[보고서]초강력분 밀 품종육성 체계 확립 및 불량환경 저항성 분자육종연구

서용원

보고서 정보

주관연구기관

고려대학교
Korea University

연구책임자

서용원

참여연구자

이라파눌라칸 , 이용진 , 김상헌 , 윤진석 , 정우주 , 고찬섭 , 김재호

보고서유형

최종보고서

발행국가

대한민국

언어

한국어

발행년월

2021-02

과제시작연도

2020

주관부처

농촌진흥청
Rural Development Administration(RDA)

등록번호

TRKO202100009771

과제고유번호

1395063110

사업명

차세대바이오그린21(R&D)

DB 구축일자

2021-09-18

키워드

밀.경질밀.불량환경 저항성.품질.분자육종.wheat.hard wheat.abiotic stress tolerance.end-use quality.molecular breeding.

표 Identification and selection of genes via multiple sequence alignment
표 Classification of gluten genes (HMW-GS) based on sequence information
표 Typical structure of LMW-GS . Sig: signal pepetide, N-ter: N terminal domain, Rep: repetitive domain, C-ter: C terminal domain
표 Signal peptide sequences of LMW-GS
표 N terminal sequences of LMW-GS
표 Examples of sequence alignment of Glu-A3(353bp, 441bp), Glu-B3(548bp, 557bp), and Glu-D3(394bp, 397bp)
표 Phenotypes of ‘KOMAC9’ at (A) flowering, (B) ripening stage and (C) seeds
표 Representative conserved sequences of 700 selected LMW-GS genes that are suit to design primer pairs. (A): Signal peptide sequences, (B): DNA sequences for signal peptides
표 Comparison of the LMW-GS PCR product between (A) agarose gel electrophoresis and (B) capillary electrophoresis
표 High resolution capillary electrophoresis systems to analyze various lengths of PCR products of LMW-GSs
표 Correlation analysis of LMW-GS profiling and quality parameter results
표 PCA analysis of LMW-GS profiling and quality parameter results
표 Freezing damage of Korean wheat cultivars during winter (‘17-’18)
표 Head selection and succeed generation of high-quality wheat bred lines
표 Comparison of agronomic traits between Korean cultivars and freezing-tolerant bred lines. (A) Plant height, (B) Spike length, (C) Yield, (D) Liter weight, (E) 1000-kernel weight, and (F) Heading date
표 (A) Head selection and (B, C, D) succeed generation of high-quality wheat bred lines
표 ‘18-’19 plot test in (A) the mid region (Deokso, Kyonggi-do) and (B) the south region (Jeongeup, Jeonbuk) of Korea
표 Number of lines of each ELITE population
표 Field evaluation of the ELITE popluations in the mid region (left: Deokso, Gyeonggi-do, 2020-3-3) and the south region (right: Jeongeup, 2020-5-11) of Korea for cultivar registration
표 Seed increase and field evaluation of (A)‘KOMAC5’, (B)‘KOMAC7’and (C)‘KOMAC9’for cultivar registration (left: 2020-5-11, right: 2020-6-3)
표 Phenotypes of ‘KOMAC5’ at (A) flowering, (B) maturing stage and (C) seeds
표 Phenotypes of ‘KOMAC7’ at (A) flowering, (B) maturing stage and (C) seeds
표 A schematic representation of developing markers specific to each short arm of chromosome 1. The boxes represent each procedure, and the utilized database, program, and germplasms and the genes filtered through each step are shown in the left and right side of the arrows, respectively. First, the gene sequences located on the short arm of wheat chromosome 1 were obtained from the International Wheat Genome Sequencing Consortium (IWGSC) RefSeq v1.0 (http://wheat-urgi.versailles.inra.fr/), 1,364 for 1AS, 1,520 for 1BS, and 1,344 for 1DS. These genes were used as queries for basic local alignment search tool (BLAST)n search against the rye draft genome (IPK Lo7 v3). The obtained rye sequences were reciprocally BLAST-searched against the wheat genome, and the homoeologs meeting the criteria of e-value 95%, and sequence length > 1,500 bp were selected, leaving 331 homoeologs. Multiple sequence alignment was conducted, the homoeologs containing at least two consecutive SNPs in each genome were selected, and each subgenome-specific PCR primer was designed with the GSP program (https://probes.pw.usda.gov/GSP/). The specificity of the developed markers was validated using wheat chromosome 1 null-tetrasomic aneuploid lines and wheat-rye chromosome addition lines. Following the marker specificity test, the recombinant inbred lines (RILs) developed in this study were screened with the developed markers, from which T1RS.1BL translocation lines were selected
