@article{oai:repository.naro.go.jp:00001363,
 author = {FUNATSUKI, Wakako and 船附, 稚子},
 journal = {北海道農業研究センター研究報告, Research BUlletin of the NARO Hokkaido Agricultural Research Center},
 month = {Nov},
 note = {The annual consumption of strong wheat flour in Japan now exceeds two million tons. Although consumers are demanding foods made with domestic flour, domestic production of strong flour is less than 1% of total consumption. The use of blended flour consisting of medium-soft flour and extra-strong flour improves the quality of various foods such as bread, chinese yellow-alkaline noodles, and instant noodles. However, there are few strong wheat cultivars and no extra-strong wheat cultivars in Japan. About half of the total amount of wheat produced in Japan is culitivated in Hokkaido, a cold region of Japan. It is therefore essential to breed strong and extra-strong wheat varieties suitable for this region. High-molecular-weight (HMW) glutenin subunits (GSs) that confer a strong dough property and good bread-making quality have been intensively studied. HMW-GS 5+10, encoded by the gene Glu-D1 , plays an important role in dough properties appropriate for bread-making and has been used as a protein marker in selection of strong wheat varieties. On the other hand, the allelic form, HMW-GS 2+12, is widely present in Japanese wheat cultivars and is not greatly involved in strong dough properties. Although it is known that the compositions of low-molecular-weight (LMW) GSs are important factors for strong dough properties of wheat, it has not been determined which LMW-GSs confer an extra-strong dough property and how those LMW-GSs interact with HMW-GSs. The aim of this study was to identify LMW-GSs associated with a strong dough property and to develop DNA markers linked to such LMW-GSs genes. In this study, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) was performed even in segregating populations to enable intensive monitoring of glutenin components, since the difficulty in resolving LMW-GSs is a bottleneck in research on LMW-GS. 1. Identification of LMW-GSs associated with a strong dough property A near-isogenic line (NIL) in which LMW-GSs GL1 and GL2 are introduced from a Canadian Western Extra-Strong wheat cultivar, 'Glenlea', into a Japanese spring wheat cultivar, 'Harunoakebono', has much stronger dough property than does 'Harunoakebono'. GL1 and GL2 are therefore LMW-GSs associated with the strong dough property of the 'NIL'. Protein analysis for BC_5F_2 progenies derived from a cross between 'Harunoakebono' and 'Glenlea' showed that GL1 and GL2 are cosegregated and that HA1 of 'Harunoakebono' is an allelic form to GL1/GL2. Protein analysis for F_2 progenies derived from a cross between 'Chinese Spring' and 'Glenlea' and for ditelocentric lines of 'Chinese Spring' revealed that GL1 and GL2 are derived from the short arm of chromosome 1B (probably Glu-B3 locus). However, these LMW-GSs have not been found to correspond to any allele previously reported. Using a hard red winter wheat line, 'KS831957', with an extra-strong dough property and a Japanese wheat cultivar with a medium dough property as well as their progenies, 'Kachikei 32', 'Kachikei 33' and 'Kachikei 34', we identified LMW-GSs, KS1-KS5, associated with the extra-strong dough property. KS2 and KS3 of 'KS831957' comigrated with GL1 and GL2, respectively, in SDS-PAGE. KS3 was resolved into four protein spots in 2D-PAGE, and only two with higher pls are segregated in 'Kachikei 34'. Therefore, the two spots were referred to as KS3a subunit. 2D-PAGE for F_7 RILs derived from a cross between 'Kachikei 32' and 'Kachikei 34' showed that KS2 and KS3a are cosegregated and that KS2/KS3a is an allelic form to HS1 of 'Horoshirikomugi' and 'Kachikei 32'. Flour evaluation tests for F_6 RILs revealed that KS2/KS3a and HMW-GS 5+10 show interaction effects on the strong dough property. 2D-PAGE analysis showed that KS2, KS3a and HS1 are resolved similarly to GL1, GL2 and HA1, respectively. Furthermore, the N-terminal amino acid sequences of all protein spots comprising KS2, KS3a, GL1, and GL2 were identical to 'SHIPGLERPSQQQPLPP', which is the most-frequently analyzed sequence of LMW-GS and is classified as LMW-s (LMW-GS starting with serine). All N-terminal amino acid sequences of spots for HS1 and HA1 that we could determine are also the same as the above sequence. 