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.     

Variations of Single Nucleotide Polymorphism （SNP） of Hordoindoline a Gene （hina） and Its Relationships with Kernel Hardness in 92 North American Barley Varieties （Hordeum vulgare L.）

Grain texture in barley （Hordeum vulgare L.） is an important quality character. The single nucleotide polymorphisms （SNPs） in Hordoindoline a （hina） gene and its relationship with hardness index among North American harley cultivars were investigated. The hina genes in 36 cultivars were sequenced for the SNP analyses, and 17 sets of SNP primers were designed to detect the SNP variations of hina locus in 92 North American barley cultivars. SNP detection indicated that there were four haplotypes in the hina genes of 92 barley cultivars, and haplotype 01 and 02 were shared by 68 and 14 cultivars, respectively, suggesting that there was a very limited diversity in hina genes among North American barley cultivars. Despite the wide range in hardness exists in 92 barley cultivars, however, unlike wheat, where a clear relationship has been demonstrated between a number of SNPs in the wheat hardness genes and quality （soft or hard wheat）, there was no such relationship for barley. The genotypes used in this study demonstrated that there was a low level of polymorphism in hina gene in North American barley cultivars and these polymorphisms had no impact on grain hardness.     

Comparative Analysis of Hina Gene Sequences in Wild （Hordeum spontaneum） and Cultivated （H. vulgare） Barleys

The Hina gene is one of the two known Hin genes for hardness, and its RNA expression is correlated with grain hardness and dry matter digestibility variation. In this study, only one clone of Hina gene was obtained from one barley accession. A total of 121 Hina gene sequences were isolated from 121 wild barley {Hordeum spontaneum) accessions in Israel, Iran, and Turkey, and then their molecular characteristics were compared with 97 Hina gene sequences from 74 cultivated barley (H. vulgäre) lines in Europe and 23 landrace (H. vulgäre) with global distribution and other 26 Hina gene sequences from cultivated barleys (H. vulgäre) with unknown global distribution. C/s-acting regulatory element (CARE) searching revealed that there were different types of regulatory element for the Hina gene in wild and landrace/cultivated barleys. There were six consistent cw-acting binding sites in wild and landrace/cultivated barleys, whereas 8 to 16 inconsistent TATA-boxes were observed. In addition, three special elements (E2Fb, Spl, and boxS) were only observed in wild barley, while one (AT 1 -motif) was only found in landrace/cultivated barley. Forty-four deduced amino acid sequences of HINA from wild and landrace/cultivated barleys were obtained by deleting repetitive amino acid sequences, and they were clustered into two groups on the basis of Neighbor-Joining analysis. However, there was no obvious difference in the amino acid sequences of HINA between wild and landrace/cultivated barleys. Comparing to protein secondary structure of wheat PINA, it was indicated that HINA also existed a signal peptide. In addition, HINA was a hydrophilic protein on the basis of the protein properties and composition.     

SNP Analysis and Haplotype Identification in Chymotrypsin Inhibitor-2 （CI-2） Gene of Barley

Barley chymotrypsin inhibitor-2 （CI-2） was considered to be a promising candidate for enhancing the nutritional value of other cereals by increasing its concentration as it is rich in lysine than any other storage protein. Also, it was proposed that CI-2 might play an important role in the inhibition of proteolytic enzymes from pests or pathogens as CI-2 can strongly inhibit chymotrypsin and subtilisin. In this study, a total of 93 CI-2 gene sequences were isolated from wild and cultivated barley. 48 SNPs and 4 indels were detected across the entire sequences. The frequency of SNPs in the noncoding region （1 out of 9 bases） was slightly higher than that in the coding region （1 out of 10.7 bases）. In all, 33.3% of the candidate cSNPs resulted in amino acid changes. As a total, the 24 cSNPs resulted in 15 amino acid changes. Ten distinguishable haplotypes were detected, among which 3 haplotypes were shared in the most barley accessions, whereas the rest of the haplotypes appeared at a lower frequency. In addition, three haplotypes （haplotype 4, 8, and 9） were unique for single accessions. These results suggested that low diversity at the CI-2 locus was detected among the cultivated and wild barley.     