표 Physical and genetic mapping of the subgenome-specific markers (SSMs). The numbers on the left side of the chromosomes represent the position of the markers and the genes on the chromosomes: kbp in 1A, 1B, and 1D, or cM in 1R. Cen; centromere, kbp; kilobase pairs, cM; centimorgan
표 Validation of the marker specificity using wheat nullisomic-tetrasomic aneuploid lines and wheat-rye chromosome addition lines. The marker names indicate each subgenome-specific marker (SSM) in a homoeologous relationship with the same number
표 Screening recombinant inbred lines (RILs) with subgenome-specific markers (SSMs). HR1491, HR1492, and HR1773 (translocation lines), and HR1498, HR1778, and HR2382 (non-translocation lines) indicate RILs developed by crossing “Keumgang” (non-translocated wheat) and “Seri 82” (T1RS.1BL). A, B, D, and R represent each SSM, and SSM-1 to SSM-10 indicate the markers in a homoeologous relationship with the same number
$Analysis of seed storage protein subunits of “Keumgang,” “Seri 82,” and RILs. (a) Glutenin subunit analysis using sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The bands indicating subunits of HMW-GS are shown on the right side. HMW-GS: high molecular weight-glutenin subunit, LMW-GS: low molecular weight-glutenin subunit. (b) Gliadin subunit analysis using aluminum lactate-polyacrylamide gel electrophoresis. α/β, γ, ω-5, and ω-1,2 represent each fraction of gliadin. The arrow indicates one of the α/β gliadins in HR1492 and HR1773$ 표 Analysis of seed storage protein subunits of “Keumgang,” “Seri 82,” and RILs. (a) Glutenin subunit analysis using sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The bands indicating subunits of HMW-GS are shown on the right side. HMW-GS: high molecular weight-glutenin subunit, LMW-GS: low molecular weight-glutenin subunit. (b) Gliadin subunit analysis using aluminum lactate-polyacrylamide gel electrophoresis. α/β, γ, ω-5, and ω-1,2 represent each fraction of gliadin. The arrow indicates one of the α/β gliadins in HR1492 and HR1773
표 Classification of 35 Korean wheat cultivars depending on freezing tolerance
표 (A) Phenotypes during cold acclimation and regeneration following the freezing treatment. (B) Survival rate of 4 wheat cultivars after freezing treatment at -10℃ for 20 h. Error bars represent the standard deviation and mean values do not share the same letters (a and b) are considered significant difference between the cultivars. (C) MDA concentration of 4 wheat cultivars at normal temperature, mild freezing (-10℃), and severe freezing (-18℃). Error bars represent the standard deviation and the letters above each bar indicate significant differences for each condition among the days after cold acclimation (one-way-ANOVA, p<0.05)
표 Observation of changes in (A) Total chlorophyll content, (B) Free proline content, and (C) Total soluble sugar content in the shoots of four wheat cultivars. Error bars represent the standard deviation and the letters above each bar indicate significant differences for each condition among the days after cold acclimation (one-way-ANOVA, p<0.05). The italic letters indicate statistically differences between the cultivars (two-way-ANOVA, Duncan’s post-hoc test, p<0.05)
