Molecular characterisation of two novel starch granule proteins 1 in wild and cultivated diploid A genome wheat species

Starch synthase IIa, also known as starch granule protein 1 (SGP-1), plays a key role in amylopectin biosynthesis. The absence of SGP-1 in cereal grains is correlated to dramatic changes in the grains’ starch content, structure, and composition. An extensive investigation of starch granule proteins in this study revealed a polymorphism in the electrophoretic mobility of SGP-1 between two species of wheat, Triticum urartu and T. monococcum; this protein was, however, conserved among all other Triticum species that share the A genome inherited from their progenitor T. urartu. Two different electrophoretic profiles were identified: SGP-A1 proteins of T. urartu accessions had a SDS–PAGE mobility similar to those of tetraploid and hexaploid wheat species; conversely, SGP-A1 proteins of T. monococcum ssp. monococcum and ssp. boeoticum accessions showed a different electrophoretic mobility. The entire coding region of the two genes was isolated and sequenced in an attempt to explain the polymorphism identified. Several single nucleotide polymorphisms (SNPs) responsible for amino acid changes were identified, but no indel polymorphism was observed to explain the difference in electrophoretic mobility. Amylose content did not differ significantly among T. urartu, T. monococcum ssp. boeoticum and T. monococcum ssp. monococcum, except in one accession of the ssp. boeoticum. Conversely, several interspecific differences were observed in viscosity properties (investigated as viscosity profiles using a rapid visco analyzer—RVA profiles) of these cereal grains. T. monococcum ssp. boeoticum accessions had the lowest RVA profiles, T. urartu accessions had an intermediate RVA profile, whereas T. monococcum ssp. monococcum showed the highest RVA profile. These differences could be associated with the numerous amino acid and structural changes evident among the SGP-1 proteins.     

Fractionation of wheat gliadin and glutenin subunits by two-dimensional electrophoresis and the role of group 6 and group 2 chromosomes in gliadin synthesis

Summary Subunits of wheat endosperm proteins have been fractionated by two-dimensional electrophoresis. To determine which subunits in the two-dimensional electrophoretic pattern belong to gliadin or glutenin the endosperm proteins have also been fractionated by a modified Osborne procedure and by gel filtration on Sephadex G-100 and Sepharose CL-4B prior to separation by two-dimensional electrophoresis.The control of production of five major grain protein subunits is shown to be determined by chromosomes 6A, 6B and 6D by comparing two-dimensional electrophoretic protein subunit patterns of aneuploid lines of the variety Chinese Spring. From these and previous studies it is concluded that some , and gliadins (molecular weights by SDS-PAGE 30,000 to 40,000) are specified by genes on the short arms of homoeologous Group 6 chromosomes, the gliadins (molecular weights by SDS-PAGE 50,000 to 70,000) are specified by genes on the short arms of homoeologous Group 1 chromosomes and the glutenin subunits (molecular weights by SDS-PAGE > 85,000) are specified by genes on the long arms of homoeologous Group 1 chromosomes.No major gliadins or glutenin subunits were absent when any of the chromosomes in homoeologous Groups 2, 3, 4, 5 or 7 were deleted. However two gliadins whose presumed structural genes are on chromosome 6D were absent in aneuploid stocks of Chinese Spring carrying two additional doses of chromosome 2A. Two out of thirty-three intervarietal or interspecific chromosome substitution lines examined, involving homoeologous Group 2 chromosomes, lacked the same two gliadins. All the subunits in the other thirty-one chromosome substitution lines were indistinguishable from those in Chinese Spring. It is therefore concluded that the major variation affecting gliadin and glutenins in wheat is concentrated on the chromosomes of homoeologous Groups 1 and 6 but Group 2 chromosomes are candidates for further study.An endosperm protein controlled by chromosome 4D in Chinese Spring is shown to be a high molecular weight globulin.     

