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Application of next-generation sequencing technology for genetic analysis and pre-breeding of rice

REYES, Vincent Pamugas 名古屋大学

2021.11.15

概要

イネの収量を劇的に増加させた1960年代の「緑の革命」の後、イネの収量は伸び悩んでいる。2050年までに世界の人口は90億人を超えると考えられており、イネの収量増加は喫緊の課題である。これらの形質の改良のためには、品種改良、栽培技術の開発、灌漑施設の整備などさまざまなアプローチが考えられる。品種改良の面では、次世代DNAシーケンス(next generation sequencing、NGS)技術により有用遺伝子の同定が迅速に行われるようになり、さらなる発展が期待されている。本研究では、NGS技術を用いたイネの遺伝子型決定(genotyping-by-sequencing、GBS)の育種現場での活用を目指し、収量の遺伝解析および育種材料の遺伝子型決定を行った。

 GBSとは、制限酵素の隣接配列のみを解読するなどして、サイト数を減らしてゲノム解読を行うことで、より多検体のDNAを解読する手法を指す。自殖性作物においては、染色体あたり200個程度のDNAマーカーがあれば遺伝解析および選抜には十分である。本研究において使用したGBSの実験系は、バーコードと呼ばれる検体認識用の配列を持つアダプターに制限酵素KpnIとMspIにより切断した検体DNAをライゲーションし、その産物をまとめてPCR増幅する手法であり、NGS用ライブラリの作成法としては簡便であり、さらに事前のDNA多型情報が不要であるという利点がある。この方法を基本として用い、さらにイルミナ社NGSで利用可能なインデックス配列を組合せ、12交配組合せ、2000検体以上をまとめて解析できる系を新たに開発し、イネnested association mapping集団の遺伝子型決定に利用した。

 イネの収量に関与する量的形質遺伝子座(QTL、quantitative trait loci)解析を行うために、indica背景を持つ高収量品種である「北陸193号(H193)」とjaponicaである「台中65号(T65)」の交雑に由来する組換え自殖系統を作出し、GBSにより遺伝子型を決定した。イネの収量に関与する形質は、ソース、シンクおよびソース-シンク間の転流に関与するものの3つに分類できる。H193は、T65よりも高い収量を示し、シンク形質では1穂粒数・穂数、ソース形質ではバイオマス・茎葉重、転流関連では2次枝梗の種子稔性でT65よりも大きな値を示した。また、収穫指数もH193の方が高い値を示した。本研究のマッピング集団においては雑種不稔によると思われる稔実率の低下が多くの系統で見られ、この不稔には染色体9と染色体12の2箇所のQTLの相互作用が関与していると考えられたため、これらの領域は収量に関与するとみなさないこととした。2020年の標準肥料区において、穂重に関する有意なQTLは検出されなかった。そこで、閾値を下げて探索したところ、LOD値が2を越える4箇所のQTLが存在したが、それらの全てでT65アレルが収量を増加させており、H193由来の穂重増加QTLは検出されなかった。1穂粒数では、染色体4のSPIKE座の領域でH193が形質値を増加させるQTLが検出された。しかしながら、穂数において、このSPIKE近傍のQTLはH193が形質値を減少される効果をもっていた。H193アレルが穂数を増加させるQTLは検出されなかった。sd1のH193アレルはバイオマス・茎葉重・稈長を増加させる効果を示したが、収穫指数は低下させていた。染色体1には、T65アレルがバイオマスを増加させるsd1ではない新規QTLが見出された。1次枝梗数に関しては、染色体8のWFP領域と、染色体1のsd1近傍にQTLが検出された。このうち、染色体体1のQTLは新規の可能性がある。2次枝梗数に関しては、染色体4にH193アレルが形質値を増加させ新規QTLを見出した。H193の高収量を特徴付ける要因である穂数や収穫指数に関与するH193由来の有用アレルが見つからなかったことから、H193の高収量は多くの遺伝要因が高度に調整された結果達成されたものであり、効果が小さい多数のQTLや、遺伝子間の相互作用が複雑に関与していると考えられた。

 WISH(Wonder rice Initiative for food Security and Health)プロジェクトは名古屋大学の研究者を中心に行われてきたプロジェクトで、組織的な交配とDNAマーカー選抜により、Grain number 1a(Gn1a)やWealthy farmer’s panicle(WFP)の1穂粒数増加アレルを世界の品種に導入する戻し交雑育種が進められてきた。WISHプロジェクトで育成された材料(BC3F4世代)について、形質調査を行った。調査した系統の多くで2次枝梗数が増加するGn1aアレルの効果や1次枝梗数が増加するWFPアレルの効果を確認した。また、Gn1aとWFPの両座で粒数増加アレルを導入した系統においてはその両方の効果が見られた。ほとんどの系統が、1穂粒数以外の形質では反復親と同等の形質を示したが、WFPの導入により穂数は減少するため、収量の増加についてはトレードオフを考慮する必要があることが明らかとなった。さらに、BC3F5世代において、GBSによる全ゲノム遺伝子型の決定を行った。その結果、Gn1aやWFPが正しく導入されていることを確認した。さらに、反復親ゲノムの回復割合(recurrent parent genome recovery)を推定した結果、ほとんどの系統で85%以上が反復親型になっており、表現型と遺伝子型の両方において、WISH系統が反復親の遺伝的背景を持つことが示された。

 以上のように、本研究ではGBS法の改善を行った上で、幅広い材料に適用し、遺伝解析や交雑育種における選抜におけるGBSの有用性を示した。たとえば戻し交雑育種の現場において、各世代でGBSによる全ゲノム選抜を行うことにより、効率的育種システムを実現可能である。NGSの解析コストは年々低下しており、将来はGBSが実際の育種の現場に広く普及することが期待される。

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