영남대학교 제주대학교

Trends of Smart Breeding in Fruit Trees 환경적응형 스마트 과수 품종의 국내외 개발 동향과 발전방안

윤해근1, 안순영1, 김선애1, 김대현2, 최철3, 송관정4

1영남대학교 원예생명과학과, 2국립원예특작과학원 과수과

3경북대학교 원예과학과, 4제주대학교 원예학과

2016. 5. 27.

Contents

Fruit tree breeding history and achievements in Korea

Fruit tree genomics

- Comparative genomics and genotyping by sequencing (GBS)

Marker-assisted selection

Transcriptomes and differentially expressed genes (DEGs)

Fruit Tree Breeding History

After the establishment of Agricultural Demonstration Station in 1906

1906 19701960 1980 1991 2016

Introduction of new cultivars from foreign countries

Establishment of nursery stock breeding technology

Cross breeding/Local var. collection

'Danbae' (1st by crossing)- Selection of phylloxera-resistant rootstock

Apple : Spur type with early ripening- Dwarfing apple, double grafting

Active Systemic breeding programs

홍로, 황금배, 월미 등

구분 출 원 등 록

채소 15작물 183품종 12작물 108품종

과수

12작물 148품종 10작물 96품종

배, 복숭아, 사과, 유자, 살구, 자두, 매실,

자두×살구, 참다래, 포도, 감귤, 감

배, 복숭아, 사과, 자두, 자두×살구, 참다

래, 포도, 살구, 매실, 감귤(유자, 감 제

외)

화훼 23작물 654품종 20작물 573품종

- 중국(16): 등록완료(배 14, 사과 2), 미국 : 등록완료(사과 1)

- 경북기술원(사과 1) 청도복숭아시험장(복숭아 3)

- 충북 포도연구소(포도 3), 강원기술원 (포도 6)

- 강원대학교 : 포도 6 품종 등

국내 품종보호 출원·등록 현황(’98~’14년)

지자체 및 대학 등록

국외 품종보호 출원·등록 현황(’01~’15.6.)

Fruit Tree Breeding History

Main objectives of selection• Fruit Quality

• Pest and disease resistances

• High and regular cropping

+ few specific or new ones

• Adaptation to various region conditions

• Adaptation to warmer and drier conditions (global warming)

• Resistance to new or region-specific pests and diseases

• High nutritional value , low allergenicity

Main characteristics of selection

농업전망 2016(한국농촌경제연구원)

Importance of Smart Breeding

Rapid Aging and Decline of Agricultural Population

농가호수, 농가인구, 농림업취업자 동향 및 전망

Importance of Smart Breeding

Rapid Aging and Decline of Agricultural Population

농업전망 2016(한국농촌경제연구원)

농업소득과 농업경영비 비중 전망

Importance of Smart Breeding

Global Warming

2070년현재

Importance of Smart Breeding

Global Warming : Great Potential Threat to Competitiveness Recover

- Deterioration of Fruit Quality (Poor Peel Coloration, Short Shelf Life,

Poor Pollination or Excess Pollination, Increased Sink & Source Competition, etc.)

재배 온도별 ‘후지’ 사과 착색 비교

Importance of Smart Breeding

Global Warming : Great Potential Threat to Competitiveness Recover

- Expanding of Plant Growth Period (Encountering Abiotic and biotic Stress)

- 사라질 과실류 : 사과, 포도, 커피, 아보카도 등

과수류 감소 병해 과수류 증가 병해

○ 배 적성병, 흑성병

(봄철기상 병 발생에 불리)

○ 사과 탄저병, 겹무늬썪음병, 갈반병

○ 감 탄저병, 둥근낙엽병

포도줄기혹병 주홍날개꽃매미 갈색여치

Concept of Smart Breeding

Cultivar Improvement for Smart Fruit Industry (Labor or Energy-saving)

- Biotic Stress Resistance (Disease or Insect Resistance)

- Abiotic Stress Resistance (Low or High temp., Soil pH, or Drought Resistance)

- Others (Dwarfness, Compactness, Seedless, Self-pruning, Self-compatibility, etc.)

