Single Nucleotide Polymorphisms (SNPs) Discovery and Linkage Disequilib-rium (LD) in Forest Trees

With completion of the Populus genome sequencing project and the availability of many expressed sequence tags （ESTs） databases in forest trees, attention is now rapidly shifting towards the study of individual genetic variation in natural populations. The most abundant form of genetic variation in many eukaryotic species is represented by single nucleotide polymorphisms （SNPs）, which can account for heritable inter-individual differences in complex phenotypes. Unlike humans, the linkage disequilibrium （LD） rapidly decays within candidate genes in forest trees. Thus, SNPs-based candidate gene association studies are considered to be a most effective approach to dissect the complex quantitative traits in forest trees. The present study demonstrates that LD mapping can be used to identify alleles associated with quantitative traits and suggests that this new approach could be particularly useful for performing breeding programs in forest trees. In this review, we will describe the fundamentals, patterns of SNPs distribution and frequency, summarize recent advances in SNPs discovery and LD and comment on the application of LD in the dissection of complex quantitative traits in forest tress. We also put forward the outlook for future SNPs-based association analysis of quantitative traits in forest trees.

The Mining of Citrus EST-SNP and Its Application in Cultivar Discrimination

Single nucleotide polymorphisms （SNPs） are the most abundant sequence variations found in plant genomes and are widely used as molecular genetic markers in cultivar identification and genetic diversity studies. The objective of this study was to identify SNP markers useful for discrimination of citrus cultivars, since large numbers of expressed sequence tags （ESTs） of sweet orange are available from the National Center for Biotechnology Information （NCBI）. We now have the opportunity to discover SNP markers suitable for determining the haplotypes with which to distinguish very closely related cultivars and to assess genetic diversity within or between related species of citrus. SNPs and small insertions/deletions （Indels） from ESTs of sweet orange and satsuma were identified by the in silico SNP discovery strategy. 55 296 EST sequences of sweet orange and 2 575 of satsuma retrieved from the NCBI repository were mined for potential SNPs. Cleaved amplified polymorphic sequences （CAPS） and sequencing approaches were used to validate putative SNPs in a sample of 30 citrus accessions. A total of 3 348 putative SNPs were identified based on the abundance of sequences and haplotype cosegregation. Of these 3 348 SNPs, the transitions, transversions and Indels ratios were 47.9, 36.1 and 16.0%, respectively. The SNPs occurred on average at a frequency of 1 per 164 bp in the coding region of citrus. 14 SNPs were randomly selected and genotyped according to 30 citrus accessions including 23 accessions of sweet orange; 11 SNPs displayed polymorphism with an average polymorphism information content （PIC） of 0.20 among 30 citrus accessions. The genetic diversity present in sweet orange was low, so the 14 SNP markers failed to discriminate different cultivars of sweet orange, but they did succeed in distinguishing accessions of inter-species of citrus. In this study, SNPs were mined from EST sequences of sweet orange and satsuma, which displayed potential capability as molecular markers to discriminate inter-species accessions of citrus. It is anticipated that these putative SNPs could be applied in citrus genetics research and breeding.

Prostate cancer research in China

Prostate cancer （PCa） research in China has been on a rocketing trend in recent years. The first genome-wide association study （GWAS） in China identified two new PCa risk associated single nucleotide polymorphisms （SNPs）. Next generation sequencing is beginning to be used, yielding novel findings： gene fusions, long non-coding RNAs and other variations. Mechanisms of PCa progression have been illustrated while various diagnosis biomarkers have been investigated extensively. Personalized therapy based on genetic factors, nano-medicine and traditional Chinese medicine has been the focus of experimental therapeutic research for PCa. This review intends to shed light upon the recent progress in PCa research in China and points out the possible breakthroughs in the future.

