(1) 유전자원 관리, 품종감별, 종자순도 검정 (2) 종 분류, 비교유전체 분석, 계통분석, 변이분석 (3) 유전자지도작성, QTL 동정 (4) 유전자/QTL 분리 (5) 육종목표 형질 간접선발에 이용 : MAS, MABC, MARS, GAS(GS) 분자표지의 육종적 활용 14/41 III. DNA 표지 이용 육종
(1) 유전자원 관리, 품종감별, 종자순도 검정
(2) 종 분류, 비교유전체 분석, 계통분석, 변이분석
(3) 유전자지도작성, QTL 동정
(4) 유전자/QTL 분리
(5) 육종목표 형질 간접선발에 이용
: MAS, MABC, MARS, GAS(GS)
분자표지의 육종적 활용
14/41
III. DNA 표지 이용 육종
연관 마커
유전자 마커
Major gene/QTL 일 경우
염색체
1 2 3
목표형질 관여 유전자(major gene)또는 major QTL
목표형질 관여
minor QTLs
형질 표지에 따른 DNA 마커의 구분
염기서열 정보Minor QTLs 일 경우
ㆍㆍ4
(1) 목표에 적합한 marker(표지) 의 선정
- 품종/계통/변이 목적 다양한 마커
특정 (유전자) 마커
- 선발(MAS)목적 목표형질과 연관된 마커
parents간 다형성이 있는 마커
- 유전자지도 작성/유전자분리 목표 부위의 마커
(2) 품종 또는 분석집단의 screening with markers
(3) Statistical analysis
- NTSYS, Structure
- MAPMAKER, JoinMap
- QTL mapper 등
(4) 결과 해석
공통 과정
1) 유전자원 관리, 품종감별, 종자순도 검정2) 종 분류, 비교유전체 분석, 계통분석, 변이분석
(1) 목표에 적합한 마커 선정- 품종/계통/변이 목적 다양한 마커: SSR, AFLP, STS, SNP 등 특정 (유전자) 마커: 특정 유전자의 변이를
보기 위해서 유전자의 염기서열을 토대로마커를 제작 – CAPS, STS, SNP
(2) 품종/종/계통의 screening with markers* DNA Fingerprinting
(3) Statistical analysis- NTSYS , Structure 등
(4) 결과 해석
과 정
유전자원의 중복성 조사
Dendrogram of 47 samples generated
By clustering of dissimilarity coefficients
Values using 63 RAPD fragments.
(Virk et al, 1995. TAG 90:1049-1055)
85개의 SSR 마커에 의한벼 japonica 51개 품종감별
엽록체 DNA 마커 염기서열 분석을 통한 234개 품종 구분
Genetics 169(2005): 1631
RFLP 마커를 이용한 야생벼 종간 유전유사성 비교
Wilson et al, 1999. Genetics 153: 453–473
Rice RiceMaize
Rice-Maize
Figure 4 The results of colinearity tests. For each panel, the 10 columns represent the 10 chromosomes. The
standard chromosome is shown in light blue, with the centromere in royal blue; the vertical axis represents the
centimorgan location on the standard chromosome. Significant colinearities between the standard and tester
chromosome are shown on the tester chromosomes in either red (P < 0.005) or dark blue (P < 0.05). (Gray
lines) Cross-hybridizing markers that do not comprise significant colinear regions. (Gaut, 2001. Genome
Research 11:55)
Colinearity between Maize Chromosomes
centromere
(Current Opinion in Plant Biology, 8:155, 2005)
GRASS genomes
Jackson et al(2011) New Phytologist 191: 915–925
Diagram of the evolutionary relationship of several major crop species showing polyploidy events (red octagons), relative genome sizes to rice (size of circles), and percentage of transposable elements (color of circle)
25/48
Genetics 162: 941–950 (2002)
Molecular Evidence on the Origin and Evolution of Glutinous Rice
Glutinous rice contains a G to T mutation at the 5 splice site of Wx intron 1 that
leads to incomplete post-transcriptional processing of Wx pre-mRNA.
3) 유전자지도 (QTL 지도) 작성
과정 – 식물재료 집단 : F2, BCF1, RILs, 다수의 품종
- DNA 마커- 집단 검정- Softwares: Mapmaker, JoinMap 등
Mapping of ges gene on chromosome 7
Chromosome 7
ge phenotpyes
벼
유전자
지도
- 분리집단, RILs, 다양한 품종 이용- 마커이용 양적형질 유전자지도 작성, 연관 마커 선발
QTL(양적형질유전자좌) 지도 작성_대학원 과정
Fig. 3. QTLs detected for agronomic and PNUE-associated characters. QTLs inside rectangular boxes stand for QTLs detected under low N-fertilizer (5 kgN/10a) condition, and the other QTLs are for ordinary N-fertilizer (10 kgN/10a)condition.
