DNA sequence organisation in avian genomes

By means of renaturation kinetics of DNA of the three avian species Cairina domestica, Gallus domesticus and Columba livia domestica the following major DNA repetition classes were observed: a very fast reannealing fraction comprising about 15% of the DNA, a fast or intermediate reannealing fraction that makes up 10%, and a slow reannealing fraction of about 70%, which apparently renatures with single copy properties. — Comparing the reassociation behaviour of short (0.3 kb) and long (>2 kb) DNA fragments of duck and chicken it becomes apparent that only 12% (duck) and 28% (chicken) of the single copy DNA are interspersed with repetitive elements on 2 to 3 kb long fragments. The lengths of the repetitive sequences were estimated by optical hyperchromicity measurements, by agarose A-50 chromatography of S₁ nuclease resistant duplexes and by electron microscopic measurements of the S₁ nuclease resistant duplexes. It was found that in the case of the chicken DNA the single copy sequences alternating with middle repetitive ones are at least 2.3 kb long; the interspersed moderate repeats have a length average of at least 1.5 kb. The sequence length of the moderate repeats in duck DNA is smaller. The results show that the duck and the chicken genomes do not follow the short period interspersion pattern of genome organisation, characteristic of the eucaryotic organisms studied so far.     

Characterization of Allomyces genome

Allomyces arbuscula DNA isolated from whole cells (bulk DNA) is composed of a major (alpha) and two minor components (beta & gamma) with buoyant densities in neutral CsCl corresponding to 1.721, 1.710 and 1.702 g/cm3, respectively. The DNA obtained from purified nuclei contains alpha component only. The beta component corresponds to mitochondrial DNA. The gamma component is also extra-nuclear but has not been characterized. The reassociation kinetics of sheared, bulk and nuclear DNA show that (i) 25 % bulk and 10% of nuclear DNA reanneal very rapidly and contain highly repeated sequences; (ii) moderately repeated sequences, accounting for 15% of both bulk and nuclear DNA, have a sequence complexity of approximately 7.2-10(6) daltons and are repeated about 320 times; (iii) the slow reannealing fraction accounts for about 60% of the genome and has kinetic properties similar to single copy sequences. The sequence complexity of this fraction was determined in relation to that of Escherichia coli. After a correction for the size of the repeated sequences the genome size of A. arbuscula was calculated to be 1.7-10(10) daltons.     

