Quality traits. Waxy mutants
References
1. A PCR-based marker for selection of starch and potential noodle quality in wheat. Briney A, Wilson R, Potter RH, Barclay I, Crosbie G, Appels R, Jones MGJ. In: Molecular Breeding, 1998, 4(5):427-433.
A strong association between the absence of the granule-bound starch synthase (GBSS) protein for the 4A chromosome of wheat and Japanese Udon noodle quality has been previously described. The aim of this study was to identify a molecular marker linked to the GBSS 4A locus which could be used to identify wheat with the desired texture for Udon noodles. PCR primers were designed to target this gene which gave a 440 bp PCR band, corresponding to the presence or absence of the 4A GBSS gene. Of the 268 genotypes screened with these primers, 267 were correctly identified using the PCR primers. The remaining genotype was shown to be heterogeneous for the marker. The PCR marker test developed has advantages over existing methods used to screen for Udon noodle starch quality as it enables high throughput, accurate tests to be carried out on leaves of young seedlings or mature seed and identify breeding lines that are heterogeneous for the 4A allele which will allow for reselections. Application of this PCR test will speed up selection for Udon noodle quality genotypes and reduce breeding costs for production of noodle wheat varieties.
2. Application of a high-throughput antibody-based assay for identification of the granule-bound starch synthase Wx-B1b allele in Australian wheat lines. Gale KR, Panozzo JF, Eagles HA, Blundell M, Olsen H, Appels R. In: Australian Journal of Agricultural Research, 2001, 52(11-12):1417-1423.
An enzyme-linked immunosorbent assay (ELISA) for the discrimination of Wx-B1a and Wx-B1b genotypes at the granule-bound starch synthase I (GBSSI) or waxy locus of hexaploid wheat (Triticum aestivum L.) was adapted to a high-throughput, 96-well microtitre plate format. This test is applicable to the direct analysis of starch, flour, or crushed grain and requires less than 1 grain to perform. Several hundred samples may be routinely analysed in one day. The assay was validated using quantitative trait locus (QTL) analysis of a doubled haploid mapping population of the cross Cranbrook (Wx-B1a)/Halberd (Wx-B1b). This demonstrated that the assay unambiguously identified 153 of 161 lines analysed, with a highly significant QTL (LRS value 270) accounting for 83% of ELISA variation, at the Wx-B1 locus on chromosome 4AL. In addition, measurement of total GBSSI variation using a non-isoform-specific GBSSI detection monoclonal antibody also gave a significant QTL (LRS of 84, accounting for 42% of ELISA variation) at the Wx-B1 locus. Application of the assay to crude flour extracts of 8 grains for each of 1093 progeny from 4 crosses segregating at the Wx-B1 locus permitted the unambiguous scoring of lines as pure Wx-B1a or pure Wx-B1b. The scoring by ELISA was strongly related to the flour swelling volume of the lines, thus demonstrating the utility of this high-throughput screening method for the faster, more efficient development of Australian noodle wheats.
3. Waxy wheats: Origin, properties, and prospects Graybosch RA. In: Trends in Food Science & Technology, 1998, 9(4):135-142
Starch amylose is synthesized through the activity of the granule-bound starch synthase (GBSS). In wheat (Triticum aestivum L.), there are three structural genes encoding isoforms of GBSS. Naturally occurring mutations (null alleles) resulting in the loss of one or more GBSS isoforms have recently been identified. The presence of one or two GBSS null alleles results in the production of starch with reduced amylose content. Reduced amylose wheats have been termed `partial waxy'. Wheats with three GBSS null alleles produce essentially amylose-free, or waxy, starch. Partial waxy wheats are sources of flours with optimal quality characteristics in certain Asian wet noodle products. In addition, partial waxy wheats are essential to the development of waxy wheats with acceptable agronomic performance. Biochemical features of starch from waxy wheats are similar to those of waxy maize. Waxy wheats may find application in the production of modified food starches, as blending wheats for the formulation of superior noodle flours, and as a means to manipulate amylose contents in substrates for extrusion. Flour from waxy wheats may also be used to extend the shelf-life of baked goods, without a concomitant dilution of wheat gluten. Finally, waxy wheat may increase profitability to gluten manufacturers by providing a co-product with added value.
4. Development of robust PCR-based DNA markers for each homoeo-allele of granule-bound starch synthase and their application in wheat breeding programs. McLauchlan A, Ogbonnaya FC, Hollingsworth B, Carter M, Gale KR, Henry RJ, Holton TA, Morell MK, Rampling LR, Sharp PJ, Shariflou MR, Jones MGK, Appels R.In: Australian Journal of Agricultural Research, 2001, 52(11-12):1409-1416.
