講演内容 牧草とそのゲノム研究の最近の状況講演者グループの成果を中心に バイオマス資源作物の重要性 米国スイッチグラスのゲノム研究 ススキ属植物の有用性とゲノム研究の必要性 講演者グループの研究内容米国 DOE 予算による国際共同研究など

牧草およびセルロース系バイオマス

資源作物の世界におけるゲノム解

析の状況ならびに海外との国際共

同研究の取組み

北海道大学北方生物圏フィールド科学センター

山田 敏彦

講演内容

• 牧草とそのゲノム研究の最近の状況

講演者グループの成果を中心に

• バイオマス資源作物の重要性

• 米国スイッチグラスのゲノム研究

• ススキ属植物の有用性とゲノム研究の必要性

• 講演者グループの研究内容

米国ＤＯＥ予算による国際共同研究など

草地の各種多様な機能

家畜飼料としての生産機能

地力増進

土壌・水保全

生物相保全

炭素保持

景観保全

レジャー・スポーツのアメニティー

牧草の種類

寒地型牧草

暖地型牧草

イネ科牧草

マメ科牧草

イタリアンライグラス，ペレニアルライグラス，

チモシー，オーチャードグラス，トールフェスク，

メドウフェスク，ケンタッキーグルーグラス

シロクローバ，アカクローバ，アルファルファ

イネ科牧草

マメ科牧草

ギニアグラス，バヒアグラス，ダリスグラス，ロー

ズグラス

サイラトロなど

T

ritic

u

m

Poaceae

Pooideae

Bambusoideae Chloridoideae Panicoideae

Triticodae Poodae _Oryzodae

Aveneae

Panicodae Andropogonodae

Triticeae Poeae Oryzeae Chlorideae Paniceae Maydeae Andropogoneae

O

ryza

E

le

us

ine

P

enni

se

tum

Se

tar

ia

T

ri

ps

ac

um

Ze

a

Sor

ghum

Sac

char

um

Se

cal

e

H

or

de

um

A

ven

a

F

est

u

ca

Lo

lium

イネ科の分類

ペレニアルライグラス

世界中の温暖地の草

地に広く栽培され、良

質な永年生牧草である。

再生力は旺盛であるが、

越冬性，耐暑性，耐干

性に劣る。放牧利用に

適している。また、初期

生育が良好で、緑化

・芝生用にも広く利用さ

れている。

英名：perennial ryegrass

和名：ホソムギ

学名：

Lolium perenne

L.

核DNA量：

4.16pg/2C (イネ 0.88 pg/2c)

自家不和合性(S,Z遺伝子座）

2n=2x=14 同質４倍体品種あり

イタリアンライグラス

1～越年性。初期生育

が旺盛で、採草で利用

される。本州以南では

重要な冬作飼料作物

である。

英名：Italian ryegrass

和名：ネズミムギ

学名：

Lolium multiflorum

Lam.