2. Identification of candidate genes corresponding to LMW-GSs associated with a strong dough property Reverse transcription-polymerase chain reactions (RT-PCRs) using primers specific to an LMW glutenin gene, S-type2F/S-type978R, were performed for 'Harunoakebono', 'NIL' and 'Glenlea' and amplified a novel LMW-s glutenin gene, GLEN42K, for 'Glenlea' and 'NIL' and another LMW-s glutenin gene, HARU48K, for 'Harunoakebono'. The nucleotide sequence of HARU48K had 99% identity to that of Y17845, which is a gene corresponding to the 42-kDa subunit of 'Yecora Rojo' associated with good bread-making quality. Alignment of the deduced amino acid sequences of GLEN42K and HARU48K revealed that the identity is 86.2% and that GLEN42K has a shorter repetitive domain and a motif of fifteen continuous glutamine residues in the repetitive domain. The deduced amino acid sequences of the two genes included 'SHIPGLERPSQQQPLPP'. All available LMW-s glutenin gene candidates including the deduced amino acid sequence 'SHIPGLERPSQQQPLPP' are thought to lack nucleotides corresponding to several residues in the N-termini of signal peptides. Thus, HARU48K and GLEN42K are the first full-sized LMW-s glutenin genes including the deduced amino acid sequｅnce 'SHIPGLERPSQQQPLPP'. Genomic PCR using primers, s-F1/s-R2, designed on the basis of inner sequences of the genes was carried out for the BC_5F_2 progenies and amplified two DNA fragments that correspond to GLEN42K and HARU48K . The two DNA fragments are respectively cosegregated with LMW glutenin components comprising GL1/GL2 and the allelic components comprising HA1. GLEN42K is therefore a candidate gene encoding at least one protein spot comprising GL1/GL2, and HARU48K is thought to be a gene encoding at least one protein spot comprising HA1. There is also a possibility these LMW-s genes are linked to genes directly encoding GL1/GL2 and HA1, respectively. RT-PCR using S-type2F/S-type978R was performed for 'KS831957', 'Horoshirikomugi' and 'Kachikei 34' and amplified a candidate gene for KS2/KS3a, KANS2, and another candidate gene for HS1, HORO1. The partial inner sequences of the two genes that were amplified by genomic PCR cosegregated with KS2/KS3a and HS1, respectively, in F_6 RILs. It was also found that KANS2 has almost the same sequence as that of GLEN42K and that HORO1 and HARU48K perfectly coincide. HA1 of 'Harunoakebono', HS1 of 'Horoshirikomugi', and the 42-kDa subunit of 'Yecora Rojo' are thought to be homologous LMW-GS in that their candidate genes have very high identities and that they are resolved similarly in electrophoresis analysis. These LMW-GSs might all be chain extenders to accelerate glutenin polymer formations as predicted for the 42-kDa subunit of 'Yecora Rojo'. GL1/GL2 of 'Glenlea' and KS2/KS3a of 'Horoshirikomugi' are also thought to be homologous since they have similar characteristics on the basis of genes and proteins. The fact that GL1/GL2 (KS2/KS3a) has greater effects on strong dough properties than does HA1 (HS1) might be caused by the structure as chain extenders and their higher amount level of GL1/GL2 (KS2/KS3a). In addition, interactions between a hydrogen-bond state caused by the glutamine motif and the high-dimensional structure of GL1 and/or GL2 (KS2 and/or KS3a) might affect gluten formation. Furthermore, these probable characteristics of GL1/GL2 (KS2/KS3a) may explain the effects of interaction between KS2/KS3a and HMW-GS 5+10 on the strong dough property. We hypothesized that a wheat variety carrying GL1/GL2 (KS2/KS3a) and HMW-GS 5+10 could have an extra-strong dough property. 3. DNA marker to detect genes corresponding to LMW-GSs associated with a strong dough property and its application to a breeding program The inner DNA fragment of GLEN42K and KANS2 that is amplified by genomic PCR in this study could be used as a DNA marker in marker-assisted selection for breeding varieties carrying GL1/GL2 or KS2/KS3a. The DNA marker may also be useful for detecting LMW glutenin components similar to GL1/GL2 and KS2/KS3a in other wheat varieties. A small-scale trial examination of selection showed that varieties carrying both the 5+10 marker and KS2/KS3a (GL1/GL2) marker tend to have stronger dough properties than do varieties with only the 5+10 marker. It is therefore thought that extra-strong varieties could be bred by selection using a convenient PCR method.},
 pages = {1--86},
 title = {小麦粉の生地物性を強める低分子量グルテニンタンパク質の同定とその育種的利用に関する研究},
 volume = {183},
 year = {2005},
 yomi = {フナツキ, ワカコ}
}