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

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1 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

2 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

3 開陽台 ( 中標津町 )

4 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

5 牧草の種類寒地型牧草イネ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マメ科牧草シロクローバ, アカクローバ, アルファルファ暖地型牧草イネ科牧草ギニアグラス, バヒアグラス, ダリスグラス, ローズグラスマメ科牧草サイラトロなど

6 イネ科の分類 Poaceae Pooideae Bambusoideae Chloridoideae Panicoideae Triticodae Poodae Oryzodae Panicodae Andropogonodae Triticeae Aveneae Poeae Oryzeae Chlorideae Paniceae Maydeae Andropogoneae Saccharum Sorghum Zea Tripsacum Setaria Pennisetum Eleusine Oryza Lolium Festuca Avena Hordeum Secale Triticum

7 ペレニアルライグラス英名 :perennial ryegrass 和名 : ホソムギ学名 :Lolium perenne L. 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

8 イタリアンライグラス英名 :Italian ryegrass 和名 : ネズミムギ学名 :Lolium multiflorum Lam. 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自家不和合性 (S,Z 遺伝子座 ) 2n=2x=14 同質 4 倍体品種あり

9 International Lolium Genome Initiative (ILGI) AV-PBC, Australia IGER, UK INRA, France NARCH/YPDES, Japan P150/112 one-way pseudo-test cross population LG1 LG2 LG3 LG4 LG5 LG6 LG7 Gramineae anchor probes (barley, oat, wheat, rice RFLP clones ) Rice cdnas of RGP 0 cm 50 cm 100 cm 150 cm 90 cm e33t50800 Xc1239a.1* e41t47500 e33t62180, Xablpg83 Xcdo580 e41t47146 e41t50365, Xbcd Xcdo98 e33t62130 e41t50131 ose e41t50323, Xpsr601* Xcdo105.1 Xbcd1072.1** e33t50180, Xpsr162 Xbcd738 e41t47180 e33t50175 Xcdo202 e41t59188 e33t61133 Xcdo456 e41t cm 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 Xc390 e33t48178 e41t50800* Xcdo460*** e41t50540* e41t47990* e41t47115* e41t50124 e33t50310 e33t61335 e33t48210 Xc1239a.2* Xc919* 114 cm Xcdo328.2*** Xr1613** e41t47400 e41t4798* Xcdo920***, Xcdo244*** Xcdo328.1*, Xpsr394*** Xpsr370* got/3***, Xwg889*** Xbcd828*** e41t47162* e33t50280** e33t62147*, Xcdo345*** e33t50149* Xwg110* Xr2702B.1* Xc1164 e38m50348 e38t50350 Xbcd1421***, Xpsr580* Xpsr305*** Xc764.1 Xr1538* Xr3182, Xcdo241* e33t50247 e41t50710 pgm*, Xpsr922 Xr2880* Xcdo542.2* Xcdo122* Xr2847.2*** Xr2847.1*** e41t47132 e33t cm e38t50420 Xcdo795 Xrbcs e33t5088, Xpsr144 e38t50640, Xcdo938 Xpsr163 e38t50218, Xcdo38.2 Xc913A Xc e33t62402 Xcdo1387, Xablpg60 e35t59310 e41t47208 e33t5065 e41t47148, Xcdo20 e38t5074 e41t50320 e33t50144 e38t50263 e33t61228 e38t cm e36t50375 e33t50163 e41t47750 e33t50112 e41t50590, Xablpg26y e41t47198 e41t50960, Xcdo459 Xablpg224, Xc e33t50189 Xbcd1087 e41t47445 Xablpg26x Xcdo412 osrb e38t50311 Xr1751 e33t62210 e38t50189* e33t50147* e33t62101* e41t50200*, Xr2710**** e40t50268, Xcdo400**** Xrz404* Xcdo395 Xcdo542.1 e40t5092 e38t47515 e41t4769, Xcdo962 e38t50169 e41t4790 mze e38t50480 e41t47498 Xpsr131, Xpsr113 Xc37*** e33t50990 Xcdo516 e33t62980 e38t50344 Xcdo686 Xcdo590, Xcdo497 Xcdo1380*, Xcdo204 Xablpg303, Xcdo470 e33t62283 Xbcd880 e38t47290 e41t4761, Xpsr119 e35t59465 Xcdo545 osw Xr1562 e41t47600 e35t59420 Xpsr150 e33t62108, Xrz144 Xcdo99 Xr1394A e33t6270, Xbcd349 Xcdo385.1 Xc30.1, Xbcd147 e41t50144 e33t50405 e33t50120 e33t6272, Xpsr154 Xpsr690** 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: ,2002) e40t50131 e36t4888 e38t cm e33t62760 e38t50244 e41t47520 e33t62272 lp1 e38t50388 e41t47300 acp/2 e41t50970 e41t cm

