본 연구는 국립농업과학원 농업유전자원센터와 일본 NIAS가 보유하고 있는 밀 유전자원 중 한국, 일본, 중국원산 밀 재래종 유전자원 486자원을 대상으로, 국내 적응성판별을 위한 성숙기 특성과 제빵 특성평가를 위하여 분자마커와 SDS-PAGE를 이용하여 고분자글루테닌 조성을 분석하였으며, 또한 이에 대한 원산지별 유전적 다양성을 조사하고 유용자원을 선발하였다. 결과는 다음과 같다. 첫째, SDS-PAGE와 DNA marker를 이용한 고분자글루테닌 분석결과 기존의 보고와 조성패턴이 일치하는 자원은 전체의 381자원으로 78.6%이고, 이중 24자원은(5.6%)는 재래종 유전자원 특성상 불균일성으로 인해 한 allele에 중첩된 band pattern을 보이는 자원이었으며, 13자원(3.2%)은 기존보고에서 볼 수 없는 새로운 band pattern을 보이는 자원으로 비슷한 유형별로 그룹화해 추후 DNA sequencing 분석이 필요할 것으로 생각된다. 둘째, 한국과 일본 자원의 경우 Glu-A1과 Glu-B1 locus에서 Glu-A1c (null)와 Glu-B1b (7+8) allele이 가장 높은 빈도를 보였으나, 중국 자원의 경우 Glu-A1a (2*)와 Glu-B1c (7*+9)가 가장 높은 빈도를 보여 그 양상이 달랐다. 셋째, Glu-B1d (6+8)과 Glu-B1e (20), Glu-D1b(3+12), Glu-D1c(4+12)는 한국자원만 가지고 있는 특이 allele이었고, Glu-B1f (13+16)과 Glu-D1e (2+10)의 경우 오직 중국자원만 가지고 있는 독특한 allele로서 원산지별로 고유한 allele이 존재하였다. 넷째, 고분자 글루테닌 subunit 조합을 기준으로 하여 PIC 및 UPGMA 분석을 통해 유전적 다양성을 분석한 결과 중국자원(PIC value 0.53)의 경우 한국, 일본 자원(PIC value 0.35)보다 유전적 다양성이 높은 것으로 나타났다. 또한 UPGMA tree상 한국, 일본의 유전자원 대부분이 Group I 에 속하는 것과 달리 중국자원의 대부분은 GroupⅡ에 속해 양국과는 구별되는 특성을 보였다. 다섯째, 총 291자원에 대한 Glu-1점수평가를 실시한 결과, 총 26자원이 10점 만점 자원이었다. 이중 중국원산 자원이 23자원, 한국원산 자원은 3자원이었다. 선호되는 allele인 Glu-D1d를 가진 자원은 만점 자원을 제외하고 총 16점이 있었다. 또한 전체 자원 중에는 6점의 자원이 147자원으로 가장 많았고, 8점 이상의 비교적 우수한 조성의 자원은 84자원이었다. 여섯째, 선발된 만점 자원의 출수기, 성숙기를 조사한 결과, 6월 상순 이전에 성숙한 자원은 16자원으로 특히 이중 3자원(K151847, K151865, K151962)은 극조숙성인 국내품종들의 숙기와 비슷한 5월 하순 성숙해 특히 이용가능성이 높을 것으로 기대된다.

A wheat mutant of low-molecular-weight glutenin (LMW-GS) “Gunji-2” at Glu-B3 locus was derived among the double haploid lines. Gunji-2 was derived from F1 plants of Keumkang and Olgeuru crosses using the wheat × maize system according to the procedures of Inagaki and Mujeeb-Kazi at International Maize and Wheat Improvement Center (CIMMYT). Deletion of Glu-B3 LMW-GS proteins was found by allele specific DNA marker, one dimensional SDS-PAGE and two dimensional gel electrophoresis (2-DGE). Tandom mass spectrometry (MS/MS) was used to obtain direct evidence of LMW-GS deletion. In addition, we examined the basic agronomic traits, protein content, dough properties of mixing and bread loaf volumeof Gunji-2 and parental wheat cultivars grown for two years. This mutant will represent a valuable resource in quality test for specific allele or gene at Glu-B3 locus.

