Chromosome mapping and phylogenetic analysis of the cytosolic acetyl-CoA carboxylase loci in wheat

The cytosolic isoform of plant acetyl-CoA carboxylase is a multidomain enzyme involved in the synthesis of very-long-chain fatty acids and in secondary metabolism. Chromosome mapping of wheat identified one locus containing cytosolic acetyl-CoA carboxylase genes (Acc-2) and a related partially processed pseudogene (Psi-Acc-2) in the distal region of the long arm of wheat homoeologous group 3 chromosomes. Multiple copies of the Acc-2 genes, whose presence was suggested by sequence analysis, are likely to be arranged in tandem repeats. At least three out of five genes cloned from hexaploid wheat map to this locus. Another locus containing Acc-2--related sequences is present in the distal region of the long arm of chromosome 5D. The identity of the hybridizing DNA present at this locus remains unknown. A system based on PCR-cloning and DNA sequence analysis of acetyl-CoA carboxylase genes was developed to address various phylogenetic and systematics questions in grasses. It was applied to reconstruct the phylogeny of the Acc-2 genes from D- and S-genome Aegilops and A-genome Triticum diploid species, AABB- and AAGG-genome tetraploid wheat, and AABBDD-genome hexaploid wheat, as well as from rye and barley. The combined cytogenetic and molecular evolution approach allowed assignment of gene sequences included in phylogenetic analysis to specific loci on homoeologous chromosomes. Recurring gene duplication followed by chromosome translocation and/or possible loss of some gene copies, as well as loss of introns, occurred in the gene family in different plant lineages. Two major Acc-2 clades appeared before the divergence of barley and rye. Nucleotide substitution rates in different parts of the Acc-2 gene were assessed. This analysis of the Acc-2 loci provides detailed information regarding evolutionary events at a low--copy-number locus containing important functional genes. These events are likely to be common and to play a significant role in shaping grass genomes.     

Organization of the histone H3 genes in soybean, barley and wheat

Several variants of the replacement histone H3 genes from soybean, barley and wheat have been cloned and sequenced. Analysis of segregating populations in barley and soybean, as well as analysis of clones isolated from a soybean genomic library, suggested that these genes are dispersed throughout the genome. Several genes contain introns located in similar positions, but of different lengths and sequence. Comparison of mRNA levels in different tissues revealed that the intron-containing and intronless genes have different expression patterns. The distribution of the introns in the histone H3 genes across several plant species suggests that some of the introns might have been lost during the evolution of the gene family. Sequence divergence among introns and gene-flanking sequences in cloned gene variants allowed us to use them as specific probes for localizing individual gene copies and analyzing the genomic distribution of these variants across a range of genotypes.Journal paper No. J-16127 of the Iowa Agriculture and Home Economics Experiment Station, Ames, IowaMention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the United States Department of Agriculture and does not imply its approval to the exclusion of other products that may be suitable     

Simultaneous mutation detection of three homoeologous genes in wheat by High Resolution Melting analysis and Mutation Surveyor?

Background  

TILLING (Targeting Induced Local Lesions IN Genomes) is a powerful tool for reverse genetics, combining traditional chemical mutagenesis with high-throughput PCR-based mutation detection to discover induced mutations that alter protein function. The most popular mutation detection method for TILLING is a mismatch cleavage assay using the endonuclease CelI. For this method, locus-specific PCR is essential. Most wheat genes are present as three similar sequences with high homology in exons and low homology in introns. Locus-specific primers can usually be designed in introns. However, it is sometimes difficult to design locus-specific PCR primers in a conserved region with high homology among the three homoeologous genes, or in a gene lacking introns, or if information on introns is not available. Here we describe a mutation detection method which combines High Resolution Melting (HRM) analysis of mixed PCR amplicons containing three homoeologous gene fragments and sequence analysis using Mutation Surveyor^? software, aimed at simultaneous detection of mutations in three homoeologous genes.     