- Attractiveness for consumers : Color and content of compounds

Difficulties in fruit tree breeding

Long juvenility & a fruiting a year

Heterozygosity

- Time, Space, Labor consuming

Difficulties in fruit tree breeding

노균병 저항성 포도 품종 육성

- Crossing (Montpellier), screening/selection (Colmar)

- Ampelographic collections : American species

- No spray : high pressure, Wood cuttings

V. vinifera x V. rotundifolia

F1

BC1 x V. vinifera

BC2, BC3,,,,,

Resistant to DMSimilar to Muscadine

엽형은 유럽종과 유사, 저항성 감수성 개체 혼재

x V. vinifera

교배 및 후대 검정 모식도

Difficulties in fruit tree breeding

흰가루병 저항성 유전 분석

V. vinifera x V. rotundifolia

- F1 : 700 hybrids (불임 : 화분의 감수분열 저해),

90% 불임, 10% ovule 부분 임성, 여교잡(V. vinifera)에 이용

- BC1 : 10-40 plants

유전적 불균형 (75% V. vinifera, 25% Muscadine)

상위적 불균형, 치사 혹은 불임, 내병성

BC2 with another V. vinifera

BC3 - BC4 - BC5 - BC6 : 2.5% Muscadine 특성 발현

|

Selfing - R:S = 75:25

Run1 - 단일 유전자

Screening System for Disease resistance

노균병 저항성 검정

1. Leaf disk inoculation : 잎의 괴사 크기 및 포자 형성량 조사

2. 포자 형성량 조사 : sonication(vortex), cell counting

0

1

2

3

4

5

6

7

8

9

1 2 3 4 5 6

No of sporangiospores

Leve

l of

resi

stan

ce

Concept of Smart Breeding

Smart Breeding Process : Molecular breeding and Increased efficiency

- Comparative Genomics and Transcriptomics :

Automatic Data Collection and Calculation

- Application of MAS for Efficient Conventional Breeding

- Breeding Design (Gene-editing)

Concept of Smart Breeding

1. Comparative genomics (Genome-wide association study)

Relative Genome Sizes

Plant Genome Composition:Junk vs. Genes

PotatoCanola

TomatoSoy

Wheat

Corn

Grass

RiceMoss

ArabidopsisHuman

valuable gene space

Small Portion

repetitive “junk” DNA

Plant Genomes Can Be Larger Than The Human Genome

Nuclear genome size in different species

1 Expressed in Megabases (1MB: 1,000,000 bases)

Concept of Smart Breeding

1. Comparative genomics (Genome-wide association study)

New Technologies Have Produced Explosion of Data

1990 2000 2010

CombinatorialChemistry

HumanGenome

The Internet

Mergers and Acquisitions

Growth in Clinical Trials

External Research Partnerships

Medical Data Growth

MetabolicPathways

Proteins

Pharmacogenomics

ESTs

HTS

SNPsPetabytesofData

Source: Jeff Augen, IBM

Next Generation Data ExplosionThe Sanger method of sequencing caused a revolution in every corner of biology. With the recent emergence ofNext Generation Sequencing, it is happening all over again.

Fruit tree genomes currently in the process : Pear (Pyrus communis )Sweet Cherry (Prunus avium)Coffee (Coffea caneophora)Chinese Chestnut (Castanea mollissima)European plum (Prunus domestica)Red Raspberry (Rubus idaeus )Sangiovese Grapevine (Vitis vinifera)

Sequenced Plant Genomes

22 fruit trees or woody plants / 93 Crops

Blue line : Added from previous one

- Blueberry, Cranberry, Kiwi, Coffee, Jujube, Oil palm

ㅍ ㅍ ㅍ ㅍ ㅍㅍ

Dicots

Columbine, Sugar beetAsterids Blueberry, Cranberry, Kiwi, Coffee, Tomato, Chilli Pepper Potato

Rosids

GrapeRose Gum TreeMalpighiales Poplar, Flax, Castor Bean, Cassava, Rubber Tree

Eurosids 1

Dwarf BirchCucumber, Melon, Water MelonWoodland Strawberry, Apple, Pear, Cannabis, Peach,Chinese Plum/Mei, JujubeMedicago, Chickpea, Lotus japonicus, Soybean,Pigeon Pea, Common bean