Genetic elimination of a starch granule protein, SGP-1, of wheat generates an altered starch with apparent high amylose

A starch granule protein, SGP-1, is a starch synthase bound to starch granules in wheat endosperm. A wheat lacking SGP-1 was produced by crossing three variants each deficient in one of three SGP-1 classes, namely SGP-A1, -B1 or -D1. This deficient wheat (SGP–1 null wheat) showed some alterations in endosperm starch, meaning that SGP-1 is involved in starch synthesis. Electrophoretic experiments revealed that the levels of two starch granule proteins, SGP-2 and -3, decreased considerably in the SGP-1 null wheat though that of the waxy protein (granule-bound starch syn- thase I) did not. The A-type starch granules were deformed. Apparent high amylose level (30.8–37.4%) was indicated by colorimetric measurement, amperometric titration, and the concanavalin A method. The altered structure of amylopectin was detected by both high- performance size-exclusion chromatography and high-performance anion exchange chromatography. Levels of amylopectin chains with degrees of polymerization (DP) 6–10 increased, while DP 11–25 chains decreased. A low starch crystallinity was shown by both X-ray diffraction and differential scanning calorimetry (DSC) analyses because major peaks were absent. Abnormal crystallinity was also suggested by the lack of a polarized cross in SGP-1 null starch. The above results suggest that SGP-1 is responsible for amylopectin synthesis. Since the SGP-1 null wheat produced novel starch which has not been described before, it can be used to expand variation in wheat starch. Received: 30 April 1999 / Accepted: 9 November 1999     

Genetic control of amylose content in wheat endosperm starch and differential effects of three Wx genes

The endosperm starch of the wheat grain is composed of amylose and amylopectin. Genetic manipulation of the ratio of amylose to amylopectin or the amylose content could bring about improved texture and quality of wheat flour. The chromosomal locations of genes affecting amylose content were investigated using a monosomic series of Chinese Spring (CS) and a set of Cheyenne (CNN) chromosome substitution lines in the CS genetic background. Trials over three seasons revealed that a decrease in amylose content occurred in monosomic 4A and an increase in monosomic 7B. Allelic variation between CS and CNN was suggested for the genes on chromosomes 4A and 7B. To examine the effects of three Waxy (Wx) genes which encode a granule-bound starch synthase (Wx protein), the Wx proteins from CS monosomics of interest were analyzed using SDS-PAGE. The amount of the Wx protein coded by the Wx-B1 gene on chromosome arm 4AL was reduced in monosomic 4A, and thus accounted for its decreased amylose content. The amounts of two other Wx proteins coded by the Wx-A1 and Wx-D1 genes on chromosome arms 7AS and 7DS, respectively, showed low levels of protein in the monosomics but no effect on amylose content. The effect of chromosome 7B on the level of amylose suggested the presence of a regulator gene which suppresses the activities of the Wx genes.     

Waxy protein deficiency and chromosomal location of coding genes in common wheat

Deficiency of the wheat waxy (Wx) proteins (Wx-A1, Wx-B1 and Wx-D1) was studied in 1,960 cultivars derived from several countries. Gel electrophoretic analyses revealed that the null allele for the Wx-A1 protein occurred frequently in Korean, Japanese and Turkish wheats but was relatively rare in cultivars from other countries and regions. About 48% of the wheats deficient for the Wx-B1 protein were from Australia and India. One Chinese cultivar lacked the WxD1 protein. While 9 Japanese cultivars were deficient in both the Wx-A1 and Wx-B1 proteins, no cultivars lacked both the Wx-A1 and Wx-D1 proteins, both the Wx-B1 and Wx-D1 proteins or all three Wx proteins. Two-dimensional gel electrophoresis revealed polymorphisms of the three Wx proteins that varied according to isoelectric points or molecular weight. The Wx-A1 gene coding the Wx-A1 protein and the Wx-B1 gene coding the Wx-B1 protein were localized in the distal regions of chromosome arms 7AS and 4AL, respectively, by deletion mapping using the deletion lines developed in the common wheat cultivar Chinese Spring.     