Glutenin is the major factor responsible for the unique viscoelastic dough characterisitcs of wheat flour, which determine mixing and bread baking performance(X.Shan et al, 2007). And early maturity is one of the most important cultural characteristic in Korea because of its winter cropping system. This study is to reveal the genetic properties of Asian wheat landrace collection originated from 6 separate regions such as Korea, China, Japan, Afganistan, Iran, Pakistan, Caucasus, and Middle East. Using germplasms maintained in National Agrobiodiversity Center, RDA, Korea, the variations in morphological character and HMW glutenin subunit composition were investigated. In this study, Glu-A1c(null), Glu-B1b(7+8) and Glu-D1a(2+12) alleles are the most frequent in Asian landrace wheats. When it comes to unique composition, Glu-B1aj(8) and Glu-D1q(2+11) subunits are only in Afghanistan wheat. And Glu-B1k(22), Glu-D1l(12), Glu-D1m(10) subunits are only in accessions from Pakistan, Korea, and China, respectively. The accessions from Iran and Caucasus have the highest PIC value(0.57), which shows wheat origin region has high genetic diversity. Grouping by UPGMA anlysis of combination of Glu-1 allele, most accessions from Afghanistan, Korea, and Japan were in the same group despite of geological distance. Contrasively, many germplasms originated from China, Caucasus, and Middle East were in the other same group. The evaluation of bread baking quality by Glu-1 scoring system, 34 accessions are perfect 10. 16 samples from China and 1 Afghanistan among them were also matured before early June, suitable to Korean cropping system. Especially, 3 accessions(K151847, K151865, K151962) had extremely early maturity, ripened before late May. These genetic resources having good gluten quality and early maturity are expected to be used for Korea wheat breeding system.

Low-molecular-weight glutenin subunits (LMW-GS) play a crucial role in the processing quality of wheat flour. They are encoded multi gene family located at the Glu-A3, Glu-B3 and Glu-D3 on the short arm of chromosome 1A, 1B and 1D respectively. Typical LMW-GSs are composed of three parts including a short N-terminal domain, a relatively short repetitive domain and a C-terminal domain. Further, typical LMW-GS sequences are divided into LMW-s, LMW-m and LMW-i types, on the basis of the first amino acid of the mature proteins (serine, methionine and isoleucine, respectively). Although it is known that the allelic variation of LMW-GSs affect the properties of dough, it is still not clear which LMW-GSs confer better bread-making quality because of the larger number of expressed subunits and their overlapping mobility with abundant gliadin proteins. Therefore, it is important to characterize LMW-GS genes and develop functional markers to identify different LMW-GS alleles for application in wheat breeding. In this review, we discuss the various aspects of LMW-GS, including their structural characteristics, the development of marker, relationship between LMW-GSs and bread wheat quality, and genetic engineering of the LMW-GSs.

This study is to raise the utilization of genetic resource of wheat (Triticum aestivum) landrace by evaluating genetic variation related to end use quality of the Far East. Allelic composition of HMW-glutenin subunits encoded by genes of Glu-1 loci associated with bread baking quality was investigated in 324 wheat landrace genetic resources originated from Korea, China, and Japan. The most frequent combination of HMW-glutenin subunits were Glu-A1c, Glu-B1b in Korean and Japanese resources, but Glu-A1a and Glu-B1c were the most in Chinese resources. By using the Glu-1 score system, 24 accessions were evaluated as 10 out of 10. As for genetic diversity, represented by polymorphic information content(PIC) index, the level of variation of Korean landrace(0.245) was lower than that in China(0.569) and Japan(0.294). When it comes to unique composition, Glu-B1f(13+16) and Glu-D1f(2+10) subunits are only in Chinese resources. Glu-B1d(6+8), Glu-B1e(20), Glu-D1b(7+8), and Glu-D1c (7+9) subunits are only in Korean resources. Consequently, this study showed that Chinese landrace collection was the most highly diverse and had distinctive characteristics compared to Korean and Japanese one. Especially, some resources having preferable genetic stock or high Glu-1 score can be valuable for wheat breeding program.