自家不和合性(S,Z遺伝子座）

100 cM 0 cM 50 cM 150 cM LG2 Xcdo456 e41t4782 e41t50380 e35t59220, Xcdo38.1 e33t62225 e41t50231 Xcdo405* e33t62515 e41t47225 Xcdo385.2 e35t59112, Xrz395 Xr3349, Xcdo365, Xpsr126 e33t50133, Xbcd808 e33t62460, Xcdo1328 e36t48595, Xpsr901 e33t62113 e35t59575 e41t50240, Xcdo1417 Xbcd1823 e33t62340 Xbcd135, Xc600.1 e33t62620 Xpsr540.1 e40t50334 e40t49173, Xcdo36 Xpsr546 Xpsr1316 Xc472 Xr738 Xc556, Xc498, Xpsr10 (Gli-2)* Xc847 129 cM LG4 e38t4785 e33t61228 e38t50263 e33t50144 e41t50320 e38t5074 e41t47148, Xcdo20 e33t5065 e41t47208 e35t59310 e33t62402 Xcdo1387, Xablpg60 Xc1176.1 Xc913A e38t50218, Xcdo38.2 e38t50640, Xcdo938 Xpsr163 e33t5088, Xpsr144 Xrbcs Xcdo795 e38t50420 e33t6178 e41t47132 Xr2847.1*** Xr2847.2*** Xcdo122* Xcdo542.2* Xr2880* pgm*, Xpsr922 e41t50710 e33t50247 Xr3182, Xcdo241* Xr1538* Xc764.1 Xpsr305*** Xbcd1421***, Xpsr580* e38t50350 e38m50348 Xc1164 Xr2702B.1* 136 cM LG5 e36t50375 e33t50163 e41t47750 e33t50112 e41t50590, Xablpg26y e41t47198 e41t50960, Xcdo459 Xablpg224, Xc1176.2 e33t50189 Xbcd1087 e41t47445 Xablpg26x Xcdo412 osrb e38t50311 Xr1751 e33t62210 e38t50189* e33t50147* e33t62101* e41t50200*, Xr2710**** e40t50268, Xcdo400**** Xrz404* 96 cM LG3 114 cM Xc919* Xc1239a.2* e33t48210 e33t61335 e33t50310 e41t50124 e33t50149* e33t62147*, Xcdo345*** e33t50280** e41t47162* got/3***, Xwg889*** Xbcd828*** Xcdo920***, Xcdo244*** Xcdo328.1*, Xpsr394*** Xpsr370* e41t4798* e41t47400 Xr1613** Xcdo328.2*** e41t47115* e41t47990* e41t50540* Xcdo460*** e41t50800* e33t48178 Xc390 Xwg110* LG7 122 cM e41t47395 e41t50970 acp/2 e41t47300 e38t50388 lp1 e33t62272 e41t47520 e38t50244 e33t62760 e33t6272, Xpsr154 Xpsr690** e33t50120 e33t50405 e41t50144 Xc30.1, Xbcd147 Xcdo385.1 e33t6270, Xbcd349 Xr1394A Xcdo99 e33t62108, Xrz144 e35t59420 e41t47600 Xr1562 osw Xcdo545 e35t59465 e41t4761, Xpsr119 e38t47290 Xpsr150 e33t50800 Xc1239a.1* e41t47500 e33t62180, Xablpg83 Xcdo580 e41t47146 e41t50365, Xbcd1072.2 Xcdo98 e33t62130 e41t50131 ose e41t50323, Xpsr601* Xcdo105.1 Xbcd1072.1** e33t50180, Xpsr162 Xbcd738 e41t47180 e33t50175 Xcdo202 e41t59188 e33t61133 LG1 90 cM LG6 Xcdo395 Xcdo542.1 e40t5092 e38t47515 e41t4769, Xcdo962 e38t50169 e41t4790 e38t50480 e41t47498 Xpsr131, Xpsr113 e33t50990 Xcdo516 e33t62980 e38t50344 Xcdo686 Xcdo590, Xcdo497 Xcdo1380*, Xcdo204 Xablpg303, Xcdo470 e33t62283 Xbcd880 mze Xc37*** e38t50510 124 cM e40t50131 e36t4888

The reference map for the p150/112 cross based on segregation data from 240 molecular markers covering 811 cM on seven linkage groups.

(Genome 45: 282-295,2002)

International Lolium Genome Initiative (ILGI)

・AV-PBC, Australia

・IGER, UK

・INRA, France

・NARCH/YPDES, Japan

Gramineae anchor probes

(barley, oat, wheat, rice

RFLP clones )

Rice cDNAs of RGP

P150/112

主要なイネ科草種のゲノム比較

SNPマーカーによるマッピング

Cogan et al. (2006) MGG, 276, 101-112 Gene-associated SNP

NA₆ x AU₆集団におけるマッピング

Hirata et al. (2006) TAG, 113, 270-279 395個のゲノム ライブラリー由来 SSRマーカー 218個のSSRマーカー のマッピング

イタリアンライグラス

Susanne Barth group , Teagasc , Ireland:

Whole genome shotgun sequencing and assembly to

access the gene rich portion of the genome of Lolium

perenne L.