Cloning and partial characteristics of the barley D-hordein sequence]

A number of clones containing major endosperm-specifically transcribed gene copies were selected from a cDNA library developed on the basis of barley endosperm mRNA. Approx. 30% of the recombinant clones carried sequences homologous to mRNA of various cereal storage proteins. Some of them appeared to be related to cDNA clones of wheat and barley storage proteins. The typical insert length ranged from 0.3 to 1.7 kB. A couple of clones among them were selected which revealed positive hybridization with all probes used. The positive signals disappeared after stringent washing of the filters. The nucleotide sequences of two representatives of the group were determined and corresponding amino acid sequence deduced after subsequent computer analysis. The comparison with known cereal storage protein genes revealed relatively high homology level with the central part of wheat high molecular weight (HMW) glutenine subunit genes. The fact suggests the cloned gene to belong to barley D-hordein family.     

Sequence and tissue-specific expression of a putative peroxidase gene from wheat (Triticum aestivum L.)

We have used a cDNA clone encoding a pathogen-induced putative wheat peroxidase to screen a genomic libary of wheat (Triticum aestivum L. cv. Cheyenne) and isolated one positive clone, lambda POX1. Sequence analysis revealed that this clone contains a gene encoding a putative peroxidase with a calculated pI of 8.1 which exhibits 58% and 83% sequence identity to the amino acid sequence of the turnip (Brassica rapa) peroxidase and a pathogen-induced putative wheat peroxidase, respectively. The two introns in the wheat gene are at the same positions as introns in the peroxidase genes of tomato and horseradish. Results of S1-mapping experiments suggest that this gene is neither pathogen-nor wound-induced in leaves but is constitutively expressed in roots.     

一个小麦ω-醇溶蛋白基因同源序列的分离与序列分析

通过基因组PCR克隆了小麦济南177的一个ω-醇溶蛋白基因同源序列（ω1236）,该序列包括部分5′、3′ 侧翼序列和全部可能的编码序列,没有内含子,但在第87、117、125、160、198、313、357和365氨基酸残基处有终止密码子,所有8个终止密码的形成都是碱基转换的结果。比较分析发现,该序列与一个ω-醇溶蛋白基因序列（AB059812）有98%的同源性。推导的氨基酸序列表明该基因符合禾谷类醇溶蛋白的特点。系统进化分析表明,该序列与小麦ω醇溶蛋白在进化上亲缘关系较近,与α-,β-,γ-醇溶蛋白基因关系较远。ω1236推导的氨基酸序列编码了一个可能的47.2 kDa的蛋白质。转化大肠杆菌发现,在IPTG诱导后最初2 h,有小肽段产生,这说明在该基因序列中可能存在终止密码,这与测序结果是一致的。该研究为用PCR技术克隆ω醇溶蛋白基因和进一步研究ω醇溶蛋白基因的结构和功能积累了资料。     

Association of allelic variation in two NPR1-like genes with Fusarium head blight resistance in wheat

Fusarium head blight (FHB) is a destructive disease of wheat and barley. In wheat it is mainly caused by the fungal pathogens Fusarium graminearum and Fusarium culmorum. We report the identification and evaluation of candidate genes for quantitative FHB resistance. These genes showed altered expression levels in the moderately resistant winter wheat genotypes Capo and SVP72017 after inoculation with F. graminearum. Amongst others, a NPR1-like gene was identified. Sequence analysis of this gene fragment revealed a high level of variation between the parents of a doubled haploid population. Single nucleotide polymorphism and polymerase chain reaction markers were developed and two homoeologous genes were mapped on the long arms of chromosomes 2A and 2D, respectively. Markers for both genes had significant effects on FHB resistance in a diverse collection of 178 European winter wheat cultivars evaluated in multi-environmental field trials after spray inoculation with F. culmorum. These results revealed that allelic variation in two homoeologous NPR1-like genes is associated with FHB resistance in European winter wheat. Markers for these genes might therefore be used for marker-assisted breeding programs.     

