小麦果聚糖合成酶基因6-SFT-A单核苷酸多态性分析及其定位

 【目的】小麦果聚糖合成酶基因6-SFT是果聚糖合成过程中的关键酶基因,研究6-SFT-A的多态性,分析其与小麦苗期抗旱性的关系,并进行遗传定位。【方法】以苗期抗旱性不同的30份六倍体小麦和4份小麦A基因组供体种乌拉尔图小麦为材料,通过直接测序分析6-SFT-A的单核苷酸多态性（SNP）及其与抗旱性的关系;开发基因分子标记,利用RIL群体（偃展1号×内乡188）对该基因进行遗传定位。【结果】在30份六倍体小麦材料中检测到14个核苷酸多态性位点,包括13个SNP和1个InDel,平均234 bp检测到一个多态性位点,仅在1 727和1 781 bp 2个位点检测到非同义突变;在4份乌拉尔图小麦中检测到28个SNP和4个InDel,其频率明显高于普通小麦。该基因的内含子1、2、3和外显子3为变异富集区,其它区域变异较小,外显子2变异最小,π值为0。34份材料分为3种单倍型,HaplⅠ主要包括中等抗旱材料和水敏感材料,Hapl Ⅲ中主要包括强抗旱材料和中等抗旱材料。利用RIL群体将该基因定位于染色体4A的标记Xcwm-27与Xwpt688之间,遗传距离分别为5.3和7.9 cM。【结论】单倍型分析表明,小麦果聚糖合成酶基因6-SFT-A单倍型与小麦苗期抗旱性有一定的相关性。     

Quantitative modulation of polycomb silencing underlies natural variation in vernalization

Arabidopsis thaliana accessions have adapted to growth in a wide range of climates. Variation in flowering and alignment of vernalization response with winter length are central to this adaptation. Vernalization involves the epigenetic silencing of the floral repressor FLC via a conserved Polycomb (PRC2) mechanism involving trimethylation of Lys(27) on histone H3 (H3K27me3). We found that variation for response to winter length maps to cis polymorphism within FLC. A rare combination of four polymorphisms localized around the nucleation region of a PHD-Polycomb complex determines a need for longer cold. Chromatin immunoprecipitation experiments indicate that these polymorphisms influence the accumulation of H3K27me3 in Arabidopsis accession Lov-1, both at the nucleation site and over the gene body. Quantitative modulation of chromatin silencing through cis variation may be a general mechanism contributing to evolutionary change.     

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.     

Positive natural selection in the human lineage

Positive natural selection is the force that drives the increase in prevalence of advantageous traits, and it has played a central role in our development as a species. Until recently, the study of natural selection in humans has largely been restricted to comparing individual candidate genes to theoretical expectations. The advent of genome-wide sequence and polymorphism data brings fundamental new tools to the study of natural selection. It is now possible to identify new candidates for selection and to reevaluate previous claims by comparison with empirical distributions of DNA sequence variation across the human genome and among populations. The flood of data and analytical methods, however, raises many new challenges. Here, we review approaches to detect positive natural selection, describe results from recent analyses of genome-wide data, and discuss the prospects and challenges ahead as we expand our understanding of the role of natural selection in shaping the human genome.     