A case-control study about the association between vascular endothelial growth inhibitor gene polymorphisms and breast cancer risk in female patients in Northeast China

Objective： The inhibition of the neovascularization in tumors is a potential therapeutic target of cancer. Vascular endothelial growth inhibitor （VEGI） is a member of the TNF superfamily which has the ability to suppress the formation of new vessels in tumors. In order to study the association between VEGI gene polymorphisms and breast cancer risk, a case-control study was conducted in Chinese Han women in Northeast China. Methods： Our study involved 708 female breast cancer patients and 685 healthy volunteers. Four SNPs of VEGI gene were analyzed through the polymerase chain reaction-restriction fragment length polymorphism （PCR-RFLP） method. The association between VEGI gene polymorphisms and breast cancer risk was analyzed in our study. The relation between VEGI gene variants and clinical features of breast cancer including lymph node （LN） metastasis, esl^ogen receptor （ER）, progestrogen receptor （PR）, tumor protein 53 （1953）, human epidermal growth factor receptor 2 （Her-2） and triple negative （ER-/PR-/Her-2-） status was analyzed as well. Results： We found that the CT genotype and T allele of rs6478106 were more frequent in patients than in controls. There was also a statistical difference in the distribution of Crs6478106Grs4263839 haplotype between patients and controls. In addition, SNP rs6478106 and rs4979462 were related with the Her-2 status. Conclusions： Our results suggest that VEGI gene variants may be related to the breast cancer risk and the clinical features of breast cancer in Chinese Han women in Northeast China.

Genetic Variants in MMP9 and TCF2 Contribute to Susceptibility to Lung Cancer

Objective：The Wnt signaling pathway is crucial for pulmonary development and differentiation;dysregulation of the Wnt signaling pathway may impair lung function.Indeed,single nucleotide polymorphisms （SNPs） of Wnt pathway-related genes have been suggested as risk factors for certain types of cancers.In this study,we aimed to evaluate the influence of SNPs in Wnt-related genes （TCF2,MMP9） on susceptibility to lung cancer.Methods：Polymorphisms of TCF2 rs4430796,MMP9 rs2250889,and MMP9 rs17576 were studied in Han Chinese subjects,including 135 patients with lung cancer and 176 controls,using the Sequenom MassARRAY platform.The association of genotypes with susceptibility to lung cancer was analyzed using odds ratio （OR）,with 95% confidence interval （95% CI） and χ2.Results：The three SNPs （rs4430796,rs2250889,and rs17576） were found to be significantly associated with an increased risk of lung cancer.The AA genotype and AG＋AA genotype of rs4430796 showed a significantly increased susceptibility to lung cancer compared with the GG genotype （adjusted OR=6.03,95% CI：1.30-28.09,P=0.022;5.55,95% CI：1.20-25.58,P=0.028）.Compared with the rs17576 GG genotype,the AG and AG＋AA genotypes were also associated with a significant risk （adjusted OR=2.65,95% CI：1.60-4.37,P≤0.001;2.57,95% CI：1.59-4.19,P≤0.001） whereas the rs2250889 CG and CG＋GG genotypes had 2.97-fold （95% CI：1.81-4.85;P≤0.001） and 2.80-fold increased associations with lung cancer （95% CI：1.73-4.54;P≤0.001）,respectively,compared with the rs2250889 CC genotype.Furthermore,the association of rs4430796 with lung cancer became insignificant （P0.05） after adjusting for gender and rs2250889.Conclusion：The three SNPs may play a role in the predisposition of members of the Han Chinese population to lung cancer.

Expression analysis,single nucleotide polymorphisms within SIRT4 and SIRT7 genes and their association with body size and meat quality traits in Qinchuan cattle

Silent information regulator 2 （Sir2） proteins, or sirtuins, are nicotine adenine dinucleotide （NAD）-dependent deacetylases that connect metabolism with longevity in lower organisms. In mammals, there are seven Sir2 homologs, namely, silent information regulators （SIRT1-7）. SIRT4 and SIRT7 genes play a crucial role in regulating lipid metabolism, cellular growth and metabolism. This suggests that they are potential candidate genes for affecting body size and meat quality traits in animals. Hence, this study aimed to detect genetic variations of both SIRT4 and SIRT7 bovine genes in Qinchuan cattle, and to evaluate the effect of these variations on economically important body size and meat quality traits. Expression analysis using quantitative real-time PCR （qPCR） indicated that SIRT4 and SIRT7 were broadly expressed in all thirteen studied tissues. The expression of SIRT4 was higher in liver, muscle, and in subcutaneous fat tissue. In the case of SIRT7, the expression was higher in lung, abomasum, and subcutaneous fat. Using DNAsequencing, a total of three single nucleotide polymorphisms （SNPs） were identified within SIRT4 and SIRT7 genes in 468 Qinchuan cattle. These included one novel SNP within 3＇ untranslated regions （UTR） of SIRT4 （SNP1： g. 13915A〉G） and two novel synonymous substitutions in SIRT7 （SNP2： g.3587C〉T and SNP3： g.3793T〉C）. Statistical analyses indicated that all three SNPs could significantly influence some body size and meat quality traits in Qinchuan cattle. These novel findings will provide a background for application of bovine SIRT4 and SIRT7 genes in the selection program of Chinese cattle.