Torben Asp group, Aarhus University, Denmark:

De novo genome sequencing of perennial ryegrass

(Lolium perenne)

Hongwei Cai group,Japan Grassland Agriculture and

Forage Seed Association：

Development of SNP markers for Lolium species

SNP markers development using the next generation sequencing data from L.

temulentum, L. persicum and Italian ryegrass

Plant and Animal Genome XX

(2012)の発表から

チモシー

英名：timothy

和名：オオアワガエリ

学名：Phleum pratense L.

北海道で最も広く栽培

されている重要な牧草

である（約７割）。再

生力にはやや劣るが、

越冬性はかなり優れる。

牛の嗜好性が高く、主

として採草用として利

用されている。

栽培種は六倍体である

(2n=6x=42)。

170 200 230 260 0.60 0.65 0.70 0.75 0.80 ト ッ プ 交配後代系統 2 か年合計乾物収量 (kg/ a) SSRマーカーに基づいた遺伝距離

種子親－テスター間遺伝距離と

トップ交配後代の収量

雑種強勢を利用した品種育成 育種母集団

DNAマーカーによるグルーピン

相反循環選抜への移行の提案

Festuca spp. Lolium spp.

good regrowth high nutritive value

superior persistence and abiotic-stress tolerance

X

Selection by genotype using DNA marker

Effective introgression of favorable traits

Lolium Festuca

Linked with stress resistance genes from Festuca

For example…..

Introgression breeding using DNA-marker selection

backcrossing

DNA markers to distinguish between different species are required for MAS in hybrid breeding.

Species specificity of marker is important factor for hybrid breeding in which multiple genotypes are involved.

Concept of intron-flanking EST markers

using comparative genomics

Intron-flanking EST marker

Wei et al. (2005) Theoretical and Applied Genetics

Comparative anchor-tagged sequence (CATS) marker

Fredslund et al. (2006) BMC Genomics

PCR-based landmark unique gene (PLUG) marker

Ishikawa et al. (2007) BMC Genomics

unique gene

genome sequence

(e.x. rice, sorghum) cDNA (EST)

sequence

(forage grass and the related species)

exon intron _exon

Genotype A

Wheat Rice

LG1 LG3

Insertion-deletion

(Indel) marker Cleavage amplified polymorphic sites _{(CAPS) marker etc.} SNPs

Detection of polymorphisms in intron regions Lolium/ Festuca LG3 Estimation of genomic loci Genotype B primer primer PCR with the

target genome DNA

Specificities of intron-flanking EST markers

to Lolium and Festuca genomes

Genotyping of L. perenne and F. pratensis (each 4 cultivars x 8 plants )

Specificity index, S value was calculated

= maximum difference of fragment frequency

200bp Os06g06090 AFLp = 0.97, AFFp = 0.19 Diffrence of F_A =|0.97 - 0.19 | = 0.78, Diffrence of F_B = |0 - 0.81 | = 0.81 _{∴ SLp =0.78, SFp =0.81} Severodvinskaya Reveille Riikka Yatsugatake D-13 Pokoro Harusakae Pradel Tammisto

L. perenne F. pratensis M M M B A BFLp = 0, BFFp = 0.81

Intron-flanking EST markers were developed by screening using each one cultivar F. pratensis and L. perenne

The 2011 Eastern Japan Earthquake and Tsunami

on 11

_{March 2011}

Failure of Cooling Systems at Fukushima I Nuclear

Power Plant after Earthquake and Tsunami 2011

Renewable energy

Wind power

Solar energy

Biorefinery

Liquid transportation fuel value-added chemicals

Feedstock

production

バイオ燃料

電力

リグノセルロース系バイオマスの変換

Native grass with high root and shoot yield compared to other grasses in USA. High yield is achieved with low fertilizer inputs.

Great potential to improve yield through breeding, biotechnology and management.

Why switchgrass as a main biofuel crop in USA?