Eurosids 2

Cotton, Chocolate, Citrus, Orange, Clementine mandarin, PapayaArabidopsis thaliana, Arabidopsis lyrata, Brassica rapa, Capsella rubella, Thellungiella parvula, T. salsuginea, T. halophila, Cleome sp

Mono-cots

Date Palm, Oil palm, Banana

Grasses Rice, Brachypodium, Barley, Wheat, Moso Bamboo,Maize/Corn, Sorghum, Foxtail Millet

Sequenced plant genomes

For genome-wide SNP discovery,

27 apple cultivars (24 Cherry, 56 Peach) were

re-sequenced=>Array with the Illumina Genome Analyzer II.

SNPs were identified using SoapSNP.

Concept of Smart Breeding

2. GBS (Genotyping-By-Sequencing - Marker Generation Protocol

1. Sample Plants –progeny and two of the parents ‘Hongro’ and ‘Alps otome’

2. Extract DNA and Digest with restriction enzymes - Digested with ApeKI

3. Ligate barcoded adaptors - Ligated with T4 DNA ligase using sticky-ended adaptors

4. Pool and amplify – Pooled in 96-plex and PCR-amplified

5. Sequence on Illumina HiSeq2500 - 164,859,738 reads

6. Process reads with TASSEL-GBS pipeline

7. Align reads and call SNPs

Concept of Smart Breeding

2. GBS - Marker Generation Protocol

GBS is better than microarray

Microarray is costly and time consuming

GBS data increase in value over the time : Improvement of reference genome, alignment algorithms and genotype callers

Urgent need for improved genotype callingand imputation algorithms

• By using TASSEL 4.0

• Composed of 1,540 SNPsgenerated spanning 1,371 cMover 17 LGs.

• Average marker density of 1 marker per 0.89 cM

GBS (‘Hongro’ x ‘Alps otome’)

SNP callingSNP-based linkage map of the H X A progeny

Concept of Smart Breeding

3. Marker assisted selection

– Mapping of major genes and QTLs :

• Resistance

• Fruit quality

• Tree architecture

• …

– Functional genomics

• Candidate genes (ACO, ACS, Exp7, Araf…)

• cDNA chips

– Gene cloning (Rvi6-Vf/apple; Ma peach, …)

Concept of Smart Breeding

3. Marker assisted selection

Crop Number of marker

Apple 93

Pear 18

Peach 23

Almond 7

Sweet cherry 2

Sour cherry 4

Apricot 1

Strawberry 12

Raspberry 10

Blackberry 0

Rose 14

Table and references compiled by Dr. Nadozie Oraguzie and Dr. Richard Bell

Marker -Trait associations known in Rosaceae Crops

• SNP-based linkage map of the M432 progeny. • Composed of 2,579 molecular markers, including 2,272 SNPs generated with

the IRSC array, 306 SSRs and the S-locus, spanning 1,282.2 cM over 17 LGs. Antanaviciute et al. BMC Genomics 2012

Progeny of apple rootstock ‘M.27’ X ‘M.116’

유연관계분석, 품종구굽, 양친구명, 유전자지도작성, QTL 등Oh et al. JPB (2016)

Tolerance to coldness in peach cultivars

UBC344-12

UBC342-6

UBC196

UBC123UBC221-2

Un

UBC317-2

LRR

PDRRP

LRRRP

CYP GST

CYP : cytochrome P450GST : glutathione S-transferase TAULRR : Leucine-rich repeatPDR : pleiotropic drug resistance 12RP: ribosomal protein