0
50
100
150
200
250
cM
PNUE
Days to headingGrain yield Straw yield Harvest index
Nitrogen concentration Of grain Nitrogen concentration Of straw Nitrogen content of shoot
0
50
100
150
200
250
300
cM
7RM125
RM481
RM82
RM248
RM420
RM234RM18RM118
RM346RM336
RM255
RM119
RM142
RM317RM349RM348RM127RM280RM131RM124
4RM133
RM510
RM527
RM275
6
RM247
12RM20ARM4A
RM101
RM277
RM19
RM270RM17
11RM332RM167
RM202RM287RM229
RM209
RM187
RM206
RM224
RM468
3
RM251
RM175
RM523RM132RM489
RM545
RM282RM554
RM16
RM135
RM422
RM130RM143
RM22
10RM216RM239RM467
RM184
RM590
RM110RM109
RM53
RM71
RM438
RM327
RM341RM475
RM263RM318RM112RM250
RM207
2
RM319
RM84
RM323RM283
RM600RM312
RM128RM237
RM315RM165RM104
RM5
1RM153
RM413RM593RM267RM159RM122
RM249RM289RM146RM430RM440
RM173RM161
RM421RM538
RM87RM31
5
RM350
8RM25RM72RM126RM342A
RM284
RM264RM477
RM228
9RM316
RM219RM105
RM288
RM328
RM108RM278RM242RM201RM215RM245
4) 지도에 기초한 유전자 분리 (QTL 분리는 대학원과정)
과정: - 목표형질 주위의 정밀 유전자지도 작성- 거대 DNA clone 분석- candidate gene 추정- 형질전환 후 확인 (RNAi, overexpression, complementation)
Stay-green mutant
Natural senescence
Dark-induced senescence
9 days
WT
sgr
WT sgr
Cha et al. (2002)
TAG 104:526-532
Fine Mapping of Stay-green Gene
2.0
3.0
2.4
1.8
2.1
8.7
5.5
RM160
RG570
C1263
RG662
sgr
C482
T4
RM189
RG662
E4055, RM3808
S21134
RM3636
sgr
E10960C12220RM1553
C482
0.7
0.7
0.3
0.3
0.3
1.5
Stay-green locus region
150 kb
Fine-mapping using japonica-indica polymorphism in AP005314
(140.4 kb)
Point mutation resides within 4.3 kb region
Recombinants among 860 F3-tested F2 plants
SSR SSR AFLP SSR SSR AFLP DraI (CAPS) SSR SSRphenotype F2 # RM1553 5314-94K5314-100.7K5314-101.6K5314-102.4K5314-103.3KS20246-105.9KRM3636-138k RM3808
A 729 A A A A A A A H HA 854 A A A A A A H H HB 350 B B B B B B B H HB 844 H H B B B B B B BH 53 H H H H H H H A AH 140 H H H H H H H A AH 186 H H H H H H H A AH 277 A A A A H H H H HH 292 H H H H H H H A AH 445 H H H H H H H A AH 916 H H H H H H H A A
j (ccg)6 - (caaca)4-j (ct)6-j - 2 cutI (ccg)10 22 (caaca)5-I (ct)10-I aaaagctc 1 cut
12 22 5 8 8 ~660 bp
101.6 ~ 105.9K = 4.3kb
31 2 1 1 0 0 1 9 45
AP005314
sgr (Mutant)
AP005314
Hwacheong-wx
AP005314
sgr F2 (844)
AP005314
Milyang23
(indica)
Point mutation in Exon2 of S20246in the stay-green mutant
[ gtg (valine) atg (methionine)]
Genic MS-chalky endosperm mutant
Hwacheongbyeo(left), Hwacheong-gms (mutant), and mapping
of the mutant gene, ms-h(t), Koh et al. (1999) Euphytica 106(1): 57-
62
Chromosome 9
ms-h(t)
AP005564
AC0137596
AC0137593
1
36856
48920
1 44918-5642571196-77219
9483 -4105
Overlapped and splitted sequence
SSR592(5564)
2462g
77483-
79978
5564p
31264-
31419
5564v
18717-
18876
7596b7596f
93271-
93413
78633-
78820
0cM 0 0 0.1 0 0
12469-
12864
5564s 5564d
971-1262
7596c 7596d 7596e
39255-
39390
43299-
43376
57848-
58068
0.4 000
5564m 5564t
65857-
66021
43884-
44305
7596a 7593a
90649-
90826
65187-
65466Pop. size: 1051
0 0 00
ms-h (<60 kbp)
21637195
UGPase2)
1187512535
Core histone H2A/H2B/H3/H4
3411256
Expressed protein-related
1459416900
NAMprotein
1) NAM : No apical meristem
2) UGPase: UTPglucose-1-phosphate uridylyltransferase
1947921824