Mutations in wheat starch synthase II genes and PCR-based selection of a SGP-1 null line

Wheat (Triticum aestivum L.) starch synthase II, which is also known as starch granule protein 1 (SGP-1), plays a major role in endosperm starch synthesis. The three SGP-1 proteins, SGP-A1, B1 and D1, are produced by three homoeologous SSII genes, wSSII-A, B, and D. Lines carrying null alleles for each SGP-1 protein have previously been identified. In this report, the mutations occurring in each wSSII gene were characterized, and PCR-based DNA markers capable of detecting the mutations were developed. In the null wSSII-A allele, a 289 bp deletion accompanied by 8 bp of filler DNA was present near the initiation codon. A 175 bp insertion occurred in exon 8 of the null wSSII-B allele. The insertion represented a recently discovered miniature inverted-repeat transposable element (MITE) named Hikkoshi that was first found in a wheat waxy gene. A 63 bp deletion was found at the region surrounding the junction of the fifth exon and intron of the null wSSII-D allele. Based on this information, we designed primer sets to enable us to conduct allele-specific amplifications for each locus. The applicability of these primer sets for breeding programs was demonstrated by reconstructing a line lacking all three SGP-1 proteins using marker-assisted selection. These markers will also be useful in breeding programs aimed at obtaining partial mutants missing one or two SGP-1 proteins.     

Two-step one-dimensional SDS-PAGE analysis of LMW subunits of glutelin

Summary Analysis of intergeneric substitution lines in hexaploid wheats by a two-step electrophoretic method of protein separation revealed that low-molecular-weight (LMW) subunits of glutelin in Triticum longissimum, T. Umbelullatum, Elytrigia elongata (2 x) were controlled by chromosomes/chromosome arms 1S ^l , 1U, and 1ES, respectively. A LMW glutelin band in Secale montanum was detected but its chromosomal location could not be determined. Genes controlling gliadins and HMW subunits of glutelin were also located on chromosome 1S ^l in T. longissimum.The term glutelin refers to the polymeric prolamins of cereals, e.g., glutenins in wheat, HMW, and 75-k secalins in rye     

The peroxidase isozymes of the wheat kernel: tissue and substrate specificity and their chromosomal location

Summary Peroxidase isozymes were studied in the Triticum aestivum L. kernel and in nullisomic-tetrasomic and ditelocentric combinations of Chinese Spring wheat. Analyses were carried out on different parts of dry kernels (embryo plus scutellum and endosperm) using polyacrylamide and starch gel electrophoresis, different electrophoretic buffer systems and various staining methods. The peroxidase isozymes showed a low substrate-specificity and a high tissue-specificity. The embryo plus scutellum and the endosperm always presented different peroxidase patterns. Endosperm peroxidases were associated with chromosome arms 7DS, 4BL and 7AS; whereas the embryo plus scutellum isozymes were related to chromosome arms 3AL, 3BL and 3DS. The different results obtained using various electrophoretic techniques are due to the buffer system used. All staining procedures employed revealed the same peroxidase isozymes.     

Interaction between genes controlling a new group of glutenin subunits in bread wheat

Summary One-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of reduced total protein extracts from the endosperm of hexaploid wheat revealed a new set of faintly-stained bands, having slower electrophoretic mobility than the high-molecular-weight (HMW) glutenin subunits. These new bands have been termed the E group of glutenin subunits. Analysis of aneuploid stocks of Chinese Spring wheat has shown that three of the E bands, in order of increasing electrophoretic mobility, are controlled by genes on the short arms of chromosomes 1B, 1A and 1D, respectively. The E bands are expressed only in the presence of the long arm of chromosome 1B indicating an interaction between two or more genes involved in their production in wheat endosperm. The gene on the short arm of chromosome 1D controlling an E subunit recombined freely with Tri-D1 and the centromere but not at all with Gli-D1, indicating additional complexity at the Gli-DI locus in wheat.     

