哈尔滨和湛江雄性褐家鼠Gsk3β差异甲基化区域DNA序列多态性分析与调控功能鉴定

糖原合成酶激酶-3β（glycogen synthase kinase 3β, Gsk3β）基因能够参与多条细胞周期信号传导通路,从而通过调控细胞分裂过程,影响组织与器官的发育。本实验室前期研究发现,与湛江褐家鼠种群相比,哈尔滨褐家鼠种群在秋冬季存在睾丸发育受抑制的现象,并通过基因组与甲基化组联合分析,在Gsk3β基因内含子区域筛选到1个差异甲基化区域（differentially methylated region, DMR）。为分析该DMR 的DNA序列多态性及其在Gsk3β基因表达调控中的可能作用,本研究选取了哈尔滨（n=52）和湛江（n=39）共91个性成熟褐家鼠睾丸样品。采用PCR测序方法在群体水平上分析了该DMR的DNA序列多态性,在2个褐家鼠亚种的分化特征,并选取2个群体中具有代表性的单倍型,通过荧光素酶基因报告系统在293T细胞中检测该DMR不同单倍型的调控活性。结果表明,该DMR存在2个SNP位点,共组成3个单倍型、6个基因型。卡方检验表明,其频率在2个群体间发生显著分化（单倍型,P=1.13E-29;基因型,P=1.15E-14）。Gsk3β基因的-2 218/+238 bp区具有明显启动子活性（P<0.05）;哈尔滨和湛江2个单倍型都显示显著的沉默子活性（P<0.05）,同时表现显著的调控活性差异（P<0.05）,表明这2个单倍型可以在293T细胞中作为沉默子抑制Gsk3β基因的启动子活性。2个单倍型调控活性差异,可能与SNP导致的转录因子结合位点的获得/丢失,以及2个单倍型在不同种群中甲基化状态差异有关。本研究结果表明,该DMR可能通过调控褐家鼠Gsk3β基因表达,在睾丸发育过程中发挥作用。2个种群主要单倍型的调控活性差异,及其甲基化状态差异可能是2个种群睾丸发育表型差异的重要调控因素之一。     

云南洞密蛛的种群遗传结构初探

云南洞密蛛Trogloneta yunnanense（Song&Zhu, 1994）是生活在云贵高原的一种洞穴蜘蛛。本文基于6个洞穴种群159只个体样本的线粒体COⅠ基因,初步探讨了该物种的种群结构和遗传多样性特征。研究结果表明：1)159个样本共检测到16个单倍型,种群间无共享单倍型,遗传多样性呈现出总体高、种群内低、单倍型多样性高、核苷酸多样性低的模式;2)种群间遗传分化极显著,种群内变异小,种群间F_ST值均在0.9以上（P<0.01）,种群间基因流小（Nm=0.01）,种群间变异占总变异的95.75%,种群内变异仅占4.25%;3)中性检验和核苷酸错配分布检验显示,云南洞密蛛未经历种群扩张,群体大小保持稳定。推测洞穴隔离和地下极端环境条件可能维持着云南洞密蛛的种群规模稳定,同时促进其种群间的遗传分化。     

云南中缅树鼩线粒体细胞色素b和D-loop区遗传多样性研究

对3个地方种群的49只样本的线粒体Cyt b基因全序列（1140 bp）及33只样本的控制区D-loop基因区段（745 bp）进行了序列测定。结果表明：Cyt b基因多态性位点有47个,其中单变异位点位点23个,简约信息位点24个。共定义了24个单倍型,其中种群间的共享单倍型有2个（8.33%）,其余均为某个种群所特有,单倍型多样度范围为0.80952（剑川种群）~0.91532（禄劝种群）,核苷酸多样度指数介于0.00326（禄劝种群）~0.00635（剑川种群）之间;D-loop基因多态性位点有18个,其中单变异位点8个,简约信息位点10个。共定义了16个单倍型,无种群间的共享单倍型,单倍型多样度范围为0.76615（禄劝种群）~0.93333（丽江种群）,核苷酸多样度指数介于0.00269（禄劝种群）~0.00583（丽江种群）之间。从各单倍型的TCS网络进化图显示横断山种群位于分支的末端,表现出中缅树鼩由南向北的扩散模式,支持＂岛屿起源＂假说。     

