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.     

Differential effects of Wx-A1, -B1 and -D1 protein deficiencies on apparent amylose content and starch pasting properties in common wheat

Waxy (Wx) protein is a granule-bound starch synthase (GBSS) responsible for amylose production in cereal endosperm. Eight isolines of wheat (Triticum aestivum L.) having different combinations of presence and absence of three Wx proteins, Wx-A1, -B1, and -D1, were produced in order to elucidate the effect of Wx protein deficiencies on the apparent amylose content and starch-pasting properties. An improved SDS gel electrophoresis showed that ’Bai Huo’ (a parental wheat) carried a variant Wx-B1 protein from an allele, Wx-B1e. Thus, wheat lines of types 1, 2, 4, and 6 examined in this study contained a variant Wx-B1 allele and not the standard allele, Wx-B1a. The results from 3 years of experiments using 176 lines derived from two cross-combinations showed that apparent amylose content increased the least in type 8 (waxy) having no Wx proteins and, in ascending order, increased in type 5 (only the Wx-A1 protein is present) <type 7 (Wx-D1) <type 6 (Wx-B1) <type 3 (Wx-A1 and -D1) <type 4 (Wx-A1 and -B1) <type 2 (Wx-B1 and -D1) <type 1 (three Wx proteins). However, Tukey’ s studentized range test did not detect significant differences in some cases. Densitometric analysis suggested that the amylose content was related to the amount of the Wx protein in the eight types. Parameters in the Rapid Visco-Analyzer test and swelling power were correlated to amylose content. Consequently, amylose content and pasting properties of starch were determined to be influenced the most by the lack of the Wx-B1 protein, followed by a lack of Wx-D1, and leastly by the Wx-A1 deficiency, which indicated the presence of differential effects of the three null alleles for the Wx protein. Received: 1 February 1999 / Accepted: 10 April 1999     

Identification of genetic loci affecting amylose content and agronomic traits on chromosome 4A of wheat

 Chromosome 4A of wheat carries the Wx-B1 gene encoding the granule-bound starch synthase involved in amylose synthesis in the endosperm. To determine the pleiotropic effects of this locus and effects of independent QTLs on agronomic traits, genetical analysis of chromosome 4A was conducted using 98 single-chromosome recombinant substitution lines derived from a cross of Chinese Spring and Chinese Spring (Kanto107 4A) with a low amylose content due to the null Wx-B1b allele. For amylose content, most of the genetic variation was explained by the allelic difference at the Wx-B1 locus. An additional QTL of minor effect was mapped in the 6.2-cM Xbcd1738/Xcdo1387 interval on the short arm, where the allele from Kanto107 led to an increase in amylose content. Field trials over two seasons revealed a pleiotropic effect of Wx-B1, or else the effect of a closely linked QTL, on ear emergence time. A QTL linked to Wx-B1 was detected for plant height. For plant yield and its components, there was no evidence for significant main effects associated with Wx-B1 or adjacent regions. One plant-yield QTL was identified by RFLP markers on the short arm and this was identical to QTLs controlling spikelet number/ear and grain weight/ear. At these QTLs for agronomic traits, alleles from Kanto107 contributed to an earlier emergence time, a height reduction and an yield increase. Received: 10 August 1998 / Accepted: 3 November 1998     

Molecular genetic characteristics of the <Emphasis Type="Italic">Wx-B1e</Emphasis> allele from common wheat and applicability of the DNA markers for its identification

Molecular genetic characterization of the Wx-B1e allele identified by the authors of the study in the common wheat cultivar Korotyshka was performed. The 804-bp Wx-B1e fragment was cloned and sequenced. Comparison of the sequence obtained with that for the wild-type allele of common wheat (Wx-B1a) demonstrated that Wx-B1e carried the 34-bp insertion, 8-bp deletion, and 23 nucleotide substitutions. BLAST analysis revealed the highest homology with the nucleotide sequences of Wx genes from Triticum spelta and Triticum durum. The amplification variants of four Wx-B1 molecular markers, applied worldwide for testing the collections for different Wx allelic variants, are demonstrated.     

Transposon insertion resulted in the silencing of <Emphasis Type="Italic">Wx-B1n</Emphasis> in Chinese wheat landraces

Key message

A novel Wx-B1 allele was characterized; a transposon insertion resulted in the loss of its function, which is different from the previously reported gene silencing mechanisms at the Wx-B1 locus.

