Genomic and genie sequence variation in synthetic hexaploid wheat(AABBDD)as compared to their parental species

In order to understand the genomic changes during the evolution of hexaploid wheat,two sets of synthetic hexaploid wheat from hybridization between maternal tetraploid wheat (AABB) and paternal diploid goat grass(DD)were used for DNA-AFLP and single strand conformation polymorphism (SSCP) analysis to determine the genomic and genie variation in the synthetic hexaploid wheat.Results indicated that more DNA sequences from paternal diploid species wen eliminated in the synthetic hexaploid wheat than from maternal tetraploid wheat,suggesting that genome from parental species of lower ploidity tends to be eliminated preferentially.However,sequence variation detected by SSCP procedure was much lower than those detected by DNA-AFLP.which indicated that much less variation in the genie regions occurred in the synthetic hexaploid wheat.and sequence variations detected by DNA-AFLP could be derived mostly from non-coding regions and repetitive sequences.Our results also indicated that sequence variation in 4 genes can be detected in hybrid F1.which suggested that this type of sequence variation could be resulted from distant hybridization.It was interesting to note that 3 out of the 4 genes were mapped and clustered on the long alTll of chromosome 2D,which indicated that variation in genic sequences in synthetic hexaploid wheat might not be a randomized process.     

Genomic and genic sequence variation in synthetic hexaploid wheat （AABBDD） as compared to their parental species

In order to understand the genomic changes during the evolution of hexaploid wheat, two sets of synthetic hexaploid wheat from hybridization between maternal tetraploid wheat （AABB） and paternal diploid goat grass （DD） were used for DNA-AFLP and single strand conformation polymorphism （SSCP） analysis to determine the genomic and genic variation in the synthetic hexaploid wheat. Results indicated that more DNA sequences from paternal diploid species were eliminated in the synthetic hexaploid wheat than from maternal tetraploid wheat, suggesting that genome from parental species of lower ploidity tends to be eliminated preferentially. However, sequence variation detected by SSCP procedure was much lower than those detected by DNA-AFLP, which indicated that much less variation in the genic regions occurred in the synthetic hexaploid wheat, and sequence variations detected by DNA-AFLP could be derived mostly from non-coding regions and repetitive sequences. Our results also indicated that sequence variation in 4 genes can be detected in hybrid F1, which suggested that this type of sequence variation could be resulted from distant hybridization. It was interesting to note that 3 out of the 4 genes were mapped and clustered on the long arm of chromosome 2D, which indicated that variation in genic sequences in synthetic hexaploid wheat might not be a randomized process.     

Speciation- induced heritable cytosine methylation changes in polyploid wheat

To study possible epigenetic changes accompanying polyploid speciation, genomic DNA from natural polyploid wheats and their putative diploid progenitors were digested with a pair of isoschizomers Hpa II / Msp I and hybridized to 21 different types of low-copy DNA sequences. It was found that cytosine methylation changes were abundant in natural polyploid wheats after their speciation. The hybridization of the same set of sequences to a synthetic hexaploid wheat along with its parental lines indicated that the extensive DNA methylation changes already existed in the early generations (S5, S6 and Sy) of this plant. Moreover, the high similarity of the changed restriction fragment length polymorphism (RFLP) patterns among three randomly chosen individual plants suggested that the methylation changes occurred even earlier, and/or were of a nonrandom nature. The changed patterns were stably inherited in the three successive selfed generations. Though methylation changes are probably a genome-wide occurrence, they appeared to be confined to the specific types of DNA sequences. The possible implications of the rapid and extensive cytosine methylation changes for several attributes of allopolyploid genome evolution, such as genetic diploidization and gene diversification, are discussed .     