Isolation of a D-genome specific repeated DNA sequence fromAegilops squarrosa

Three repeated sequence clones, pAS1(1.0 Kb), pAS2(1.8 Kb) and pAS12(2.5 Kb), were isolated fromAegilops squarrosa (Triticum tauschii). The inserts of the three clones did not hybridize to each other. Two of the clones, pAS2 and pAS12, contain repeated sequences which were distributed throughout the genome. The clone pAS1 sequence was more restricted and was located in specific areas on telomeres and certain interstitial sites along the chromosome length. This cloned sequence was also found to be restricted to the D genome at the level ofin situ hybridization. The pAS1 sequence will be useful in chromosomal identification and phylogenetic analysis. All three clones will allow assessment of genome plasticity inAegilops squarrosa. Nuclear DNA content varies over a range of 10,000 fold among all organisms (Nagl et al., 1983). Among angiosperms, at least a 65-fold range in genome size occurs in diploid species (Sparrow, Price and Underbrink, 1972; Bennett, Smith and Heslop-Harrison, 1982). This DNA variation has been reported within families, genera, and species (Rothfels et al., 1966; Rees and Jones, 1967; Miksche, 1968; Price, Chambers and Bachmann, 1981). Much of the interspecific variation in genome size among angiosperms appears to be due to amplification and/or deletion of DNA within chromosomes. The variation in genome size does not appear to result in changes in the number of coding genes (Nagl et al., 1983). While the number of coding genes, with the exception of rDNA in specific examples, appears to remain constant, the remaining non-coding regions are quite flexible. This non-coding DNA encompasses over 99% of the plant genome and consists of sequences that exist as multiple copies throughout the genome and are identified as repeated DNA sequences (Flavell et al., 1974). Flavell et al. (1974) have reported that increasing genome size in higher plants is associated with increasing repetitive DNA amounts. Subsequent reports have substantiated this correlation (Bachmann and Price, 1977; Narayan, 1982). In various cereals, heterochromatin, which has been demonstrated to be correlated with the location of specific repeated DNA sequences, has been positively correlated with genome size (Bennett, Gustafson and Smith, 1977; Rayburn et al., 1985). Furuta, Nishikawa and Makino (1975) found significant DNA content variation among different accessions ofAegilops squarrosa L. This species contains the D genome, a pivotal genome in several polyploid species and also found in hexaploid wheat (AABBDD). The importance of this genome to the study of bread wheat genomes makes the mechanism(s) of this genomic plasticity of particular interest. In order to determine which sequences are varying, one must first have a way to identify specific types of chromatin and/or DNA. Specific types of chromosome banding such as C- and N-banding have been used to identity types of chromatin in previous studies. C-banding of the D genome results in very lightly staining bands whose pattern is somewhat indistinct. N-banding alternatively has been shown to be useful in identifying certain chromosomes of hexaploid wheat but is limited by the lack of major bands in the D genome (Endo and Gill, 1984). Specific DNA sequences have been isolated fromTriticum aestivum cultivar “Chinese Spring” (hexaploid wheat). However, these sequences are representatives of the A and/or B genomes of hexaploid wheat and are not found in significant quantities in the D genome (Hutchinson and Lonsdale, 1982). Various other repeated DNA sequences have been successfully isolated from rye (Bedbrook et al., 1980) and identified on rye chromosomes (Appels et al., 1981; Jones and Flavell, 1982). Certain of these sequences are found in wheat genomes, but the sequences are representative of only a minor fraction of the D genome (Bedbrook et al., 1980; Rayburn and Gill, 1985). The purpose of this report is to describe three distinct repeated DNA sequences isolated fromA. squarrosa (D genome). Two clones appear to be distributed throughout the total genome, and the third clone is restricted to specific sites along the chromosomes. This latter clone will prove useful in cytologically defining the D genome chromosomes. These sequences appear representative of two types of repeated DNA genome organization: 1) sequences distributed throughout the genome and 2) specific arrays of repeated sequences. The availability of such repeated DNA sequence clones along with the known intraspecific DNA content variation inA. squarrosa will allow the study of genomic plasticity of this species.     

An integrated molecular linkage map of diploid wheat based on a <Emphasis Type="Italic">Triticum boeoticum</Emphasis> × <Emphasis Type="Italic">T. monococcum</Emphasis> RIL population

Diploid A genome species of wheat harbour immense variability for biotic stresses and productivity traits, and these could be transferred efficiently to hexaploid wheat through marker assisted selection, provided the target genes are tagged at diploid level first. Here we report an integrated molecular linkage map of A genome diploid wheat based on 93 recombinant inbred lines (RILs) derived from Triticum boeoticum × Triticum monococcum inter sub-specific cross. The parental lines were analysed with 306 simple sequence repeat (SSR) and 194 RFLP markers, including 66 bin mapped ESTs. Out of 306 SSRs tested for polymorphism, 74 (24.2%) did not show amplification (null) in both the parents. Overall, 171 (73.7%) of the 232 remaining SSR and 98 (50.5%) of the 194 RFLP markers were polymorphic. Both A and D genome specific SSR markers showed similar transferability to A genome of diploid wheat species. The 176 polymorphic markers, that were assayed on a set of 93 RILs, yielded 188 polymorphic loci and 177 of these as well as two additional morphological traits mapped on seven linkage groups with a total map length of 1,262 cM, which is longer than most of the available A genome linkage maps in diploid and hexaploid wheat. About 58 loci showed distorted segregation with majority of these mapping on chromosome 2A^m. With a few exceptions, the position and order of the markers was similar to the ones in other maps of the wheat A genome. Chromosome 1A^m of T. monococcum and T. boeoticum showed a small paracentric inversion relative to the A genome of hexaploid wheat. The described linkage map could be useful for gene tagging, marker assisted gene introgression from diploid into hexaploid wheat as well as for map based cloning of genes from diploid A genome species and orthologous genes from hexaploid wheat.     