Abstract

The waxy protein composition of 53 Chinese wheat landraces was analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional gel electrophoresis; of these, 10 did not show the expression of Wx-A1 (four accession) or Wx-B1 (six accessions) protein. The results of molecular marker detection revealed that the Wx-B1 allele (Wx-B1n) showed normal expression, inconsistent with the findings of SDS-PAGE for the Xiaobaipi accession. Further cloning of the 9160-bp region covering the Wx-B1 coding region and 3′-downstream region revealed that a 2178-bp transposon fragment had been inserted at 2462 bp within the tenth exon of Wx-B1n ORF, leading to the absence of Wx-B1 protein. Sequence analysis indicated that the insertion possessed the structural features of invert repeat and target repeat elements, we deduced that it was a transposon. Further PCR analysis revealed that this fragment had moved, but not copied itself, from 3B chromosome to the current location in Wx-B1n. Therefore, the reason for the inactivation of Wx-B1n was considerably different from those for the inactivation of Wx-B1b, Wx-B1k, and Wx-B1m; to our knowledge, this kind of structural mutation has never been reported in Wx-B1 alleles. This novel allele is interesting, because it was not associated with the deletion of other quality-related genes included in the 67 kb region lost with the common null allele Wx-B1b. The null Wx-B1n might be useful for investigating gene inactivation and expression as well as for enriching the genetic resource pool for the modification of the amylose/amylopectin ratio, thereby improving wheat quality.

     

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.     

Differential effects of the null alleles at the three Wx loci on the starch-pasting properties of wheat

The amylose/amylopectin ratio and the pasting properties of wheat starch are important in producing marketable flour products, especially Japanese noodles. To determine if null mutations at the three Wx loci confer differences in starch-pasting viscosity, we analyzed the variation associated with the null mutations in three separate sets of recombinant substitution lines of chromosomes 7A, 4A and 7D produced from crosses between Chinese Spring and three single-chromosome substitution lines carrying the null Wx alleles. Differential effects of null alleles at the three Wx loci on starch-pasting properties were revealed. With respect to chromosome 4A, the effect of the Wx-B1b allele, giving a higher peak and breakdown viscosity, was unambiguous. In addition, a QTL of minor effect was identified near the centromere on the short arm. The presence or absence of the Wx-A1 protein gave some variation in peak and breakdown viscosity, but the effects of Wx-Alb were much smaller than those of the Wx-Blb allele. Associated effects of the Wx-D1 locus were detected for the breakdown viscosity as the null Wx-D1b allele produced a higher viscosity than the wild-type Wx-D1a. While negative correlations between amylose content and breakdown viscosity were common in the three populations, the null mutations at the Wx loci produced some variation independent of amylose content. The genetic variation detected for breakdown viscosity was more evident than that for peak viscosity in all three recombinant populations. Received: 20 July 1999 / Accepted: 7 October 1999     

黄淮冬麦区不同时期主推品种淀粉合成酶基因分子标记鉴定

利用普通小麦直链淀粉合成酶GBSS基因及支链淀粉关键合成酶SSⅡ基因的特异分子标记鉴定了黄淮冬麦区自20世纪50年代以来253份主推品种,发现8份品种缺失Wx-B1基因,其中5份材料（小偃168、秦麦1号、83S502、山东935031、山东928802）是新发现的缺失Wx-B1基因的小麦品种,未发现Wx-A1、Wx-D1基因缺失类型品种;所鉴定253份品种的SSⅡ基因均为野生型,未发现缺失突变类型。通过对8份缺失Wx—B1基因品种直链淀粉含量分析,发现这些品种的直链淀粉含量差异较大．变异范围为19．9％-33．0％,其中豫麦47、83S502和中育5号3个品种的直链淀粉含量较低,分别为19．9％、21．3％和26．4％,可以用于优质面条小麦品质改良。     

Molecular genetic characteristics of the Wx-B1e allele from common wheat and applicability of the DNA markers for its identification

Molecular genetic characterization of the Wx-B1e allele identified by the authors of the study in the common wheat cultivar Korotyshka was performed. The 804-bp Wx-B1e fragment was cloned and sequenced. Comparison of the sequence obtained with that for the wild-type allele of common wheat (Wx-B1a) demonstrated that Wx-B1e carried the 34-bp insertion, 8-bp deletion, and 23 nucleotide substitutions. BLAST analysis revealed the highest homology with the nucleotide sequences of Wx genes from Triticum spelta and Triticum durum. The amplification variants of four Wx-B1 molecular markers, applied worldwide for testing the collections for different Wx allelic variants, are demonstrated.     