Prostate cancer risk-associated genetic markers and their potential clinical utility

Prostate cancer （PCa） is one of the most common cancers among men in Western developed countries and its incidence has increased considerably in many other parts of the world, including China. The etiology of PCa is largely unknown but is thought to be multifactorial, where inherited genetics plays an important role. In this article, we first briefly review results from studies of familial aggregation and genetic susceptibility to PCa. We then recap key findings of rare and high-penetrance PCa susceptibility genes from linkage studies in PCa families. We devote a significant portion of this article to summarizing discoveries of common and Iow-penetrance PCa risk-associated single-nucleotide polymorphisms （SNPs） from genetic association studies in PCa cases and controls, especially those from genome-wide association studies （GWASs）. A strong focus of this article is to review the literature on the potential clinical utility of these implicated genetic markers. Most of these published studies described PCa risk estimation using a genetic score derived from multiple risk-associated SNPs and its utility in determining the need for prostate biopsy. Finally, we comment on the newly proposed concept of genetic score; the notion is to treat it as a marker for genetic predisposition, similar to family history, rather than a diagnostic marker to discriminate PCa patients from non-cancer patients. Available evidence to date suggests that genetic score is an objective and better measurement of inherited risk of PCa than family history. Another unique feature of this article is the inclusion of genetic association studies of PCa in Chinese and Japanese populations.

Identification of SNPs involved in regulating a novel alternative transcript of P450 CYP6ER1 in the brown planthopper

Cytochrome P450-mediated metabolic resistance is one of the major mecha- nisms involved in insecticide resistance. Although the up-regulation of cytochrome P450 plays a vital role in insecticide metabolism, the molecular basis for the transcriptional regulation of cytochrome P450 remains largely unknown. The P450 gene CYP6ERI, has been reported to confer imidacloprid resistance to the brown planthopper, Nilaparvata lugens. Here, we identified a novel alternative transcript of CYP6ER1 （transcript A2） that had different expression patterns between resistant and susceptible populations, and was more stable after insecticide induction. The promoter of this transcript was sequenced and multiple single nucleotide polymorphisms （SNPs） were detected in individuals from susceptible and resistant field-collected populations. Resistant alleles of four SNPs were found to significantly enhance the promoter activity of the CYP6ER1 transcript A2. Elec- trophoretic mobility shift assays （EMSAs） revealed that these SNPs might regulate the binding of transcription factors to the promoter. Our findings provide novel evidence re- garding the transcriptional regulation of a metabolic resistance-related gene and may be useful to understand the resistance mechanism ofN. lugens in the field.

Is KPNB3 Locus Associated With Schizophrenia?

Objective To reconfirm the association of KPNB3 with schizophrenia in Chinese population. Methods Two single nucleotide polymorphisms （SNPs）, rs2588014 and rs626716 trios consisting of fathers, mothers, and affected offsprings at the KPNB3 locus, were genotyped in 304 Chinese Han family with schizophrenia. These 2 SNPs were detected by PCR-based restriction fragment length polymorphism （RFLP） analysis. The Hardy-Weinberg equilibrium for genotypic distributions was estimated by the goodness-of-fit test. The UNPHASED program was used to perform transmission disequilibrium test （TDT）, haplotype analysis, and pair-wise measure of linkage disequilibrium （LD） between these 2 SNPs. Results The genotypic distributions of both rs2588014 and rs626716 were in the Hardy-Weinberg equilibrium （P〉0.05）. The TDT revealed allelic association with rs626716 （χ^2 =9.31, P=0.0023） but not with rs2588014 （χ^2 =3.44, P=0.064）. The global P-value was 0.0099 for 100 permutations. The haplotype analysis also showed a disease association （χ^2 =25.97, df=3, P=0.0000097）. Conclusion The present study provides further evidence in support of the KPNB3 association with schizophrenia in Chinese population.