Northern Upland Southern Upland Northern Lowland Southern Lowland

スイッチグラスの分布

バイオマス大

草丈高

湿潤、温暖な冬の

地域に分布

四倍体

(2n=4x=36)

バイオマス小

草丈低

乾燥、寒い冬の地域に分布

八倍体(2n=4x=72)

C

₄

植物、２つのエコタイプが存在する

Chloroplast trnL (UAA) intron DNA でエコタイプの

分類可能 （Missaoui et al. 2006)

“Association Mapping of Cell Wall Synthesis Regulatory Genes and Cell Wall Quality in Switchgrass“

"Linkage Analysis Appropriate for Comparative Genome Analysis and Trait Selection in Switchgrass“

"Developing Association Mapping in Polyploid Perennial Biofuel Grasses"

"Translational Genomics for the Improvement of Switchgrass" "The Hunt for Green Every April: Factors Affecting Fitness in Switchgrass"

"The Role of Small RNA in Biomass Deposition and Perenniality in Andropogoneae Feedstocks"

http://genomicscience.energy.gov/pubs/switchgrassreport.pdf

Switchgrass Research Group: Progress Report

Foxtail millet

Sweet

Sorghum

資源作物のゲノム研究の

対象植物

Brachypodium

distachyon

Insights into Switchgrass Genome Structure and

Organization

A total of 330,297 high quality BAC-end sequences (BES) were generated, accounting for 263.2 Mbp (16.4%) of the switchgrass genome. Analysis of the BES identified 279,099 known repetitive elements, >50,000 SSRs and 2,528 novel repeat elements, named switchgrass repetitive elements (SREs). A total of 48,000 clones from each library were organized into pools and superpools (~7X coverage) and established an efficient qPCR-based screening system. 300 BACs carrying cell wall and defense response-related genes were selected and 176 are sequenced to full-length providing complete genomic sequences of rice orthologs of 259 kinases, 118 gylcosyltransferases, 84 glycoside hydrolases and 13

ethylene response factors (ERFs). Comparative mapping of coding regions from 100 full-length BAC sequences and 330K BES revealed high levels of synteny with the grass genomes sorghum, rice, maize and Brachypodium.

Plant and Animal Genome XX

(2012)の発表から

Genetic Improvement of Switchgrass Feedstock for Biofuel

Production

Ｔransgenic switchgrass plant biomass showed a 30% reduction in lignin content and 57% more efficient in fermentable sugar release for biofuel production.

Genomics to Feed a Switchgrass Breeding Program

The switchgrass breeding program operated by USDA-ARS includes (1)

development of single nucleotide polymorphic (SNP) markers within candidate genes that are associated with endogenous genetic variation for lignin and

fermentability, (2) development of SNP markers within genes for flowering time and developmental traits for the purpose of creating reproductively compatible upland and lowland populations, and (3) development of robust and repeatable SNP marker systems and breeding methods that can be used to implement genomic selection (GS).

Characterization of the Genetic Diversity of Switchgrass

Using Genotyping by Sequencing

About 1000 individuals were genotyped using GBS. Using a creative SNP

calling method designed for species without reference genomes, more than one million high density SNP markers were generated.

ミスカンサス

Crop Average Producti-vity (MT ha−1 year−1) Ethanol yield (liter ha−1) Seasonal water requirements (cm year−1) Tolerance to drought Nitrogen Require-ments (kg ha−1 year−1) Corn 3,800 (total) 50-80 low 90-120 _{Grain 7} 2,900 _{Stover 3} 900 Sugarcane 80 (wet) 9,950 (total) 150-250 moderate 0-100 _{Sugar 11} 6,900 _{Bagasse 10} 3,000

Miscanthus 15-40

Advantage of Perennial Grasses

for Biomass Production

A low demand for nutrient inputs

Higher yields on relatively poor quality land

Longer persistency

Increase in soil carbon content

Nitrogen Use Efficiency Theory for Perennials

Translocation from rhizomes to growing shoot

Dry shoots harvested, nutrients stay in rhizomes Translocation to rhizomes as shoot senesces Spring and Summer Fall Winter M iner al nut ri ent s M iner al nut ri ent s

C accumulation rate into soil in Aso,

Kumamoto, Japan

503 kg C ha

-1

284kg C ha

-1

yr

-1

1.8 times higher in grassland than in forest

Cryptomeria japonica forest plantation Miscanthus sinensis grassland

Geographical Distribution of the Miscanthus spp.