Distribution of puroindoline alleles in bread wheat cultivars of the Yellow and Huai valley of China and discovery of a novel puroindoline a allele without PINA protein

Kernel hardness is one of the most important factors determining the milling and processing quality of bread wheat (Triticum aestivum L.). In the present study, 267 wheat cultivars and advanced lines from the Yellow and Huai Valley of China, CIMMYT, Russia and Ukraine were used for identification of SKCS (Single Kernel Characterization System) hardness and puroindoline alleles. Results indicated that Pinb-D1b is the most popular genotype in wheat cultivars from the Yellow and Huai Valley, Russia and Ukraine, whereas PINA null is a predominant genotype in wheat cultivars and advanced lines from CIMMYT. Molecular characterization of PINA-null alleles indicated that one Chinese landrace Chiyacao had the allele Pina-D1l with a single nucleotide C deletion at position 265 in Pina coding region based on sequencing results, and 35 of 39 PINA-null alleles belonged to Pina-D1b according to PCR amplification with the sequence-tagged site (STS) marker Pina-N developed previously. The remaining three cultivars (Jiangdongmen, Heshangtou and Hongquanmang from China) with PINA-null alleles were characterized at the DNA level by a primer walking strategy, and the results showed that all three cultivars with PINA-null alleles possessed a uniform 10,415-bp deletion from −5,117 bp to +5,298 bp (ATG codon references zero), designated as Pina-D1r. Correspondingly, an STS marker Pina-N2 with an expected fragment size of 436-bp spanning the 10,415-bp deletion was developed for detection of the Pina-D1r allele. This study provided a useful molecular marker for straightforward detection of one of the PINA-null alleles and would also be helpful to further understand the molecular and genetic basis of kernel hardness in bread wheat.     

Molecular differentiation of null alleles at <Emphasis Type="Italic">ZCCT</Emphasis>-<Emphasis Type="Italic">1</Emphasis> genes on the A,B, and D genomes of hexaploid wheat

A dominant allele of the vernalization gene Vrn-2 is the wild type conferring winter growth habit, whereas a recessive vrn-2 allele confers spring growth habit. The recessive vrn-2 allele is mutated due to the deletion of the complete gene (a null form) or alternation of a key amino acid in the VRN-2 protein (a nonfunctional form) in diploid wheat or tetraploid wheat. VRN-2 is also denoted ZCCT due to the presence of a zinc finger and a CCT domain in its protein. There are two paralogous ZCCT genes at the VRN-2 locus in diploid Triticum monococcum and three paralogous ZCCT genes on each of the A and B genomes in tetraploid wheat, but little is known about the allelic variation in VRN-2 in hexaploid wheat. In the study reported here, we performed a one-shot PCR to simultaneously amplify the promoter regions of the three ZCCT-1 genes from hexaploid wheat, including the 302-bp fragment from ZCCT-A1, the 294-bp fragment from ZCCT-B1, and the 320-bp fragment from ZCCT-D1. Each amplicon could be differentiated by electrophoresis in an acrylamide/bisacrylamide gel. This PCR marker for different lengths of the three ZCCT-1 genes was used to search for null alleles in hexaploid wheat. A null allele was found in each of ZCCT-A1, ZCCT-B1, and ZCCT-D1 among 74 cultivars and genetic stocks of U.S. hexaploid wheat. Among 54 Chinese wheat cultivars, breeding lines, and landraces, we identified three accessions carrying a single null allele at ZCCT-A1, three accessions carrying a null allele at ZCCT-B1, and one accession carrying a double null allele at both ZCCT-A1 and ZCCT-D1. The potential application of these natural ZCCT-1 mutant materials in wheat breeding programs and studies on the genetics of wheat is discussed.     

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.     

GSTT1 and GSTM1 gene polymorphisms in European and African populations

Glutathione S-transferases (GSTs) are a superfamily of detoxificant enzymes. Pharmacogenomic studies have revealed interethnic differences in GST allelic frequencies. This study is focused on GSTT1 (gene deletion, rs17850155, rs2234953, and rs11550605) and GSTM1 (gene deletion) gene frequency distributions in two population samples of Europe origin (Italy, n = 120; Spain, n = 94) and two population samples of Africa origin (Cameroon, n = 126; Ethiopia, n = 153). Detection of GSTT1 and GSTM1 null genotypes was performed by multiplex PCR analysis, while the other GSTT1 gene polymorphisms were detected using allele specific PCR and sequencing. GSTT1 and GSTM1 null frequencies in the samples analyzed fit with the variability range observed in European and African populations, respectively. The SNP analysis in GSTT1 gene did not highlight any nucleotide substitution in 493 individuals analyzed. The comparisons among GSTM1 and GSTT1 null phenotype frequencies in worldwide populations show different patterns between Asians, Africans, and Europeans. Important insights into the effects of GSTM1 and GSTT1 gene deletions on the pathogenesis of human diseases have been hypothesized. Detailed studies on the geography of GST variants could therefore increase knowledge about the relationship between ethnicity and the prevalence of certain diseases.     