Rapid Elimination of Low-Copy DNA Sequences in Polyploid Wheat: A Possible Mechanism for Differentiation of Homoeologous Chromosomes

To study genome evolution in allopolyploid plants, we analyzed polyploid wheats and their diploid progenitors for the occurrence of 16 low-copy chromosome- or genome-specific sequences isolated from hexaploid wheat. Based on their occurrence in the diploid species, we classified the sequences into two groups: group I, found in only one of the three diploid progenitors of hexaploid wheat, and group II, found in all three diploid progenitors. The absence of group II sequences from one genome of tetraploid wheat and from two genomes of hexaploid wheat indicates their specific elimination from these genomes at the polyploid level. Analysis of a newly synthesized amphiploid, having a genomic constitution analogous to that of hexaploid wheat, revealed a pattern of sequence elimination similar to the one found in hexaploid wheat. Apparently, speciation through allopolyploidy is accompanied by a rapid, nonrandom elimination of specific, low-copy, probably noncoding DNA sequences at the early stages of allopolyploidization, resulting in further divergence of homoeologous chromosomes (partially homologous chromosomes of different genomes carrying the same order of gene loci). We suggest that such genomic changes may provide the physical basis for the diploid-like meiotic behavior of polyploid wheat.     

Rapid genome evolution revealed by comparative sequence analysis of orthologous regions from four triticeae genomes

Bread wheat (Triticum aestivum) is an allohexaploid species, consisting of three subgenomes (A, B, and D). To study the molecular evolution of these closely related genomes, we compared the sequence of a 307-kb physical contig covering the high molecular weight (HMW)-glutenin locus from the A genome of durum wheat (Triticum turgidum, AABB) with the orthologous regions from the B genome of the same wheat and the D genome of the diploid wheat Aegilops tauschii (Anderson et al., 2003; Kong et al., 2004). Although gene colinearity appears to be retained, four out of six genes including the two paralogous HMW-glutenin genes are disrupted in the orthologous region of the A genome. Mechanisms involved in gene disruption in the A genome include retroelement insertions, sequence deletions, and mutations causing in-frame stop codons in the coding sequences. Comparative sequence analysis also revealed that sequences in the colinear intergenic regions of these different genomes were generally not conserved. The rapid genome evolution in these regions is attributable mainly to the large number of retrotransposon insertions that occurred after the divergence of the three wheat genomes. Our comparative studies indicate that the B genome diverged prior to the separation of the A and D genomes. Furthermore, sequence comparison of two distinct types of allelic variations at the HMW-glutenin loci in the A genomes of different hexaploid wheat cultivars with the A genome locus of durum wheat indicates that hexaploid wheat may have more than one tetraploid ancestor.     

Origin, dispersal and genomic structure of a low-copy-number hypervariable RFLP clone in Triticum and Aegilops species

The genome of common wheat has evolved through allopolyploidization of three ancestral diploid genomes. A previously identified restriction fragment length polymorphism (RFLP) marker, pTag546, has the unique feature of showing hypervariability among closely related common wheat cultivars. To understand the origin and the mode of dispersal of this hypervariable sequence in the wheat genome, the distribution and structure of the homologous sequences were studied using ancestral diploid species, tetraploid disomic substitution lines and synthetic hexaploid lines. Comparative Southern blot and PCR analyses suggested that pTag546 homologs in the tetraploid and hexaploid wheat were derived from the S genome of Aegilops speltoides. Some pTag546 homologs were found to have transposed to A and D genomes in polyploid wheat. Evidence of transposition and elimination in some synthetic hexaploid lines was also obtained by comparing their copy numbers with those in the parental lines. Southern blot analysis of a genomic clone using a contiguous subset of sequences as probes revealed a core region of hypervariability that coincided with the region containing pTag546. No obvious structural characteristics that could explain the hypervariability, however, were found around the pTag546 sequence, except for accumulation of small repetitive sequences at one border. It was concluded that pTag546 increased its copy number through yet unknown mechanism(s) of transposition to various chromosomal locations over the period of allopolyploid evolution and during the artificial genome manipulation in wheat.     