표 Antioxidant enzyme activities under cold treatment in four Korean wheat cultivars are represented in U/mg proteins. (A) SOD (superoxide dismutase), (B) APX (ascorbate peroxidase), (C) GPX (glutathione peroxidase), (D) POD (peroxidase), and (E) CAT (catalase). Error bars represent the standard deviation and the letters above each bar indicate significant differences for each condition among the days after cold acclimation (one-way-ANOVA, p<0.05). The italic letters indicate statistically differences between the cultivars (two-way-ANOVA, Duncan’s post-hoc test, p<0.05)
표 Gene expression of cold acclimation-related genes in 4 wheat cultivars. (A) VRN1, (B) VRN2, (C) CBF3, and (D) CBF12. Error bars represent the standard deviation and the letters above each bar indicate significant differences for each condition among the days after cold acclimation (one-way-ANOVA, p<0.05). The italic letters indicate statistically differences between the cultivars (two-way-ANOVA, Duncan’s post-hoc test, p<0.05)
표 Gene expression of cold stress resistance-related genes in four wheat cultivars. (A) Wcs120, (B) Wdhn13, (C) Wrab17, and (D) Wrab19. Error bars represent the standard deviation and the letters above each bar indicate significant differences for each condition among the days after cold acclimation (one-way-ANOVA, p<0.05). The italic letters indicate statistically differences between the cultivars (two-way-ANOVA, Duncan’s post-hoc test, p<0.05)
표 Survival rate distribution of the genotyping population after winter. DS: Deokso, JJ: Jeonju, MY: Milyang
표 Flowering date distribution of the genotyping population. DS: Deokso, JJ: Jeonju, MY: Milyang
표 Stem length distribution of the genotyping population. DS: Deokso, JJ: Jeonju, MY: Milyang
표 Spike length distribution of the genotyping population. DS: Deokso, JJ: Jeonju, MY: Milyang
표 Awn length distribution of the genotyping population. DS: Deokso, JJ: Jeonju, MY: Milyang
표 Liter weight distribution of the genotyping population. DS: Deokso, JJ: Jeonju, MY: Milyang
표 Thousand kernel weight distribution of the genotyping population. DS: Deokso, JJ: Jeonju, MY: Milyang
표 Yield distribution of the genotyping population. DS: Deokso, JJ: Jeonju, MY: Milyang
표 Population structure analysis of 288 genotyping popoluation
표 Phylogenetic tree analysis of (A) MPK, (B) MPKK, (C) MPKKK gene family in the Triticeae
표 Gene expression of (A) MAPK, (B) MPKK, (C) MP3K genes of wheat under normal and cold-treated conditions
표 Left: Gene expression of MAPK30, MAPK43 and MAPK3 under cold treatment. Right: Gene expression of MAPK and MAPK43 under cold treatment
표 Functional annotation of the genes encoding TaMAPK3 interacting proteins
표 TaMAPK3 interacts with CBFIVd-D9, ICE41 and ICE87 in yeast. Yeast were grown on SD/-Leu/-Trp(-LW) or SD/-Leu/-Trp/-His/-Ade supplemented with X-α-gal (-LWHA+X-α-gal) medium. The empty pGBKT7 and pGADT7 vectors were used as negative control
표 Subcellular localization of TaMAPK3, TaCBFIVd-D9, ICE41 and ICE 87. Empty pMDC43 vector serving as control and GFP-recombinant vectors were transiently expressed in N. benthamiana leaves through agroinfiltration and GFP fusion proteins localization was visualized through confocal laser scanning microscope
표 BiFC interaction of between TaMAPK3 and TaCBFIVd-D9/TaICE41/TaICE87. Empty pGTQL1211YN (YFPN) and pGTQL1221YC (YFPC) were used as control interacting partners. Agrobacterium harboring TaMAPK3-YFPC vector was co-infiltrated with Agrobacterium containing YFPN-fused TaCBFIVd-D9/TaICE41/TaICE87 vectors into tobacco leaf epidermal cells. YFP fusion proteins localization was visualized through confocal laser scanning microscope
표 TaMAPK3-mediated phosphorylation of its interacting proteins, TaCBFIVd-D9, TaICE41 and TaICE87. Reactions lacking the specified components (-) were used as controls. TaMAPK3 was activated with recombinant His-MBP-TaMKK5DD protein. Recombinant proteins were separated by 10% SDS-PAGE after incubation in protein kinase buffer containing [γ-32P]-ATP. Phosphorylated TaCBFIVd-D9, TaICE41 and TaICE87 were detected by autoradiography after gel electrophoresis (top panel). Recombinant TaMKK5DD and TaMAPK3 were detected by Coomassie brilliant blue (CBB) staining (bottom panel)