Smart Breeding in Citrus

Some Achievements in Korea

- New Release of High Sweetness Cultivars by CRI/NIHHS/RDA

- New Release of High Peel Color Cultivars by JARES

<내병성, 이병성 및 F1 실생묘에 적용한 SNP에 대한 genotyping>

하례조생 탐나는봉신예감

- Development of DNA markers linked to some traits

<감귤 교잡배 선발을 위한 SSR markers의 적용>

Smart Breeding in Citrus

Some Achievements in Korea

Smart Breeding in Citrus

Some Achievements in Advanced Countries

- Development of Seedless Triploids and Red Flesh Cultivars in Italy and Spain

Smart Breeding in Citrus

Some Achievements in Advanced Counties

- Application of SNP and SSR markers in Spain

Ruby +

Ruby -

Genotyped by KASPar Technology

positive / negative controlspositive / negative samples

Smart Breeding in Citrus

Some Achievements in Advanced Counties

- Genome Mapping in SpainF 0.0

mCrCIR02D09 11.4

CiC5785-01 44.7CX2004 46.7CX6F23 49.5

CI_1 55.3CENTROMERE 56.9

CiC6278-01 57.0CI_2 58.7

CiC3440-07 67.2mCrCIR07D05 75.6mCrCIR03C08 82.2

CIBE6006 124.0mCrCIR05A05 125.2

JK-TAA41 131.9L 138.9

F 0.0

CIBE6147 14.4CiC4827-01 20.5

CiC2110-01 28.8MEST057 32.2

CID0806 55.2CIBE5720 58.4

CI_1 58.9CENTROMERE 60.7

CI_2 61.6MEST539 61.8

CiC4581-01 63.7MEST001 70.6

CID6193 92.2

JK-taa15 119.7

L 128.5

F 0.0CI_1 4.8

CENTROMERE 6.2CiC2635-06 6.4

CI_2 6.8CiC4033-01 7.0

MEST191 10.9CiC4993-03 13.9

MEST132 26.9

MEST346 56.9CiC2128-01 61.2

MEST322 70.1CiC3056-02 72.5

CID5874 73.3

mCrCIR01C06 88.9MEST123 92.0

L 99.8

F 0.0

CiC4954-02 8.4CiC5327-03 14.9

CID0245 20.9CI_1 22.3

CiC1380-05 22.4CENTROMERE 23.1

CI_2 25.6CiC1135-01 33.0

MEST104 40.5

CX6F06 60.5

CiC5842-02 77.3

CIBE2493 97.4mCrCIR06A12 98.7

CID5485 107.4CX6F03 108.4

CiC2417-04 108.9

L 119.9

F 0.0CiC4240-04 7.1CiC1757-02 12.1

CI_1 15.1CENTROMERE 16.1

CiC5261-01 16.7CI_2 16.8

CiC2840-01 17.0CiC2824-01 23.0CF-ACA01 24.4

CiC3740-02 43.9

MEST146 65.6

mCrCIR03G05 75.1CiC0446-01 77.8

CiC6213-07 84.5mCrCIR02D04b 85.7

CIBE3255 89.5

F 0.0CX6F24 2.1

CiC4876-07 2.7

CiC5087-01 15.9

MEST494 29.0

mCrCIR07F11 49.6CI_1 50.3

CENTROMERE 52.2CI_2 53.8

CiC4620-07 54.2

CiC0046-02 63.7

CiC2768-01 73.3

CiC5089-06 80.8

L 87.5

F 0.0cms04 3.5

mCrCIR01F04a 5.9CiC0640-03 12.8

mCrCIR07B05 31.7

MEST502 43.5CI_1 50.9

CENTROMERE 54.2CI_2 56.7

CiC1208-01 58.2CiC4853-01 65.3

mCrCIR02A09 98.6CiC1749-05 103.0CiC4790-02 106.1

L 118.0

F 0.0

MEST107 8.9CiC1444-03 13.6

MEST473 15.8MEST202 20.6

CiC3674-02 23.6CiC4877-04 24.7

CiC2401-02 46.6

mCrCIR03B07 83.4CiC3361-04 94.3

CI_1 95.0CENTROMERE 96.4

CI_2 97.6Ci07C07 98.0

CID0591 115.6

LG1 LG2 LG3

LG4 LG5 LG6

LG7 LG8 LG9

F 0.0

MEST369 20.0

MEST370 50.5CID6314 65.7CID6286 74.8CID4225 86.3CID5376 88.2

MEST470 88.8CI_1 89.1

CENTROMERE 90.6CI_2 93.9

CiC0868-01 102.8CX0124 110.3

CID4894 116.3

MEST131 179.3L 186.3

Limits of the use of markers in selection

Many researches, results, QTLs ….BUTNo (few) use in selection

Main reasons:- Low marker density (SSR)

• gaps• weak precision on the QTL mapping

- Lack of information on the allelic diversity

- Lack of information on background and

environmental effects

- So far, lack of cheap and high throughput

genotyping tools

Apple Breeding by D

NA Inform

ation

…. Whole genom

e sequences available for apple, peach, and … strawberry

GWA (Genom

e Wide A

ssociation) Mapping

One of the primary goals of genomics research is to establish relationships between genotypes and phenotypes.