大石鸡边缘种群的遗传结构

大石鸡（Alectoris magna）分布于青海西部、东部,甘肃中部和宁夏西部的干部和半干旱区,由于环境变化,其种群向甘肃南部森林被砍伐的地区扩散,形成涟缘种群。本研究采用聚合酶链式反应（PCR）和直接测序的方法获得了采自甘肃的大石鸡一个边缘种群和两个中心地理种群共39个个体的线粒体DNA（mtDNA）控制区基因（D－loop）456－457个核苷酸的基因序列,16个变异位点（占整个序列的3.5%）有15个单倍型。边缘扩散种群有3种单倍型,M6与另两个种群共有,单倍型频率为0.108,而M4和M5为其特有,单倍型频率分别为0.081和0.027。边缘种群的单倍型比率和遗传多样性分别为37.5%和0.549。中心地理种群1和种群2各有7个单倍型,除M6炳种群共有,其余为自所特有,单倍型比率分别为46.7%和50.0%,遗传多样性分别是0.729和0.786。边缘种群的单倍型比率和遗传多样性均低于中心地理种群。     

基于nrDNA GapC基因内含子序列的资源冷杉遗传多样性研究

通过nrDNA GapC基因内含子序列的测序和分析,揭示资源冷杉遗传多样性水平和种群分化的强弱并推断其进化历史。资源冷杉3个种群的34个个体共获得70条GapC基因内含子序列,有8个核苷酸变异位点,鉴别出12种单倍型。银竹老山、大院和舜黄山种群分别有10种、6种和7种单倍型;有7个个体得到了2种以上的单倍型。资源冷杉物种水平的单倍型多样性（h）为0.817 0,种群的在0.683 3～0.883 1之间;物种水平的核苷酸多样性（π）为0.003 90,种群的在0.002 63～0.003 82之间。种群遗传分化研究结果（G_st=0.103,p<0.05）说明种群间存在显著的遗传分化,分子方差分析（AMOVA）结果也证明虽然大多数的核苷酸多态性（88.64％,p<0.001）来源于种群内个体间的变异,但是仍有显著性比例存在种群间（11.36%,p<0.05）。Tajima’s D、Fu &; Li’s D、Fu &; Li’s Fs中性检验结果都表明资源冷杉在物种和种群水平上都不拒绝中性进化。资源冷杉单倍型的谱系没有出现地区特异性谱系分支,核苷酸不配对分析（mismatch analysis）结果表明没有发生近期的群体扩张,资源冷杉的N_st（0.131）与G_st（0.103）没有显著性差异（p>0.05）,表明种群没有明显的地理结构。推测现存资源冷杉是相对较近的时间内片断化的产物。     

云南4个地区高山姬鼠线粒体控制区种群遗传分化研究

对云南4个种群38个个体的线粒体控制区（D-loop）905 bp的核苷酸序列遗传变异进行分析,探讨了高山姬鼠种群遗传结构和分化。在905 bp D-loop基因的碱基序列中,共发现了57个变异位点（全变异的6.30%）,共定义了23个单倍型,其中有一个单倍型（Hap1）为横断山3个种群（中甸、丽江和剑川）所共享,其余22个单倍型均为各个种群所特有。分子变异分析（AMOVA）表明,种群间的遗传变异占33.7%,种群内的遗传变异占66.3%。FST统计结果表明,除昆明种群和横断山种群之间差异显著（P〈0.05）,其它地理种群间的差异均不显著（P〉0.05）,说明昆明种群与横断山种群之间出现了明显的遗传分化。     