이차원 전기영동 분석을 이용하여 국내 밀 32 품종의 HMW-GS 단백질 발현의 정성 및 정량적인 분석을 통해 품 HMW-GS 발현 정도를 평가하여 국내 밀 품종 육성의 초 자료로 활용하고자 수행하였다. 평균 HMW-GS 스팟 수는 11.78개였으며, Glu-A1 1.31개, Glu-B1 5.53개, 그리고 Glu-D1에서 4.94 개였다. Glu-B1과 Glu-D1에서는 subunit에 따른 단백질 스팟 수가 차이가 없기 때문에, Glu-A1에서는 1과 2* subunit을 지닌 품종이 null allele 품종에 비하여 단백질 스팟 수가 많았다. 단백질 스팟 수는 조경밀이 18개로가장 많았으며, 다홍밀은 7개로 제일 적었다. 단백질의 상대적인 발현량을 조사한 결과 평균 0.44로 대비 품종인 Chinese Spring에 비하여(1.0) 낮았고, 고분밀이 1.11로 가장 높았으며, 은파밀이 0.24로 가장 낮았다. 단백질 스팟수와 발현량을 이용한 유연관계 분석 결과, 국내 밀 품종을 6개 그룹으로 분류할 수 있었다.

Allelic variations in glutenin and puroindolines of 26 Korean wheat cultivars were evaluated to determine their effects on the physicochemical properties of flour and quality of white salted noodles. Cultivars carrying Pina-D1b and Pinb-D1b exhibited a coarser particle size of wheat flour and a higher ash and damaged starch content than those with Pina-D1a and Pinb-D1a. Glu-B1b, Glu-D1f, Glu-B3d and Pina-D1a alleles exhibited lower protein content than other alleles. Glu-A1c, Glu-B1b, Glu-D1f Glu-B3d, Glu-B3i and Pinb-D1b alleles appeared to be related to a lower SDS-sedimentation volume than other alleles. In dough rheological properties, Glu-A1a and Glu-D1d alleles showed a longer mixing time on the mixograph and maximum dough height but Glu-A3e and Glu-B3i alleles had a lower mixing time on the mixograph and a lower maximum dough height than other alleles at Glu-1 and Glu-3 loci. Regarding the quality of white salt noodles, about 10% of the variations in the hardness of cooked noodles were explained by Glu-A1 and Glu-B3 loci. Hardness rankings of cooked noodles were Glu-A1a > Glu-A1c > Glu-A1c at the Glu-A1 locus. Glu-B3h showed higher cooked noodle hardness (5.10 N) than other alleles at the Glu-B3 locus (< 4.66 N).

국내 밀 육종 시스템의 빵용 밀 육종 선발의 효율성을 높이고자 국내산 밀 품종을 이용하여 Glu-1과 Glu-3의 DNA 표지인자를 이용한 유전자형 확인과 multiplex-PCR을 이용하여 Glu-1과 Glu-3을 분석하였다. 글루텐닌 발현에 연관된 유전자 서열 특이적 분자표지인 Glu-1과 Glu-3의 밴드 패턴은 SDS-PAGE를 이용하여 분석한 글루텐닌 조성 분석 결과와 일치하였으며, multiplex-PCR을 이용하여 Glu-1 분석하였고, Glu-3의 경우 제빵 적성에 연관되거나 국내 품종에서 특이적으로 발현되는 Glu-A3와 Glu-B3의 분석이 가능하였다. 이러한 결과는 국내 밀의 유전적 다양성을 확보하기 위한 자원 도입을 위한 평가와 육종 초기세대 계통에서 글루텐닌의 유전적 분석에 이용이 가능하기 때문에 국내 밀 육종 시스템에서 유용하게 적용될 것으로 생각한다.

