24种香稻品种甜菜碱醛脱氢酶2基因突变位点的分析及分子标记开发

根据香型与非香型水稻甜菜碱醛脱氢酶2基因(badh2)在第2、第4内含子、第7外显子3处序列差异和第2外显子1处序列差异,分别设计了两类检测badh2第7和第2外显子突变的功能性分子标记引物M7和M2;利用两类引物,分别对属于第7外显子突变的香型水稻W香99075和第2外显子突变的香型水稻武香14、非香型水稻以及两种香稻分别与非香稻杂交的F1植株基因组DNA进行PCR检测后发现,M7和M2引物完全能够分别被用于以第7和第2外显子突变的香稻作为亲本,进行分子标记辅助培育香稻新品种的研究.M7引物综合考虑了badh2内含子和外显子两方面突变情况而设计的.以非香稻261S、分别发生第7和第2外显子突变的香稻品种W香99075和武香14为对照,使用M7和M2引物,对本实验室收集的另外22份香稻品种进行badh2突变位点检测,结果可将这些香稻分为badh2第2外显子突变类型、第7外显子突变类型和外显子未发生突变类型,同时明确了大多目前在上海等周边地区种植的香稻品种的badh2所属的突变位点.开展本研究为利用分子标记辅助选育香型水稻新品种研究奠定了重要的基础.     

猪产仔数分子标记及其效应分析

初步确定了2个新的猪产仔数分子标记,雌激素受体基因ESR的第8外显子处的ESRB位点、催乳素受体基因的第7外显子的FSHRB位点。通过比较和分析多个产仔数的效应,初步确定了4个有利于产仔数提高的分子标记基因型,基因位点ESR、FSHRB的基因型BB的产仔数显著地高于AB、AA型;位点ESRB、PRLR的基因型AA的产仔数显著地高于AB、BB型;4个基因位点多态性与仔猪生长性能、母猪乳头数不存在显著的影响。     

人肝细胞癌中抑癌基因PTEN/MMAC1/TEP1的突变分析

PTEN／MMAC1／TEP1(PTEN)是新近分离到的抑癌基因,在多种肿瘤中存在突变。我们检测了34例人肝细胞癌中PTEN基因第5外显子和第8外显子的突变。采用聚合酶链方法以内含子引物扩增第5和第8外显子,继之以单链构象多态性和测序技术分析PTEN基因突变。有4例肝细胞癌SSCP显示异常条带并经测序证实存在突变。2例发生于第4内含子,突变位点相同;另两例发生于第8外显子,其中1例碱基颠换导致PTEN蛋白产物304位半胱氨酸突变为甘氨酸。     

番茄OVATE基因调控果实梨形发育

ＰｒｏｃＮａｔｌＡｃａｄＳｃｉＵＳＡ ,2 0 0 2 ,99:1 330 2～ 1 330 6早期研究表明番茄果实的梨形性状由位于第二染色体的一个隐性基因ｏｖａｔｅ所控制 ,ｏｖａｔｅ基因位于分子标记ＴＧ645附近 ,以此标记为探针筛选番茄ＢＡＣ文库将该基因定位于一个长为 1 0 5ｋｂ的ＤＮＡ片段内 ,进一步的分子标记定位表明该基因在含有 8个ＯＲＦ的 55ｋｂＤＮＡ内。通过ＰＣＲ扩增了圆形番茄这一区域ＤＮＡ并进行测序 ,结果表明突变体的ＯＲＦ6中有三处发生了突变。ＯＲＦ6由两个外显子和 1个内含子组成 ,有一个点突变和一个 2ｂｐ的插入或缺失突变…     

牛FSHR基因第10外显子单核苷酸多态性及其与双胎性状的关系

以秦川牛和荷斯坦奶牛的双胎母牛和单胎母牛为实验材料 ,以牛的FSHR基因的第 10个外显子作为标记牛双胎性状的候选基因 ,用SNP法进行了多态检测 .结果发现 ,在秦川牛的双胎母牛中突变率 6 0 % (6 10 ) ,而在单胎母牛中突变率为 2 0 % (2 10 ) ;在荷斯坦奶牛中 ,双胎母牛突变率为31 2 5 % (5 16 ) ,单胎母牛突变率为 6 6 7% (1 15 ) ;由此可见双胎牛和单胎牛二者之间FSHR基因的第 10个外显子的突变率差异明显 .这表明 ,选择FSHR基因的第 10个外显子有可能作为双胎性状的候选基因 .序列分析发现 ,在FSHR基因的第 15 0 6位碱基发生了突变 (T→C) ,但氨基酸没有发生变化 .     

