Cloning of three genes involved in the flavonoid metabolic pathway and their expression during insect resistance in Pinus massoniana Lamb.

Pinus massoniana Lamb. is an important timber and turpentine-producing tree species in China. Dendrolimus punctatus and Dasychira axutha are leaf-eating pests that have harmful effects on P. massoniana production. Few studies have focused on the molecular mechanisms underlying pest resistance in P. massoniana. Based on sequencing analysis of the transcriptomes of insect-resistant P. massoniana, three key genes involved in the flavonoid metabolic pathway were identified in the present study (PmF3H, PmF3'5'H, and PmC4H). Structural domain analysis showed that the PmF3H gene contains typical binding sites for the 2OG-Fe (II) oxygenase superfamily, while PmF3'5'H and PmC4H both contain the cytochrome P450 structural domain, which is specific for P450 enzymes. Phylogenetic analysis showed that each of the three P. massoniana genes, and the homologous genes in gymnosperms, clustered into a group. Expression of these three genes was highest in the stems, and was higher in the insect-resistant P. massoniana varieties than in the controls. The extent of the increased expression in the insect-resistant P. massoniana varieties indicated that these three genes are involved in defense mechanisms against pests in this species. In the insect-resistant varieties, rapid induction of PmF3H increased the levels of PmF3'5'H and PmC4H expression. The enhanced anti-pest capability of the insect-resistant varieties could be related to temperature and humidity. In addition, these results suggest that these three genes maycontribute to the change in flower color during female cone development.

A genome-wide association study of growth trait-related single nucleotide polymorphisms in Chinese Yancheng chickens.

Chicken (Gallus gallus) growth traits are important economic traits, and many studies have been conducted on genetic selection for body weight. However, most of these studies have detected functional chromosome mutations or regions by conventional molecular markers or gene chips. In this study, we performed a new genome-wide association study using specific-locus amplified fragment sequencing (SLAF-seq) technology in purebred Yancheng chickens. Single nucleotide polymorphisms (SNPs) that were significantly associated with phenotypic traits were identified by GAPIT-compressed mixed linear models. Eighteen SNP markers reached 5% Bonferroni genome-wide significance. A region spanning 72.3 to 82.1 Mb on GGA4 had a strong influence on growth traits. Four genes (FAM184B, KCNIP4, MIR15A, and GLI3) were closely associated with body weight. Some SNPs were coincident with previously reported quantitative trait locus regions. Our results would promote the researches of Chinese chickens and accelerate the utilization of Chinese chicken, specially Yancheng chicken.

Correlation between natriuretic peptide receptor C (NPR3) gene polymorphisms and hypertension in the Dai people of China.

Hypertension affects one-fifth of the world population. Genome-wide association studies (GWAS) have identified several single nucleotide polymorphisms (SNPs) that correlated with hyper-tension in large samples. However, the genetic mutations leading to hypertension might differ among various populations, as they have different origins and are subjected to different environmental pressures. Therefore, additional studies are urgently needed to verify the GWAS findings across different populations. This study focused on the natriuretic peptide receptor C gene (NPR3), one of the hypertension-positive genes identified in a GWAS of an East Asian population. The correlation analysis between NPR3 and hypertension was replicated in 450 Chinese Dai (235 patients vs 215 controls) and 484 Chinese Mongolian (211 patients vs 273 controls) individuals. The positive SNP identified by GWAS analysis and three other tag SNPs representing the NPR3 linkage disequilibrium (LD) block regions were selected for genotyping. The results revealed that the rs1173766 polymorphism was associated with the occurrence of hypertension (χ(2) = 6.87, P = 0.0088), and that the T allele should be protective in the Dai ethnic group. Consider-ing a close LD block at the 3' end of the NPR3 gene in the East Asian population, we speculate that there might be a mutation in the last five exons or the 3' untranslated region of NPR3 that could change the structure or expression of the NPR3 gene. However, in the Mongolian ethnic group, these SNPs were not associated with the incidence of hypertension, suggesting population heterogeneity for the genetic factors that contribute to hypertension.

Development and characterization of SSR markers from Pinus massoniana and their transferability to P. elliottii, P. caribaea and P. yunnanensis.

Pinus massoniana (Masson's pine) is a widespread tree species in central and southern China and northern Vietnam; it is valued for rosin and paper production. Despite the significant economic value of Masson's pine, little work has been done on its molecular genetics. We developed 318 SSR primers from genome sequences of P. massoniana, and we identified 10 polymorphic markers. The number of alleles in the population of P. massoniana that we examined ranged from two to four, and the Shannon diversity index ranged from 0.150 to 1.133. Cross-species transferability of the 318 SSRs was also analyzed in the slash pine (Pinus elliottii), the Caribbean pine (Pinus caribaea) and the Yunnan pine (Pinus yunnanensis); 15, 10, and 10 primer pairs generated polymorphic amplification, respectively. These sets of polymorphic SSR markers will be useful for population genetics studies of P. massoniana, for genetic identification of interspecific hybridization, and for phylogeographic studies of Pinus spp.

Genetic variations of ISL1 associated with human congenital heart disease in Chinese Han people.

Congenital heart disease (CHD) is the most common birth abnormality, but the etiology of CHD is unknown. ISL1 may play a fundamental role in cardiac morphogenesis, and mutations of this gene could cause CHD. To evaluate whether genetic variations of ISL1 are associated with CHD in Chinese Han people, polymerase chain reaction restriction fragment-length polymorphism and SNaPshot were used to examine 9 polymorphisms of ISL1 in 233 patients with CHD as well as 288 healthy controls. We found that one SNP (rs1017) in ISL1 was significantly associated with simple CHD. Genetic variation of ISL1 was confirmed to be associated with the risk of CHD. ISL1 is related to the atrial septal defect group and the ventricular septal defect group, and the genotypes were associated with the occurrence of CHD in the dominant mode of inheritance. We concluded that rs1017 contributed to the risk of CHD in Chinese Han people, and ISL1 may be involved in the formation and development of the heart.

mRNA expression profiles of calmodulin and liver receptor homolog-1 genes in chickens.

Calmodulin (CALM), a calcium-binding protein, is expressed in the hypothalamic-pituitary-gonadal axis; it plays a pivotal role in the reproductive system by regulating gonadotropin-releasing hormone signaling. Downstream of hypothalamic-pituitary-gonadal signaling pathways, liver receptor homolog-1 (LRH-1) is involved in female gonadal hormone synthesis. In the chicken, although the two genes are known to be associated with reproductive traits, the interaction between gonadotropins and gonadal steroids remains unclear. We used quantitative real-time PCR to quantify the tissular (hypothalamus, pituitary, ovary, liver, kidney, oviduct, heart) and ontogenetic (12, 18, 32, and 45 weeks) mRNA expression profiles of CALM and LRH-1 in Erlang Mountainous chickens to determine their roles in the endocrine control of fertility, and compared these profiles with expression in Roman chickens. We found that the relative expressions of CALM and LRH-1 genes had the highest levels in the pituitary and ovary at 32 weeks. The expression level of CALM mRNA in the pituitary of Roman chickens was significantly higher than that in Erlang Mountainous chickens at 32 and 45 weeks, while the LRH-1 transcript level in the ovaries of Roman chickens was significantly lower than that of Erlang Mountainous chickens at 32 and 45 weeks. In summary, the transcript levels of CALM and LRH-1 genes are associated with chicken reproductive traits; in addition, we found that the CALM gene is the key regulator in the hypothalamic-pituitary-gonadal signaling network.