Genotype-phenotype associations form the basis of genomics-assisted breeding programs that aim to accelerate the breeding of improved varieties.

Improving fruit and wine: what does genomics have to offer, Sean Myles(2013)

Vv Pathogenesis-related protein R major form

Cluster2: 2412genes

metaboli

c

process

67%

cellular

compone

nt

biogen…

cellular

process

63%

response

to

stimulus

29%

organelle

part

45%

macromol

ecular

complex

29%

organelle

68%

membrane

-enclosed

lumen

14%

envelope

13%

Disease resistance related transcriptomes

0h 24h

Cluster1: 2392genes

Vv glycine-rich RNA-binding protein GRP1A-like

4. Transcriptomics : 과실발육 성숙, 내재해성(병해충, 기후, 양수분)

무핵 유전자원 전사체 분석 및 candidate gene 분석 Cluster analysis

10,837개의 차등 발현 유전자들은 발현패턴의 변화에 따라 9개의 cluster로 구분되었음

Cluster 3과 4에 포함되는 유전자들은 개화기를 전후로 뚜렷한 발현의 변화를 보였음

무핵 유전자원 전사체 분석 및 candidate gene 분석

Cluster 3에 포함되는 2,592개 유전자들의 KEGG 분석 결과

Phenylpropanoid biosynthesis pathway 상에 7개 유전자가 위치하였음

Cluster 3에 포함되는 유전자들의 KEGG 분석 결과

- Gene and genome and editing - CRISPR/CAS9

4. Elucidating functions of DEGs

□ 저항성 유전자 분리

- 식물 : 241, 병원균 : 138(비병원유전자: 23, 병해: 120)

- Gene classes : 16

- Manually curated R-Genes : 112

- Putative R-Genes, collected from NCBI Protein : 9639

- Putative R-Genes, predicted from NCBI UniGene : 24919

- Putative R-Genes, predicted from Pythozome : 68509

- Total number of Plant Genes : 106373

- 프로모터 : 벼 병 방어 반응 조절 ‘스위치 유전자’를 발견

- NBS-LRR receptor kinase 등

I. The CNL class :

a coiled-coil domain, a nucleotide binding site and a leucine-rich

repeat (CC-NB-LRR)

II. The TNL class :

Toll-interleukin receptor-like domain, a nucleotide binding site and a

leucine-rich repeat (TIR-NB-LRR)

III. The RLP class, acronym for receptor-like protein : a receptor serine–

threonine kinase-like domain, and an extracellular leucine- rich

repeat (ser/thr-LRR)

IV. The RLK class : a kinase domain, and an extracellular leucine-rich

repeat (Kin-LRR)

V~VI. Mlo and Asc-1.

2) Six distinct classes of R-genes in plants (based on the presence of specific domains)

S

MVfRPS5-like1059 NBSC

C

LR

R

LR

R

LR

R

M

VfRPS5-like1833 NBSCC

LR

R

LR

R

VfRPS5-like4135 NBSCC

LR

RL

RR

LR

R

M

M

VfRPS5-like4832 NBSCC

LR

RL

RR

LR

RL

RR

M

VfRPS5-like6172 NBSCC

LR

R

LR

R

LR

R

VfRPS5-like13564 NBSCC

CC

LR

RL

RR

M

VfRPS5-like20585 NBSCC

LR

RL

RR

LR

R

LR

R

M

VfRPS5-like55532 NBSCC

LR

R

CC

M

VfRPS5-like62178 NBSCC

LR

RL

RR

M

Rela

tive

exp

ress

ion

-3

-2

-1

0

1

2

3

4

5

0 1 6 12 24 48

a

Transformation with R-gene constructIn Arabidopsis and grapevines

- Gene and genome and editing - CRISPR/CAS9

우리나라 자생머루 5종이 분포하고 있음

A. brevipedunculata내병충성, 내재해성

Euvitis (2n=38)

