Rapid mapping of candidate genes for cold tolerance in Oryza rufipogon Griff. by QTL-seq of seedlings

Cold stress is a major problem in rice production. To rapidly identify genes for cold tolerance in Dongxiang wild rice(DWR, Oryza rufipogon Griff.), sequencing-based bulked segregant analysis of QTL-seq method was used to resequence the extremely resistant(R) and susceptible(S) bulks of a backcross inbred lines(BILs) population(derived from Oryza sativa×O. rufipogon) and their parents. Single nucleotide polymorphisms(SNP)-index graphs and corresponding Δ(SNPindex) graphs(at 99 and 95% confidence levels) for R-and S-bulks detected a total of 2 609 candidate SNPs, including 58 candidate cold-tolerance genes. Quantitative real-time PCR analysis revealed that 5 out of the 58 candidate genes had significant differences in expression between O. sativa and O. rufipogon. Structural variation and functional annotations of the 5 candidate genes were also analyzed, and allowed us to identify 2 insertion-deletion(InDel) markers(12-7 and 12-16) that were linked with candidate genes on chromosome 12 in DWR. These results are helpful for cloning and using cold tolerance genes from common wild rice in cultivated rice.     

鱼类组蛋白核苷酸的多态性及其在杀鱼爱德华氏菌感染中的作用

组蛋白是染色体核小体的重要组分,在调控染色质结构、基因转录、个体发育等不同生物学过程中起着重要作用。为了研究鱼类组蛋白基因是否存在核苷酸的多态性以及组蛋白核苷酸多态性是否会影响鱼类的抗病力,本实验以斑马鱼和草鱼为研究对象,通过PCR扩增克隆了组蛋白H2A的全长开放阅读框;利用过表达技术、菌落平板计数、感染存活分析以及荧光定量PCR技术,研究了斑马鱼和草鱼组蛋白H2A核苷酸多态性不同变异体在杀鱼爱德华氏菌感染中的作用。在本研究中,实验发现鱼类组蛋白H2A存在着丰富的核苷酸多态性。序列分析结果显示斑马鱼和草鱼组蛋白H2A核苷酸多态性的变异体核苷酸序列相似性为90%～100%;而两两H2A核苷酸多态性变异体氨基酸序列之间最多只有3个位点存在差异。通过体内和体外抗菌实验可知,斑马鱼和草鱼组蛋白H2A核苷酸的多态性显著影响H2A的抗菌活性。此外,筛选出的抗菌组蛋白H2A核苷酸多态性的变异体在斑马鱼体内的过表达,不仅具有免疫增强的作用,还能显著增强斑马鱼对杀鱼爱德华氏菌感染的抗病力。本研究为筛选具有抗病作用的组蛋白H2A免疫保护原奠定了重要基础。     

绵羊心脏脂肪酸结合蛋白基因单核苷酸多态性分析

试验旨在探讨心脏脂肪酸结合蛋白（heart fatty acid-binding protein，H-FABP）基因在绵羊中的遗传多态性，并寻找可用于辅助选择的分子标记。本研究以滩羊（250只）及滩羊×湖羊杂交F1代（174只）为试验动物，利用SNaPshot分型技术对H-FABP基因（GenBank登录号：AY157617）的多态位点进行单核苷酸多态性（SNP）分析，统计基因频率和基因型频率，进行Hardy-Weinberg平衡性检测，计算期望杂合度（He）、多态信息含量（PIC）和有效等位基因数（Ne）等遗传多态指标，分析候选基因不同基因型与体重、体长、体高、胸围、胸深、胸宽和管围等生长性状的关联性。结果显示：①检测到9个多态位点：939[A/G]、980[G/A]、1018[T/C]、2878[C/T]、2956[C/T]、3017[G/A]、3341[G/C]、3394[T/A]、1056[-/G]，其中有6处转换、2处颠换、1处单碱基插入。②939[A/G]、980[G/A]、2956[C/T]、3341[G/C]、3394[T/A]和1018[T/C]的He为0.3200~0.4666，PIC为0.2688~0.3577；2878[C/T]、3017[G/A]位点的He为0.0283~0.1272，PIC为0.0279~0.1191，为低度多态；1056[-/G]位点的He在滩羊、滩羊×湖羊杂交F1代群体中分别为0.0120和0，PIC在滩羊、滩羊×湖羊杂交F1代群体中分别为0.0119和0；9个多态位点在滩羊、滩羊×湖羊杂交F1代群体中均符合Hardy-Weinberg平衡定律。③5个多态位点：939[A/G]、980[G/A]、2956[C/T]、3341[G/C]、3394[T/A]处于紧密连锁（D'>0.99），将H-FABP基因分为3个单倍型：AA、AB和BB。④在41只滩羊×湖羊杂交F1代羊中，BB单倍型在各生产性状上具有最大值，但各单倍型间差异不显著（P>0.05）。结果表明，绵羊H-FABP基因具有丰富的遗传多态性，939[A/G]、980[G/A]、2956[C/T]、3341[G/C]、3394[T/A]5个位点紧密连锁，BB单倍型可能是与绵羊生产性状相关的优势单倍型。     