PCR-based assays with co-dominant or dominant allele-specific STS (sequence tagged sites) markers and sodium-dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) have been used in this study to discriminate Glu-1 and Glu-3 alleles in Korean wheat cultivars. Three alleles were identified at each high molecular weight glutenin (Glu-1) homoeologous locus and 10 alleles were identified at low molecular weight glutenin (Glu-3) homoeologous locus in Korean wheat cultivars. At Glu-1 loci, wheat cultivars contained Glu-A1a, b and c alleles amplified a 1093, 1063 and 920bp fragments, respectively, Glu-B1b, c and f alleles discriminated with 707(Glu-B1b and f) and 662bp(Glu-B1c) fragments and Glu-D1a, d and f alleles amplified a 576(Glu-D1d) and 612bp(Glu-D1a and f) fragments, respectively. At Glu-3 loci, wheat cultivars contained Glu-A3c, d and e alleles amplified a 573, 967 and 158bp fragments, respectively. Glu-B3d, g, h and i alleles gave a 662, 853, 1022 and 621bp, respectively and Glu-D3a, b and c alleles amplified a 884(Glu-D3a and b) and 338bp(Glu-D3c) fragments. A multiplex-PCR assay was also established which permitted the discrimination of the major Glu-1 and Glu-3 to determine glutenin compositions in a single PCR reaction and agarose gel assay because these systems could be useful to select DNA-based identification of wheat lines carrying good breadmaking performance in Korean wheat breeding programs. Three multiplex-PCR for Glu-1 and Glu-3, Glu-A1c + Glu-B1acf + Glu-D1d, Glu-A3ac + Glu-A3d + Glu-A3e and Glu-B3d + Glu-B3fg + Glu-B3h were established in this study.

In order to improve rice dough functionality, we cloned 4 kinds of high-molecular-weight glutenin subunit (HMW-GS) genes from bread wheat, ‘Jokyeong’. Among them, we first examined Dx5 gene to generate marker-free transgenic rice for advanced quality processing of bread and noodles. The GluB1 promoter was inserted into binary vector for seed specific expression of the Dx5 gene. Two expression cassettes comprised of separate DNA fragments containing only the high-molecular-weight glutein subunit (HMW-GS) protein (Dx5) and hygromycin phosphotransferase II (HPTII) resistance genes were introduced separately to tumefaciens EHA105 strain for co-infection. Each EHA105 strain harboring Dx5 or HPTII was infected to rice calli at 3: 1 ratio of Dx5 and HPTII, respectively. Then among 66 hygromycin-resistant transformants, we obtained two transgenic lines inserted both with Dx5 and HPTII gene to rice genome. We reconfirmed integration of the Dx5 and HPTII genes into the rice genome by Southern blot analysis. Wheat Dx5 transcriptsin rice seeds was examined with semi-quantitative RT-PCR. Finally, the marker-free plants containing only Dx5 gene were successfully screened at T1 generation. This result also provides that co-infection system with two expression cassettes could be efficient strategy to generate marker-free transgenic rice plants.

Gluten is the main functional component of wheat, and is the main source of the viscoelastic properties in a dough. One of the gluten group is glutenin, which is composed of high molecular weight (HMW) and low molecular weight (LMW) subunits. The HMW glutenin subunits (HMW-GS) have been shown to play a crucial role in determining the processing properties of the grain. They are encoded by the Glu-1 loci located on the long arms of homeologous group one chromosomes, with each locus comprising two genes encoding x- and y-type subunits. The presence of certain HMW subunits is positively correlated with good bread-making quality. The highly conserved N- and C- terminal contaning cystein residues which form interand intra-chain disulphide bonds. This inter chain disulphide bonds stabilize the glutenin polymers. In contrast, the repetitive domains that comprise the central part of the HMW-GS are responsible for the elastic properties due to extensive arrays of interchain hydrogen bonds. In this review, we discuss HMW-GS, HMW-GS structure and gluten elasticity, relationship between HMW-GS and bread wheat quality and genetic engineering of the HMW-GS.

미성숙 종자로부터 추출된 전체 RNA를 이용하여 합성한 cDNA와 LMW-GS 특이 프라이머세트를 이용하여 43개의 LMW-GS 유전자를 분리하였다. 각각의 유추 아미노산은 상동성이 높은 20개의 시그널 펩타이드, N-말단 영역, 반복서열영역 그리고 C-말단 영역을 가지며 C-말단 영역에 분자내 혹은 분자간 이황화 결합을 형성하는 전형적인 8개의 시스테인을 가지고 있었다. 이들 시스테인의 위치는 첫번째, 일곱번째를 제외하고는 보존되어 있었다. Ikeda