入侵性食叶害虫椰子织蛾的单倍型多样性分析

【目的】分析危害棕榈科Palmae植物的一种重要入侵食叶害虫椰子织蛾Opisina arenosella单倍型在原产地和入侵地的分布特点,揭示椰子织蛾入侵我国的虫源信息。【方法】利用线粒体COI基因分析16个地理种群共计172个样本,比较椰子织蛾印度种群和入侵地(中国、马来西亚和泰国)种群的遗传关系。【结果】片段长度为625 bp的172条序列共鉴定出12个单倍型,包含15个变异位点,构成2个明显的单倍型分支,其中一个分支由11个单倍型(IN1～IN11)组成,均来自印度种群,单倍型IN1是6个印度种群的共享单倍型,IN2～IN11为独享单倍型;另一个分支为单倍型HAP,由来自中国、马来西亚和泰国的种群共享;HAP与11个来自印度的单倍型IN1～IN11均存在4个变异位点。【结论】入侵地区的椰子织蛾种群来自同一基因型或者具有相同的入侵源;椰子织蛾种群入侵后受环境选择压力,在新栖息地产生新的突变或杂交。     

基于线粒体Cytb序列的3个宽体金线蛭群体遗传多样性分析

对江苏省扬州高邮、苏州张家港、泰州海陵3个地区宽体金线蛭自然群体的线粒体Cytb基因全序列进行了测定和分析。结果显示:宽体金线蛭Cytb基因全长1 146 bp,样本的A、G、T、C平均含量分别为27.57%、15.77%、43.49%、13.17%,A+T含量(71.06%)明显高于G+C含量(28.94%)。在77个宽体金线蛭样本中,共检测到96个多态位点,其中有58个简约信息位点,获得47个单倍型,共享单倍型数目1个。3个宽体金线蛭群体的平均单倍型多样性、核苷酸多样性、平均核苷酸差异数分别为0.9781、0.0133、15.20,其中泰州海陵群体的单倍型多样度(0.987 7)和核苷酸多样度(0.013 1)最高。3个群体间的遗传分化指数F_(st)为0.065 1～0.175 6,且差异显著(P0.05),不同群体间存在一定的遗传分化。分子方差分析(AMOVA)表明,11.13%的变异来自群体间,88.87%的变异来自群体内。用邻接法构建的单倍型系统发育进化树显示,不同地理群体的个体呈交错分布,没有形成明显的地理谱系。中性检验和核酸不配对分布结果表明,3个宽体金线蛭野生群体总体大小保持相对稳定,没有发生明显的种群扩张。     

青稞耐低氮相关类甜蛋白基因HvTOND1克隆和亚细胞定位研究

为了探索青稞耐低氮相关类甜蛋白基因HvTOND1的基因和蛋白结构特点,为青稞耐低氮分子机制研究提供基础.以青稞'昆仑14'叶片为材料,根据植物基因组数据库Gramene(http://www.gramene.org/)中的大麦HvTOND1(HORVU5Hr1G005300)基因和启动子区域序列设计引物,通过PCR获得...     

中国乌骨鸡品种BCDO2基因多态性和肤色鉴定

为分析β-胡萝卜素脱氧酶2(β-carotene dioxygenase2, BCDO2)基因在我国乌骨鸡中的变异情况，采用PCR方法分别扩增和测序了我国5个乌骨鸡品种(丝羽乌骨鸡、东乡绿壳蛋鸡、金湖乌凤鸡、余干乌骨鸡和新兴竹丝鸡3号)、白耳黄鸡(黄肤色)和崇仁麻鸡(白肤色)的BCDO2基因，并与GenBank数据库中的原鸡序列进行系统发育分析。结果显示：7个品种215条序列，总计发现6个多态位点，分别为6273091(G-A)、6273129(A-G)、6273229(C-T)、6273240(C-T)、6273307(A-G)和6273672(A-G)。基于6个多态点界定了3种单倍型，定义为Hap1、Hap2和Hap3。其中只有白耳黄鸡、崇仁麻鸡和丝羽乌骨鸡有1种单倍型，东乡绿壳蛋鸡、金湖乌凤鸡、余干乌骨鸡和新兴竹丝鸡3号均有2种单倍型。系统发育分析显示：Hap1和Hap2只与红色原鸡聚为一类，Hap3和多个原鸡聚为一类。BCDO2基因多态位点能够很好地区分黄白肤色的品种，但很难区分乌骨鸡品种。多个原鸡在我国乌骨鸡品种形成过程中都做出了贡献。该研究结果为我国乌骨鸡遗传资源的保护、选育和鉴定工作提供了遗传背景信息。     