中国地方绵羊群体TYRP1基因遗传变异研究

本研究旨在了解酪氨酸酶相关蛋白1（tyrosinase related protein 1，TYRP1）基因在中国地方绵羊群体内的遗传变异，以及TYRP1基因突变与不同毛色表型绵羊群体的相关性。通过直接测序法和PCR-RFLP技术对10个中国地方绵羊群体进行单核苷酸多态性（SNP）检测，利用Beagle、PLINK和POPGENE等软件对突变位点数据进行单倍型构建、连锁不平衡分析和遗传变异研究。突变位点检测结果表明，在绵羊TYRP1基因内识别了13个SNPs，其中位于TYRP1基因外显子上的10个SNPs位点，除个别位点在大尾寒羊、中国美利奴羊和岷县黑裘皮羊中没有发生突变外，其他突变位点在所有绵羊品种中均出现不同程度变异，说明中国地方绵羊群体具有较高的遗传多样性。单倍型分析结果表明，所有样本中共有42个单倍型，优势单倍型0000000000（245/918）、0100000001（91/918）在所有绵羊群体中均存在，除单倍型0101100000（93/918）在中国美利奴羊中没有出现，单倍型0001000001（69/918）在岷县黑裘皮羊、哈萨克羊群体中没有出现外，在其他群体中均存在。连锁分析结果表明，10个SNPs在所有样本中均存在2个连锁模块。群体遗传变异分析表明，中国地方绵羊群体具有较高水平的群体内遗传变异，各绵羊品种间存在明显的遗传分化模式，且各品种遗传关系与其品种传统分类结果基本一致。本研究为进一步研究TYRP1基因对绵羊毛色遗传性状的影响提供了参考依据。     

Single nucleotide polymorphisms in the growth hormone gene of Oreochromis niloticus and their association with growth performance

Polymorphisms in the growth hormone (GH) gene that is associated with the growth rate of farmed fish have been the target of many breeding programmes. The present study aimed to identify single nucleotide polymorphisms (SNPs) in GH gene regions to evaluate the association of SNP variations with the growth rate of two Nile tilapia: Oreochromis niloticus (Linnaeus, 1758) strains. The targeted regions were amplified, sequenced, aligned and screened for the presence of SNPs; thereafter, performance tests were used to check for the association between SNPs and weight. Allele and genotype frequencies were estimated for each SNP and genotype. Genotype blocks or sets of SNP genotypes and frequencies were also estimated. Association between SNPs and growth rate was statistically evaluated using a univariate linear mixed model that included both fixed and random effects. A total of 10 SNPs were identified, nine in the proximal promoter and one located in the 5′ UTR, forming 10 genotype blocks. In all weight recordings, five genotype blocks were significantly associated with the highest weights. Single nucleotide polymorphisms 6‐10 were also found to be significantly associated with growth (p‐value < .05). Genotypes with higher additive genetic values for weight were identified in the Chitralada strain, suggesting a possible impact of these additive effects of the GH SNP genotype on the growth rate of Nile tilapia. These findings may be used as part of marker‐assisted selection in tilapia breeding programmes.     

Association of TNF-α, IL-10 and IL-6 promoter polymorphisms in pulmonary tuberculosis patients and their household contacts of younger age group

Single nucleotide polymorphisms (SNPs) of cytokine genes have been found to be involved in the clinical outcome of Tuberculosis. The present study was aimed to identify the high risk genotypes in Tuberculosis patients and their household contacts. A total of 490 subjects were studied which includes 150 active pulmonary tuberculosis patients (APTB), 190 household contacts (HHC) and 150 healthy controls (HC). The SNPs of TNF-α (-308A/G), IL-10(-1082G/A) and IL-6(-174G/C) were performed by ARMs PCR. The IL-10 GA genotype showed significant association in APTB and HHC and was 2.3 times higher risk in APTB and 3.7 times in HHC compared to HCs. The A allele was found to be significantly associated with the risk of disease. The CC genotype of IL-6 was found to be significantly associated in APTB and an insignificant positive association in HHCs. The multifactor dimensionality reduction (MDR) analysis indicated that the genotypes of IL-6 were showing high risk with GA genotype of IL-10. In conclusion the gene interaction may be useful for identification of genotypes as biomarkers to distinguish high risk individuals.     