猪STCH基因的Bi-PASA遗传标记及多态性研究

微粒体应激 70蛋白三磷酸腺苷酶 (STCH)基因属于应激 70蛋白基因伴侣家族 ,在机体免疫反应和疾病抵抗力等方面起重要作用。根据人和小鼠STCH基因的保守序列设计引物 ,PCR扩增到猪STCH基因第5外显子 4 4 5bp片段。序列测定显示 ,猪STCH基因与人和小鼠STCH基因分别具有 87 13%和 80 4 5 %的同源性。通过测定和比较中国梅山猪、欧洲约克夏猪及PIC商品猪的STCH基因序列 ,发现在猪STCH基因编码区第 5外显子 10 5 0位点上存在一个单碱基突变位点。利用双向特定等位基因PCR扩增法 (Bi PASA)建立了检测猪STCH基因变异的遗传标记 ,并用该标记分析了STCH基因在中国家猪 (梅山猪、荣昌猪和金华猪 )、欧洲家猪 (约克夏猪、大白猪 )、商品猪 (PIC合成系 )以及欧洲野猪的基因频率和多态性。本研究建立的Bi PASA遗传标记和基因变异信息 ,将为进一步分析猪STCH基因变异与经济性状的相关分析提供基础资料。     

马铃薯晚疫病抗性基因分子标记检测及抗性评价

马铃薯是重庆优势特色作物之一,但其安全生产受到晚疫病的严重威胁。马铃薯生产中种植抗病品种是防控马铃薯晚疫病最为经济有效和环境友好的途径。为了了解来自国内外不同种质的晚疫病抗性基因组成以及确定在重庆市具有晚疫病抗性的马铃薯品种(系),本研究以218份来自国内外的品种(系)为材料,进行了6个晚疫病抗性(R)基因分子标记检测,同时进行了田间晚疫病抗性评价及室内接种鉴定和筛选。研究结果显示,6个R基因的分子标记在供试材料中均有分布,但分子标记的组成不尽相同,主要分为4大类。第Ⅰ类含有具有广谱抗性基因的RB,晚疫病抗性评价表现为中抗以上;第Ⅲ类缺失R2 family基因标记,绝大部分表现为晚疫病敏感型;第Ⅱ类和第Ⅳ类中分别主要含有3个R基因(R2 Family+R3a+R3b)标记类型和4个R基因(R1+R2 Family+R3a+R3b)标记类型,这两类材料中表现出一定比例的晚疫病抗性水平,但第Ⅳ类中出现抗性表现的比例高于第Ⅱ类。结果说明含有RB基因标记贡献了较高的晚疫病抗性,缺失R2 famlily基因标记的材料可能不利于晚疫病抗性,利用这些基因标记辅助筛选有助于提高重庆地区晚疫病抗性育种效率。本研究评价了218份马铃薯材料6个重要R基因组成,并筛选出重庆地区表现抗性的多个材料,为新品种(系)的推广应用以及抗病育种选育提供了科学依据,同时为发掘新的抗病基因提供了遗传资源。     

大口黑鲈IGFBP1基因SNPs的筛选及与生长性状的关联分析

本研究使用PCR技术扩增得到2 743 bp长度的大口黑鲈IGFBP1基因序列,序列含4个外显子和3个内含子,编码263个氨基酸。采用直接测序法在IGFBP1基因上筛选到4个SNPs位点,分别位于该基因核苷酸序列中的第147个、第791个、第2 436个和第2 676个碱基,其中2个位点位于外显子上,A-141C位点发生了同义突变,C-791T发生了错义突变,随机检测同批430尾实验个体中4个位点的基因型,均符合Hardy-Weinberg定律。其中A-2436G和C-2676T位点完全连锁,组成单倍型标记H1。分析H1标记中的AB和AA基因型实验群体,显示两者在尾柄长、全长指标上存在显著差异(p0.05)。综合分析表明,H1标记可用于大口黑鲈分子辅助育种研究。     