小麦A/B染色体组SSR标记在新小麦合成前后的比较研究

微卫星分子标记已广泛用于普通小麦遗传和进化研究。由于人工合成小麦与小麦品种之间存在高的遗传多样性,人工合成小麦已被大量应用于小麦分子标记工作中。但是,目前还缺乏人工合成小麦的异源六倍化过程对微卫星影响的研究。本研究直接比较了四倍体小麦与节节麦远缘杂交并经染色体加倍获得人工合成小麦前后,位于普通小麦A/B染色体组不同染色体臂上的66个特异引物揭示的微卫星位点的保守性和可转移性。结果表明,除了一个引物在新合成小麦中扩增出供体亲本没有的新带,一个引物在节节麦扩增出的产物在新合成小麦中消失,其他的所有微卫星引物的扩增产物在小麦合成前后是保守的,没有变异发生。所有的引物能够在四倍体小麦中扩增出微卫星产物,四倍体小麦中的扩增产物也出现在新的人工合成小麦中;有70%的引物能够在节节麦扩增出产物,其中的绝大多数产物也出现在新的人工合成小麦中。因此,普通小麦A/B染色体组的这些微卫星引物除了在人工合成小麦的A/B染色体组中扩增出产物,还能在其D染色体组中扩增出产物,也就是说,这些引物对人工合成小麦而言,并非是A/B染色体组特异的。根据该研究结果,讨论了小麦微卫星的可转移性和特异性问题,重点讨论了在应用人工合成小麦构建的遗传群体进行微卫星分子标记中的应用价值及其应该注意的问题。     

从CIMMYT引进的人工合成六倍体小麦D染色体组微卫星分子标记的遗传差异

采用微卫星（SSR）分子标记技术,选用23个D染色体组特异性引的对来自CIMMYT的26份人工合成六倍体小麦D染色体组的遗传多样性进行了分析。研究发现,26份材料在D染色体组上存在丰富的等位基因变异（92个）,平均每个基因座为4个。遗传距离计算结果也显示,26份材料D染色体组之间具有较大的遗传差异,平均遗传距离高达0．4955。因此,人工合成六倍体小麦D染色体组中存在丰富的遗传多样性,可以作为拓宽普通小麦遗传基础的新的遗传变异来源。研究还发现,由同一个粗山羊草基因型与不同硬粒小麦杂交合成的人工合成六倍体小麦（如合成种17和18）在所用检测的23个基因座中有3个存在差异,说明小麦在多倍化后,供体基因组在重复序列区域会发生遗传分化。     

Exploring the diploid wheat ancestral A genome through sequence comparison at the high-molecular-weight glutenin locus region

The polyploid nature of hexaploid wheat (T. aestivum, AABBDD) often represents a great challenge in various aspects of research including genetic mapping, map-based cloning of important genes, and sequencing and accurately assembly of its genome. To explore the utility of ancestral diploid species of polyploid wheat, sequence variation of T. urartu (A^uA^u) was analyzed by comparing its 277-kb large genomic region carrying the important Glu-1 locus with the homologous regions from the A genomes of the diploid T. monococcum (A^mA^m), tetraploid T. turgidum (AABB), and hexaploid T. aestivum (AABBDD). Our results revealed that in addition to a high degree of the gene collinearity, nested retroelement structures were also considerably conserved among the A^u genome and the A genomes in polyploid wheats, suggesting that the majority of the repetitive sequences in the A genomes of polyploid wheats originated from the diploid A^u genome. The difference in the compared region between A^u and A is mainly caused by four differential TE insertion and two deletion events between these genomes. The estimated divergence time of A genomes calculated on nucleotide substitution rate in both shared TEs and collinear genes further supports the closer evolutionary relationship of A to A^u than to A^m. The structure conservation in the repetitive regions promoted us to develop repeat junction markers based on the A^u sequence for mapping the A genome in hexaploid wheat. Eighty percent of these repeat junction markers were successfully mapped to the corresponding region in hexaploid wheat, suggesting that T. urartu could serve as a useful resource for developing molecular markers for genetic and breeding studies in hexaploid wheat.     

Transferability of wheat microsatellites to diploid Triticeae species carrying the A, B and D genomes

Hexaploid wheat (Triticum aestivum L em Thell) is derived from a complex hybridization procedure involving three diploid species carrying the A, B and D genomes. In this study, we evaluated the ability of microsatellite sequences from T. aestivum to be revealed on different ancestral diploid species more or less closely related, i.e. to test for their transferability. Fifty five primer pairs, evenly distributed all over the genome, were investigated. Forty three of them mapped to single loci on the hexaploid wheat genetic map although only 20 (46%) gave single PCR products; the 23 others (54%) gave more than one band with either only one being polymorphic, the others remaining monomorphic, or with several co-segregating polymorphic bands. The other 12 detected two (9) or three (3) different loci. From the 20 primer pairs which gave one amplification pro- duct on hexaploid wheat, nine (45%) also amplified products on only one of the diploid species, and seven (35%) on more than one. Four microsatellites (20%) which mapped to chromosomes from the B genome of wheat, did not give any amplification signal on any of the diploid species. This suggests that some regions of the B genome have evolved more rapidly compared to the A or D genomes since the emergence of polyploidy, or else that the donor(s) of this B genome has(have) not yet been identified. Our results confirm that Triticum monococcum ssp. urartu and Triticum tauschii were the main donors of the A and D genomes respectively, and that Aegilops speltoides is related to the ancestor(s) of the wheat polyploid B genome. Received: 21 June 2000 / Accepted: 15 November 2000     