刺参(Apostichopus japonicus)重要经济性状相关SNP标记的验证分析

本研究在前期构建的刺参(Apostichopus japonicus)高密度遗传连锁图谱和QTL分析的基础上，筛选出26个与体长、体重、体宽、棘刺总数、抗病力相关的SNP位点，设计出可用于HRM检测的SNP扩增引物13对。在扩大群体中利用HRM小片段法对这13个刺参重要经济性状相关的候选SNP位点进行分型和多态性检测，并结合扩大群体的相关性状数据进行了QTL位点验证。多态性结果显示，13个位点中有3个单态性位点，其余10个多态性位点中有3个位点为低等位多态性，7个位点为中等位多态性。10个多态性位点的最小等位基因频率(MAF)介于0.016(SNP113)~ 0.332(SNP160)之间，平均值为0.173；各位点的观测杂合度(Ho)介于0.031(SNP113)~0.818(SNP9)之间，平均值为0.433；期望杂合度(He)介于0.031(SNP113)~0.834(SNP9)之间，平均值为0.402；多态信息含量(PIC)介于0.030(SNP113)~0.393(SNP160)之间，平均为0.248，有6个位点偏离Hardy- Weinberg平衡。QTL验证结果表明，SNP40和SNP160位点为与生长(体长、体重、体宽)相关的位点，各位点的优势基因型分别为SNP40(CC)和SNP160(AA)；SNP88、SNP112和SNP126这3个位点为与抗病力相关的位点，各位点的优势基因型为SNP88(CC)、SNP112(AA)和SNP126(TT)。基于这5个位点构建生长和抗病二倍型，发现二倍型K1(CC AA TT)抗病力最强，S1(CC AA)、S3(CC AC)在生长方面优势显著，相关研究结果可为分子标记辅助育种在生产中应用提供基础数据。     

亚洲棉EST-SNP的挖掘及其在陆地棉中的验证

【目的】随着不同棉种序列数据库的逐步完善以及高通量测序技术的发展,棉花单核苷酸多态性(Single nucleotide polymorphism,SNP)标记开发可利用的公共数据资源逐步增加。【方法】本研究基于陆地棉祖先基因组的现代种亚洲棉表达序列标签(Expressed sequence tag,EST)数据库,利用CAP3对亚洲棉EST数据库进行拼接。拼接获得7 187个重叠群(Contig),再利用Quality SNP软件进行SNP位点分析。【结果】在807条含有4条以上EST序列的Contig中查找到2 690个SNP位点。通过筛选次要等位基因频率大于30%的位点,获得953个可靠度较高的候选SNP,通过电子筛选,最终获得可用于陆地棉分析的SNP 149个,利用位点特异性聚合酶链式反应以及酶切扩增多态序列验证了EST-SNP的准确性。【结论】本研究证实基于亚洲棉EST数据库挖掘用于陆地棉研究的EST-SNP切实可行,并有望将EST-SNP用于陆地棉遗传图谱构建、重要性状的基因定位以及分子标记辅助育种。     

Development of a core set of KASP markers for assaying genetic diversity in Brassica rapa subsp. chinensis Makino

Single‐nucleotide polymorphisms (SNPs) are rapid, economical and reliable genotyping tools. Non‐heading Chinese cabbage (Brassica rapa L. subsp. chinensis Makino) is now an economically important vegetable crop worldwide. In this study, 1,167 SNPs were evaluated for 7polymorphism among 70 representative non‐heading Chinese cabbage inbred lines using a Kompetitive Allele Specific PCR (KASP) genotyping assay. On the basis of identified polymorphisms and the results of a principal component analysis, we selected 50 core SNPs that were balanced sufficiently to provide adequate information for genetic identification. The core SNPs were used for construction of a neighbour‐joining dendrogram that separated the 70 inbred lines into four main groups and several subgroups corresponding to Caixin, Heiyebaicai, Huangxinwu, Naibaicai, Taitsai, Pak‐choi, and Wutatsai. This categorization was superior to that achieved using a dataset of 479 polymorphic SNPs. To confirm the utility of the core SNP markers in genetic identification, we tested their stability and resolution using 162 commercial hybrid cultivars. The SNPs, which represent a cost‐effective, accurate marker set for germplasm analysis and cultivar identification, are suitable for molecular marker‐assisted breeding in non‐heading Chinese cabbage.     

Development of a kompetitive allele‐specific PCR marker for selection of the mutated Wx‐D1d allele in wheat breeding

Waxy (Wx) protein is a key enzyme for synthesis of amylose in endosperm. Amylose content in wheat grain influences the quality of end‐use products. Seven alleles have been described at the Wx‐D1 locus, but only two of them (Wx‐D1b, Wx‐D1e) were genotyped with codominant markers. The waxy wheat line K107Wx1 developed by treating ‘Kanto 107’ seeds with ethyl methanesulphonate carries the Wx‐D1d allele. However, no molecular basis supports this nomenclature. In the present study, DNA sequence analysis confirmed that a single nucleotide polymorphism in the sixth exon of Wx‐D1 changed tryptophan at position 301 into a termination codon. Based on this sequence variation, a PCR‐based KASP marker was developed to detect this point mutation using 68 BC₈F₁ plants and 297 BC₈F₂ lines derived from the cross ‘Ningmai 14’*9/K107Wx1. Combined with codominant markers for the Wx‐A1 and Wx‐B1 alleles, waxy and non‐waxy near‐isogenic lines were distinguished. The KASP marker was efficient in identifying the mutant allele and can be used to transfer waxiness to elite lines.