(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