Molecular characterization of Ph1 as a major chromosome pairing locus in polyploid wheat

The foundation of western civilization owes much to the high fertility of bread wheat, which results from the stability of its polyploid genome. Despite possessing multiple sets of related chromosomes, hexaploid (bread) and tetraploid (pasta) wheat both behave as diploids at meiosis. Correct pairing of homologous chromosomes is controlled by the Ph1 locus. In wheat hybrids, Ph1 prevents pairing between related chromosomes. Lack of Ph1 activity in diploid relatives of wheat suggests that Ph1 arose on polyploidization. Absence of phenotypic variation, apart from dosage effects, and the failure of ethylmethane sulphonate treatment to yield mutants, indicates that Ph1 has a complex structure. Here we have localized Ph1 to a 2.5-megabase interstitial region of wheat chromosome 5B containing a structure consisting of a segment of subtelomeric heterochromatin that inserted into a cluster of cdc2-related genes after polyploidization. The correlation of the presence of this structure with Ph1 activity in related species, and the involvement of heterochromatin with Ph1 (ref. 6) and cdc2 genes with meiosis, makes the structure a good candidate for the Ph1 locus.     

Analysis of enzymatically amplified beta-globin and HLA-DQ alpha DNA with allele-specific oligonucleotide probes

Allelic sequence variation has been analysed by synthetic oligonucleotide hybridization probes which can detect single base substitutions in human genomic DNA. An allele-specific oligonucleotide (ASO) will only anneal to sequences that match it perfectly, a single mismatch being sufficient to prevent hybridization under appropriate conditions. To improve the sensitivity, specificity and simplicity of this approach, we used the polymerase chain reaction (PCR) procedure to enzymatically amplify a specific segment of the beta-globin or HLA-DQ alpha gene in human genomic DNA before hybridization with ASOs. This in vitro amplification method, which produces a greater than 10(5)-fold increase in the amount of target sequence, permits the analysis of allelic variation with as little as 1 ng of genomic DNA and the use of a simple 'dot blot' for probe hybridization. As a further simplification, PCR amplification has been performed directly on crude cell lysates, eliminating the need for DNA purification.     

Analysis of DNA methylation variation in wheat genetic background after alien chromatin introduction based on methylation-sensitive amplification polymorphism

During the process of alien germplasm introduced into wheat genome by chromosome engineering, extensive genetic variations of genome structure and gene expression in recipient could be induced. In this study, we performed GISH （genome in situ hybridization） and AFLP （amplified fragment length polymorphism） on wheat-rye chromosome translocation lines and their parents to detect the identity in genomic structure of different translocation lines. The results showed that the genome primary structure variations were not obviously detected in different translocation lines except the same 1RS chromosome translocation. Methylation sensitive amplification polymorphism （MSAP） analyses on genomic DNA showed that the ratios of fully-methylated sites were significantly increased in translocation lines （CN12, 20.15%; CN17, 20.91%; CN18, 22.42%）, but the ratios of hemimethylated sites were significantly lowered （CN12, 21.41%; CN17, 23.43%; CN18, 22.42%）, whereas 16.37% were fully-methylated and 25.44% were hemimethylated in case of their wheat parent. Twenty-nine classes of methylation patterns were identified in a comparative assay of cytosine methylation patterns between wheat-rye translocation lines and their wheat parent, including 13 hypermethylation patterns （33.74%）, 9 demethylation patterns （22.76%） and 7 uncertain patterns （4.07%）. In further sequence analysis, the alterations of methylation pattern affected both repetitive DNA sequences, such as retrotransposons and tandem repetitive sequences, and low-copy DNA.     

Cloning and characterization of a C genome-specific repetitive sequence inAegilops caudata L.

pAeca212 is 204 bp in length, and the G + C content is 51%. It disperses on all seven chromosome pairs ofAegilops caudata except centromeres and secondary constrictions. Compared with the 316893 DNA sequences registered in Genbank/EMBL/DDJB/PDB, pAeca212 is a new C-genome specific repetitive sequence. The results of genomic specificity analysis of pAece212 show that there are no hybridization signals detected in all donor Poaceae plants except in rye. pAeca212 is a very useful molecular marker in the study of the origin of Triticeace and the detection of C chromatin in wheat background.     

Molecular verification of the integration of Tripsacum dactyloides DNA into wheat genome through wide hybridization

RAPD and RFLP analyses of double haploid lines which derived from hybridization between hexaploid wheat (Triticum aestivum L.2n=42) and eastern gamagrass (Tripsacum dactyloides L.2n=4x=72) are reported.Two of the 340 Operon primers have been screened,which stably amplified Tripsacum dactyloides (male parent) specific bands in the double haploid lines.These results confirm the fact that Tripsacum dactyloides DNA has been integrated into wheat genome by sexual hybridization at molecular level.This idea has been further testified by RFLP analysis.Application and potentials of transferring Tripsacum dactyloides DNA into wheat genome by sexual hybridization in wheat breeding are discussed.     

