Cloning and characterization of a gene encoding wheat starch synthase I

 A cDNA clone, and a corresponding genomic DNA clone, containing full-length sequences encoding wheat starch synthase I, were isolated from a cDNA library of hexaploid wheat (Triticum aestivum) and a genomic DNA library of Triticum tauschii, respectively. The entire sequence of the starch synthase-I cDNA (wSSI-cDNA) is 2591 bp, and it encodes a polypeptide of 647 amino-acid residues that shows 81% and 61% identity to the amino-acid sequences of SSI-type starch synthases from rice and potato, respectively. In addition, the putative N-terminal amino-acid sequence of the encoded protein is identical to that determined for the N-terminal region of the 75-kDa starch synthase present in the starch granule of hexaploid wheat. Two prominent starch synthase activities were demonstrated to be present in the soluble fraction of wheat endosperm by activity staining of the non-denaturing PAGE gels. The most anodal band (wheat SSI) shows the highest staining intensity and results from the activity of a 75-kDa protein. The wheat SSI mRNA is expressed in the endosperm during the early to mid stages of wheat grain development but was not detected by Northern blotting in other tissues from the wheat plant. The gene encoding the wheat SSI (SsI-D1) consists of 15 exons and 14 introns, similar to the structure of the rice starch synthase-I gene. While the exons of wheat and rice are virtually identical in length, the wheat SsI-D1 gene has longer sequences in introns 1, 2, 4 and 10, and shorter sequences in introns 6, 11 and 14, than the corresponding rice gene. Received: 5 June 1998 / Accepted: 29 September 1998     

The organization of genes tightly linked to the Ha locus in Aegilops tauschii, the D-genome donor to wheat.

The grain hardness locus, Ha, is located at the distal end of the short arm of chromosome 5D in wheat. Three polypeptides, puroindoline-a, puroindoline-b, and grain softness protein (GSP-1), have been identified as components of friabilin, a biochemical marker for grain softness, and the genes for these polypeptides are known to be tightly linked to the Ha locus. However, this region of the chromosome 5D has not been well characterized and the physical distance between the markers is not known. Separate lambda clones containing the puroindoline-a gene and the puroindoline-b gene have been isolated from an Aegilops tauschii (the donor of the D genome to wheat) genomic lambda library and investigated. Considerable variation appears to exist in the organization of the region upstream of the gene for puroindoline-b among species closely related to wheat. Using in situ hybridization the genes for puroindoline-a, -b, and GSP-1 were demonstrated to be physically located at the tip of the short arm of chromosome 5 of A. tauschii. Four overlapping clones were isolated from a large-insert BAC library constructed from A. tauschii and of these one contained genes for all of puroindoline-a, puroindoline-b, and GSP-1. The gene for puroindoline-a is located between the other two genes at a distance no greater than approximately 30 kb from either gene. The BAC clone containing all three known genes was used to screen a cDNA library constructed from hexaploid wheat and cDNAs that could encode novel polypeptides were isolated.     

Cloning and characterization of wheat PDI (protein disulfide isomerase) homoeologous genes and promoter sequences

The genomic and cDNA sequences of three PDI homoeologous genes located on chromosomes 4A, 4B and 4D of bread wheat and their promoters were cloned and sequenced. The three sequences showed a very high conservation of the coding region and of the exon/intron structure, which consisted of ten exons. The comparison of wheat sequences with those of rice and Arabidopsis showed a significant conservation of the exon/intron structure across the three species. The expression of each gene was analysed by RT-PCR in different plant tissues (roots, coleoptiles, spikelets, leaves and developing caryopses). All the genes showed a higher expression in developing caryopses than in other analysed tissues, wherein some differences were detected. The promoter sequences of the three genes possessed some regulatory motifs typical of endosperm specific expression.     

Complementation of sugary-1 phenotype in rice endosperm with the wheat isoamylase1 gene supports a direct role for isoamylase1 in amylopectin biosynthesis

To examine the role of isoamylase1 (ISA1) in amylopectin biosynthesis in plants, a genomic DNA fragment from Aegilops tauschii was introduced into the ISA1-deficient rice (Oryza sativa) sugary-1 mutant line EM914, in which endosperm starch is completely replaced by phytoglycogen. A. tauschii is the D genome donor of wheat (Triticum aestivum), and the introduced fragment effectively included the gene for ISA1 for wheat (TaISA1) that was encoded on the D genome. In TaISA1-expressing rice endosperm, phytoglycogen synthesis was substantially replaced by starch synthesis, leaving only residual levels of phytoglycogen. The levels of residual phytoglycogen present were inversely proportional to the expression level of the TaISA1 protein, although the level of pullulanase that had been reduced in EM914 was restored to the same level as that in the wild type. Small but significant differences were found in the amylopectin chain-length distribution, gelatinization temperatures, and A-type x-ray diffraction patterns of the starches from lines expressing TaISA1 when compared with wild-type rice starch, although in the first two parameters, the effect was proportional to the expression level of TaISA. The impact of expression levels of ISA1 on starch structure and properties provides support for the view that ISA1 is directly involved in the synthesis of amylopectin.     