Organization of the histone genes in the rainbow trout (Salmo gairdnerii)

Summary Twelve clones containing histone genes were isolated from a genomic trout library constructed in the vector Charon 4A. Each of the clones was found to contain a conserved 10.2-kb Eco RI fragment that contained one copy of each of the histones in the order H4-H2B-H1-H2A-H3, all of which are transcribed from the same strand. Genomic Southern blots indicate that these clusters are representative of the vast majority of the histone genes in the trout. Tandemly linked clusters were not found. Approximately 145 copies of this cluster are present in a trout sperm cell. Sequence analysis has shown the genes to be without introns and to show strong selection for codons ending in C or G. Consensus signals similar to those found in other histone genes are present in the flanking regions.     

Isolation and characterization of the three Waxy genes encoding the granule-bound starch synthase in hexaploid wheat.

Complete genomic DNA sequences of three homoeologous Waxy structural genes, located on the chromosomes 7A, 4A, and 7D in hexaploid wheat (Triticum aestivum L. cv. Chinese Spring), were separately determined and analyzed. Those structural genes in lengths from start to stop codon were 2781bp in Wx-7A, 2794bp in Wx-4A, and 2862bp in Wx-7D, each of which consisted of 11 exons and ten introns. They were closely similar to one another in the nucleotide sequences, with 95.6-96.3% homology in mature protein regions, 88. 7-93.0% in transit-peptide regions, and 70.5-75.2% in the introns. These wheat Waxy genes were GC-rich when compared with standard values for plant genomes reported so far. This was reflected in the extremely high G/C occupation frequency at the third position of the codons in the coding regions. The sequence divergence in the exon regions was mostly due to the substitution of nucleotides, whereas that found in the introns was attributed to substitution, insertion and/or deletion of nucleotides. Only the Wx-4A gene contained a trinucleotide insertion (CAA) in the region encoding the transit peptide. Most of the substitutions observed in the exon regions were categorized as synonymous, and higher sequence similarities (96.5-97. 4%) were conserved at the protein level. The phylogenetic tree obtained in terms of the amino acid sequence variations showed a well-resolved phylogenetic relationship among wheat Waxy genes and those from other plants.     

Molecular evolution of receptor-like kinase genes in hexaploid wheat. Independent evolution of orthologs after polyploidization and mechanisms of local rearrangements at paralogous loci

Hexaploid wheat is a young polyploid species and represents a good model to study mechanisms of gene evolution after polyploidization. Recent studies at the scale of the whole genome have suggested rapid genomic changes after polyploidization but so far the rearrangements that have occurred in terms of gene content and organization have not been analyzed at the microlevel in wheat. Here, we have isolated members of a receptor kinase (Lrk) gene family in hexaploid and diploid wheat, Aegilops tauschii, and barley (Hordeum vulgare). Phylogenetic analysis has allowed us to establish evolutionary relationships (orthology versus paralogy) between the different members of this gene family in wheat as well as with Lrk genes from barley. It also demonstrated that the sequences of the homoeologous Lrk genes evolved independently after polyploidization. In addition, we found evidence for gene loss during the evolution of wheat and barley. Analysis of large genomic fragments isolated from nonorthologous Lrk loci showed a high conservation of the gene content and gene organization at these loci on the homoeologous group 1 chromosomes of wheat and barley. Finally, sequence comparison of two paralogous fragments of chromosome 1B showed a large number of local events (sequence duplications, deletions, and insertions), which reveal rearrangements and mechanisms for genome enlargement at the microlevel.     