小麦染色体配对基因Ph1结构剖析及调控机制研究进展

近年来,关于小麦抑制部分同源染色体配对基因Ph1的研究有了突破性进展.本文对该基因的结构和调控机理的最新研究进行综述.通过创造和分子标记鉴定Ph1缺失突变体,利用分子生物学及比较基因组学技术,该基因位点被界定于5BL上一个2.5 Mb的区域内,含有一个类cdk基因簇,且在该类cdk基因簇中插入一个亚端粒异染色质片段.细胞学研究显示,Ph1基因通过控制亚端粒的互作启始染色体识别和配对伙伴选择.与此同时,生物信息学揭示,这些类cdk基因与人类和老鼠的cdk2基因高度同源,它们与细胞周期中DNA复制、染色质凝集、碱基错配修复等事件相关.减数分裂时,该基因位点通过"感知"染色体的同源性程度而触发染色质的构象变化,从而控制染色体的配对和重组.此外,小麦中可能存在一种与Ph1相关的类似于酵母中的粗线期检查点机制.预测未来的研究将可能集中在Ph1对染色体同源性的"感知"机制、Ph1的开启与关闭、植物减数分裂重组的忠实性及减数分裂过程的检查点机制等方面.     

Comparative analysis and evolutionary significance of the HMG1 gene in crucian carp,blunt snout bream,and their polyploid progeny

The full-length mRNA of the high mobility group protein 1 coding gene (HMG1) was obtained by RACE-PCR from red crucian carp (Carassius auratus red var.),blunt snout bream (Megalobrama amblycephala),and their triploid and tetraploid progeny.The sequence contained an open reading frame of 579 nucleotides coding for 193 amino acids.The nucleotide identity of HMG1 was higher between the tetraploid hybrid and the maternal red crucian carp (99%) than between the tetraploid hybrid and the paternal blunt snout bream (97%).The nucleotide identity between the triploid hybrids and each parent (95%) was lower than that between the parents (98%).The protein identity between the tetraploid hybrid and each parent (100%) was higher than that between the triploid hybrid and each parent (97%).Our results suggest that interspecific hybridization generates a shock to the HMG1 gene in triploid hybrids,causing divergence of nucleotides.The HMG1 protein of the tetraploid hybrids was consistent with that of its parents,which reduced the barrier of cross incompatibility between alleles,providing the basis for the bisexual fertile tetraploid hybrids forming a new polyploid species in nature.The secondary and tertiary structures of the HMG1 protein contain eight helices,three switches,two DNA-binding domains in the N-terminus,and a long acidic tail in the C-terminus.Together,these data suggest that the HMG1 protein plays a role of protein-DNA interactions,facilitating various DNA-dependent activities in the nucleus.We also investigated the phylogeny of fish,amphibian,reptilian,bird,and mammalian HMG1 proteins.Our results suggest that HMG1 is an ancestral protein that has been highly conserved.These data provide clues as to how interspecific hybridization may form polyploid hybrids.     

Genetic diversity revealed by genomic SSR and EST-SSR markers among common wheat, spelt and compectum

In this study, two SSR molecular markers, named genomic-SSR and EST-SSR, are used to measure the genetic diversity among three hexaploid wheat populations, which include 28 common wheat ( Triticum aestivum L. ), 13 spelt ( Triticum spelta L. ),and 11 compactum ( Triticum compactum Host. ). The results show that common wheat has the highest genetic polymorphism, followed by spelt and then compactum. The mean genetic distance between the populations is higher than that within a population, and similar tendency is detected for individual genomes A, B and D. Therefore, spelt and compactum can be used as potential germplasms for wheat breeding, especially for enriching the genetic variation in genome D. As compared with spelt, the genetic diversity between common wheat and compactum is much smaller, indicating a closer consanguine relationship between these two species. Although the polymorphism revealed by EST-SSR is lower than that by genomic-SSR, it can effectively differentiate diverse genotypes as well. Together with our present results, it is concluded that EST-SSR marker is an ideal marker for assessing the genetic diversity in wheat. Meanwhile, the origin and evolution of hexaploid wheat is also analyzed and discussed.     