外生菌根真菌土生空团菌种群遗传多样性与结构研究

土生空团菌Cenococcum geophilum是生态系统中广泛分布的外生菌根真菌,具有丰富的遗传多样性和重要的生态功能。为揭示土生空团菌的种群遗传多样性和结构,本研究对采集自中国10个森林地点的桦木科Betulaceae、壳斗科Fagaceae和松科Pinaceae植物根系219份样品高通量测序的土生空团菌ITS2序列进行分析。结果表明4 380条土生空团菌ITS2序列共划分为137个单倍型（Hap_1-Hap_137）,其中Hap_1是3科植物的优势单倍型并分布于所有地点,有48个单倍型分布于少数几个地点,但有88个单倍型仅分布于单个地点。非参数（Kruskal-Wallis）检验结果表明地点对土生空团菌单倍型的丰度和多样性具有显著影响,而宿主植物科对其无显著影响。分子方差分析（AMOVA）结果显示土生空团菌在10个地理种群间和3个植物科种群间的遗传分化均不明显,而遗传分化主要来自于种群内的个体差异。基于K-2P遗传距离的邻接法（NJ）树显示10个地理种群可以划分为3支,壳斗科土生空团菌种群的遗传距离比桦木科和松科远。土生空团菌单倍型组成在不同地点间和不同植物科间均有明显差异,有些土生空团菌单倍型具有地点和植物科的偏好性。研究结果为进一步揭示土生空团菌遗传多样性的生态功能提供科学依据。     

基于amy基因的中国野桑蚕遗传多样性及其与家蚕的系统发育关系

为探索中国野桑蚕Bombyx mandarina的遗传多样性及其与家蚕B. mori的系统发育关系, 采用PCR产物直接测序法（少数样本克隆测序）获得34个家蚕和野桑蚕样本淀粉酶基因amy序列片段（715 bp）。分析发现56个多态性位点, 鉴定出28种单倍型（haplotype）; 核苷酸多样性π=0.01390±0.00103, 单倍型多样度Hd=0.988±0.011。核苷酸不配对分析（mismatch analysis）和Fu’s Fs 检测表明中国野桑蚕曾发生过种群扩张。分子方差分析（AMOVA）表明, 遗野桑蚕传差异主要在种群内, 种群间和地理组群间差异不显著。聚类树上34个样本聚为3枝/3蔟, 野蚕和家蚕都不按地理区域或系统（类型）聚类, A枝由来自不同地区的野蚕和不同类型的家蚕混合构成, 并且进一步分成3个亚枝, 每一亚枝也同时包含家蚕和野蚕, B枝由3个家蚕和1个野蚕混合构成, C枝全部由来自不同地区的野蚕构成。网络分析没有发现“祖先单倍型”和优势单倍型。结果提示, 淀粉酶基因是一个多态性丰富的分子标记, 中国野桑蚕遗传多样性十分丰富, 据此推测家蚕起源于多种生态类型混杂的野桑蚕。     

沙葱萤叶甲对蜕皮激素响应的转录组分析

为建立沙葱萤叶甲Gauleruca daurica成虫对蜕皮激素（20-hydroxyecdysone,20E）响应的转录组数据库,挖掘对20E响应的基因以及代谢和信号通路,并在转录组水平探讨20E调控生殖滞育的分子机制,本研究采用Illumina HiSeqTM4000高通量测序平台对20E及二甲基亚砜（dimethyl sulfoxide,DMSO）处理后的沙葱萤叶甲成虫进行了转录组测序,共获得80 313个unigene;与阴性对照DMSO相比,共获得201个差异表达基因,其中106个上调、95个下调。GO和KEGG富集分析表明,多数差异表达功能基因富集于各种代谢通路,其中核黄素代谢（riboflavin metabolism）、溶酶体（lysosome）和泛酸与乙酰辅酶A合成（pantothenate and CoA biosynthesis）通路显著富集（q<0.05）。结果表明,20E可能通过影响多种代谢通路调控沙葱萤叶甲的生殖滞育。     