10 主要なイネ科草種のゲノム比較コムギ 6.0 pg ペレニアルライグラス 2.1 pg トウモロコシ 2.5 pg イネ 0.4 pg

11 SNP マーカーによるマッピング Cogan et al. (2006) MGG, 276, Gene-associated SNP NA 6 AU 6 NA 6 x AU 6 集団におけるマッピング

12 イタリアンライグラス Hirata et al. (2006) TAG, 113, 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

13 Plant and Animal Genome XX (2012) の発表から 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

14 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)

15 トップ交配後代系統 2 か年合計乾物収量 (kg/a) 種子親 - テスター間遺伝距離とトップ交配後代の収量 SSRマーカーに基づいた遺伝距離 Tanaka et al., Crop Sci 51, , 2011

16 相反循環選抜への移行の提案育種母集団グループ内循環選抜 DNA マーカーによるグルーピングループ内循環選抜ヘテロティックグループ A ヘテロティックグループ B 選抜個体テスト交配によるグループ間の選抜個体雑種強勢を利用した品種育成次選抜サイクル次選抜サイクル Tanaka et al. Crop Sci. 51: (2011)

introgression of favorable traits Lolium Festuca Linked with stress resistance genes from Festuca DNA markers to distinguish

17 Introgression breeding using DNA-marker selection Lolium spp. Festuca spp. X good regrowth high nutritive value For example.. backcrossing superior persistence and abiotic-stress tolerance Selection by genotype using DNA marker Effective introgression of favorable traits Lolium Festuca Linked with stress resistance genes from Festuca 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

18 Tamura et al. (2009) TAG 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 unique gene Ishikawa et al. (2007) BMC Genomics Estimation of genome sequence (e.x. rice, sorghum) exon genomic loci intron exon Lolium/ Rice Wheat cdna (EST) Festuca LG1 LG3 LG3 sequence primer primer (forage grass PCR with the and the related target genome DNA species) Detection of polymorphisms in intron Genotype A regions Genotype B SNPs Insertion-deletion (Indel) marker Cleavage amplified polymorphic sites (CAPS) marker etc.

Specificities of intron-flanking EST markers to Lolium and Festuca genomes Intron-flanking EST markers

of fragment frequency Os06g06090 200bp A L. perenne F.

19 Specificities of intron-flanking EST markers to Lolium and Festuca genomes Intron-flanking EST markers were developed by screening using each one cultivar F. pratensis and L. perenne Genotyping of L. perenne and F. pratensis (each 4 cultivars x 8 plants ) Specificity index, S value was calculated = maximum difference of fragment frequency Os06g bp A L. perenne F. pratensis Reveille Riikka Yatsugatake D-13 Pokoro Harusakae Pradel Tammisto M M M B Severodvinskaya AF Lp = 0.97, A F Fp = BF Lp = 0, B F Fp = Diffrence 0.19 of F A = = 0.78, 0.81 Diffrence of F B = = 0.81 S Lp = 0.78, S Fp = 0.81

20 The 2011 Eastern Japan Earthquake and Tsunami on 11 th March 2011 Failure of Cooling Systems at Fukushima I Nuclear Power Plant after Earthquake and Tsunami 2011

21 Renewable energy Solar energy Wind power Geothermal energy Biomass energy

22 Biorefinery Feedstock production Liquid transportation fuel value-added chemicals

23 リグノセルロース系バイオマスの変換コーンストーバーフィードストックの生産収集調整加水分解酸, 酵素ガス化高温, 低酸素分解微生物糖リグニン合成ガスバイオガスバイオ燃料電力バイオ由来化学物質スイッチグラス熱分解触媒, 熱, 圧力バイオオイルバイオ由来素材抽出機械的, 化学的炭素リッチ鎖分離機械的, 化学的植物体熱雑種ポプラ

24 Why switchgrass as a main biofuel crop in USA? 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.

25 Chloroplast trnl (UAA) intron DNA でエコタイプの分類可能 (Missaoui et al. 2006) スイッチグラスの分布 C 4 植物 2つのエコタイプが存在する Lowlands: 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) Uplands: 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) Northern Upland Southern Upland Northern Lowland Southern Lowland

26 Switchgrass Research Group: Progress Report January 2012 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"

27 資源作物のゲノム研究の対象植物 Foxtail millet Brachypodium distachyon Sweet Sorghum

28 PNAS 108: (2011)

29 Plant and Animal Genome XX (2012) の発表から Insights into Switchgrass Genome Structure and Organization A total of 330,297 high quality BAC-end sequences (BES) were generated, accounting for 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.

30 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.

31 ミスカンサス (Miscanthus spp.)