표 The expression cassette specific primers to screen transgenic plants
표 (A) A vector map used for Agrobacterium-mediated Brachypodium transformation. (B) TaMAPK3 transgenic T0 plants before harvest. (C) NPTII strip test for screening transgenic T1&T2 plants
표 Comparison of TaMAPK3 expression in the wild type and TaMAPK3-overexpressing transgenic plants, OE5 and OE8
표 (A) Phenotype of wild type (WT) and TaMAPK3 overexpressing transgenic lines (OE5 and OE8) before and after freezing treatment at -10℃ for 3 h. (B) Survival rate of WT, OE5 and OE8 after freezing treatment followed by 2 weeks of regeneration. (C) Proline content in total shoots of WT, OE5 and OE8 at 0 d and 7 d after the beginning of cold treatment at 4℃
표 Gene expression of cold response-related genes, (A) BdCBF1, (B) BdCBF2, (C) BdCBF3, (D) BdCOR15a, (E) BdCOR413 and (F) BdCOR414 under cold treatment
표 (A) A Venn diagram depicting the number of up-regulated DEGs of 1BL.1RS and non-1RS. The up-regulated DEGs of (B) 1BL.1RS and (C) non-1RS were analyzed with MapMan 3.5.1. The color code scale is based on the log2 of the fold change values of each gene
표 GO (Gene Ontology) annotation of the up-regulated DEGs of 1BL.1RS and non-1RS. BP (biological process), MF (molecular function) and CC (cellular component) were represented in a row. x-axis and y-axis indicate GO function and the number of GO annotations, respectively
표 Chromosomal distribution of the up-regulated DEGs. Red and green bars represent DEGs from 1BL.1RS and non-1RS, respectively
표 Cold stress-responsive transcription factors in 1BL.1RS translocation.
표 Validation of 1RS specific gene expression using Real-time PCR
표 Comparison of plant morphology after drought treatment
표 Comparison of root morphology after drought treatment
표 Changes of soluble sugar, proline and malondialdehyde contents under drought treatment. Black bars show “control” plants and red bars indicate “treated” plants under complex stress
표 Comparison of the antioxidant enzyme activities under complex stress. Black and red boxes show “control” plants and “treated” plants, respectively
표 Information of the genes used for RT-PCR in this study
표 Expression of the photosynthesis-related genes. Black boxes show “control” plants and red boxes show “treated” plants under drought stress. Each bar show relative expression of alanine aminotransferase (a), NAD-dependent-malic enzyme (b), pepcase carboxylase (c), pyruvate orthophosphate dikinase (d) and rubisCO (e)
표 Expression of the antioxidant related genes. Black boxes show “control” plants and red boxes show “treated” plants under drought stress. Each bar shows relative expression of (a) superoxide dismutase and (b) peroxidase
표 Expression of the stress response-related genes. Black boxes show “control” plants and red boxes show “treated” plants under drought stress. Each bar show relative expression of (a) abscisic stress-ripening, (b) heat stress transcription factor, (c) chopper chaperone and (d) pathogen-related gene
표 Expression of the inhibitor related genes. Black boxes show “control” plants and red boxes show “treated” plants under drought stress. Each bar shows relative expression of (a) protease and (b) xylanase related gene
표 Expression of the transcription factor-related genes. Black boxes show “control” plants and red boxes show “treated” plants under stress. (a) 금강, (b) 금강/Seri82(1B.1R), (c) 금강/TAM201(1A.1R), (d) 금강/Siete Cerros66 (1D.1R)