白菜类作物开花时间的全基因组关联分析

【目的】解析白菜类作物开花时间的调控位点,定位白菜类作物开花时间相关的候选基因,为白菜类作物抽薹开花时间的遗传改良提供依据。【方法】以116份白菜类作物组成的自然群体作为研究材料,分别种植在温室与露地2个独立的环境中,进行开花时间调查。同时,提取试验材料的DNA样品进行深度为1.2x的重测序,对测序数据用Pooled Mapping法进行过滤、与参考基因组比对,获得全基因组高密度SNP集合。经过条件过滤后,对高质量的SNP集合进行生物信息分析,包括试验材料的群体结构分析和全基因组连锁不平衡分析。从高质量的SNP集合中,随机挑选出2 000个变异位点,用Phy ML软件以最大似然法对116份试验材料进行系统发育树分析。用全部的高质量SNP集合位点通过软件Haploview进行全基因组连锁不平衡分析。最后,将高质量的SNP集合与开花时间数据结合,通过TASSEL和GAPIT软件包以及R程序语言进行全基因组关联分析。根据强关联峰值信号点位置和连锁不平衡区间定位开花时间候选位点,再通过白菜与同源物种拟南芥的基因共线性关系以及基因功能注释分析来预测白菜类作物开花时间相关的候选基因。【结果】不同种植条件下、不同类型的白菜类作物在开花时间上存在广泛差异。试验材料在露地环境下的开花时间高峰期明显早于温室环境下的材料;试验材料在露地环境下的开花时间总体表现出偏正态分布,而在温室环境下,开花时间各个阶段呈现出较为均衡的分布。温室与露地环境下的开花时间呈显著正相关。通过生物信息学分析最终得到的高质量SNP位点共103万个。试验材料的群体结构分析表明在系统发育树上各亚群内部分布较为集中,不同亚群之间的分布与材料的地理起源密切相关。全基因组衰减平均LD为2.3 kb,表明在116份白菜类作物构建的群体内存在较为频繁的重组和突变。对不同条件下的开花时间进行全基因组关联分析,用复合模型检测到54个(P4)强关联峰值信号点,一般模型检测到87个(P5)。通过进一步分析强关联信号点的连锁不平衡(linkage disequilibrium,LD)区段,得到存在强连锁关系(r20.33)的峰值信号点共33个(温室环境下27个,露地环境下19个)。其中,在温室与露地环境下的共定位位点13个。根据33个关联候选位点,再通过白菜与同源物种拟南芥的基因共线性关系以及基因功能注释分析筛选出白菜类作物开花时间相关的候选基因14个,其中温室与露地环境下共定位候选基因3个(FUL、PHYB和FPF1)。在露地条件下定位到开花关键基因FT1。【结论】不同条件下开花时间的相关性分析表明,遗传效应在开花早晚中起着决定性作用。全基因组关联分析共鉴定出33个与开花时间相关的显著关联信号。通过连锁不平衡分析、白菜与同源物种拟南芥的基因共线性关系以及基因功能注释分析初步鉴定出14个白菜类作物开花时间相关的候选基因。     

新疆小麦地方品种遗传多样性的SSR分析

为了评价新疆小麦地方品种的遗传多样性,利用42对SSR引物对75份新疆小麦地方品种进行遗传多样性分析。结果表明：在75份地方品种中42个SSR位点共检测到317个等位变异,等位变异变化范围为2~15个,平均7.55个;多态性信息指数(PIC值)变化范围为0.169~0.905,平均0.696。基因组的平均等位变异是D＞B＞A,遗传多样性指数为B＞D＞A。聚类分析表明75份供试新疆小麦材料可划分为3大类8亚类,聚类结果与材料的生态型密切相关,冬小麦地方品种和春小麦地方品种分别归属不同的类或亚类,春小麦地方品种的遗传多样性高于冬小麦地方品种,同时也反映了一定的地域特性。总之,新疆小麦地方品种具有丰富的遗传多样性,可用以拓宽育成品种的遗传基础。     

Diversity of Growth Habits and Their Association with VRN Allele of 81 American Wheat Lines

Eighty-one wheat accessions including 50 southern regional performance nursery （SRPN） lines and 31 northern regional performance nursery （NRPN） lines from the United States were tested to evaluate the growth habit by chilling treatments and to estimate the VRN allele variation with 19 pairs of published VRN primers. Two spring wheat accessions and 44 semi-spring wheat accessions were confirmed based on their chilling days＇ requirement and polymorphism was found at VRN loci. The Vrn-A1 allele had the highest frequency in the RPN accessions and VA1-CAPs markers identified growth habit of RPN lines. No polymorphism was found at the VRN3 loci and some polymorphism at the region of promoter and the first intron of VRN1 was not always consistent to growth habit in the wheat RPN accessions. The existence of variation in VRN alleles suggested that singly using the dominant Vrn allele is possible to extend the diversity of wheat accessions and improve their adaption to different environments in autumn-sowing region. This information will be useful for the cultivars exploitation and wheat breeding program.     

Diversity of Growth Habits and Their Association with VRN Allele of 81 American Wheat Lines

Eighty-one wheat accessions including 50 southern regional performance nursery (SRPN) lines and 31 northern regional performance nursery (NRPN) lines from the United States were tested to evaluate the growth habit by chilling treatments and to estimate the VRN allele variation with 19 pairs of published VRN primers. Two spring wheat accessions and 44 semi-spring wheat accessions were confirmed based on their chilling days' requirement and polymorphism was found at VRN loci. The Vrn-A1 allele had the highest frequency in the RPN accessions and VA1-CAPs markers identified growth habit of RPN lines. No polymorphism was found at the VRN3 loci and some polymorphism at the region of promoter and the first intron of VRN1 was not always consistent to growth habit in the wheat RPN accessions. The existence of variation in VRN alleles suggested that singly using the dominant Vrn allele is possible to extend the diversity of wheat accessions and improve their adaption to different environments in autumn-sowing region. This information will be useful for the cultivars exploitation and wheat breeding program.     