32 Crop Corn Average Productivity (MT ha 1 year 1 ) Ethanol yield (liter ha 1 ) 3,800 (total) Grain 7 2,900 Stover Sugarcane 80 (wet) 9,950 (total) Sugar 11 6,900 Bagasse 10 3,000 Seasonal water requirements (cm year 1 ) Tolerance to drought Nitrogen Requirements (kg ha 1 year 1 ) low moderate Miscanthus ,600-12, low 0-15 Poplar 5-11 Agave ssp ,500-3,400 3,000-10, moderate high 0-12 Somerville et al. (2010) Science 329:

33 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 Effect on stability and cover value for wildlife

34 Nitrogen Use Efficiency Theory for Perennials Spring and Summer Fall Winter Mineral nutrients Mineral nutrients Translocation from rhizomes to growing shoot Translocation to rhizomes as shoot senesces Dry shoots harvested, nutrients stay in rhizomes

plantation 503 kg C ha -1 284kg C ha -1 yr -1 1.

35 C accumulation rate into soil in Aso, Kumamoto, Japan Miscanthus sinensis grassland Cryptomeria japonica forest plantation 503 kg C ha kg C ha -1 yr times higher in grassland than in forest Toma et al. GCB Bioenergy, in press

36 Geographical Distribution of the Miscanthus spp. Ogi( 荻オギ ) Susuki( 薄ススキ ) (Clifton-Brown et al. 2008)

37 Use of Miscanthis plants in Japan Roof materials of traditional houses Animal feeds Component of manure Traditional house with a thatched roof

38 Tourist attraction World heritage Shirakawa-go Village Gokayama Village

39 Grazing Burning Cutting for forage Biotic pressures involved maintenance of semi-natural Miscanthus grassland

40 Famous Miscanthus grassland in Japan Aso (Kumamoto) Soni (Nara) Kawatabi (Miyagi) Sugadaira (Nagano)

41 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.

42 Susuki Miscanthus sinensis Ogi 2n=38 2n=4x=76 Miscanthus sacchariflorus Compact roots Natural hybrid Rhizomes Triploid (3n=57) M, x giganteus Giants Miscanthus High biomass production (30-45 t/ha/yr) Hybrid vigor High density Collection of new natural hybrids and artificial crosses will be important.

43 Why Use Miscanthus C4 photosynthesis High photosynthesis level at low temperature High energy ratio (output/input) Perenniality Disadvantage of M. x giganteus High establishment costs of sterile triploid Narrow genetic background Less winter hardiness, especially first winter at established year

44 M. sacchariflorus collection in Japan

First genetically modified Miscanthus grass

transformed Miscanthus callus GFP-illuminated

46 First genetically modified Miscanthus grass developed Transformation system of Miscanthus sinensis by reporter gfp (green fluorescence protein) gene GFP-illuminated genetically transformed Miscanthus callus GFP-illuminated genetically transformed Miscanthus leaf Wang et al. GCB Bioenergy 3, (2011)

47 Transgenic Miscanthus works Altering the content of components of cell wall by transgenic approach (carbohydrates and lignin) Introduction of fructan biosynthesis genes Down-regulation of Lignin genes by RNAi CaMV 35S pro Fructan biosyntehsis gene NOS ter CaMV 35S pro NOS ter Transgenic plants with ability to synthesis of fructan (increasing carbohydrate and reduction of lignin content) Starch into chlorophyll Photosyntesis Lignin biosynthesis genes( CAD, COMT )partial sequences Transgenic plants with reduced lignin content (alter of components of cell wall) RNAi H H lignin (C 6 H 10 O 5 ) n Reduction of リグニン含量の減少 lignin content O G O F O F O F O F O F Fructan into vacuole O F RNAi RNAi G lignin S lignin

source for microbes) Value-added materials Point 2 High assimilation selectivity of glucose

48 Carbon recyclable refinery system Point 1 New catalyst Inedible biomass biodegradation Refinery Selective refinery Carbon recycle Cellulose hydrolysate (glucose as a main carbon source for microbes) Value-added materials Point 2 High assimilation selectivity of glucose Bioplastics manufacturing Point 3 Microbial factory One-pot polymerization under mild reaction conditions

49 ABS (Access and Benefit Sharing) Country of origin (mainly developing countries) Benefit sharing Enterprise Benefit Resources Exploitation Chemical Product etc 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.

50 Collection of Miscanthus seeds through Japan Agricultural Cooperatives (JAs) under the contracts of MTA by EBI grant Red color number: seeds with MTA from JA Blue color number: seeds with no MTA now (asking JA to issue it)

International collaboration DOE and USDA Fund New Project for Biomass Genomics Research, 2011-2014 Quantifying Phenotypic and Genetic Diversity of Miscanthus sinensis as a Resource for

51 International collaboration DOE and USDA Fund New Project for Biomass Genomics Research, 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

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ñ{ï 01-65 191252005.2 19 *1 *2 *3 19562000 45 10 10 Abstract A review of annual change in leading rice varieties for the 45 years between 1956 and 2000 in Japan yielded 10 leading varieties of non-glutinous lowland