DNA condensation with polyamines. II. Electron microscopic studies

Approximately 75% of the wheat and rye genomes consist of repeated sequence DNA. Three-quarters of the non-repeated or few copy sequences in wheat are less than 1000 base-pairs long, whilst in rye approximately half of the non-repeated or few copy sequences are in this size class. Most of the remaining non-repeated or few copy sequences appear to be a few thousand base-pairs long.In this paper a somewhat novel approach has been used to quantitatively analyse the linear organisation of the large proportion of repeated sequence DNA as well as the non-repeated DNA in the wheat and rye genomes. Repeated sequences in the genomes of oats, barley, wheat and rye have been used as probes to distinguish and isolate four different groups of repeated sequences and their neighbouring sequences from the wheat and rye genomes. Radioactively labelled wheat or rye DNA fragments ranging from 200 to over 9000 nucleotides long were incubated separately with large excesses of denatured unlabelled oats, barley, wheat and rye DNAs to C_ot values which enable all the repeated sequences of the unlabelled DNA to renature. The following parameters were then determined from the proportions of total labelled DNA in fragments which had at least partially renatured. (1) The proportions of the repeated sequences in the labelled DNAs that were able to hybridise to each unlabelled DNA; (2) the mean distance apart of the hybridising sequences on the longer labelled fragments; and (3) the proportion of the genome in which the hybridising sequences were concentrated. Analysis of these results, together with those of separate experiments designed to quantitatively estimate the nature of sequences unable to reanneal with the repeated sequences of each of the probe DNAs, have enabled schematic maps to be drawn which show how the repeated and non-repeated sequences are arranged in the wheat and rye genomes.Both genomes are constructed from millions of relatively short sequences, most of them considerably shorter than 3000 base-pairs. This structure was recognised because adjacent sequences can be distinguished by their frequency of repetition (i.e. repeated or non-repeated) or by their evolutionary origin. Approximately 40 to 45% of the wheat genome and 30 to 35% of the rye genome consists of short non-repeated sequences interspersed between short repeated sequences. Approximately 50% of the wheat genome and 60% of the rye genome consists of tandemly arranged repeated sequences of different evolutionary origins. It is postulated that much of this complex repeated sequence DNA could have arisen from amplification of compound sequences, each containing repeated and non-repeated sequence DNA.Short repeated sequences with a number average length of around 200 base-pairs and which occupy about 20% of the wheat and rye genomes are related to repeated sequences also found in oats and barley. They are concentrated in 60 to 70% of the wheat and rye genomes, being interspersed with different short repeated sequences and a significant proportion of the short non-repeated sequences.Rye chromosomes contain more DNA than wheat chromosomes. This is principally, but not entirely, due to additional repeated sequence DNA. Many quantitative changes appear to have occurred in both genomes, possibly affecting most families of repeated sequences, since wheat and rye diverged from a common ancestor. Both species contain species-specific repeated sequences (24% of rye genome; 16% of wheat genome) but a large proportion of these are closely interspersed with repeated sequences found in both genomes.     

Cell organisation,sulphur metabolism and ion transport-related genes are differentially expressed in Paracoccidioides brasiliensis mycelium and yeast cells

Background

Bread wheat (Triticum aestivum) is an important staple food. However, wheat gluten proteins cause celiac disease (CD) in 0.5 to 1% of the general population. Among these proteins, the α-gliadins contain several peptides that are associated to the disease.

Results

We obtained 230 distinct α-gliadin gene sequences from severaldiploid wheat species representing the ancestral A, B, and D genomes of the hexaploid bread wheat. The large majority of these sequences (87%) contained an internal stop codon. All α-gliadin sequences could be distinguished according to the genome of origin on the basis of sequence similarity, of the average length of the polyglutamine repeats, and of the differences in the presence of four peptides that have been identified as T cell stimulatory epitopes in CD patients through binding to HLA-DQ2/8. By sequence similarity, α-gliadins from the public database of hexaploid T. aestivum could be assigned directly to chromosome 6A, 6B, or 6D. T. monococcum (A genome) sequences, as well as those from chromosome 6A of bread wheat, almost invariably contained epitope glia-α9 and glia-α20, but never the intact epitopes glia-α and glia-α2. A number of sequences from T. speltoides, as well as a number of sequences fromchromosome 6B of bread wheat, did not contain any of the four T cell epitopes screened for. The sequences from T. tauschii (D genome), as well as those from chromosome 6D of bread wheat, were found to contain all of these T cell epitopes in variable combinations per gene. The differences in epitope composition resulted mainly from point mutations. These substitutions appeared to be genome specific.