Resistance to wheat leaf rust and stem rust in Triticum tauschii and inheritance in hexaploid wheat of resistance transferred from T. tauschii.

Twelve accessions of Triticum tauschii (Coss.) Schmal. were genetically analyzed for resistance to leaf rust (Puccinia recondita Rob. ex Desm.) and stem rust (Puccinia graminis Pers. f.sp. tritici Eriks. and E. Henn.) of common wheat (Triticum aestivum L.). Four genes conferring seedling resistance to leaf rust, one gene conferring seedling resistance to stem rust, and one gene conferring adult-plant resistance to stem rust were identified. These genes were genetically distinct from genes previously transferred to common wheat from T. tauschii and conferred resistance to a broad spectrum of pathogen races. Two of the four seedling leaf rust resistance genes were not expressed in synthetic hexaploids, produced by combining tetraploid wheat with the resistant T. tauschii accessions, probably owing to the action of one or more intergenomic suppressor loci on the A or B genome. The other two seedling leaf rust resistance genes were expressed at the hexaploid level as effectively as in the source diploids. One gene was mapped to the short arm of chromosome 2D more than 50 cM from the centromere and the other was mapped to chromosome 5D. Suppression of seedling resistance to leaf rust in synthetic hexaploids derived from three accessions of T. tauschii allowed the detection of three different genes conferring adult-plant resistance to a broad spectrum of leaf rust races. The gene for seedling resistance to stem rust was mapped to chromosome ID. The degree of expression of this gene at the hexaploid level was dependent on the genetic background in which it occurred and on environmental conditions. The expression of the adult-plant gene for resistance to stem rust was slightly diminished in hexaploids. The production of synthetic hexaploids was determined to be the most efficient and flexible method for transferring genes from T. tauschii to T. aestivum, but crossing success was determined by the genotypes of both parents. Although more laborious, the direct introgression method of crossing hexaploid wheat with T. tauschii has the advantages of enabling selection for maximum expression of resistance in the background hexaploid genotype and gene transfer into an agronomically superior cultivar.     

The structure and expression of the wheat starch synthase III gene. Motifs in the expressed gene define the lineage of the starch synthase III gene family

The endosperm of hexaploid wheat (Triticum aestivum [L.]) was shown to contain a high molecular weight starch synthase (SS) analogous to the product of the maize du1 gene, starch synthase III (SSIII; DU1). cDNA and genomic DNA sequences encoding wheat SSIII were isolated and characterized. The wheat SSIII cDNA is 5,346 bp long and contains an open reading frame that encodes a 1,628-amino acid polypeptide. A putative N-terminal transit peptide, a 436-amino acid C-terminal catalytic domain, and a central 470-amino acid SSIII-specific domain containing three regions of repeated amino acid similarity were identified in the wheat gene. A fourth region between the transit peptide and the SSIII-specific domain contains repeat motifs that are variable with respect to motif sequence and repeat number between wheat and maize. In dicots, this N-terminal region does not contain repeat motifs and is truncated. The gene encoding wheat SSIII, designated ss3, consists of 16 exons extending over 10 kb, and is located on wheat chromosome I. Expression of ss3 mRNA in wheat was detected in leaves, pre-anthesis florets, and from very early to middle stage of endosperm development. The entire N-terminal variable repeat region and the majority of the SSIII-specific domain are encoded on a single 2,703-bp exon. A gene encoding a class III SS from the Arabidopsis genome sequencing project shows a strongly conserved exon structure to the wheat ss3 gene, with the exception of the N-terminal region. The evolutionary relationships of the genes encoding monocot and dicot class III SSs are discussed.     

Purification, characterization, and cDNA structure of isoamylase from developing endosperm of rice