(Clifton-Brown et al. 2008)

Susuki（薄、ススキ）

Use of Miscanthis plants in Japan

 Roof materials of traditional houses

 Animal feeds

 Component of manure

Traditional house with a thatched roof

Tourist attraction

World heritage

Biotic pressures involved

maintenance of semi-natural

Miscanthus grassland

Burning

Grazing

Famous Miscanthus grassland in Japan

Aso (Kumamoto)

Kawatabi (Miyagi）

Soni (Nara)

Miscantus

×

giganteus

(Giant Miscanthus)

Triploid natural hybrid:

M. sinensis x

M. sacchariflorus

Introduction to Denmark in 1935 from Japan as an

ornamental variety

Potential energy crop since oil crisis happened in 1970.

×

High biomass production

（30-45 t/ha/yr)

Miscanthus sinensis Miscanthus sacchariflorus

Compact roots Rhizomes

Triploid (3n=57) M, x giganteus Giants Miscanthus Hybrid vigor High density “Susuki” _2n=38 “Ogi” 2n=4x=76

Natural hybrid

Collection of new natural

hybrids and artificial crosses will be important.

Why Use Miscanthus

 C4 photosynthesis

 High photosynthesis level at low temperature

 High energy ratio (output/input) 22-50

 Perenniality

Disadvantage of M. x giganteus

 High establishment costs of sterile triploid

 Narrow genetic background

 Less winter hardiness, especially first winter

GFP-illuminated genetically transformed Miscanthus leaf GFP-illuminated genetically

transformed Miscanthus callus

First genetically modified Miscanthus grass

developed

Transformation system of Miscanthus sinensis by reporter gfp (green fluorescence protein) gene

H ligninH lignin G lignin S lignin リグニン含量の減少 RNAi RNAi RNAi

Transgenic Miscanthus works

CaMV 35S pro Fructan biosyntehsis gene NOS ter

CaMV 35S pro

Lignin biosynthesis genes( CAD, COMT )partial sequences NOS ter

Down-regulation of Lignin genes by RNAi Introduction of fructan biosynthesis genes

Transgenic plants with ability to synthesis of fructan (increasing carbohydrate and reduction of lignin content)

Altering the content of components of cell wall by transgenic approach

（carbohydrates and lignin）

Transgenic plants with reduced

lignin content (alter of components of cell wall) Reduction of lignin content O F O F O F O F O G O F O F (C₆H₁₀O₅) _n Photosyntesis

Starch into chlorophyll

High assimilation selectivity of glucose

Cellulose hydrolysate

（glucose as a main carbon source for microbes）

manufacturing

Bioplastics

Microbial factory

• One-pot polymerization

• under mild reaction conditions

Point 1 New catalyst Point 3 Point 2 Selective refinery Inedible biomass biodegradation

Carbon recycle

ABS (Access and Benefit Sharing)

Resources

Exploitation

Benefit sharing

The Nagoya Protocol on Access to Genetic Resources and the Fair and Equitable Sharing of Benefits Arising from their Utilization to the Convention on Biological Diversity was adopted by COP 10 meeting on 29 October 2010 in Nagoya, Japan.

Collection of Miscanthus seeds through Japan Agricultural

Cooperatives (JAs) under the contracts of MTA by EBI grant

2 1 3 4 5 6 7 ₈ 9 10 11 12 13 14 15 16 23 18 19 20 21 22 25 2 4 26 30 29 28 27 31 17

Red color number: seeds

with MTA from JA

Blue color number: seeds

with no MTA now

(asking JA to issue it)

32 ₃₃ 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 51 52 53 54 55 56 57 58 50

International collaboration

Quantifying Phenotypic and Genetic Diversity of Miscanthus sinensis as a Resource for Knowledge-Based Improvement of M. ×giganteus (M. sinensis × M. sacchariflorus)

INVESTIGATORS: Erik J. Sacks, Joe Brummer, Megan Hall, Stephen Long,

Junhua Peng, Toshihiko Yamada, and Chang Yeon Yu

INSTITUTIONS: University of Illinois; Colorado State University; University of

California, Berkeley; Wuhan Botanical Garden; Hokkaido University; Kangwon National University

DOE and USDA Fund New Project for