The fine-scale structure of recombination rate variation in the human genome

The nature and scale of recombination rate variation are largely unknown for most species. In humans, pedigree analysis has documented variation at the chromosomal level, and sperm studies have identified specific hotspots in which crossing-over events cluster. To address whether this picture is representative of the genome as a whole, we have developed and validated a method for estimating recombination rates from patterns of genetic variation. From extensive single-nucleotide polymorphism surveys in European and African populations, we find evidence for extreme local rate variation spanning four orders in magnitude, in which 50% of all recombination events take place in less than 10% of the sequence. We demonstrate that recombination hotspots are a ubiquitous feature of the human genome, occurring on average every 200 kilobases or less, but recombination occurs preferentially outside genes.     

玉米ZmEXO70s基因家族鉴定及苗期耐热性关联分析

【目的】鉴定玉米EXO70s（ZmEXO70s）基因家族成员,分析其遗传变异与玉米耐热性的关联性,为揭示其在玉米耐热方面的功能和分子机制打下基础。【方法】以拟南芥EXO70s（AtEXO70s）基因家族成员序列为参考,利用MaizeGDB和NCBI数据库从玉米全基因组中鉴定出ZmEXO70s基因家族成员,对其进行基因结构及系统进化分析,根据其在系统发育进化树上的位置对其进行系统命名,并基于转录组测序（RNA-Seq）数据分析ZmEXO70s基因家族成员在不同组织及非生物胁迫下的表达模式。最后,鉴定玉米自交群体AM368中ZmEXO70s基因SNP位点,结合玉米AM368群体苗期耐热存活率进行基因家族关联分析。【结果】从玉米全基因组序列中共鉴定出36个ZmEXO70s基因,分布在10条染色体上,长度为228～3726 bp,编码75～1241个氨基酸残基,蛋白分子量为8.6～136.6 kD,理论等电点（pI）介于4.5～9.9,大部分蛋白pI小于7.0。拟南芥、水稻和玉米的EXO70s蛋白被分为9组（A组～I组）,其中ZmEXO70s蛋白在各组中均有分布。大多数ZmEXO70s基因不含内含子,只有少数基因含有内含子,且内含子数量不同。36个ZmEXO70s蛋白共含8种基序（Motif 1～Motif 8）,主要集中在C端,说明这些蛋白端序列较保守,且Motif 1～Motif 8组成Exo70保守结构域。ZmEXO70s基因在不同组织中的表达水平均存在明显差异,其中ZmEXO70B3基因在雄穗、花药、叶片和花丝中高表达,ZmEXO70C1a基因只在雄穗和花药中高表达,ZmEXO70D2b基因在胚乳和萌发种子中低表达,ZmEXO70G1c基因在花丝和萌发种子中低表达。有23个ZmEXO70s基因至少可响应一种非生物胁迫,说明这些ZmEXO70s基因参与非生物胁迫响应过程。ZmEXO70D2a和ZmEXO70E1基因的遗传变异与玉米苗期耐热性具有显著相关性（P≤0.01）。【结论】ZmEXO70s基因家族成员在系统发育进化上较保守,其基因组复制事件可能发生在禾本科植物分化后,且大多数ZmEXO70s基因被保留,仅部分基因被丢失。ZmEXO70s基因可能在非生物胁迫响应过程中发挥重要作用,ZmEXO70D2a和ZmEXO70E1基因可作为调控玉米苗期耐热性重要候选基因。     

黄瓜cpSSR引物的多态性与通用性

从黄瓜叶绿体基因组序列中设计15对以A或T为重复单元的微卫星引物,其中12对处于基因组非编码区,3对位于基因编码区。这15对引物在21份黄瓜材料中均有扩增产物,但只有非编码区的微卫星表现出位点多态性。每对引物可检测1~3个等位基因,平均2.0个,引物的多样性值(polymorphic information content PIC)从0至0.484,平均值0.202。15对引物在甜瓜和南瓜中均能通用,13对可在西瓜中通用。这些叶绿体引物可用于黄瓜及其近缘种的叶绿体遗传研究。     