NAM1)
protein
2505225603
Hypothet.protein
2762528773
Globulin-like
protein-related
4305342539
Putative photosystem II protein
6055959761
TC 75636
7192571178
TC 80014
8864485500
Unknown
Protein
(TC 80014)
Fine linkage and physical map surrounding the ms-h locus
1 bp (GA) change in the final nucleotide
at the 3’ splice junction of the 14th intron
Mutation site in UGPase1 gene(UDP-glucose pyrophosphorylase )
<Woo et al. 2008. Plant J. 54: 190-204>
exon 14intron14
exon 15 exon 16intron15
AGAA (single nucleotide substitution)
Wild
Mutant
3' 5'
74-bp
dCAPS marker analysis
F2 plants genotype 2 2 1 2 1 2 1 1 2 2 1 2 3 3 3 3
F2 plants phenotype Fertile Male-Sterile
◄undigested◄digested
F2 plants genotype 2 2 1 2 1 2 1 1 2 2 1 2 3 3 3 3
F2 plants phenotype Fertile Male-Sterile
◄undigested◄digested
Comparison of dCAPS marker genotypes with phenotypes in F2
plants from Hwacheong ms-h Hwacheong cross.
Genotype 1: MS-h/MS-h, Genotype 2: MS-h/ms-h,
Genotype 3: ms-h/ms-h
Figure 5. Comparison between empty vector control and UGPase-RNAi plant (A: panicle, B: pollen).
BA
Figure 6. Comparison between empty vector control and UGPase-complemented gms mutant. (A: panicle, B: pollen).
A B
5) 형질표지 및 간접선발gene tagging and marker-assisted selection (MAS)
MAS: 표현형을 직접 선발하지않고 대신에 단백질이나
DNA마커를 이용하여 표현형을 간접 선발하는 방식
(1) 목표에 적합한 마커 선정- 분석집단 또는 분석대상 품종들에 다형성이 있는
마커(2) 분리집단 또는 품종의 screening with markers(3) 형질의 유전자지도 작성
목표형질과 연관있는 마커 선발
형질표지 과정
간접선발 과정
(1) 목표형질과 연관 마커 다수 예비 선발(2) 분리집단 양친의 다형성 검정 마커 선정(3) 선발 마커로 분리 집단 검정 연관된 마커 allele을 가지고
있는 개체 또는계통 선발
1. naturally occurring / neutral
2. heritability = 1 (no environm. effect)
3. usually codominant (identification of all genotypes)
4. can be done from DNA extracts of small plants
(often before target characters are actually expressed)
5. non-dustructive
6. simultaneous assays for multiple targets
* Disadvantages;
1. expensive
Advantages of MAS
MAS will be useful for
1. Low heritability characters (normally require progeny
testing)
2. Traits expensive to score (e.g. secondary metabolites)
3. Multiple characters (e.g. sequential disease screening)
* gene pyramiding
4. Recessive characters
(1) 형질 표지 방법
3. 목표 형질의 후보유전자 탐색
4. 공분리와 형질전환 실험을 통하여 유전자 확인
5. 유전자 염기서열 기반 마커 개발
형질 연관 마커
유전자 기반 마커
1. 목표 형질과 마커와의 연관 검정 : 지도작성,
association test, QTL 분석
2. 목표부위(유전자 또는 QTL)의 정밀 유전자지도
작성 : 염기서열 정보 (DB이용 또는 직접 분석)
를 이용하여 마커 개발 또는 이미 개발된 마커
탐색
Methods for identifying molecular marker(s) linked to gene of interest
1. Single plant based mapping - very reliable - time consuming - best choice for genes with H(heritability)<1
(QTLs) 2. Bulked segregant analysis (BSA)
- rapid - ideal for genes with H=1 (e.g. major genes)
3. Nearly isogenic line (NIL) analysis - rapid - ideal for cases where NILs already exist
P1P2ⅹ(RR) (rr)
F1 (Rr)
F2
F2 DNA bulk
DNA extractionfrom each plant
(RR) (rr)(Rr)
RRbulk
rrbulk
P1 P2
(RR) (rr)RRbulk
rrbulk
Screening of bulks
with markers
P1 P2
(RR) (rr)RRbulk
rrbulk
not linked linked
if
RR plants rr plants RR plants rr plants
Marker genotype
of each F2 plant
Bulked-segregant analysis (BSA) - 형질 또는 유전자형별로 개체나 품종들을 grouping하여 DNA bulk를 만든다.