安徽淮河水系黄鳝群体遗传多样性及其遗传结构

利用线粒体Cyt b基因全序列(1138 bp)对安徽淮河水系黄鳝6个地理群体(阜南Fn、颍上Ys、平圩Pw、怀远Hy、凤阳Fy、明光Mg)165个样品进行遗传多样性和遗传结构分析。共检测到变异位点74个、单倍型25个,平均A+T含量(54.8%)显著大于G+C(45.2%)含量。平均单倍型多样性和平均核苷酸多样性分别为0.787、0.01882。各群体的遗传分化指数FST为0.01933~0.81352、基因流Nm为0.11461~26.36650,分子方差分析(AMOVA)显示44.1%的变异来自群体间,表明淮河水系黄鳝地理群体间存在较高程度的遗传分化。单倍型系统进化树和进化网络图揭示安徽淮河黄鳝6个群体的个体组成2个遗传差异明显的谱系。错配分布和中性检验结果表明安徽淮河黄鳝群体历史上较为稳定,无明显群体扩张。     

The influence of drought stress on malt quality traits of the wild and cultivated barleys

As a major abiotic stress, drought causes instability and deterioration of malt barley quality. There is distinct difference among barley cultivars in the responses of the main malt quality traits to drought stress. In the previous study, we identified some Tibetan wild barley accessions with relatively less change of malt quality traits under drought. In this study, we examined the impact of drought stress during grain filling stage on grain weight and several important malt quality traits, including total protein content, β-glucan content, limit dextrinase activity, β-amylase activity, and protein fractions in four barley genotypes(two Tibetan wild accessions and two cultivars). Drought treatment reduced grain weight, β-glucan content, and increased total protein content, β-amylase activity. These changes differed among barley genotypes and treatments, and are closely associated with grain filling process and kernel weight. All the results indicated Tibetan wild barley had great potential for developing drought tolerant barley cultivars. Relatively stable kernel weight or filling process under water stress should be highlighted in malt barley breeding in order to reduce the effect of water stress on malt barley quality.     

基于28S rDNA D2区序列的陕南凹缘菱纹叶蝉分子系统学与遗传多样性研究

为探讨陕南桑园凹缘菱纹叶蝉(Hishimonus sellatus Uhler)系统关系与遗传多样性,对陕南7个主要蚕桑产区共132头凹缘菱纹叶蝉的 28S rDNA基因D2区序列进行核苷酸多样性和遗传差异分析,同时研究单倍型之间的分子系统关系和网络进化图。结果表明,总群体共存在多态性位点63个,单倍型8个,优势单倍型H_1占单倍型总数的74.2%,遗传分化程度较低。分子方差分析显示:种群内变异占总组分的98.94%,差异不显著,且其基因流系数Nst和种群地理距离之间不存在相关性(R~2=0.019 7),说明各个地理种群之间存在渐渗杂交。     

青稞类钙调素蛋白基因CML19的克隆、序列分析及原核表达

根据鉴定得到青稞类钙调素蛋白基因CML19的序列设计引物,以青稞叶片的cDNA为模板,扩增得到目的片段,对目的基因序列进行鉴定和分析,进一步构建该基因的原核表达载体pET28a-CML19,转入大肠杆菌BL21(DE3),利用IPTG诱导外源蛋白质表达并检测。结果表明,扩增得到CML19的CDS序列全长为447bp,编码的蛋白质含有148个氨基酸,分子质量为16.46ku,理论等电点(pI)为4.40,总平均亲水性(GRAVY)为-0.176,不稳定系数为49.59,存在典型的EF手结构域;系统进化分析表明,青稞CML19与山羊草具有较近的亲缘关系;原核表达蛋白结果显示,重组蛋白主要以包涵体形式存在于沉淀中。初步阐明青稞CML19基因的序列特征,为进一步制备抗体、探讨CML19基因的功能奠定基础。     

我国不同品种黑羽鸡MC1R基因多态性研究

为了研究我国地方黑羽鸡品种MC1R基因的多态性,采用PCR的方法扩增了我国7个黑羽鸡品种(狼山鸡、寿光鸡、东乡绿壳蛋鸡、盐津乌骨鸡、余干乌骨鸡、鸿光黑鸡和新杨黑鸡)的MC1R基因,并对不同品种鸡MC1R基因序列的多态性进行生物信息学分析。结果显示：7个品种MC1R基因编码区全长均为945 bp,共编码315个氨基酸。7个品种210条序列,总计发现10个多态位点,位于编码区的有9个,其中有7个导致编码氨基酸发生变化,共界定了14种单倍型,定义为Hap1—Hap14,其中东乡绿蛋鸡和寿光鸡有2种单倍型,其他品种单倍型数都在2个以上。系统发育树分化为两个分支,分支I仅包含新杨黑鸡,分支II包含其他6个黑羽鸡品种。我国黑羽鸡MC1R基因表现出丰富的多态性,中国本土黑羽鸡品种和国外血缘黑羽品种存在MC1R基因多态位点上存在差别。该研究结果为我国黑羽地方鸡遗传资源的种质保护、品种选育和鉴定工作提供了参考。     