표 Expression of the hormone signaling and protection factor related genes. Black boxes show “control” plants and red boxes show “treated” plants under stress. (a) 금강, (b) 금강 /Seri82(1B.1R), (c) 금강/TAM201(1A.1R), (d) 금강/Siete Cerros66 (1D.1R)
표 Motif analysis and Phylogenetic analysis of 24 TaASR genes. (A) Motif analysis using MEME (B) Phylogenetic relationship TaASRs and other ASR family
표 Expression profiles of TaASR genes under PEG and ABA treatment via real-time PCR. (A) 100μM ABA (B) 25% PEG6000 treatment
표 Generation of transgenic Brachypodium plants. (A): Schematic vector map of TaASR gene. (B)-(E): Generating of transgenic plants
표 Overexpression of TaASR2D enhanced the drought tolerance of Brachypodium plants. (A) The phenotypes of transgenic and wild type (WT) plants under drought stress. (B-G) The physiological drought stress tolerance assay of transgenic and WT plants under drought stress. (B) Survival rate of transgenic and WT plants were examined after 15 days of rewatering, (C) Relative water content of transgenic and WT plants under normal and 10 days of drought stress conditions, (D) Water loss rate of transgenic and WT plants was measured at the indicated time points, (E) Total chlorophyll content of transgenic and WT plants under normal and 10-day drought stress conditions, (F) Ion leakage of transgenic and WT plants under normal and 10-day drought stress conditions, and (G) MDA content of transgenic and WT plants under normal and 10-day drought stress conditions
표 Dissection of wheat spike (Right). Comparison of “Keumgang” in related to DAF0, DAF4, DAF9 and dissected parts. Numbers [#1 - #5] start from rachilla
표 Gene list of stress related genes
표 Expression of the starch synthesis-related genes. (A) Starch synthesis mechanism and heat map of 10 genes and (B) qRT PCR analysis of relative genes. Black boxes show “control” plants and gray boxes show “treated” plants under stress. ⁎ denotes p < 0.05, ⁎⁎ denotes p<0.01 and ⁎⁎⁎ denotes p < 0.001
표 Expression of the cell cycle related genes. (A) Cell cycle mechanism and heat map of 10 genes and (B) qRT PCR analysis of relative genes. Black boxes show “control” plants and gray boxes show “treated” plants under stress. ⁎ denotes p < 0.05, ⁎⁎ denotes p<0.01 and ⁎⁎⁎ denotes p < 0.001
표 Result of 2DE analysis. (A) Overall 2DE profiling of Gluten (glutenins & gliadins) at different stress periods (DAT3, DAT6, DAT10) as obtained from different regions (5 regions from top to bottom) in a single spike, (B) Selected spots that shows different profiling as evaluated by treated periods and different regions within a spike
표 Result of PCR product obtained by using ‘Keumgang’ genomic DNA. Primers were designed by using ORF of cloned TraesCS1D02G317301 cDNAs. P1: primer1, P2: primer2, P3: primer3
표 Analysis of motifs in 1D glutenin gene promoter
표 Analysis of motifs in ‘Keumgang’1D glutenin gene (TraesCS1D02G317301) promoter. Motifs are marked in different colors
표 A complete TaHMW1D gene with the promoter , UTR and CDS region from c.v. Keumgang gDNA. (A) Promoter region : promoter sequence information. Each color box indicated transcription binding sites. (B) CDS region : CDS sequence information. Purple : Signal region, Blue : N-terminal region, Black : repetitive region, Brown : C-terminal region
표 Information of SPA gene
표 Expression of SPA transcription factor gene. Black bars indicate “control” plants and red bars indicate “treated” plants under stress. (a) Spike region #1, (b) Spike region #2, (c) Spike region #3, (d) Spike region #4, (e) Spike region #5
표 Crosses made and breeding population test
표 The results of crossing each year
표 The results of breeding population each year
표 The condition of yield test

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초강력분 밀 품종육성 체계 확립 및 불량환경 저항성 분자육종연구
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