The absence of expression of the granule-bound starch synthase I (GBSSI) allele from chromosome 4A of wheat is associated with improved starch quality for making Udon noodles. Several PCR-based methods for the analysis of GBSS alleles have been developed for application in wheat. A widely applied approach has involved a simple PCR followed by electrophoretic separation of DNA products on agarose gels. The PCR amplifies one band from each of the loci on chromosomes 4A (Wx-B1), 7A (Wx-A1), and 7D (Wx-D1), and the band from the Wx-B1 locus is diagnostic for the occurrence of the null Wx-B1 allele that is associated with improved starch quality. The reliable detection of the null Wx-B1 allele has been important in identifying wheat breeding lines. Allele-specific PCR has also been used to successfully detect the occurrence of the null Wx-B1 allele. In the present paper the various protocols were evaluated by testing a segregating double haploid population from a cross between Cranbrook and Halberd and the tests gave good agreement in different laboratories. The application of the DNA-based tests applied in wheat breeding programs provides one of the first examples of a molecular marker selection for a grain quality trait being successfully applied in an Australian wheat breeding program.
5. Registration of D-null "Bai Huo" waxy wheat germplasm. . Morris CF, Konzak CF. In: Crop Science, 2000, 40(1):304-305.
6. A polymorphic microsatellite in the 3' end of 'waxy' genes of wheat, Triticum aestivum. Shariflou MR, Sharp PJ. In: Plant Breeding, 1999, 118(3):275-277.
Potential polymorphism of an (AT)n microsatellite at the 3' end of waxy genes in
bread wheat was examined. Primers were designed from a published cDNA sequence
of a wheat waxy gene. Polymerase chain reaction (PCR) amplification of genomic
DNA from 135 mainly Australian cultivars revealed eight alleles on chromosome
7A. This polymorphic microsatellite is a potential codominant marker for the
Wx-A1 locus in breeding programmes. A distinguishable fragment was also
amplified from chromosome 7D. This fragment was absent where a plant was null
for the waxy gene on chromosome 7D, being a dominant marker for the Wx-D1 locus.
The primers were also useful for amplifying genomic DNA from barley, rye and
triticale and can be used to detect potential polymorphism in these species.
7. Molecular characterization of waxy mutations in wheat. Vrinten P, Nakamura T, Yamamori M. In: Molecular and General Genetics, 1999, 261(3):463-471.
To date, few mutations in wheat have been characterized at the molecular level. In this study, the mutations in the three waxy alleles in waxy wheat (Wx-Alb, Wx-Blb and Wx-Dlb) were characterized, and waxy gene expression was compared in several wheat lines, including hexaploid and tetraploid waxy lines of wheat. Southern analysis showed that the Wx-Blb allele had sustained a deletion which included the entire coding region of the Wx-Bl gene. DNA homologous to waxy gene sequences was still present in the Wx-Alb and Wx-Dlb alleles of the hexaploid waxy mutant. Transcripts of waxy alleles were also detected in both hexaploid and tetraploid mutants at 10 days post-anthesis. but the transcript level was dramatically reduced compared to that found in non-waxy lines. Isolation of cDNAs showed that transcripts were produced by both the Wx-Alb and Wx-Dlb alleles. A 23-bp deletion sustained by the Wx-Alb allele at an exon-intron junction results in the use of a cryptic splice site during mRNA processing. The deduced translation product encoded by the Wx-Alb cDNA lacks 39 amino acids, including the putative ADP-glucose binding site and a portion of the transit peptide. The C-terminal region of the deduced protein encoded by the Wx-Dlb cDNA lacks the last 30 amino acids. Comparison of the genomic sequences of the null and wild-type Wx-Dl alleles indicated that 588 bp were deleted in the Wx-Dlb mutation, and that the last 261 bp of the Wx-Dlb cDNA originated from the normally non-transcribed 3' flanking region. Like several deletion mutations characterized in other plant species, both Wx-Alb and Wx-Dlb alleles contain small DNA insertions, or filler DNA, between the deletion end-points.