云南铁壳麦D染色体组SSR标记遗传多样性研究

为进一步研究云南铁壳麦D染色体组的遗传变异,利用分布于D染色体上的29对SSR引物对31份云南铁壳麦进行了遗传多样性分析,其中22个位点具有多态性,共扩增出60个等位位点,每个引物扩增出1～4个,平均2.1个。等位变异较人工合成麦及二倍体材料(平均2.4个)和育成品种(平均2.2个)低,但较地方品种(平均1.9个)丰富;多态性信息指数变幅为0～0.639,平均为0.217;等位变异2D>5D>1D=3D>7D>4D=6D;云南铁壳麦平均遗传相似系数为0.749,变幅在0.231～1,与人工合成麦及二倍体材料之间的遗传距离较远,与育成品种间的遗传距离最近。根据遗传相似系数云南铁壳麦可划分为11大类群,类群与变种和来源地都不相一致。     

人参单一基因微卫星标记的分析

 【目的】利用人参单一基因（unigene）序列,研究人参转录区SSR的分布特征;开发单一基因微卫星（UGMS）标记,并比较人参UGMS与基因组SSR标记（G-SSR）在分布频率、多态性及通用性等方面的不同,为人参等五加科药用植物的鉴定、遗传图谱的建立等研究奠定基础。【方法】利用NCBI公共数据库的7 649条人参EST序列,筛选出含有SSR的单一基因序列,并分析人参SSR的分布;根据这些序列以及包含SSR的人参基因组序列合成的引物扩增人参不同品种和其它五加科植物。【结果】检测到总长度为2.72 Mb的4 869个单一基因。其中,488个单一基因分布有724个SSR,占人参EST的10.02%,SSR的分布密度为3.75 Kb。一至三核苷酸重复分布频率分别为29.28%、48.06%和19.06%。非编码区和编码区重复序列分别以AT/TA和AAG/CTT为主。在合成的100对UGMS和44对G-SSR引物中,分别有86和44对能够扩增出人参的PCR产物,其多态率分别为42.0%和43.2%。人参UGMS对五加科西洋参、三七和刺五加植物的通用性分别为100%、87.2%和75.6%;G-SSR分别为95.5%、72.7%和40.9%。【结论】人参基因中SSR发生频率较高,并以二核苷酸重复为主。不同基因区域内的SSR分布具有非随机性。UGMS在揭示种内多态性方面不及G-SSR,但具有较高的种间通用性。     

Sequence Polymorphisms and Phylogenetic Relationships of hina Gene in Wild Barley from Tibet, China

The hina gene encodes a HINA protein in seeds of barley (Hordeum vulgare), which was known to affect the grain hardness. 171 hina gene sequences from Tibetan wild barley accessions and worldwide were characterized. Across 1 452 nucleotides of 171 hina genes, 152 SNPs were detected, giving an average frequency of one SNP per 9.5 bases. There were 93 singleton variable sites (the nucleotide polymorphism only observed in a single accession), 59 polymorphic sites (the polymorphisms found in two or more accessions) and 8 indels. A total of 18 haplotypes were defined, and most of the barley accessions shared one gene haplotype. H. spontaneum had a wider haplotype distribution. Through the analysis of median-joining network of the 18 haplotypes, 4 haplotype groups were found, which were testified by neighbor-joining tree based on the complete sequence alignment. Extremely low level of hina gene diversity was observed in Tibetan wild barley accessions, indicating that Tibet is unlikely a center of origin for cultivated barley.     

基于谷子测序开发的SSR标记多态性检测

利用谷子和狗尾草属其它种的24个材料,对474对基于谷子基因组测序结果设计的SSR引物进行了多态性鉴定。结果显示,有96对引物在各材料之间表现出了丰富的多态性,多态性率20.25%。本研究结果所筛选的谷子SSR标记丰富了狗尾草属遗传进行分析、种质资源研究、遗传图谱构建、目的基因定位及分子标记辅助选择可利用SSR标记的数量,同时也为谷子SSR标记核心数据库的建立奠定了基础。     