Association of Matrix Metalloproteinase-9 and p53 Gene Polymorphisms with Genetic Susceptibility to No-small-cell Lung Cancer

Matrix metalloproteinase-9（MMP-9） and p53 genes play an essential role in the multi-step process of tumorigenesis in lung cancer. Single nucleotide polymorphisms（SNPs） of MMP-9 and p53 genes are associated with the risk and progression of many cancers. In this study, we evaluated the association of the R279Q polymorphism of MMP-9 or the A1/A2 polymorphism of p53 gcne with the risk of no-small-cell lung cancer（NSCLC） in Han population of Northeast China. We examined the frequency of SNPs in the two kinds of genes of 50 patients with NSCLC and 50 cancer-free controls frequency-matched by age and sex. Polymerase chain reaction-restriction fragment length polymorphism（PCR-RFLP） technique was used to determine the genotypes. The results indicate that the 279RR genotype in MMP-9 gene and the A1/A2 genotype in p53 gene show a significantly increased risk of NSCLC. Therefore, the MMP-9 279RR and p53 A1/A2 genotypes may be used as markers for susceptibility to NSCLC in Han population of Northeast China.

Genetic variants of the class A scavenger receptor gene are associated with coronary artery disease in Chinese

The class A scavenger receptor, encoded by the macrophage scavenger receptor 1 （MSR1） gene, is a pattern recognition receptor （PPR） primarily expressed in macrophages. It has been reported that genetic polymorphisms of MSR1 are significantly associated with the number of diseased vessels and coronary artery narrowing greater than 20% in Caucasians. However, whether it links genetically to coronary artery disease （CAD） in Chinese is not defined. Here, we performed an independent case-control study in a Chinese population consisting of 402 CAD cases and 400 controls by genotyping ten single nucleotide polymorphisms （SNPs） of MSR1. We found that rs416748 and rs13306541 were significantly associated with an increased risk of CAD with per allele odds ratio （OR） of 1.56 [95% confidence interval （CI） = 1.28-1.90; P 〈 0.001] and 1.70 （95% CI = 1.27-2.27; P 〈 0.001）, re- spectively. Our results indicate that genetic variants of MSR1 may serve as predictive markers for the risk of CAD / in combination with traditional risk factors of CAD in Chinese population.

Sequencing and Analysis of G HR Gene from Genomic DNA Pools of Mink

In order to study the single nucleotide polymorphisms （SNPs） of growth hormone receptor （GHR） gene in mink populations, genomic DNA pools of Minghua black minks and silver-blue minks were constructed, and the 10^th exon of GHR gene was PCR amplified from the two DNA pools and sequenced. The results showed that two SNPs were found at position 209 （T/C） and position 533 （C/A） of the 10^th exon of GHR gene in the two mink populations.

Association of Single Nucleotide Polymorphisms in IRF6 and TGFA Genes With Nonsyndromic Cleft Lip With Or Without Cleft Palate in Chinese Patients

Objective： Nonsyndromic cleft lip with or without cleft palate（NSCL/P） is a common birth defect with unclear etiology. Both genetic and environmental factors may contribute to NSCL/P. Many genes have been identified as candidate genes associated with this disease. Interferon regulatory factor 6（IRF6） gene and transforming growth factor-a（TGFA） gene seem to be crucial in the predisposition of NSCL/ P. Here we evaluated some single nucleotide polymorphisms（SNPs） loci of TGFA and IRF6 genes in Chinese nuclear families consisting of fathers, mothers and affected offspring with NSCL/P. Methods：Fifty patients of NSCL/P were confirmed by the plastic surgeons. They and their parents were included in the study, all with the informed consents. SNPs loci of TGFA and IRF6 genes were analyzed by microarray technology. Some PCR products were randomly chosen and sequenced to check microarray results. The distribution of gene type and allele frequency between patient group and parents group were compared. Then a Haplotype Relative Risk（HRR） and Transmission Disequilibrium Test（TDT） were performed. Results：The sequences of randomly selected PCR products were all consistent with the microarray results. All loci were in Hardy-Weinberg equilibrium. There were no significant differences in the distribution of genotypes and alleles between patients and their parents. Using HRR and TDT analyses the V274I of IRF6 was associated with NSCL/P, while another SNP locus oflRF6 was not. Strong evidence of linkage disequilibrium was found between the 2 SNP loci of TGFA and disease with the HRR analysis, but not with the TDT analysis. Conclusion：Our study confirms the contribution of IRF6 in the etiology of NSCL/P in populations of Asian ancestry. The association of TGFA with NSCL/P requires further research.