Protein related parameters of pan bread and white salted noodles prepared from 26 Korean wheat cultivars and 6 commercial and imported wheat flours were evaluated to elucidate the relationship between rheological properties and end-use characteristics and to determine the effects of Glu-1 and Glu-3 alleles on those characteristics in Korean wheat cultivars. SDS-sedimentation volume based on protein weight was positively correlated with mixograph parameters and maximum height of dough and also positively correlated with bread loaf volume, crumb firmness and springiness of cooked noodles. Within Glu-1 loci, 1 or 2* subunit and 5 + 10 subunits showed longer mixingtime, higher maximum height of dough, and larger loaf volume than null allele, 2.2 + 12, and 2 + 12 subunits. Cultivars with 13 + 16 subunits at Glu-B1 locus showed higher protein content and optimum water absorption of mixograph than cultivars with 7 + 8 subunits. At Glu-3 loci, Glu-A3d showed longer mixing time than Glu-A3e, and Glu-B3d and Glu-B3h had stronger mixing properties than Glu-B3i. Glu-B3h had higher loaf volume and hardness of cooked noodles than Glu-B3d. Glu-D3a had lower protein content than Glu-D3c, and Glu-D3b showed stronger mixing properties than Glu-D3a. Glu-D3c showed lower hardness of cooked noodles than others.

Allelic variations in glutenin and puroindolines of Korean wheat cultivars evaluated to determine the effects of allelic variations on physico-chemical properties of flour and qualities of white salted noodles. In grain hardness and flour yield, Pinb-D1b had higher hardness index and flour yield than Pinb-D1a alleles. Glu-B1b and Glu-D1f also had lower hardness index than other alleles at the same locus and Glu-A1c, Glu-A3e and Glu-B3i alleles showed lower flour yield than other alleles. In flour compositions, Pina-D1b and Pinb-D1b showed higher particle size, ash and damaged starch content and lower lightness of wheat flour than Pina-D1a and Pinb-D1a. Glu-A1c, Glu-B1b, Glu-D1f, Glu-B3d and Glu-B3i showed lower particle size of flour than other alleles at Glu-1 and Glu-B3 locus. Korean wheats with Glu-B1f, Glu-D1a and Glu-B3b alleles had higher damaged starch content and lower lightness of flour than wheats other alleles at the same locus. Glu-B1b, Glu-D1f, Glu-B3d and Pina-D1a showed lower protein content and Glu-A1c, Glu-B1b, Glu-D1f Glu-B3d, Glu-B3i and Pinb-D1b showed lower SDS-sedimentation volume than other alleles. Hardness of cooked noodles ranked as Glu-A1a > Glu-A1c > Glu-A1c at Glu-A1 locus. Glu-B3h showed higher hardness of cooked noodles (5.10 N) than other alleles at Glu-B3 locus (< 4.66 N).

To investigate the genetic variation of high-and low-molecular-weight glutenin subunits (BMW-GS and LMW-GS), granule-bound starch synthase I (GBSSI) and puroindoline in 24 Korean wheat cultivars. At the BMW-GS compositions, three Glu-A1 alleles, five Glu-B1 alleles and three Glu-D1 alleles were identified. The high frequency of alleles at each locus was Glu-A1c allele (15 cultivars), Glu-B1b allele (16 cultivars) and Glu-D1f allele (16 cultivars). Four alleles were identified at the Glu-A3 and Glu-B3 loci and three at Glu-D3 locus and Glu-A3d, Glu-B3d and Glu-D3a were mainly found at each Glu-3 locus. Glu-A3d, Glu-B3d, Glu-D3b or c (4 cultivars, respectively) and Glu-A3d, Glu-B3d, Glu-D3a and Glu-A3c, Glu-B3d or h, Glu-D3a (3 cultivar, respectively) were predominantly found in Korean wheats. At the GBSS compositions, 2 waxy wheat cultivars, Shinmichal and Shinmichal1, showed null alleles on the Wx loci and other cultivars were wild type in GBSS compositions. At the puroindoline gene compositions, Korean wheat cultivars carried 3 genotypes, which 10 cultivars (41.7%) were Pina-D1a and Pinb-D1a, 11 cultivars (45.8%) had Pina-D1a and Pinb-D1b and 3 cultivars (12.5%) carried Pina-D1b and Pinb-D1a. These genetic variations could present the information to improve flour and end-use quality in Korean wheat breeding programs.

Flour protein content and SDS sedimentation volume were measured for 128 wheat varieties bred in Korea and foreign countries. Allelic variation of glutenin, one of seed storage proteins, was also identified by SDS-PAGE to measure the effect of HMW-GS (hig