用高基元微卫星标记分析中国糜子遗传多样性

【目的】开发高基元(4—6)碱基重复微卫星标记,分析种质资源遗传多样性,为糜子遗传和进化研究提供理论基础。【方法】用隶属函数、主成分分析和聚类分析综合评价糜子资源表型多样性,用前期糜子转录组测序获得高基元SSR引物对地理来源差异大的糜子材料进行PCR扩增检测其多态性,用Power Marker 3.25计算遗传多样性参数,用Pop Gen 1.32计算Nei’s遗传距离,用MEGA 5.0进行聚类分析,用Structure 2.2鉴定遗传类群。【结果】96份糜子资源株高和穗长变异最丰富,多样性指数分别为2.08和1.91。PCR扩增发现,占56.29%的85对引物具多态性,其中四、五和六碱基重复引物分别为71对(83.53%)、10对(11.76%)和4对(4.7%)。85个标记扩增产物大小分布为100—450 bp,PIC值平均为0.51,Rp值为1.00—5.75,平均为3.15。四、五和六碱基重复SSR的平均Rp值分别为3.15、2.8和4.0。基于Rp值分析SSR的分布频次,发现85个标记分布区间为0—1、1—2、2—3、3—4、4—5和5—6,分别包含1(1.18%)、15(17.65%)、31(36.47%)、20(23.53%)、12(14.12%)和6(7.06%)个标记,60%(51个)的标记分布在区间2—3和3—4。用85个SSR扩增96份糜子资源,共检测到232个等位变异,每个位点检测到等位变异2—3个,平均2.7294个;62个位点产生3个变异,23个位点产生2个变异;多样性指数为0.2842—1.0633,平均为0.7708;PIC值为0.0400—0.7281,平均为0.4723。不同生态区糜子种质间的遗传距离为0.0093—0.5052(平均为0.1798),遗传一致度为0.6034—0.9907(平均为0.8485)。基于UPGMA将96个糜子基因型聚为4个群组,第一群组主要属于北方春糜子区;第二群主要属于东北春糜子区;第三群组主要属于华北夏糜子区;第四群组主要属于黄土高原春夏糜子区。遗传结构分析将96份试材划分为4个类群,分别代表黄土高原、华北、东北和北方基因库。UPGMA聚类分析和遗传结构分析结果基本一致,均与地理起源相关。【结论】在糜子中构建了85个四、五和六碱基重复微卫星标记,这些高基元SSR的引物分辨率(Rp)高,对不同基因型分辨能力强,PCR扩增多态性好;用其评估中国糜子资源的遗传差异发现,黄土高原春夏糜子区和北方春糜子区资源遗传多样性最丰富。     

Sequence Polymorphisms and Phylogenetic Relationships of hina Gene in Wild Barley from Tibet, China

The hina gene encodes a HINA protein in seeds of barley （Hordeum vulgare）, which was known to affect the grain hardness. 171 hina gene sequences from Tibetan wild barley accessions and worldwide were characterized. Across 1 452 nucleotides of 171 hina genes, 152 SNPs were detected, giving an average frequency of one SNP per 9.5 bases. There were 93 singleton variable sites （the nucleotide polymorphism only observed in a single accession）, 59 polymorphic sites （the polymorphisms found in two or more accessions） and 8 indels. A total of 18 haplotypes were defined, and most of the barley accessions shared one gene haplotype. H. spontaneum had a wider haplotype distribution. Through the analysis of median-joining network of the 18 haplotypes, 4 haplotype groups were found, which were testified by neighbor-joining tree based on the complete sequence alignment. Extremely low level of hina gene diversity was observed in Tibetan wild barley accessions, indicating that Tibet is unlikely a center of origin for cultivated barley.     

Genome-wide development of interspecific microsatellite markers for Saccharum officinarum and Saccharum spontaneum

Sugarcane has a large, complex, polyploid genome that has hindered the progress of genomic research and molecular marker-assisted selection. The user-friendly SSR markers have attracted considerable attention owing to their ideal genetic attributes. However, these markers were not characterized and developed at the genome-wide scale due to the previously lacking high-quality chromosome-level assembled sugarcane genomes. In this present study, 744305 and 361638 candidate SSRs were identified from the genomes of S. officinarum and S. spontaneum, respectively. We verified the reliability of the predicted SSRs by using 1200 interspecific SSR primer pairs to detect polymorphisms among 11 representative accessions of Saccharum, including S. spontaneum, S. officinarum, S. robustum, and modern sugarcane hybrid. The results showed that 660 SSR markers displayed interspecific polymorphisms among these accessions. Furthermore, 100 SSRs were randomly selected to detect the genetic diversity for 39 representative Saccharum accessions. A total of 320 alleles were generated using 100 polymorphic primers, with each marker ranging from two to seven alleles. The genetic diversity analysis revealed that these accessions were distributed in four main groups, including group I (14 S. spontaneum accessions), group II (two S. officinarum accessions), group III (18 modern sugarcane hybrid accessions), and group IV (five S. robustum accessions). Experimental verification supported the reliability of the SSR markers based on genome-wide predictions. The development of a large number of SSR markers based on wet experiments is valuable for genetic studies, including genetic linkage maps, comparative genome analysis, genome-wide association studies, and marker-assisted selection in Saccharum.