A High-Density SNP Genotyping Array for Rice Biology and Molecular Breeding

A high-density single nucleotide polymorphism （SNP） array is critically important for geneticists and molecu- lar breeders. With the accumulation of huge amounts of genomic re-sequencing data and available technologies for accurate SNP detection, it is possible to design high-density and high-quality rice SNP arrays. Here we report the devel- opment of a high-density rice SNP array and its utility. SNP probes were designed by screening more than 10 000 000 SNP loci extracted from the re-sequencing data of 801 rice varieties and an array named RiceSNP50 was produced on the Illumina Infinium platform. The array contained 51 478 evenly distributed markers, 68% of which were within genic regions. Several hundred rice plants with parent/F1 relationships were used to generate a high-quality cluster file for accurate SNP calling. Application tests showed that this array had high genotyping accuracy, and could be used for dif- ferent objectives. For example, a core collection of elite rice varieties was clustered with fine resolution. Genome-wide association studies （GWAS） analysis correctly identified a characterized QTL. Further, this array was successfully used for variety verification and trait introgression. As an accurate high-throughput genotyping tool, RiceSNP50 will play an important role in both functional genomics studies and molecular breeding.

Crop Breeding Chips and Genotyping Platforms： Progress, Challenges, and Perspectives

There is a rapidly rising trend in the development and application of molecular marker assays for gene map- ping and discovery in field crops and trees. Thus far, more than 50 SNP arrays and 15 different types of genotyping-by-sequencing （GBS） platforms have been developed in over 25 crop species and perennial trees. However, much less effort has been made on developing ultra-high-throughput and cost-effective genotyping platforms for applied breeding programs. In this review, we discuss the scientific bottlenecks in existing SNP arrays and GBS technologies and the strategies to develop targeted platforms for crop mo- lecular breeding. We propose that future practical breeding platforms should adopt automated genotyping technologies, either array or sequencing based, target functional polymorphisms underpinning economic traits, and provide desirable prediction accuracy for quantitative traits, with universal applications under wide genetic backgrounds in crops. The development of such platforms faces serious challenges at both the technological level due to cost ineffectiveness, and the knowledge level due to large genotype- phenotype gaps in crop plants. It is expected that such genotyping platforms will be achieved in the next ten years in major crops in consideration of （a） rapid development in gene discovery of important traits, （b） deepened understanding of quantitative traits through new analytical models and population designs, （c） integration of multi-layer -omics data leading to identification of genes and pathways responsible for important breeding traits, and （d） improvement in cost effectiveness of large-scale genotyping. Crop breeding chips and genotyping platforms will provide unprecedented opportunities to accelerate the development of cultivars with desired yield potential, quality, and enhanced adaptation to mitigate the effects of climate change.

Association of Genetic Polymorphisms of Anti-Müllerian Hormone (AMH) and Its Type II Receptor with Ovarian Hyperstimulation Syndrome

Objective To explore the association of genetic polymorphisms in the genes encoding the anti-Miillerian hormone （AMH） and its type H receptor （AMHRII） with ovarian hyperstimulation syndrome （OHSS）. Methods Using polymerase chain reaction （PCR） and DNA sequencing techniques, the exons of AMH and AMHRII were analyzed in 27 OHSS patients （OHSS group） and 22 non-OHSS patients （control group） who were applied controlled ovarian hyper- stimulation （COH）. Single nucleotide polymorphisms （SNPs） were also analyzed. Results SNPs G〉 T at position 146 of AMH exon 1 and G〉 A at position 134 of AMH exon 2 showed significant differences between the OHSS group and control group （P〈0.05）. SNP G〉 T at position 303 of AMH exon 1 showed no significant difference between the OHSS group and control group （P〉0.05）. No SNP was detected from the AMHR H exons 1 to 11 in either groups. Conclusion Genetic polymorphisms in the AMH gene may be a cause of ovarian hypersensitivity to exogenous hormone stimulation and the development of OHSS.