赤水河两种荷马条鳅属鱼类的遗传多样性及谱系生物地理学过程分析

研究以线粒体Cyt b基因为分子标记,对赤水河两种荷马条鳅属（Homatula）鱼类（红尾荷马条鳅和短体荷马条鳅）的遗传多样性及种群结构进行了分析;同时,结合了对在长江上游其他几个水系分布的同种鱼类进行比较,分析其生物地理学过程。遗传多样性分析结果表明,5个水系135尾红尾荷马条鳅Cyt b基因序列共检测出42个单倍型,单倍型多样性和核苷酸多样性分别为0.936和0.00493;其中,赤水河种群的分别为0.891和0.00208。3个水系52尾短体荷马条鳅Cyt b基因序列共检测出12个单倍型,单倍型多样性和核苷酸多样性分别0.821和0.01105;赤水河种群的分别为0.646和0.00390。基于ML和BI法构建的单倍型分子系统发育树结果表明,两物种各自构成单系,且得到较强支持。红尾荷马条鳅各种群地理格局分布明显,赤水河种群为并系类群位于分支基部。在短体荷马条鳅支系中,岷江与沱江两个水系的个体相互聚类在一起,而赤水河群体聚成一个分支。赤水河与其他水系不存在共享单倍型,表现了明显的隔离和差异性的地理分布格局。由于这些水系之间地理位置相距较远,推测这种格局的形成不是地质运动造成的水系隔离,而是历史时期水位的高低变化造成鱼类种群的扩散和隔离。错配分析支持赤水河两物种种群扩张的推断,但中性检验却并非全部支持,显示种群历史相对复杂。     

基于RNA-seq技术的青藏高原不同海拔水毛茛转录组测序及基因表达分析

以青藏高原不同海拔7个居群的水毛茛(Batrachium bungei(Steud.)L.Liou)为材料,对其进行转录组测序及基因表达分析,研究它们在极端环境下基因表达方面的适应性。结果显示,7个居群内样本间的基因表达具有较高的相似性。差异基因富集分析结果发现苯丙烷生物合成相关基因在5个差异组中均呈显著富集;此外类黄酮、类胡萝卜素、苯丙氨酸、酪氨酸、色氨酸的生物合成以及激素的信号转导、MAPK信号通路、植物与病原菌互作等相关基因大多被显著富集。与低海拔居群相比,参与类黄酮生物合成通路的相关基因(HHT1、HCT、F3′H、CHS、CYP73A、CCOAOMT5、CYP98A)在高海拔居群中均显著上调表达。研究结果表明水毛茛主要通过多途径的参与及关键基因的调控表达来适应青藏高原的高海拔环境。     

基于大豆胞囊线虫病抗性候选基因的SNP位点遗传变异分析

以我国363份栽培和野生大豆资源为材料, 对大豆胞囊线虫抗性候选基因(rhg1和Rhg4)的SNP位点(8个)进行遗传变异分析, 以期阐明野生和栽培大豆间遗传多样性及连锁不平衡水平差异。结果表明, 与野生大豆相比, 代表我国栽培大豆总体资源多样性的微核心种质及其补充材料的连锁不平衡水平较高(R²值为0.216)。在栽培大豆群体内, 基因内和基因间分别有100％和16.6％的SNP位点对连锁不平衡显著, 形成两个基因特异的连锁不平衡区间(Block)。在所有供试材料中共检测到单倍型46个, 野生大豆的单倍型数目(27)少于栽培大豆(31), 但单倍型多样性(0.916)稍高于栽培大豆(0.816)。单倍型大多数(67.4％)为群体所特有(31个), 其中15个为野生大豆特有单倍型。野生大豆的两个主要优势单倍型(Hap_10和Hap_11)在栽培大豆中的发生频率也明显下降, 推测野生大豆向栽培大豆进化过程中, 一方面形成了新的单倍型, 另一方面因为瓶颈效应部分单倍型的频率降低甚至消失。     