Production of multiple wheat-rye 1RS translocation stocks and genetic analysis of LMW subunits of glutenin and gliadins in wheats using these stocks

Summary A triple (1AL.1RS/1BL.1RS/1DL.1RS) and three double (1AL.1RS/1BL.1RS, 1AL.1RS/1DL.1RS, 1BL.1RS/1DL.1RS) wheat-rye 1RS translocation stocks were isolated from a segregating population using the Gli-1, Tri-1 and Sec-1 seed proteins as genetic markers. These stocks carried 42 chromosomes and formed the expected multivalents (frequency of 14–25%) at metaphase 1. They gave floret fertility ranging from 40–60%. These stocks were subsequently used to determine the genetic control of low-molecular-weight (LMW) glutenin subunits in Chinese Spring and Gabo by means of two-step one-dimensional SDS-PAGE. All of the B subunits and most of the C subunits of glutenin were shown to be controlled by genes on the short arms of group-1 chromosomes in these wheats. The other C subunits were not controlled by group-1 chromosomes. The triple translocation line served as a suitable third parent in producing test-cross seeds for studying the inheritance of the LMW glutenin subunits and gliadins in wheat cultivars, e.g. Chinese Spring and Orca. The segregation patterns of the LMW glutenin subunits in these cultivars revealed that the subunits were inherited in clusters and that their controlling genes (Glu-3) were tightly linked with those controlling gliadins (Gli-1). The LMW glutenin patterns d, d and e in Orca segregated as alternatives to the patterns a, a and a in Chinese Spring controlled by Glu-A3, Glu-B3 and Glu-D3 loci on chromosome arms 1AS, 1BS and 1DS, respectively, thus indicating that these patterns were controlled by allelic genes at these loci.     

Chromosomal location of genes controlling seed proteins in species related to wheat

Summary The seed proteins of Chinese Spring wheat stocks which possess single chromosomes from other plant species related to wheat have been separated by gel electrophoresis in the presence of sodium dodecyl sulphate. Marker protein bands have been detected for both arms of barley chromosome 5, chromosome E (= 1R) and B (= 2R) of rye, chromosomes A,B (= 1C^u) and C (= 5C^u) of Aegilops umbellulata and chromosomes I and III of Agropyron elongatum. These studies, and previous findings, indicate that chromosome 5 of barley, chromosome 1R of rye, chromosome I of Ag. elongatum and possibly chromosome 1C^u of Ae. umbellulata are similar to chromosomes 1A, 1B and 1D in hexaploid wheat in that they carry genes controlling prolamins on their short arms and genes controlling high-molecular-weight (apparent molecular weight greater than 86,000) seed protein species on their long arms. These findings support the idea that all these chromosomes are derived from a common ancestral chromosome and that they have maintained their integrity since their derivation from that ancestral chromosome.     

Dosage effects of the three Wx genes on amylose synthesis in wheat endosperm

Amylose synthesis in wheat endosperm is mainly controlled by the granule-bound starch synthase of about 60 kDa, the so-called waxy (Wx) protein. The Wx proteins are the product of the Wx genes at a triplicate set of single-copy homoeoloci located on chromosomes 7A (Wx-A1), 4A (Wx-B1) and 7D (Wx-D1). Using Chinese Spring and its aneuploid lines, including nullisomic-tetrasomics, tetrasomics, ditelosomics and deletion stocks, together with single-chromosome substitution lines for these chromosomes, the effects of varying the dosage of whole chromosomes and chromosome arms, as well as the effects of null alleles, upon amylose synthesis were investigated. Nullisomic 4A and the deletion of chromosome segments carrying the Wx-B1 gene reduced the amylose content by more than 3%. A reasonable agreement was found in the substitution lines. This confirms that the absence of the Wx-B1 gene, or else substitution of this gene by its null allele, has the most striking effect on decreasing amylose synthesis. The removal of chromosomes carrying either the Wx-A1 or the Wx-D1 gene reduces the amylose content by less than 2%. A similar reduction was revealed by substitution of these two genes by the null alleles. Double dosages of chromosomes 7A, 4A and 7D did not increase amylose content, while the tetrasomic chromosomes produced more of the respective Wx proteins. This suggests that a certain level of Wx gene activity or of the Wx proteins led to the maximum amount of amylose.     