Isoamylase (EC 3.2.1.68) in rice (Oryza sativa L.) was efficiently purified within a day to homogeneity, as confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), from developing endosperm by sequential use of Q Sepharose HP anion- exchange chromatography, ammonium sulfate fractionation, and TSKgel G4000SW_XL and G3000SW_XL gel filtration chromatography. Although the protein exhibited a molecular size of ca. 83 kDa on SDS-PAGE, the apparent size of the native enzyme was approximately 340 and 490 kDa on TSKgel G3000SW_XL and G4000SW_XL gel filtration chromatograms, respectively, suggesting that rice isoamylase exists in a homo-tetramer to homo-hexamer form in developing endosperm. The purified rice isoamylase was able to debranch glycogen, phytoglycogen and amylopectin but could not attack pullulan. The optimum pH and temperature for isoamylase activity were found to be pH 6.5 to 7.0 and 30 °C, respectively. The enzyme activity was completely inhibited by HgCl₂ and p-chloromercuribenzoate at 1 mM. These results indicate that rice isoamylase possesses properties which are distinct from those reported for bacterial isoamylase. Complementary-DNA clones for rice endosperm isoamylase were isolated with a polymerase-chain-reaction product as probe which was generated by primers designed from nucleotides conserved in cDNA for maize Sugary-1 isoamylase (M.G. James et al., 1995, Plant Cell 7: 417–429) and a Pseudomonas amyloderamosa gene encoding isoamylase (A. Amemura et al., 1988, J Biol Chem 263: 9271–9275). The nucleotide sequence and deduced amino acid sequence of the longest clone showed a high similarity to those of maize Surgary-1 isoamylase, but a lesser similarity to those of Pseudomonas amyloderamosa isoamylase. Southern blot analysis and gene mapping analysis indicated that the isoamylase gene exists as a single copy in the rice genome and is located on chromosome 8 of cv. Nipponbare which belongs to the Japonica rice group. Phylogenetic analysis indicated that isoamylases from maize and rice are more closely related to a number of glgX gene products of the blue green alga Synechocystis and various bacteria than to isoamylases from Pseudomonas and Flavobacterium. Hence, it is proposed that glgX proteins are classified as isoamylase-type debranching enzymes. Our tree also showed that all starch- and glycogen-debranching enzymes from plants and bacteria tested can be classified into two distinct types, an isoamylase-type and a pullulanase-type. Received: 29 October 1998 / Accepted: 10 December 1998     

Application of real-time PCR-based SNP detection for mapping of Net2, a causal D-genome gene for hybrid necrosis in interspecific crosses between tetraploid wheat and Aegilops tauschii

Available information on genetically assigned molecular markers is not sufficient for efficient construction of a high-density linkage map in wheat. Here, we report on application of high resolution melting (HRM) analysis using a real-time PCR apparatus to develop single nucleotide polymorphism (SNP) markers linked to a hybrid necrosis gene, Net2, located on wheat chromosome 2D. Based on genomic information on barley chromosome 2H and wheat expressed sequence tag libraries, we selected wheat cDNA sequences presumed to be located near the Net2 chromosomal region, and then found SNPs between the parental Ae. tauschii accessions of the synthetic wheat mapping population. HRM analysis of the PCR products from F(2) individuals' DNA enabled us to assign 44.4% of the SNP-representing cDNAs to chromosome 2D despite the presence of the A and B genomes. In addition, the designed SNP markers were assigned to chromosome 2D of Ae. tauschii. The order of the assigned SNP markers in synthetic hexaploid wheat was confirmed by comparison with the markers in barley and Ae. tauschii. Thus, the SNP-genotyping method based on HRM analysis is a useful tool for development of molecular markers at target loci in wheat.     

The Ha locus of wheat: identification of a polymorphic region for tracing grain hardness in crosses.

The grain softness proteins or friabilins are known to be composed of three main components: puroindoline a, puroindoline b, and GSP-1. cDNAs for GSP-1 have previously been mapped to group-5 chromosomes and their location on chromosome 5D is closely linked to the grain hardness (Ha) locus of hexaploid wheat. A genomic DNA clone containing the GSP-1 gene (wGSP1-A1) from hexaploid wheat has been identified by fluorescent in situ hybridization as having originated from the distal end of the short arm of chromosome 5A. A genomic clone containing the gene (wGSP1-D1) was also isolated from Aegilops tauschii, the donor of the D genome to bread wheat. There are no introns in the GSP-1 genes, and there is high sequence identity between wGSP1-A1 and wGSP1-D1 up to 1 kb 5' and 300 bp 3' to wGSP1-D1. However, regions further upstream and downstream of wGSP1-D1 share no significant sequence identity to corresponding sequences in wGSP1-A1. These regions therefore identified potentially valuable sequences for tracing the Ha locus through assaying polymorphic DNA sequences. The sequence from 300 to 500 bp 3' to wGSP1-D1 (wGSP1-D13) was mapped to the Ha locus in a mapping population. wGSP1-D13 was also tightly linked to genes for puroindoline a and puroindoline b which have been previously mapped to be at the Ha locus. In addition wGSP1-D13 was used to detect RFLPs between near isogenic soft and hard Falcon lines and in a random selection of soft and hard wheats.     