Genetic diversity revealed by genomic-SSR and EST-SSR markers among common wheat, spelt and compactum

Spelt (TriticumspeltaL .) ,oncebeingthemaincropinEurope ,hasthesamegenome (AABB DD)ascommonwheat (TriticumaestivumL .) .Althoughitiscultivatedonasmallscale ,theproduc tionofspeltisclosetoorevenexceedsthatofcom monwheatinsomeregions[1] .Speltiswidelyadapt edtoavarietyofenvironmentconditions ,itsgrainscontainlargequantitiesofproteinandaresuitableforthemanufactureofsoftbread .Therefore ,speltisre ceivingincreasingattentionforplantbreeders[2 ] .ThemostrecentstudyelucidatedthattheF1generationb…     

白菜型油菜核育性相关基因片段的克隆与序列分析

通过ＲＡＰＤ标记,所获得的与育性基因紧密连锁的基因片段进行克隆与序列分析,结果表明,该育性基因片段为与油菜小孢子发育早期ＢＰ４调控基因５８％同源,并含有一ＭＡＰＤＳ盒高度同源的保邓列。Ｓｏｕｔｈｅｒｎ杂交结果表明该基因为单拷贝基因。     

利用人工合成六倍体小麦突破小麦产量瓶颈的机会与潜力

 以人工合成小麦Syn CD780及其衍生品种川麦42与优良栽培品种杂交构建的2个重组近交系群体为材料,进行多环境（年份×地点）田间试验和产量性状QTL分析,探讨利用人工合成小麦资源突破小麦产量瓶颈的机会与潜力。结果表明：① 所考察性状均呈连续性变异和双向超亲分离。S12群体（Syn CD780×川育12）高产株系平均单产670 t/hm2,比川育12提高64%,增产缘于千粒质量的显著提高（100%）;S16群体（川麦42×川农16）高产株系平均单产79 t/hm2,比川农16增产181%,增产缘于粒数/m2和千粒质量的共同提高。② 基于S16群体实验数据,共检测到LOD＞30的产量性状QTLs 55个。其中,产量QTLs 7个,贡献率75%~273%,均来自CM42;产量构成因素QTLs 48个,贡献率78%~328%。利用人工合成小麦或其衍生品种突破四川盆地小麦产量瓶颈的潜力较大。      

小麦太谷核不育F1轮选群体扬花期与株高的遗传控制

用同一太谷核不育（Tal）小麦的不育株TaIS65（共同遗传背景）为母本,同时与纯合矮秆、杂合矮秆、纯合高秆、杂合高秆的父本杂交,配制成4种不同的组合类型。对4个杂交组合的F₁群体的扬花期和株高遗传分析表明:在Tal轮选群体中,开放授粉随机互交的轮选结果,只能使杂种后代朝着高秆、晚熟的变异方向发展,这是由于Tal材料自身的生物学特点造成的。要提高Tal轮选改良的育种效率,必须选择控制父本的花粉来源,并采取人工控制杂交措施,才能有效地克服由Tal材料自身的生物学特点带来的不利影响,实现各种有利基因型间充分的杂交重组。     

杨树泛基因组构建与基因组变异分析

【目的】杨树是重要的速生用材、生态防护和碳汇造林树种,也是林木遗传研究的模式树种。开展杨树泛基因组构建与基因组变异分析,可为杨树精准育种和林木泛基因组研究提供理论先导。【方法】 以公开发表的高质量杨树基因组序列为基础,分析不同类型的序列变异,总结变异特征,并构建基于基因和图形结构的杨树泛基因组。【结果】 本研究收集到8个杨属树种和3个二倍体或三倍体杨树杂交品种的基因组序列,3个杂交品种包含7个单倍型亚基因组序列,较好地代表了杨树4个组派的基因组特征。分析结果表明,杨树基因组间存在较多大的结构变异。在基于基因的泛基因组中,共线核心基因、非共线核心基因、次核心基因、非必需基因、特异基因占比分别为12.5%、34.9%、31.4%、16.5%、4.7%。其中,非必需基因在功能上具有较高的多样性。以基因组序列变异为基础构建杨树图形结构泛基因组,大幅提升2代测序数据的变异检测效果。通过泛基因组变异热点分析,鉴定出2个与物候关联的基因位点。【结论】 杨属基因组中存在大量染色体重排,进而增加了基因调控的多样性。杨树组/派间的基因组结构变异可能与物候适应存在关联。基于林木基因组序列的复杂性,在林木泛基因组研究中应注意基因组整合范围与研究目标相匹配,结合基因泛基因组和图形结构泛基因组结果,综合解析林木的遗传变异规律和物种演化特征。     