Genetic linkage between endosperm storage protein genes on each of the short arms of chromosomes 1A and 1B in wheat

Summary The storage proteins of the endosperm of wheat grain which are known to be controlled by genes on the short arms of the homoeologous group 1 chromosomes are (1) the -gliadins, (2) most of the -gliadins, (3) a few -gliadins and (4) the major lowmolecular-weight subunits of glutenin. Several crosses were made between varieties or genetic lines which had contrasting allelic variants for some of these proteins and which were coded by genes on chromosomes 1A or 1B. The progeny were analysed by one or more of several electrophoretic procedures. The results of all the analyses are consistent with the hypothesis that chromosomes 1A and 1B each contain just one, complex locus, named Gli-A 1 and Gli-B 1 respectively, which contain the genes for the -, - and -gliadins and the low-molecular-weight subunits of glutenin.     

Identification of wheat-Agropyron cristatum monosomic addition lines by RFLP analysis using a set of assigned wheat DNA probes

A non-radioactive digoxigenin-labelled DNA method was used successfully to identify RFLP markers in 54 Triticum aestivum cv Chinese Spring — Agropyron cristatum (2n=28, genome PPPP) P-genome monosomic addition lines. Southern analysis using a set of 14 DNA probes identifying each homoeologous chromosome arm, combined with two restriction enzymes HindIII and EcoRI, indicated that six A. cristatum chromosomes (1P, 2P, 3P, 4P, 5P and 6P) and five A. cristatum chromosome arms (2PS, 2PL, 5PL, 6PS and 6PL) have been individually added to the wheat genome. The added chromosomes of three lines were Agropyron translocated chromosomes. It was also found that two addition plants possessed an Agropyron-wheat translocation. These results showed that RFLP analysis using the set of assigned wheat probes was a powerful tool in detecting and establishing homoeology of alien A. cristatum chromosomes, or arms, added to wheat, as well as in screening the alien addition material. The creation of the monosomic addition lines should be useful for the transfer of disease-resistance genes from A. cristatum to wheat.     

The chromosomal location of a third set of malate dehydrogenase loci,Mdh-3, in wheat,barley and related species

Summary A third set of malate dehydrogenase loci have been identified and located on the short arms of homoeologous group 5 chromosomes in wheat. Allelic differences have been found at each of the three Mdh-3 loci. However, Mdh-D3 appears to be least variable, with a second allele found only in Sears' Synthetic among a survey of 42 varieties. Homoeoloci were identified on chromosome 7 (5H) of Hordeum vulgare, the short arm of 5E in Agropyron elongatum and 5U in Aegilops umbellulata.     

Chromosome structure of Triticum longissimum relative to wheat

Homoeologous pairing at meiotic metaphase I was analyzed in T. longissimum x T. aestivum hybrids in order to reconfirm the homoeologous relationships of T. longissimum chromosomes to wheat. Hybrids between T. longissimum and Chinese Spring carrying the Ph1 gene or theph1b mutation, which showed low and high pairing levels, respectively, were used. Chromosome arms associated at metaphase I were identified by C-banding. The homoeology of chromosomes 1S ^l , 2S ^l , 3S ^l , 5S ^l and 6S ^l to wheat group 1,2, 3, 5, and 6 chromosomes, respectively, was confirmed. Chromsome arms 4S ^l S and 7S ^l S showed normal homoeologous relationships to wheat. The 4S ^l L arm carries a translocated segment from 7S ^l L relative to wheat. The 7S ^l L arm seldom paired, likely because this arm lost a relatively long segment and received a very short segment in the interchange with 4S ^l L. Available data suggest that translocation 4S ^l L/7S ^l L arose in the evolution of T. longissimum, which implies that this species was not the donor of the B genome of wheat.     