Muscadine (2n=40)

일반포도, 고품질, 내병성 약

V. amurensis

V. thunbergii

V. flexuosa V. coignetiae

• 내한성, 내병성, 내충성 등• 기능성 소재로 활용• 주색이 아름답고, 투명• 독특한 풍미로 주질 우수

0

5

10

15

20

25

30

t-Resveratrol c-Resveratrol Piceatannol t-Piceid c-Piceid

Pinot NoirV. flexuosa

ug/g

fw

Stilbene compounds

새머루와 Pinot Noir 과실의 stilbene 함량 비교

과실의 Transcriptome 분석

유럽종 포도 과실과 새머루 과실에서 특이적으로 발현되는 유전자군

2ha

포도-20℃ 이하 -25℃ 이하

기준온도 이하

기준온도 이상

가평

겨울철 극최저기온과 재배지 비교 : 재배지 지도 재작성

대기온도(℃)

평균온도 한계온도

연중 4월~10월 1월

배 11~15 10~14 19~21 -20

포도 10~15 12~14 20~25 -22

복숭아 12~15 - - -20

단감 13< - - -10

떫은감 11~15 - - -15

Gene Fold Change

Campbell Early – Induced

Chalcone and stilbene synthase family protein (U1) 11.1728

RmlC-like cupins superfamily protein (U2) 8.2591

Homolog of carrot EP3-3 chitinase (U3) 7.6047

Cytochrome P450, family 94, subfamily C, polypeptide 1 (U4) 6.9283

Protein of unknown function (DUF506) (U5) 6.6627

Peroxidase superfamily protein (U6) 6.5874

Terminal EAR1-like 1 (U7) 5.8613

Jasmonate-zim-domain protein 8 (U8) 5.8295

Serine protease inhibitor, potato inhibitor I-type family protein (U9) 5.6366

Nucleic acid-binding proteins superfamily (U10) 5.4565

Muscat Baily A - Inhibited

17.6 kDa class II heat shock protein (D1) -9.0863

HXXXD-type acyl-transferase family protein (D2) -7.3965

Gibberellin 2-oxidase 8 (D3) -4.9279

Pectin lyase-like superfamily protein (D4) -4.5949

Bifunctional inhibitor/lipid-transfer protein (D5) -4.2942

HSP20-like chaperones superfamily protein (D6) -4.2279

UDP-Glycosyltransferase superfamily protein (D7) -4.1322

Pentatricopeptide repeat (PPR) superfamily protein (D8) -4.1277

Heat shock protein 18.2 (D9) -4.0918

NB-ARC domain-containing disease resistance protein (D10) -3.8961

DEGS from CE and MBA vine buds exposed to 20℃ for 24hrs

APX, BMY, CAS15A, GST, HIR, LEA, LOX, PGIP

CAD2, CBF1, STSY

14-3-3, BMY, CHI, CHS, ClpP, CW, CYB5, CYP, DFR. DHN1, FLS, Glu, GPAT, GPX, LRR, LTP,

MAPK, Mn-SOD, MT, MYB, OSM, P5CS, PAL, PR4s, PR6, PRP2, sHSP, SIRT, TIL, TIP, TLP, WRKY10

Campbell EarlyUp-regulation

MBADown-regulation

DEGs by low temperature in grapevines

Poor skin coloration of berries in ‘Aki Gueen’ grapes by high temperature

Change of fruit quality by high temperature

High temp.Low temp.

Clustering transcripts by temperature, Heatmap & Line plot

25℃ vs

30℃

25℃ vs

35℃

RNA sequencing에의한 DEGs의 KEGG 분석

포도과실(25℃ vs 35℃)의전체 KEGG pathway

Carbohydrate metabolism

Biosynthesis of secondary metabolites

Aromatics degradation

Glutathione metabolism

Methane metabolism

생리학적결과1?

생리학적결과3?

생리학적결과2?

DEGs in KEGG Pathway

잎(25℃ vs 35℃)의전체 KEGG pathway

포도 감귤사과