334份青藏高原野生大麦群体结构及连锁不平衡水平分析

利用1 389个DArT标记对334份青藏高原西藏野生大麦材料进行遗传多样性、群体结构和连锁不平衡水平分析。结果显示,参试群体DArT标记的多态性信息含量(PIC)值为0.005 6~0.500 0,平均值为0.248 9。运用贝叶斯、主坐标分析2种方法对野生大麦的群体结构进行研究。结果显示参试材料存在明显的群体分层,各个亚群中的个体不同来源、不同棱形、不同皮裸性混杂分布。以标记位点间的相关系数平方(r~2)作为衡量连锁不平衡(LD)水平的参数,在整体上,LD水平随着遗传距离的增加递减,各个染色体内也有相同的趋势,不同染色体的LD水平不同。以r~2=0.2为阈值统计,大于0.2的位点组合中,P0.001的位点组合比例为3.31%,r~2的平均值为0.454;群体的LD衰减距离为0.3cM,衰减较快。青藏高原地区野生大麦群体结构和连锁不平衡水平的研究为杂交育种和关联图谱的构建奠定了良好的基础。     

甘蓝种和芥菜型油菜细胞质的遗传多样性

对36份供试材料（15份甘蓝种材料、12份芥菜型油菜、4份不同细胞质类型的甘蓝型油菜、2份白菜型油菜及黑芥、埃塞俄比亚芥、芸芥各1份）进行叶绿体和线粒体SSR分析。7对多态性叶绿体特异的SSR引物在参试材料中共检测到31条多态性条带,每个位点等位基因为3~8个,平均4.43个,PIC值为0.234~0.711,平均为0.550。 2对线粒体特异的SSR引物检测到多态性条带数分别为3和5,PIC值分别为0.409和0.558。将线粒体与叶绿体的SSR标记合并,36份材料的遗传相似系数为0.538~1.000,在遗传相似系数 0.700 处,36份材料可明显分为5类,分别为芥菜型油菜、甘蓝型油菜和白菜型油菜、埃塞俄比亚芥和黑芥、甘蓝、芸芥,结果与传统的分类一致。芥菜型油菜内存在4种单倍型,而甘蓝仅有一种单倍型,芥菜型油菜材料间的细胞质遗传多样性高于甘蓝种材料间的细胞质多样性。     

牦牛SMAD1基因第1外显子SNPs检测及与生长性状的关联分析

研究牦牛 SMAD1基因第1外显子SNPs与生长性状的关系，寻找与牦牛生长性状相关的分子标记。采用DNA测序和单倍型分型技术，对青海牦牛 SMAD1基因进行SNPs检测及其基因分型、连锁不平衡和单倍型分析，并对多态位点的基因型及组合单倍型与牦牛生长性状的关联性进行分析。结果发现， SMAD1在第1外显子上存在5个突变位点，其中，g.35084C>T、g.35111C>T和g.35333G>A均存在2种基因型，g.35171G>A和g.35312C>T均存在3种基因型。连锁不平衡分析发现5个位点间不存在强的连锁不平衡效应，单倍型分析发现存在6种不同的单倍型，其中单倍型Hap3的发生频率最高。关联性分析表明，g.35084C>T位点与胸围显著相关，g.35111C>T位点与体斜长、胸围显著相关，g.35171G>A和g.35312C>T位点与体质量、体高、体斜长和胸围显著相关，g.35333G>A位点与胸围、体高显著相关。通过基因型组合发现，4种组合单倍型均与牦牛生长性状有着显著或极显著相关，且H3H6可能是影响牦牛生长性状的最优...     