向普通小麦导入纤毛鹅观草抗黄矮病基因的研究──Ⅰ.F_1和BC_1的产生及其细胞遗传学

为了将纤毛鹅观草Z1010对黄矮病毒株系PAV和RPV的抗性基因转入普通小麦,通过幼胚拯救,获得了纤毛鹅观草Z1010×普通小麦品种莱州953的杂种F1,以及用5个普通小麦品种（系）回交的BC1衍生系。对杂种F1及BC1植株的细胞学分析表明,纤毛鹅观草Z1010不仅对Ph基因具有很强的抑制作用,而且能使杂种F1形成未减数配子,对细胞遗传学资料的进一步分析认为,通过部分同源染色体间的交换将纤毛鹅观草Z1010的抗黄矮病基因转入小麦是可能的。     

Cloning of cDNA Sequences Encoding the Calcium-Binding Protein, Calmodulin, from Barley (Hordeum vulgare L.)

Full- and partial-length cDNAs encoding calmodulin mRNA have been cloned and sequenced from barley (Hordeum vulgare L.). Barley leaf mRNA, size-fractionated in sucrose density gradients, was used to synthesize double-stranded cDNA. The cDNA was cloned in λgt10 and screened with a synthetic, 14-nucleotide oligonucleotide probe, which was designed using the predicted coding sequences of the carboxy termini of spinach and wheat calmodulin proteins. The primary structure of barley calmodulin, predicted from DNA sequencing experiments, consists of 148 amino acids and differs from that of wheat calmodulin in only three positions. In two of the three positions, the amino acid changes are conservative, while the third change consists of an apparent deletion/insertion. The overall nucleotide sequence similarity between the amino acid coding regions of barley and vertebrate calmodulin mRNAs is approximately 77%. However, a region encoding 11 amino acids of the second Ca²⁺-binding domain is very highly conserved at the nucleotide level compared with the rest of the coding sequences (94% sequence identity between barley and chicken calmodulin mRNAs). Genomic Southern blots reveal that barley calmodulin is encoded by a single copy gene. This gene is expressed as a single size class of mRNA in all tissues of 7-day-old barley seedlings. In addition, these analyses indicate that a barley calmodulin cDNA coding region subclone is suitable as a probe for isolating calmodulin genes from other plants.     

The tallysomycin biosynthetic gene cluster from Streptoalloteichus hindustanus E465-94 ATCC 31158 unveiling new insights into the biosynthesis of the bleomycin family of antitumor antibiotics

The tallysomycins (TLMs) belong to the bleomycin (BLM) family of antitumor antibiotics. The BLM biosynthetic gene cluster has been cloned and characterized previously from Streptomyces verticillus ATCC 15003, but engineering BLM biosynthesis for novel analogs has been hampered by the lack of a genetic system for S. verticillus. We now report the cloning and sequencing of the TLM biosynthetic gene cluster from Streptoalloteichus hindustanus E465-94 ATCC 31158 and the development of a genetic system for S. hindustanus, demonstrating the feasibility to manipulate TLM biosynthesis in S. hindustanus by gene inactivation and mutant complementation. Sequence analysis of the cloned 80.2 kb region revealed 40 open reading frames (ORFs), 30 of which were assigned to the TLM biosynthetic gene cluster. The TLM gene cluster consists of nonribosomal peptide synthetase (NRPS) genes encoding nine NRPS modules, a polyketide synthase (PKS) gene encoding one PKS module, genes encoding seven enzymes for deoxysugar biosynthesis and attachment, as well as genes encoding other biosynthesis, resistance, and regulatory proteins. The involvement of the cloned gene cluster in TLM biosynthesis was confirmed by inactivating the tlmE glycosyltransferase gene to generate a TLM non-producing mutant and by restoring TLM production to the DeltatlmE::ermE mutant strain upon expressing a functional copy of tlmE. The TLM gene cluster is highly homologous to the BLM cluster, with 25 of the 30 ORFs identified within the two clusters exhibiting striking similarities. The structural similarities and differences between TLM and BLM were reflected remarkably well by the genes and their organization in their respective biosynthetic gene clusters.