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.

Molecular comparison of waxy null alleles in common wheat and identification of a unique null allele.

PCR selection markers for the identification of null waxy alleles were used to screen for waxy mutations in 168 common wheat cultivars. In all cultivars where the Wx-B1 protein was absent, the Wx-B1 allele was identical to the previously identified mutation carried by Kanto 107. Although most cultivars missing the Wx-A1 protein also carried the same Wx-A1 mutation as found in Kanto 107, all of the Turkey Wx-A1 mutants produced a different PCR fragment, implying the presence of a different mutation. Sequencing of this fragment indicated the mutation, which consisted of a 173-bp insertion in an exon, was in a different location than the previously identified Wx-A1 mutation. An 8-bp duplication of the Wx-A1 sequence flanked each end of the insertion, and an element with reverse complementary sequences was present at both ends of the insertion. These structures correspond with the features of class II transposable elements. Hence, the Turkey null Wx-A1 mutation was likely caused by the movement of a transposon, and this spontaneous mutation appears to be present in a limited geographical area.

Rapid classification of partial waxy wheats using PCR-based markers.

Mutations in the three homeologous waxy loci Wx-A1, Wx-B1, and Wx-D1 of a waxy wheat line have previously been characterized at the molecular level. Using combinations of these mutations, six types of partial waxy wheat plus wild type and waxy wheat (types 1-8) can be produced. Here, we describe primer sets for all three loci that can be used under a single set of conditions, allowing 32 lines to be characterized as types 1-8 in a single PCR run using a 96-well plate. Using multiplex PCR, mutations at the Wx-B1 and Wx-D1 loci can be identified in a single PCR, reducing the number of reactions necessary to identify and select the desired partial waxy wheat line. A single multiplex PCR can be used to detect all three mutations when products are analyzed using capillary electrophoresis on a microchip device. The PCR conditions and primers are effective with a number of cultivars from other countries, indicating that the mutations found at the Wx-A1 and Wx-B1 loci of these cultivars likely have the same origins as the mutations in the corresponding loci of the waxy wheat line used in this study. The PCR selection method described here is an easy and effective alternative to the commonly used SDS-PAGE methods for identification of null alleles.

Wheat granule-bound starch synthase I and II are encoded by separate genes that are expressed in different tissues.

Studies of waxy mutations in wheat and other cereals have shown that null mutations in genes encoding granule-bound starch synthase I (GBSSI) result in amylose-free starch in endosperm and pollen grains, whereas starch in other tissues may contain amylose. We have isolated a cDNA from waxy wheat that encodes GBSSII, which is thought to be responsible for the elongation of amylose chains in non-storage tissues. The deduced amino acid sequences of wheat GBSSI and GBSSII were almost 66% identical, while those of wheat GBSSII and potato GBSSI were 72% identical. GBSSII was expressed in leaf, culm, and pericarp tissue, but transcripts were not detected in endosperm tissue. In contrast, GBSSI expression was high in endosperm tissue. The expression of GBSSII mRNA in pericarp tissue was similar at the midpoints of the day and night periods. The GBSSII genes were mapped to chromosomes 2AL, 2B, and 2D, whereas GBSSI genes are located on group 7 chromosomes. Gel-blot analysis indicated that genes related to GBSSII also occur in barley, rice, and maize. The possible role of GBSSII in starch synthesis is discussed.

Characterization of cDNAs expressed in the early stages of microspore embryogenesis in barley (Hordeum vulgare) L.

To gain insight into the molecular events occurring in the very early stages of barley microspore embryogenesis, cDNA clones corresponding to genes differentially expressed during the early stages of microspore culture were isolated and characterized. A cDNA library established from barley microspores cultured for three days was differentially screened against probes generated from freshly isolated microspores. Three cDNAs representing genes not previously identified in barley were isolated. ECA1 (early culture abundant 1) lacked significant homology to known genes or proteins, and the transcript was only expressed during the early stages of culture. Expression was also reduced in low-density control cultures, therefore this gene may play a role in the early stages of barley microspore embryogenesis. ECGST (early culture glutathione S-transferase) had homology to parA-like genes, which are members of a newly discovered group of glutathione S-transferases (GSTs). The protein corresponding to ECGST may be important in protecting cells from oxidative stress during the culture process. ECLTP (early culture lipid transfer protein) had homology to lipid transfer proteins (LTPs), and had an expression pattern similar to that of an LTP known to be a marker of the early stages of embryogenesis in the carrot somatic embryogenesis system. The identification and characterization of the clones isolated in this study provides new information on the events involved in barley microspore embryogenesis.

Molecular characterization of waxy mutations in wheat.

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-A1h, Wx-B1b and Wx-D1b) 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-B1b allele had sustained a deletion which included the entire coding region of the Wx-B1 gene. DNA homologous to waxy gene sequences was still present in the Wx-A1b and Wx-D1b 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-A1b and Wx-D1b alleles. A 23-bp deletion sustained by the Wx-A1b 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-A1b 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-D1b cDNA lacks the last 30 amino acids. Comparison of the genomic sequences of the null and wild-type Wx-D1 alleles indicated that 588 bp were deleted in the Wx-D1b mutation, and that the last 261 bp of the Wx-D1b cDNA originated from the normally non-transcribed 3' flanking region. Like several deletion mutations characterized in other plant species, both Wx-A1b and Wx-D1b alleles contain small DNA insertions, or filler DNA, between the deletion end-points.

Characterization of a granule-bound starch synthase isoform found in the pericarp of wheat.

Waxy wheat (Triticum aestivum L.) lacks the waxy protein, which is also known as granule-bound starch synthase I (GBSSI). The starch granules of waxy wheat endosperm and pollen do not contain amylose and therefore stain red-brown with iodine. However, we observed that starch from pericarp tissue of waxy wheat stained blue-black and contained amylose. Significantly higher starch synthase activity was detected in pericarp starch granules than in endosperm starch granules. A granule-bound protein that differed from GBSSI in molecular mass and isoelectric point was detected in the pericarp starch granules but not in granules from endosperm. This protein was designated GBSSII. The N-terminal amino acid sequence of GBSSII, although not identical to wheat GBSSI, showed strong homology to waxy proteins or GBSSIs of cereals and potato, and contained the motif KTGGL, which is the putative substrate-binding site of GBSSI of plants and of glycogen synthase of Escherichia coli. GBSSII cross-reacted specifically with antisera raised against potato and maize GBSSI. This study indicates that GBSSI and GBSSII are expressed in a tissue-specific manner in different organs, with GBSSII having an important function in amylose synthesis in the pericarp.