Research of Genetic Diversity in Seven Kobresia by AFLP in Tibetan Plateau

This work analyzed the genetic diversity of Kobresia accessions at the molecular level, and further obtained the necessary information for breeding and germplasm evaluation. Genomic DNA of Kobresia was amplified with four E＋3 and M＋3 primer combinations with AFLP （amplified fragment length polymorphism）. AFLP analysis produced 164 scorable bands, of which 154 （93.96%） were polymorphic. The mean Nei＇s gene diversity index （H） was 0.2430, and the Shannon＇s information index （I） was 0.4012, indicating the abundant genetic diversity of Kobresia. The 11 Kobresia accessions from Tibetan Plateau, China, can be classified into five groups after cluster analysis based on the UPGMA （unweigbted pair group method arithmetic average） method. In general, there was abundant genetic diversity among Kobresia accessions resources, and the genetic coefficient was unrelated to their geographic latitude. Natural habitats influenced genetic differentiation of Kobresia.     

锦江河3种鳜的遗传变异及其多样性评价

为探究锦江河国家级水产种质资源保护区内鳜类遗传变异特征及其多样性水平,基于PCR扩增与测序技术对大眼鳜、斑鳜和中国少鳞鳜3个群体97尾鱼的线粒体控制区序列进行比较分析。结果显示,大眼鳜序列长度为843 bp,无变异位点,只有1种单倍型,斑鳜有852 bp和856 bp两种序列,28个多态位点和11个单倍型,中国少鳞鳜也有845 bp和846 bp两种长度,5个多态位点和5个单倍型;大眼鳜、斑鳜和中国少鳞鳜3种鳜鱼群体内的遗传距离分别为0. 000 0、0. 008 7±0. 002 0和0. 002 0±0. 000 9,单倍型多样度和核苷酸多态性分别为0. 000 0、0. 859 9、0. 736 7和0. 000 0、0. 008 7、0. 002 0。分析表明,3种鳜鱼在终止序列区和保守序列区存在种或属间差异,锦江斑鳜是一个遗传变异大,多样性丰富的稳定种群;中国少鳞鳜虽然单倍型多样度丰富,但遭受过瓶颈效应,导致其核苷酸多样性偏低;大眼鳜遗传组成单一,多样性十分贫乏,推测可能有入侵的养殖群体。锦江系梵净山国家级自然保护区内最大的河流,历史上鳜类资源丰富,但已遭严重的破坏,加强其鳜类遗传资源的研究与保护十分必要。     

Molecular analysis of cultivated naked barley (Hordeum vulgare L.) from Qinghai-Tibet Plateau in China using SSR markers

Naked barley is widely planted in Qinghai-Tibet Plateau in China and is essential for the daily life of Tibetans in those regions. In this study, the genetic diversity of 64 cultivated naked barley accessions from Qinghai-Tibet Plateau in China was analyzed using 30 mapped SSRs linked with the important traits of barley improvement. A total of 132 alleles were identified at 22 polymorphic SSR loci, with the number of each locus ranging from 2 to 15, the polymorphism information content (PIC) values ranging from 0.16 to 0.91, and with an average of 0.65. Of the selected SSRs, 13 SSR markers with high PIC value were highly efficient for the genetic analysis of Chinese barley. The accessions were divided into five main groups by cluster analysis and could be differentiated from each other. The genetic diversity in the Tibet accessions was slightly higher than in the Sichuan accessions. It is found that there were specific genes linked with the collecting sites. These results indicate the cultivated naked barley from Qinghai-Tibet Plateau in China are highly polymorphic and could be considered as an important resource bank for cultivated naked barley breeding in the future. __________ Translated from Acta Scientiarum Naturalium Universitatis Sunyatseni, 46(2): 82–86 [译自: 中山大学学报(自然科学版)]     

以色列野生二棱大麦籽粒多组分营养性状的相关性分析

为探讨起源地生态地理因素对野生二棱大麦营养性状的影响,对源自以色列不同地区9个野生二棱大麦群体的60个生态型材料的籽粒总氮、可溶性蛋白质、总淀粉含量等主要营养成分进行测定。经单因素方差分析(One-Way ANOVA)、相关性网络分析(correlation-based network analysis,CAN)和斯皮尔曼秩相关分析(Spearmanp’s Rho Correlation)等软件分析结果表明,以色列野生二棱大麦在群体、生态型水平和籽粒的主要营养成分均存在明显差异;总氮、可溶性蛋白质含量以及单粒重与起源地多个生态地理因素显著相关(P0.05)。因此得出结论,野生二棱大麦是在长期适应起源地生态地理环境的过程中,在自然选择压力下形成了其特有的营养性状遗传多样性。