Starch branching enzyme IIb in wheat is expressed at low levels in the endosperm compared to other cereals and encoded at a non-syntenic locus

Studies of maize starch branching enzyme mutants suggest that the amylose extender high amylose starch phenotype is a consequence of the lack of expression of the predominant starch branching enzyme II isoform expressed in the endosperm, SBEIIb. However, in wheat, the ratio of SBEIIb and SBEIIa expression are inversely related to the expression levels observed in maize and rice. Analysis of RNA at 15 days post anthesis suggests that there are about 4-fold more RNA for SBE IIa than for SBE IIb. The genes for SBE IIa and SBE IIb from wheat are distinguished in the size of the first three exons, allowing isoform-specific antibodies to be produced. These antibodies were used to demonstrate that in the soluble fraction, the amount of SBE IIa protein is two to three fold higher than SBIIb, whereas in the starch granule, there is two to three fold more SBE IIb protein amount than SBE IIa. In a further difference to maize and rice, the genes for SBE IIa and SBE IIb are both located on the long arm of chromosome 2 in wheat, in a position not expected from rice–maize–wheat synteny.     

Macro- and microcolinearity between the genomic region of wheat chromosome 5B containing the Tsn1 gene and the rice genome

The Tsn1 gene in wheat confers sensitivity to a proteinaceous host-selective toxin (Ptr ToxA) produced by the tan spot fungus (Pyrenophora tritici-repentis) and lies within a gene-rich region of chromosome 5B. To use the rice genome sequence information for the map-based cloning of Tsn1, colinearity between the wheat genomic region containing Tsn1 and the rice genome was determined at the macro- and microlevels. Macrocolinearity was determined by testing 28 expressed sequence markers (ESMs) spanning a 25.5-cM segment and encompassing Tsn1 for similarity to rice sequences. Twelve ESMs had no similarity to rice sequences, and 16 had similarity to sequences on seven different rice chromosomes. Segments of colinearity with rice chromosomes 3 and 9 were identified, but frequent rearrangements and disruptions occurred. Microcolinearity was determined by testing the sequences of 26 putative genes identified from BAC contigs of 205 and 548 kb in length and flanking Tsn1 for similarity to rice genomic sequences. Fourteen of the predicted genes detected orthologous sequences on six different rice chromosomes, whereas the remaining 12 had no similarity with rice sequences. Four genes were colinear on rice chromosome 9, but multiple disruptions, rearrangements, and duplications were observed in wheat relative to rice. The data reported provide a detailed analysis of a region of wheat chromosome 5B that is highly rearranged relative to rice.     

Identification and High-Density Mapping of Gene-Rich Regions in Chromosome Group 5 of Wheat

The distribution of genes and recombination in the wheat genome was studied by comparing physical maps with the genetic linkage maps. The physical maps were generated by mapping 80 DNA and two phenotypic markers on an array of 65 deletion lines for homoeologous group 5 chromosomes. The genetic maps were constructed for chromosome 5B in wheat and 5D in Triticum tauschii. No marker mapped in the proximal 20% chromosome region surrounding the centromere. More than 60% of the long arm markers were present in three major clusters that physically encompassed <18% of the arm. Because 48% of the markers were cDNA clones and the distributions of the cDNA and genomic clones were similar, the marker distribution may represent the distribution of genes. The gene clusters were identified and allocated to very small chromosome regions because of a higher number of deletions in their surrounding regions. The recombination was suppressed in the centromeric regions and mainly occurred in the gene-rich regions. The bp/cM estimates varied from 118 kb for gene-rich regions to 22 Mb for gene-poor regions. The wheat genes present in these clusters are, therefore, amenable to molecular manipulations parallel to the plants with smaller genomes like rice.     

Genomic organization, chromosomal localization, and independent expression of human cyclin D genes.

Murine cDNA clones for three cyclin D genes that are normally expressed during the G1 phase of the cell cycle were used to clone the cognate human genes. Bacteriophage and cosmid clones encompassing five independent genomic loci were partially sequenced and chromosomally assigned by an analysis of somatic cell hybrids containing different human chromosomes and by fluorescence in situ hybridization to metaphase spreads from normal peripheral blood lymphocytes. The human cyclin D1 gene (approved gene symbol, CCND1) was assigned to chromosome band 11q13, cyclin D2 (CCND2) to chromosome band 12p13, and cyclin D3 (CCND3) to chromosome band 6p21. Pseudogenes containing sequences related to cyclin D2 and cyclin D3 mapped to chromosome bands 11q13 and 6p21, respectively. Partial nucleotide sequence analysis of exons within each gene revealed that the authentic human cyclin D genes are more related to their mouse counterparts than to each other. These genes are ubiquitously transcribed in human tumor cell lines derived from different cell lineages, but are independently and, in many cases, redundantly expressed. The complex patterns of expression of individual cyclin D genes and their evolutionary conservation across species suggest that each family member may play a distinct role in cell cycle progression.