Molecular verification of the integration ofTripsacum dactyloides DNA into wheat genome through wide hybridization

RAPD and RFLP analyses of double haploid lines which derived from hybridization between hexaploid wheat (Triticum aestivum L. 2n=42) and eastern gamagrass (Tripsacum dactyloides L. 2n=4x=72) are reported. Two of the 340 Operon primers have been screened, which stably amplified 处留情 (male parent) specific bands in the double haploid lines. These results confirm the fact that Tripsacum dactyloides DNA has been integrated into wheat genome by sexual hybridization at molecular level. This idea has been further testified by RFLP analysis. Application and potentials of transferring Tripsacum dactyloides DNA into wheat genome by sexual hybridization in wheat breeding are discussed.     

应用PCR—SSCP技术从显微切割的人染色体17q11—12探针池批量分离单拷贝片段的研究

将ＤＮＡ单链构象多态（ＳＳＣＰ）检测的原理，应用于“显微切割的人染色体１７ｑ１１—１２探针池”批量分离单拷贝片段，取得了很好效果。从筛选出的７４个次级单拷贝中有效地鉴定出３７个非同源的单拷贝片段。这比传统的仅限于长度比较而确认的非同源单拷贝片段（１２个）多出２５个．其中部分已经ＤＮＡ测序证实。结果提示：采用ＳＳＣＰ技术区分相似分子量单拷贝片段的同源性，可显著地提高从“显微切割探针池”分离和克隆非同源单拷贝片段的效率。本文还将一部分单拷贝片段作为探讨，与人基因组ＤＮＡ的限制性片段作Ｓｏｕｔｈｅｒｎ印迹杂交，结果均只显示单一杂交带，说明单拷贝ＤＮＡ片段的结论是可靠的。     

籼粳杂种多倍体结实情况的初步研究

用10个籼粳交组合的二倍体和四倍体为试材，研究了加倍对每穗总粒数、每穗实数数和结实经的影响。结果表明：加倍对结实率等性能的影响因组合不同而不同，四倍体籼粳杂种的结实率显高于二倍体，加倍正反交的结实情况也有影响，要提高籼粳多倍体的结实经，应注重同时增加总数数的实数数。     

The complete genome of an individual by massively parallel DNA sequencing

The association of genetic variation with disease and drug response, and improvements in nucleic acid technologies, have given great optimism for the impact of 'genomic medicine'. However, the formidable size of the diploid human genome, approximately 6 gigabases, has prevented the routine application of sequencing methods to deciphering complete individual human genomes. To realize the full potential of genomics for human health, this limitation must be overcome. Here we report the DNA sequence of a diploid genome of a single individual, James D. Watson, sequenced to 7.4-fold redundancy in two months using massively parallel sequencing in picolitre-size reaction vessels. This sequence was completed in two months at approximately one-hundredth of the cost of traditional capillary electrophoresis methods. Comparison of the sequence to the reference genome led to the identification of 3.3 million single nucleotide polymorphisms, of which 10,654 cause amino-acid substitution within the coding sequence. In addition, we accurately identified small-scale (2-40,000 base pair (bp)) insertion and deletion polymorphism as well as copy number variation resulting in the large-scale gain and loss of chromosomal segments ranging from 26,000 to 1.5 million base pairs. Overall, these results agree well with recent results of sequencing of a single individual by traditional methods. However, in addition to being faster and significantly less expensive, this sequencing technology avoids the arbitrary loss of genomic sequences inherent in random shotgun sequencing by bacterial cloning because it amplifies DNA in a cell-free system. As a result, we further demonstrate the acquisition of novel human sequence, including novel genes not previously identified by traditional genomic sequencing. This is the first genome sequenced by next-generation technologies. Therefore it is a pilot for the future challenges of 'personalized genome sequencing'.