Conclusion

Our analysis shows that α-gliadin sequences from the three genomes of bread wheat form distinct groups. The four known T cell stimulatory epitopes are distributed non-randomly across the sequences, indicating that the three genomes contribute differently to epitope content. A systematic analysis of all known epitopes in gliadins and glutenins will lead to better understanding of the differences in toxiCity among wheat varieties. On the basis of such insight, breeding strategies can be designed to generate less toxic varieties of wheat which may be tolerated by at least part of the CD patient population.     

Map-based isolation of disease resistance genes from bread wheat: cloning in a supersize genome

The genome of bread wheat is hexaploid and contains 1.6 x 10 10 bp of DNA, of which more than 80% is repetitive sequences. Its size and complexity represent a challenge for the isolation of agronomically important genes, for which we frequently know only their position on the genetic map. Recently, new genomic resources and databases from genome projects have simplified the molecular analysis of the wheat genome. The first genes to be isolated from wheat by map-based cloning include three resistance genes against the fungal diseases powdery mildew and leaf rust. In this review, we will describe the approaches and resources that have contributed to this progress, and discuss genomic strategies that will simplify positional cloning in wheat in the near future.     

不同倍性小麦对盐胁迫的适应性差异

植物染料在工业化应用过程中存在着资源限制,目标色相不丰富、色牢度不理想、植物染料本身的鉴别和成品的鉴别等问题。为了丰富染料植物资源的来源和提高染料植物资源的利用效率,该研究对西双版纳傣族利用的染料植物及其染色工艺涉及的相关植物进行了系统调查。2014年10月到2016年1月,采用半结构式访谈法对西双版纳14个村寨的56个关键信息人进行访谈,收集信息包括使用着色植物、媒染植物和助染植物的种类、傣名、利用部位和资源来历,以及预处理和染色过程工艺条件与技术步骤;采用参与式观察法对4种色相的10个染色工艺过程进行了记录,采集了凭证标本和图像资料;对调查信息进行了整理编目。结果表明：西双版纳地区的傣族使用11种着色植物和17种助染植物;目标色相有红、黄、蓝和绿。分析了傣族染料植物资源的发掘潜力、傣族利用植物染色对于染料植物利用的应用启发。该研究详细深入地记录了西双版纳傣族使用的染料植物的种类及其相关的组合和染色的过程。该研究结果对民族民间染料植物与染色工艺的产业化应用具有重要借鉴意义,为染料植物资源筛选及其染色工艺条件优化提供了参考。     

Genome analysis at different ploidy levels allows cloning of the powdery mildew resistance gene Pm3b from hexaploid wheat

In wheat, race-specific resistance to the fungal pathogen powdery mildew (Blumeria graminis f. sp. tritici) is controlled by the Pm genes. There are 10 alleles conferring resistance at the Pm3 locus (Pm3a to Pm3j) on chromosome 1AS of hexaploid bread wheat (Triticum aestivum L.). The genome of hexaploid wheat has a size of 1.6 x 1010 bp and contains more than 80% of repetitive sequences, making positional cloning difficult. Here, we demonstrate that the combined analysis of genomes from wheat species with different ploidy levels can be exploited for positional cloning in bread wheat. We have mapped the Pm3b gene in hexaploid wheat to a genetic interval of 0.97 centimorgan (cM). The diploid T. monococcum and the tetraploid T. turgidum ssp. durum provided models for the A genome of hexaploid wheat and allowed to establish a physical contig spanning the Pm3 locus. Although the haplotypes at the Pm3 locus differed markedly between the three species, a large resistance gene-like family specific to wheat group 1 chromosomes was consistently found at the Pm3 locus. A candidate gene for Pm3b was identified using partial sequence conservation between resistant line Chul and T. monococcum cv. DV92. A susceptible Pm3b mutant, carrying a single-base pair deletion in the coding region of the candidate gene was isolated. When tested in a single cell transformation assay, the Pm3b candidate gene conferred race-specific resistance to powdery mildew. These results demonstrate that the candidate gene, a member of the coiled-coil nucleotide binding site leucine-rich repeat (NBS-LRR) type of disease resistance genes, is the Pm3b gene.     