人t-PA突变体cDNA打靶敲入小鼠ES细胞β-酪蛋白位点的研究

应用克隆的基因组序列作为同源臂 ,构建了小鼠 β 酪蛋白基因定位敲入打靶载体。短臂长 2 7kb ,包括小鼠 β 酪蛋白基因 5′端侧翼区、第 1外显子、第 1内含子、部分第 2外显子。长臂包括小鼠 β 酪蛋白基因部分第 2内含子、第 3～ 7外显子、第 3～ 6内含子、部分第 7内含子 ,长 3 4kb。人t PA突变体cDNA在第 2外显子中与小鼠 β 酪蛋白信号肽序列融合。正筛选标记neo放置在 β 酪蛋白基因第 2内含子中部 ,负筛选标记tk位于短臂外侧。ES细胞株TC 1在滋养层上培养扩增。处理ES细胞使其密度达到 2× 10 7个 /mL后 ,将 4 5 μg线性化的打靶载体DNA与 1mL细胞混匀后电击。转染的细胞在含G4 18和Gancyclovir的选择培养基中培养 ,7d后挑取 192个抗性克隆 ,扩增、提取基因组DNA ,EcoRⅠ酶切后 ,用打靶载体 5′端内侧探针进行Southern杂交 ,野生型出现 9 8kb ,而中靶的 β 酪蛋白基因由于敲入的人t PA突变体携带一个EcoRⅠ位点 ,中靶等位基因出现 6 6kb。从 78株ES细胞株中获得 1株发生正确同源重组的中靶ES细胞。     

黄淮冬麦区不同时期主推品种淀粉合成酶基因分子标记鉴定

利用普通小麦直链淀粉合成酶GBSS基因及支链淀粉关键合成酶SSⅡ基因的特异分子标记鉴定了黄淮冬麦区自20世纪50年代以来253份主推品种,发现8份品种缺失Wx-B1基因,其中5份材料（小偃168、秦麦1号、83S502、山东935031、山东928802）是新发现的缺失Wx-B1基因的小麦品种,未发现Wx-A1、Wx-D1基因缺失类型品种;所鉴定253份品种的SSⅡ基因均为野生型,未发现缺失突变类型。通过对8份缺失Wx—B1基因品种直链淀粉含量分析,发现这些品种的直链淀粉含量差异较大．变异范围为19．9％-33．0％,其中豫麦47、83S502和中育5号3个品种的直链淀粉含量较低,分别为19．9％、21．3％和26．4％,可以用于优质面条小麦品质改良。     

Development of Molecular Markers Used to Identify Two Types of Fragrant Rice and Analysis of Mutation Sites of BADH2 Gene in 24 Varieties of Fragrant Rice

Functional molecular markers M7 and M2 have been developed based on the DNA sequence differences of badh2 between fragrant rice varieties and non fragrant varieties in intron2, intron 4, exon7 and exon 2 respectively. PCR analyses on genome DNA of exon7 mutated fragrant rice Wxiang 99075, exon2 mutated fragrant rice Wuxiang14,non fragrant rice 261S and the F1 plants by M7 and M2 showed that M7 and M2 could be absolutely used to the molecular marker assisted rice breeding experiments when exon7 mutated and exon2 mutated fragrant rice varieties are used as parents. The design of M7 primers took mutations both in exons and intrones into account. Moreover, taking 261S,Wxiang 99075 and Wuxiang14 as controls, the mutation sites of badh2 in 22 fragrant rice varieties were analyzed, it was showed that fragrant rice varieties could be classified into 3 types: exon 2 mutated fragrant rice, exon 7 mutated fragrant rice and non exon mutated fragrant rice. At the same time, the mutation sites of badh2 in the main fragrant rice varieties which are grown in Shanghai and the surrounding areas have been verified. This research laid an important foundation for molecular marker assisted selection for novel fragrant rice.     