"Missing" G x E Variation Controls Flowering Time in Arabidopsis thaliana

Understanding how genetic variation interacts with the environment is essential for understanding adaptation. In particular, the life cycle of plants is tightly coordinated with local environmental signals through complex interactions with the genetic variation (G x E). The mechanistic basis for G x E is almost completely unknown. We collected flowering time data for 173 natural inbred lines of Arabidopsis thaliana from Sweden under two growth temperatures (10°C and 16°C), and observed massive G x E variation. To identify the genetic polymorphisms underlying this variation, we conducted genome-wide scans using both SNPs and local variance components. The SNP-based scan identified several variants that had common effects in both environments, but found no trace of G x E effects, whereas the scan using local variance components found both. Furthermore, the G x E effects appears to be concentrated in a small fraction of the genome (0.5%). Our conclusion is that G x E effects in this study are mostly due to large numbers of allele or haplotypes at a small number of loci, many of which correspond to previously identified flowering time genes.     

Expression Quantitative Trait Locus Mapping across Water Availability Environments Reveals Contrasting Associations with Genomic Features in Arabidopsis

The regulation of gene expression is crucial for an organism’s development and response to stress, and an understanding of the evolution of gene expression is of fundamental importance to basic and applied biology. To improve this understanding, we conducted expression quantitative trait locus (eQTL) mapping in the Tsu-1 (Tsushima, Japan) × Kas-1 (Kashmir, India) recombinant inbred line population of Arabidopsis thaliana across soil drying treatments. We then used genome resequencing data to evaluate whether genomic features (promoter polymorphism, recombination rate, gene length, and gene density) are associated with genes responding to the environment (E) or with genes with genetic variation (G) in gene expression in the form of eQTLs. We identified thousands of genes that responded to soil drying and hundreds of main-effect eQTLs. However, we identified very few statistically significant eQTLs that interacted with the soil drying treatment (GxE eQTL). Analysis of genome resequencing data revealed associations of several genomic features with G and E genes. In general, E genes had lower promoter diversity and local recombination rates. By contrast, genes with eQTLs (G) had significantly greater promoter diversity and were located in genomic regions with higher recombination. These results suggest that genomic architecture may play an important a role in the evolution of gene expression.     

Systematic Functional Study of Cytochrome P450 2D6 Promoter Polymorphisms in the Chinese Han Population

The promoter polymorphisms of drug-metabolizing genes can lead to interindividual differences in gene expression, which may result in adverse drug effects and therapeutic failure. Based on the database of CYP2D6 gene polymorphisms in the Chinese Han population established by our group, we functionally characterized the single nucleotide polymorphisms (SNPs) of the promoter region and corresponding haplotypes in this population. Using site-directed mutagenesis, all the five SNPs identified and ten haplotypes with a frequency equal to or greater than 0.01 in the population were constructed on a luciferase reporter system. Dual luciferase reporter systems were used to analyze regulatory activity. The activity produced by Haplo3(−2183G>A, −1775A>G, −1589G>C, −1431C>T, −1000G>A, −678A>G), Haplo8(−2065G>A, −2058T>G, −1775A>G, −1589G>C, −1235G>A, −678A>G) and MU3(−498C>A) was 0.7−, 0.7−, 1.2− times respectively compared with the wild type in human hepatoma cell lines(p<0.05). These findings might be useful for optimizing pharmacotherapy and the design of personalized medicine.     