SNP and haplotype identification of the wheat monomeric α-amylase inhibitor genes

Seventy-three gene sequences encoding monomeric α-amylase inhibitors were characterized from cultivated wheat “Chinese Spring”, group 6 nullisomic-tetrasomic lines of “Chinese Spring” and diploid putative progenitors of common wheat. The monomeric α-amylase inhibitors from the different sources shared very high homology (99.54%). The different α-amylase inhibitors, which were determined by the 24 single nucleotide polymorphisms (SNPs) of their gene sequences, were investigated. A total of 15 haplotypes were defined by sequence alignment, among which 9 haplotypes were found with only one single sequence sample. Haplotype H02 was found to be the main haplotype occurring in 83 WMAI sequence samples, followed by haplotype H11. The median-joining network for the 15 haplotypes of monomeric α-amylase inhibitor gene sequences from hexaploid wheats was star like, and at least two subclusters emerged. Furthermore evidence of homologous recombination was found between the haplotypes. The relationship between nucleotide substitutions and the amino acid changes in WMAI of hexaploid wheats was summarized. It was clear that only five polymorphic sites in the nucleotide sequence of WMAI resulted in amino acid variations, and that should be the reason for different structure and function of inhibitors. However, little evidence could be found that there were WMAI genes in the A genome of hexaploid wheat, whereas it could conclude from our results that the A genome diploid wheat had WMAI genes. The overall information on the monomeric α-amylase inhibitors from wheat and Aegilops strongly support the view that these inhibitors have evolved from a common ancestral gene through duplication and mutation. Ji-Rui Wang and Yu-Ming Wei are contributed equally to this paper.     

Reassociation and dissociation of cytoplasmic membrane-associated DNA

The relationship between nuclear DNA and cytoplasmic membrane-associated DNA, extracted from a human lymphocyte cell line, was examined by DNA-DNA reannealing and by dissociation of renatured molecules. Up to 2% of the total cellular DNA is found in the cytoplasm as cytoplasmic membrane-associated DNA and of this 2%, approximately 70% is comprised of repeated sequences. These sequences are homologous to only about 4% of the repeated sequences of nuclear DNA. The repeat fraction of cytoplasmic membrane-associated DNA consists of sequences which are only moderately repeated. The number of copies in the average “family” could range from about 1500 copies to as few as 25 copies. A small rapidly reannealing portion of cytoplasmic membrane-associated DNA (C₀t < 4 × 10^?3) appears to consist of sequences derived from a single “family”.About 30% of cytoplasmic membrane-associated DNA reassociates slowly with a $\text{C}{}_0\text{t}\text{1}\text{2}$ value of 223 (unique cytoplasmic membrane-associated DNA). This fraction has homology with about 11% of the unique sequences of nuclear DNA. However, unique cytoplasmic membrane-associated DNA comprises only about 0·6% of the total cellular DNA. If it is assumed that each cell has the same amount of cytoplasmic membrane-associated DNA, homology with 11% of the unique sequences of nuclear DNA suggests that different cells may have different unique nucleotide sequences in the cytoplasm.     

Homoeologous relationships of Triticum sharonense chromosomes to T. aestivum

 Homoeologous pairing at metaphase I was analyzed in standard-type, ph2b, and ph1b hybrids of Triticum aestivum (common, bread or hexaploid wheat) and T. sharonense in order to establish the homoeologus relationships of T. sharonense chromosomes to hexaploid wheat. Chromosomes of both species, and their arms, were identified by C-banding. Normal homoeologous relationships for the seven chromosomes of the S^sh genome, and their arms, were revealed, which implies that no apparent chromosome rearrangement occurred in the evolution of T. sharonense relative to wheat. All three types of hybrids with low-, intermediate-, and high-pairing level showed preferential pairing between A-D and B-S^sh. A close relationship of the S^sh genome to the B genome of bread wheat was confirmed, but the results provide no evidence that the B genome was derived from T. sharonense. Data on the pairing between individual chromosomes of T. aestivum and T. sharonense provide an estimate of interspecific homoeologous recombination. Received: 14 October 1996 / Accepted: 25 October 1996