene associated with superior meat quality in indigenous Chinese cattle.

CCAAT/enhancer-binding protein alpha (C/EBPα) is an essential transcriptional factor regulating the differentiation of adipocytes. We report a novel single nucleotide polymorphism (C271A) of the C/EBPα gene in six indigenous Chinese cattle breeds using PCR-SSCP and DNA sequencing methods. Allele frequencies were investigated and evaluated by the χ(2) test in 817 individuals; all populations were found to be in Hardy-Weinberg equilibrium. Gene heterozygosity, effective allele numbers and polymorphism information content of the C/EBPα locus varied from 0.50 to 0.54, 1.84 to 1.99 and 0.35 to 0.37, respectively. We also evaluated a potential association of the C/EBPα SNP with ultrasound traits in 555 individuals; individuals of the AA genotype had greater ultrasound backfat thickness than did genotype CC (0.36 versus 0.34 cm, P < 0.01); genotypes AA and CA had higher ultrasound marbling scores than did genotype CC (3.53, 3.52 versus 3.37, P < 0.05). Analysis based on meat quality data in another 204 Qinchuan cattle showed that animals with genotype AA had bigger loin eye areas than did genotype CA (87.10 versus 79.08 cm(2), P < 0.05). These results indicate that the C271A SNP of the C/EBPα gene could be used as a molecular marker for selecting beef cattle with superior carcass traits.

A novel RUNX2 mutation (T420I) in Chinese patients with cleidocranial dysplasia.

Cleidocranial dysplasia (CCD) is an autosomal-dominant heritable skeletal disease caused by heterozygous mutations in the RUNX2 gene. We studied a Chinese family that included three affected individuals with CCD phenotypes; the clinical features of patients with CCD include delayed closure of fontanelles, frontal bossing, dysplasia of clavicles, late tooth eruption, and other skeletal anomalies. X-ray analysis showed aplasia of the clavicles. The RUNX2 gene was studied by PCR and direct sequencing of the entire coding region and the exon-intron boundaries of the gene. A novel missense mutation (c.1259C-->T[p.T420I]) in RUNX2 gene exon 7 was identified; it was found in the affected individuals in this Chinese family, but was not present in an unaffected family member or in 100 unrelated normal controls. This is the first report that gives evidence that the T420I mutation of RUNX2 is associated with CCD, expanding the spectrum of RUNX2 mutations causing CCD.