- 후보 마커들을 검정하여 연관된 것들을 선발한다 .
목표형질과 연관된marker를 찾기 위한 간편 방법
229bp
205bp
japonica indica
Temp. Trop. Tongil indica
B1 B3
B2 B4
A B
Figure. Screening of subspecies-specific (SS) STS markers
A sample figure of screening SS STS primers using four subspecies bulk DNA
(B1: temperate japonica; B2: tropical japonica; B3: Korean indica (Tongil); B4: indica)
Difference in size amplified DNA by an SS STS marker, S7011
(1~15: japonica varieties, 16~30: indica varieties)
S07011 11.0 AP004263F ctggatccaaggcatcattc
229/20590002~90361
R cttcgctctcaccatcaaca
벼 인디카와 자포니카에 특이적인 마커 선발시 BSA 활용 예
예 1) Mapping of a gms gene on chromosome 9
Chromosome 9
(2) 질적형질 분석 및 간접선발 : 쉽다
예 2) 벼 흰등멸구 저항성
DNA marker-assisted selection
A. undesirable fruitB. round leavesC. disease resistance
desirable fruitpointed leavesdisease susceptible
Select offspring for desirable fruit and disease resistance
If disease screening is difficult and a DNA marker islinked to the gene for "disease resistance", the DNA marker can be
employed to simplify the selection process
X
desirable fruitround leavesdisease resistance
A BC A BC
BCA
IRBB7저항성(R)
주남벼감수성
(S)
예) IRBB7가 보유한 흰잎마름병 저항성 유전자 Xa7을주남벼로 이전시키기 위한 여교배 과정에 MAS 적용
(주남벼 x IRBB7)
x 주남벼
여교배 F1 개체들M5 검정 선발
(R) (R) (S)
* *
(PCR 마커: M5)
여교배 F1
M5: Xa7과 0.1cM 으로 연관된 마커F:CAGCAATTCACTGGAGTAGTGGT
R:CATCACGGTCACCGCCATATCGGA
* 여교배 F1에서는
저항성 유전자가헤테로이기 때문에감수성 밴드도 나타남
(S)
(R)
(S)
(R)
(저항성 검정)
Chr.9 Sub1A = Ethynele-response-factor-like gene
IYT1
BsaA1
(TG)
AEX1(TC)
Pro Ser
GnS2
Alu1
(AG)
Sub1A
Rice FR13A
Flooded (12 d), 60 DAT (WS)
Swarna Swarna-Sub1
Swarna-Sub1Swarna
Swarna
X
IR49830Sub1 donor
F1 X
BC1F1
Swarna
X Swarna
SwarnaSub1
BC2F1 SwarnaX
Submergence tolerant rice ‘Swarna-sub1’ (IRRI)
Sub1A - screening
Marker-assisted breedinghigh-tech version of traditional breeding
31/48
1단계-저항성 유전자원탐색: 야생종 C. baccatum 이 유일한 탄저병
(anthracnose) 저항성원 확인
2단계- 저항성 이입집단 작성: 재배종(C. annuum)과 종간교잡 및 여교잡집단 양성 (배배양)
3단계- 분자마커 개발: 유전자지도 작성, 연관분석을 통하여 마커개발
4단계- 저항성품종 육성: 여교배, 마커 및 접종 병저항성 검정, 포장특성 조사, CGMS 일대잡종 도입
세계 최초 탄저병 저항성 고추 품종 육성(고추와육종 - 식물분자육종사업단)
22/41
Early generations: Selection for cumulated R genes Later generations: Selection for agronomic traits
Pyramiding of R genes via MAS Milyang 265
(Lee et al 2010, NICS)
Donor varietyResistance against
BPH (Bph1) GRH (Grh1) BB (Xa3) RSV (Stvb-i)
Nampyoung (A) S R S R
Junam (B) S S R R
Mil 220 (C) R S S R
Nampyoung/Mil 220Grh1 Bph1
Junam/Mil 220
Xa3 Bph1
Nampyoung*2/Mil 220
Nampyoung*3/Mil 220
MAS for Bph/Grh
Junam*2/Mil 220
Junam*3/Mil 220
MAS for Xa/Bph
MAS for Bph/Grh MAS for Xa/Bph
Nampyoung*3/Mil 220 // Junam*3/Mil 220
F1
F7
Bioassay selection for Bph/Grh
Selection for quality, yield, . .