Rye chromosome arm 3RS encodes a homodimeric inhibitor of insect α-amylase

A new inhibitor of insect -amylase, designated RDAI-1, has been purified from rye (Secale cereale L.) endosperm. RDAI-1 is homologous to wheat homodimeric inhibitors. This homology is supported by their similar N-terminal amino-acid sequences, inhibitory activities towards amylases from Tenebrio molitor (Coleoptera) and human saliva, and aggregative properties in gel-filtration chromatography. The gene encoding RDAI-1, IdhaR1, is located on the short arm of chromosome 3R, which is homoeologous with wheat chromosome arms 3BS and 3DS, where the genes for homodimeric inhibitors have been previously mapped.     

Polymorphism and chromosomal location of endogenous α-amylase inhibitor genes in common wheat

Summary Polymorphism of an endogenous -amylase inhibitor in wheat was studied using iso-electric focusing followed by monoclonal antibody — based immunoblotting. Ten isoforms of the inhibitor detected in common wheat and its wild counterparts were assigned to five homoeologous loci. Three -amylase inhibitor loci (Isa-1) were identified in common wheat and located on the long arms of chromosomes 2A, 2B and 2D. In a sample of 27 bread wheats, eight durum wheats, and 12 diploid wheat relatives, amphiploids and triticales, a high resolution isoelectric-focusing separation demonstrated two active and one null allele at the Isa-A1, two alleles at the Isa-B1, one allele at the Isa-D1, four alleles at the Isa-S1, and one allele at the Isa-G1 locus. The most frequent electrophoretic pattern of common wheat cultivars consisted of two isoforms, encoded respectively by the Isa-B1b, Isa-D1 a alleles and the Isa-Alnull allele. All the durum wheats had only one inhibitor form controlled by allele Isa-B1b, which was accompanied by the null allele at the Isa-A1 locus.Contribution No. 210 of the Food Science Department, University of Manitoba     

Eyespot resistance gene Pch-1 from Aegilops ventricosa is associated with a different chromosome in wheat line H-93-70 than the resistance factor in “Roazon” wheat

Summary The hexaploid wheat line H-93-70 carries a gene (Pch-1) that has been transferred from the wild grass Aegilops ventricosa and confers a high degree of resistance to eyespot diesease, caused by the fungus Pseudocercosporella herpotrichoides. Crosses of the resistant line H-93-70 with the susceptible wheat Pané 247 and with a 7D/7Ag wheat/Agropyron substitution line were carried out and F2 kernels were obtained. The kernels were cut transversally and the halves carrying the embryos were used for the resistance test, while the distal halves were used for genetic typing. Biochemical markers were used to discriminate whether the transferred Pch-1 gene was located in chromosome 7D, as is the case for a resistance factor present in Roazon wheat. In the crosses involving Pané 247, resistance was not associated with the 7D locus Pln, which determines sterol ester pattern (dominant allele in H-93-70). In the crosses with the 7D/7Ag substitution line, resistance was neither associated with protein NGE-11 (7D marker), nor alternatively inherited with respect to protein C-7 (7Ag marker). It is concluded that gene Pch-1 represents a different locus and is not an allele of the resistance factor in Roazon wheat.     

Detection and characterization of a glutenin subunit with unusual high Mr at the Glu-A1 locus in hexaploid wheat

A hexaploid wheat landrace collected from the Baluchistan province of Pakistan was found to possess a novel high-molecular-weight glutenin subunit (HMW-GS). The subunit has a very slow electrophoretic mobility as revealed by SDS-PAGE, and its molecular weight is comparable to that of the highest molecular weight glutenin subunit (2.2 encoded in the D-genome) reported so far in hexaploid wheat varieties and landraces of Japanese origin. Evidence obtained from (PCR) gene amplification studies using the primers specific for Glu-1 loci proved that the gene coding for this novel subunit belongs to the Glu-A1 locus located on the long arm of chromosome 1A. Digestion of the amplified gene (PCR product) with restriction enzymes indicated that the novel gene differs from prevailing Glu-A1 alleles (null, 1 and 2^*) by an extra DNA fragment of approximately 600 base pairs. The results also indicated that the novel subunit is most probably a derivative of subunit 2^* that has very likely incorporated the 600-bp fragment following a process of unequal crossing over. The present findings were further substantiated by reserved phase high performance liquid chromatography (RP-HPLC) analysis.