Identification of microsatellite markers linked to leaf rust resistance gene <Emphasis Type="Italic">Lr25</Emphasis> in wheat

The leaf rust resistance gene Lr25, transferred from Secale cereale L. into wheat and located on chromosome 4B, imparts resistance to all pathotypes of leaf rust in South-East Asia. In an F₂-derived F₃ population, created by crossing TcLr25 that carries the gene Lr25 for leaf rust resistance with leaf rust-susceptible parent Agra Local, three microsatellite markers located on the long arm of chromosome 4B were found to be linked to the Lr25 locus. The donor parent TcLr25 is a near-isogenic line derived from the variety Thatcher. The most virulent pathotype of leaf rust in the South-East Asian region, designated 77–5 (121R63-1), was used for challenging the population under artificially controlled conditions. The marker Xgwm251 behaved as a co-dominant marker placed 3.8 cM away from the Lr25 locus on 4BL. Two null allele markers, Xgwm538 and Xgwm6, in the same linkage group were located at a distance of 3.8 cM and 16.2 cM from the Lr25 locus, respectively. The genetic sequence of Xgwm251, Lr25, Xgwm538, and Xgwm6 covered a total length of 20 cM on 4BL. The markers were validated for their specificity to Lr25 resistance in a set of 43 wheat genetic stocks representing 43 other Lr genes.     

Distinct breakpoints in two cases with deletion in the Yp11.2 region in Japanese population

The amelogenin gene on the Y chromosome (AMELY) is a homolog of the X chromosome amelogenin gene (AMELX), and the marker is employed for sexing in forensic casework. Deletion of the sequences in the Yp11.2 region containing the AMELY locus has been found in males from various ethnic populations. Two cases of AMELY null males found in the Japanese population had different Y haplogroups and deletion mapping. Proximal and distal breakpoints of a sample of haplogroup D2* were located in TSPYA and TSPYB arrays, respectively, suggesting that the deletion mechanism was non-allelic homologous recombination (NAHR). On the other hand, a sample of haplogroup O3a3c* had the distal breakpoint in the TSPYB array and the proximal breakpoint at position 7.94 Mb, not in the TSPYA array. The likely deletion mechanism is non-homologous end-joining. High-resolution STS mapping in the TSPYB array showed the distal breakpoints differed according to the haplogroups. The deletion length was estimated as 3.1–3.7 Mb and 1.6–1.7 Mb for the sample of haplogroup D2* and O3a3c*, respectively. These deletion events should have occurred independently.     

A new PCR-based marker on chromosome 4AL for resistance to pre-harvest sprouting in wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Pre-harvest sprouting (PHS) is a complex trait controlled by multiple genes with strong interaction between environment and genotype that makes it difficult to select breeding materials by phenotypic assessment. One of the most important genes for pre-harvest sprouting resistance is consistently identified on the long arm of chromosome 4A. The 4AL PHS tolerance gene has therefore been targeted by Australian white-grained wheat breeders. A new robust PCR marker for the PHS QTL on wheat chromosome 4AL based on candidate genes search was developed in this study. The new marker was mapped on 4AL deletion bin 13-0.59-0.66 using 4AL deletion lines derived from Chinese Spring. This marker is located on 4AL between molecular markers Xbarc170 and Xwg622 in the doubled-haploid wheat population Cranbrook × Halberd. It was mapped between molecular markers Xbarc170 and Xgwm269 that have been previously shown to be closely linked to grain dormancy in the doubled haploid wheat population SW95-50213 × Cunningham and was co-located with Xgwm269 in population Janz × AUS1408. This marker offers an additional efficient tool for marker-assisted selection of dormancy for white-grained wheat breeding. Comparative analysis indicated that the wheat chromosome 4AL QTL for seed dormancy and PHS resistance is homologous with the barley QTL on chromosome 5HL controlling seed dormancy and PHS resistance. This marker will facilitate identification of the gene associated with the 4A QTL that controls a major component of grain dormancy and PHS resistance.