Development of a simple functional marker for fragrance in rice and its validation in Indian Basmati and non-Basmati fragrant rice varieties

Fragrance development in rice has been reported due to a 8-bp deletion in the exon 7 of badh2 gene located on Chromosome 8S. Multiplex markers targeting the functional InDel polymorphism was earlier reported for genotyping fragrance trait, but the marker was observed to be inconsistent and difficult to use. We have developed a simple, co-dominant, functional marker for fragrance trait, which can be resolved in an agarose gel and validated in Basmati and non-Basmati aromatic rice varieties and in a mapping population segregated for fragrance trait. The marker targets the InDel polymorphism in badh2 gene and amplifies 95 and 103 bp fragments in fragrant and non-fragrant genotypes, respectively. The newly developed marker was highly efficient in discriminating all fragrant and non-fragrant genotypes and showed perfect co-segregation with the trait of fragrance in the mapping population. We recommend the use of this simple, low-cost marker in routine genotyping for fragrance trait in large scale breeding materials and germplasm.     

Discovery of a new fragrance allele and the development of functional markers for the breeding of fragrant rice varieties

The recessive fgr gene on chromosome 8 is associated with rice fragrance. It has been reported that this gene is a non-functional badh2 allele and that the functional Badh2 allele encoding putative betaine aldehyde dehydrogenase (BADH2) could render rice non-fragrant. Here we report the discovery of a new badh2 allele and the development of functional markers for the badh2 locus. A total of 24 fragrant and ten non-fragrant rice varieties were studied and sequenced for their Badh2/badh2 loci. Of the 24 fragrant rice varieties, 12 were found to have the known badh2 allele (badh2-E7), which has an 8-bp deletion and three single nucleotide polymorphisms (SNPs) in exon 7; the others had a novel null badh2 allele (badh2-E2), which has a sequence identical to that of the Badh2 allele in exon 7, but with a 7-bp deletion in exon 2. Both null badh2 alleles are responsible for rice fragrance. Based on sequence divergence amongst the functional Badh2 and two null badh2 alleles, we developed functional markers which can be easily used to distinguish non-fragrant from fragrant rice and to differentiate between two kinds of fragrant rice. These functional markers will find their usefulness in breeding for fragrant rice varieties via marker-assisted selection. Weiwei Shi and Yi Yang contributed equally to this work.     

The genetic origin of fragrance in NERICA1

In this study, we investigated the cause and origin of fragrance in NERICA1, a fragrant rice inbred line developed from an interspecific cross between two non-fragrant parents. The genetic cause of fragrance in NERICA1 was found to be due to a previously reported mutation in the BADH2 gene, the same allele responsible for the majority of modern fragrant rice varieties. Haplotype analysis around the BADH2 gene in NERICA1, its parents, and 95 other varieties carrying the badh2.1 allele identified the source of the badh2.1 allele in NERICA1 was a fragrant tropical japonica variety, WAB638-1, which had been growing in the vicinity of the NERICA1 nursery during varietal development. The allele-specific marker for the badh2.1 allele consistently predicted fragrance in the diverse African germplasm tested, making it very useful for marker-assisted breeding of fragrant rice varieties in Africa.     

Resequencing Reveals Different Domestication Rate for BADH1 and BADH2 in Rice (Oryza sativa)