SNP variation in the promoter of the PRKAG3 gene and association with meat quality traits in pig

ABSTRACT: BACKGROUND: The PRKAG3 gene encodes the gamma3 subunit of adenosine monophosphate activated protein kinase (AMPK), a protein that plays a key role in energy metabolism in skeletal muscle. Nonsynonymous single nucleotide polymorphisms (SNPs) in this gene such as I199V are associated with important pork quality traits. The objective of this study was to investigate the relationship between gene expression of the PRKAG3 gene, SNP variation in the PRKAG3 promoter and meat quality phenotypes in pork. RESULTS: PRKAG3 gene expression was found to correlate with a number of traits relating to glycolytic potential (GP) and intramuscular fat (IMF) in three phenotypically diverse F1 crosses comprising of 31 Large White, 23 Duroc and 32 Pietrain sire breeds. The majority of associations were observed in the Large White cross. There was a significant association between genotype at the g.-311A>G locus and PRKAG3 gene expression in the Large White cross. In the same population, ten novel SNPs were identified within a 1.3 kb region spanning the promoter and from this three major haplotypes were inferred. Two tagging SNPs (g.- 995A>G and g.-311A>G) characterised the haplotypes within the promoter region being studied. These two SNPs were subsequently genotyped in larger populations consisting of Large White (n = 98), Duroc (n = 99) and Pietrain (n = 98) purebreds. Four major haplotypes including promoter SNP's g.-995A>G and g.-311A>G and I199V were inferred. In the Large White breed, HAP1 was associated with IMF% in the M. longissmus thoracis et lumborum (LTL) and driploss%. HAP2 was associated with IMFL% GP-influenced traits pH at 24 hr in LTL (pHULT), pH at 45 min in LTL (pH45LT) and pH at 45 min in the M. semimembranosus muscle (pH45SM). HAP3 was associated with driploss%, pHULT pH45LT and b* Minolta. In the Duroc breed, associations were observed between HAP1 and Driploss% and pHUSM. No associations were observed with the remaining haplotypes (HAP2, HAP3 and HAP4) in the Duroc breed. The Pietrain breed was monomorphic in the promoter region. The I199V locus was associated with several GP-influenced traits across all three breeds and IMF% in the Large White and Pietrain breed. No significant difference in promoter function was observed for the three main promoter haplotypes when tested in vitro. CONCLUSION: Gene expression levels of the porcine PRKAG3 are associated with meat quality phenotypes relating to glycolytic potential and IMF% in the Large White breed, while SNP variation in the promoter region of the gene is associated with PRKAG3 gene expression and meat quality phenotypes.     

The inference of phased haplotypes for the immunoglobulin H chain V region gene loci by analysis of VDJ gene rearrangements

The existence of many highly similar genes in the lymphocyte receptor gene loci makes them difficult to investigate, and the determination of phased "haplotypes" has been particularly problematic. However, V(D)J gene rearrangements provide an opportunity to infer the association of Ig genes along the chromosomes. The chromosomal distribution of H chain genes in an Ig genotype can be inferred through analysis of VDJ rearrangements in individuals who are heterozygous at points within the IGH locus. We analyzed VDJ rearrangements from 44 individuals for whom sufficient unique rearrangements were available to allow comprehensive genotyping. Nine individuals were identified who were heterozygous at the IGHJ6 locus and for whom sufficient suitable VDJ rearrangements were available to allow comprehensive haplotyping. Each of the 18 resulting IGHV│IGHD│IGHJ haplotypes was unique. Apparent deletion polymorphisms were seen that involved as many as four contiguous, functional IGHV genes. Two deletion polymorphisms involving multiple contiguous IGHD genes were also inferred. Three previously unidentified gene duplications were detected, where two sequences recognized as allelic variants of a single gene were both inferred to be on a single chromosome. Phased genomic data brings clarity to the study of the contribution of each gene to the available repertoire of rearranged VDJ genes. Analysis of rearrangement frequencies suggests that particular genes may have substantially different yet predictable propensities for rearrangement within different haplotypes. Together with data highlighting the extent of haplotypic variation within the population, this suggests that there may be substantial variability in the available Ab repertoires of different individuals.