Milyang 265
F5-6
~~ SSD
Backcross에서 marker 이용 간접선발
Foreground selection
Background selection
Yield Eating quali
ty
Resistance or Tolerance
Biotic Abiotic
NP GT BB Cold
SP GC SB Drought
Yield AC Blast Lodging
GW PC Insect Salinity
Sterility Viscosity Submergency
HD
An integrated linkage map of rice showing the positions of yield, eating quality, biotic/ abiotic stress resistance QTLs based on the data from the Q-ARO database
Li et al (2012) Genet Mol Res 11: 4157-68
- alternative to MAS, - uses all marker information to calculate genomic estimated
breeding values (GEBVs) for complex traits. - Selections are made directly on GEBV without further
phenotyping.
GWAS 분석NGS, GBS
각 계통의GEBV 추정
GenomicSelection
Selection 마커셑 확립
Genomic selection (GS)
QTL 유전자탐색
Flow diagram of a genomic selection breeding program
Heffner et al(2009) Crop Sci. 49:1–12
Summary of trait associations across genomic regions and percentage of variance explained by significant locus. (a) Each row represents a trait, and each column corresponds to a genomic region containing multiple SNPs that are significantly associated with a trait. (b) The x axis represents the trait, the y axis shows the contribution (%) of significant loci. Candidate genes detected within 200 Kb region of significant loci are labelled on top of the maximum effect locus.
(a)
(b)
Zhao et al, 2011 Nat Comm
Association study between phenotypes and sequence variation in rice
43
Genomewide selection and marker-assisted recurrent selection (MARS) in the intermated B73 × Mo17 maize RILs : 223 RILs, 287 SNPs for GS, 59 significant SNP markers for MAS
Genomic (estimated) breeding value (GEBV) selection in Maize
Massman et al (2013) Crop Sci. 53:58–66
10 RILs each10 RILs each
Trait means for 11 maize populations developed by genomewide selection (GWS) and by marker-assisted recurrent selection (MARS) for a Stover Index and a Yield + Stover Index
Syngenta- Artesian Corn Hybrids
Agrisure Artesian (left) vs. base hybrid (right)
*base hybrid = Roundup ready corn hybrid
Genomics-assisted breedinghigh-tech version of traditional breeding
Pioneer-AQUAmax Corn Hybrids
가뭄저항성 옥수수 품종 개발
Selection
Genomics
Germplasm
DNA표지를 이용한 맞춤형 작물설계
품종A 품종B 품종C 품종D
DNA표지선발
우수품종
우수한 특성(유전자)
유전자 모니터링에 의한 정밀 육종
Application of MAB or GAB according to genetic characteristics
of target traits in plant breeding
Genetic characteristics Target traitsMAB or GAB(GS)
High-heritability
Major-gene controlled
• Resistance against disease / insect pests
• Some fertility-sterility traits• Some morphological traits
MAB
A few genes involved
• Morphological traits• Flowering time• Some tolerance to
submergence, cold, salt, etc
Low-heritabilityPolygenes(QTLs)
• Yield• Biomass• Quality/taste-related traits• Secondary metabolites• Tolerance to abiotic stresses• Nutrient use efficiency
GAB(GS)
31
• An Epigenome consists of a record of the chemical changes to the DNAand histone proteins of an organism; these changes can be passed down to an organism's offspring.
• Changes to the epigenome can result in changes to the structure of chromatin and changes to the function of the genome.
• The epigenome is involved in regulating gene expression, development, tissue differentiation, and suppression of transposable elements.
• Unlike the underlying genome which is largely static within an individual, the epigenome can be dynamically altered by environmental conditions.
5. Epigenomics의 육종적 이용
• DNA methylation• Chromatin remodelling• Histone modification• RNA interference/interactions
The phenotype = genotype + epigenotype + environment.
(SNPs/InDels/TEs)
Springer (2013) Trends in Genet 29(4): 241-247
Heritable epigenetic variation이면 육종적 이용 가능