BADH1 and BADH2 are two homologous genes, encoding betaine aldehyde dehydrogenase in rice. In the present study, we scanned BADHs sequences of 295 rice cultivars, and 10 wild rice accessions to determine the polymorphisms, gene functions and domestication of these two genes. A total of 16 alleles for BADH1 and 10 alleles for BADH2 were detected in transcribed region of cultivars and wild species. Association study showed that BADH1 has significant correlation with salt tolerance in rice during germination stage, the SNP (T/A) in exon 4 is highly correlated with salt tolerance index (STI) (P<10⁻⁴). While, BADH2 was only responsible for rice fragrance, of which two BADH2 alleles (8 bp deletion in exon 7 and C/T SNP in exon 13) explain 97% of aroma variation in our germplasm. Theses indicate that there are no overlapping functions between the two homologous genes. In addition, a large LD block was detected in BADH2 region, however, there was no large LD blocks in a 4-Mb region of BADH1. We found that BADH2 region only showed significant bias in Tajima’s D value from the balance. Extended haplotype homozygosity study revealed fragrant accessions had a large LD block that extended around the mutation site (8 bp deletion in exon 7) of BADH2, while both of the BADH1 alleles (T/A in exon 4) did not show large extended LD block. All these results suggested that BADH2 was domesticated during rice evolution, while BADH1 was not selected by human beings.     

Genetic and molecular basis of fragrance in rice

Fragrance or aroma in rice is considered as a special trait with huge economic importance that determines the premium price in global trade. With the availability of molecular maps and genome sequences, a major gene for fragrance (badh2) was identified on chromosome 8. An 8-bp deletion in the exon 7 of this gene was reported to result in truncation of betaine aldehyde dehydrogenease enzyme whose loss-of-function lead to the accumulation of a major aromatic compound, 2-acetyl 1-pyrroline (2AP) in fragrant rice. However, several studies have reported exceptions to this mutation and indicated the involvement of other genetic loci in controlling fragrance trait. These studies emphasize the need to characterize the fragrance and its underlying factors in a wide range of genetic resources available for this trait. This review summarizes the new insights gained on the genetic and molecular understanding of fragrance in rice.     

Discovery of a new fragrance allele and development of functional markers for identifying diverse fragrant genotypes in rice

Discovery of new fragrance alleles provides important genetic resources for breeding fragrant rice. In this study, a hybrid complementation test demonstrated the association of a new fragrance allele without mutation in the coding region with flavor formation in a fragrant rice variety Nankai 138. The new allele (badh2-p-5′UTR) has a 3-bp deletion in the 5′ untranslated region and an 8-bp insertion in the promoter (?1,314 site upstream from the initiation codon). Surprisingly, we found that there is also an 8-bp insertion in the promoter of the badh2-E7 allele. We developed a new sequence tagged site functional marker to identify the badh2-p-5′UTR and badh2-E7 alleles according to the 8-bp insertion in their promoters. A cleaved amplified polymorphic sequence (AluI) functional marker targeting a common base substitution in the intron 2 of three badh2 alleles, viz. badh2-p-5′UTR, badh2-E7 and badh2-E2, was developed to identify diverse genotypes for fragrance in rice. Based on the results of sequence alignments among the three badh2 alleles, we suggest that the badh2-E7 and badh2-p-5′UTR alleles may have the same genetic origin. In addition, the genetic distance between the badh2-E7 and badh2-p-5′UTR alleles may be closer than that between the badh2-E2 and the badh2-p-5′UTR alleles, or between the badh2-E2 and the badh2-E7 alleles.     

Identification of microsatellite markers for fragrance in rice by analysis of the rice genome sequence

Several chemical constituents are important to the fragrance of cooked rice. However, the chemical compound 2-acetyl-1-pyrroline (AP) is regarded as the most important component of fragrance in the basmati- and jasmine-style fragrant rices. AP is found in all parts of the plant except the roots. It is believed that a single recessive gene is responsible for the production of fragrance in most rice plants. The detection of fragrance can be carried out via sensory or chemical methods, although each has their disadvantages. To overcome these difficulties, we have identified an (AT)₄₀ repeat microsatellite or simple sequence repeat (SSR) marker for fragrant and non-fragrant alleles of the fgr gene. Identification of this marker was facilitated through use of both the publicly available and restricted access sequence information of the Monsanto rice sequence databases. Fifty F₂ individuals from a mapping population were genotyped for the polymorphic marker. This marker has a high polymorphism information content (PIC = 0.9). Other SSR markers linked to fragrance could be identified in the same way of use in other populations. This study demonstrates that analysis of the rice genome sequence is an effective option for identification of markers for use in rice improvement.