8. The genes encoding granule-bound starch synthases at the waxy loci of the A, B, and D progenitors of common wheat.. Yan L, Bhave M, Fairclough R, Konik C, Rahman S, Appels R. In: Genome, 2000, 43(2):264-272
Three genes encoding granule-bound starch synthase (wx-TmA, wx-TsB, and wx-TtD) have been isolated from Triticum monococcum (AA), and Triticum speltoides (BB), by the polymerase chain reaction (PCR) approach, and from Triticum tauschii (DD), by screening a genomic DNA library. Multiple sequence alignment indicated that the wx-TmA, wx-TsB, and wx-TtD genes had the same extron and (or) intron structure as the previously reported waxy gene from barley. The lengths of the three wx-TmA, wx-TsB, and wx-TtD genes were 2834 bp, 2826 bp, and 2893 bp, respectively, each covering 31 bp in the untranslated leader and the entire coding region consisting of 11 exons and 10 introns. The three genes had identical lengths of exons, except exon1, and shared over 95% identity with each other within the exon regions. The majority of introns were significantly variable in length and sequence, differing mainly in length (1-57 bp) as a result of insertion and (or) deletion events. The deduced amino acid sequence from these three genes indicated that the mature WX-TMA, -TSB, and -TTD proteins contained the same number of amino acids, but differed in predicted molecular weight and isoelectric point (pI) due to amino acid substitutions (13-18). The predicted physical characteristics of the WX proteins matched the respective proteins in wheat very closely, but the match was not perfect. Furthermore the exon5 sequences of the wx-TmA, wx-TsB, and wx-TtD genes were different from a cDNA encoding a waxy gene of common wheat previously reported. The striking difference was that an insertion of 11 amino acids occurred in the cDNA sequence that could not be observed in the exons of the A, B, and D genes. It was noted, however, that the 3' end of intron4 of these genes could account for the additional 11 amino acids. The sequence information from the available waxy genes identified the intron4-exon5-intron5 region as being diagnostic for sequence variation in waxy. The sequence variation in the waxy genes provides the basis for primer design to distinguish the respective genes in common wheat, and its progenitors, using PCR.
9. Differential effects of Wx-A1,-B1 and-D1 protein deficiencies on apparent amylose content and starch pasting properties in common wheat. Yamamori M, Quynh NT. In: Theoretical and Applied Genetics, 2000, 100:32-38
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 Hue' (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.
10. A PCR-based DNA marker for detection of mutant and normal alleles of the Wx-D1 gene of wheat . Shariflou MR, Hassani ME, Sharp PJ. In: Plant Breeding, 2001, 120(2):121-124.
To assist waxy wheat breeding a DNA marker was developed to discriminate mutant and normal alleles at the Wx-D1 locus. This polymerase chain reaction-based marker distinguishes the mutant from the normal allele by targeting the previously reported deletion basis of the mutant. The marker codominantly identifies the normal allele of the Wx-D1 gene from the mutant allele originated from the Chinese landrace 'Baihoumai'. However, attempts with a number of primer combinations targeting this deletion failed to amplify the corresponding fragment from an unrelated wheat line (NP150) that has a mutant null allele at the same locus. This indicates that NP150 has a different mutant allele from that of 'Baihoumai'. This marker is a useful tool to identify wheat cultivars with mutant and normal alleles of the Wx-D1 gene, and is used in marker-assisted selection of the Wx-D1 gene in our waxy wheat breeding programme.
11. Production of all eight genotypes of null alleles at 'waxy' loci in bread wheat, Triticum aestivum L.. Zhao XC, Sharp PJ. In: Plant Breeding, 1998, 117:488-490.
12. Isolation and characterization of the three Waxy genes encoding the granule-bound starch synthase in hexaploid wheat.. Murai J, Taira T, Ohta D. In: Gene, 1999, 234(1):71-79.
Complete genomic DNA sequences of 3 homoeologous Waxy structural genes, located on chromosomes 7A, 4A and 7D in hexaploid wheat (Triticum aestivum cv. Chinese Spring), were separately determined and analysed. Lengths of structural genes, from start to stop codon, were 2781 bp in Wx-7A, 2794 bp in Wx-4A and 2862 bp in Wx-7D, and each comprised 11 exons and 10 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 Wx-4A 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. Nucleotide sequence data have been deposited under DDBJ/EMBL/GenBank accession numbers AB019622 (Wx-7A), AB019623 (Wx-4A) and AB019624 (Wx-7D).
13. Nucleotide sequence of a wheat (Triticum aestivum L.) cDNA clone encoding the waxy protein.. Clark JR, Robertson M, Ainsworth CC. In: Plant Molecular Biology, 1991, 16(6):1099-1101.
This cDNA (sequence given) was isolated from a cDNA library derived from mRNA isolated from grains of cv. Chinese Spring harvested 20 days post anthesis by screening with a barley waxy protein cDNA. It contained an open reading frame of 1845 nucleotides encoding a deduced protein of 615 amino acids with a MW of 67.7 kDa.