DEP1、Gn1a和qSW5組合應用調控水稻穗部性狀

溫一博，陳淑婷，徐正進，孫健，徐銓

、和組合應用調控水稻穗部性狀

1沈陽農業(yè)大學林學院，沈陽 110866；2沈陽農業(yè)大學水稻研究所，沈陽 110866

【】水稻是重要的糧食作物，為全球超過一半的人口提供主食。穗部性狀是影響水稻產量的主要因素，挖掘調控穗部性狀的優(yōu)異基因組合，為提高水稻產量提供聚合育種策略?！尽恳詮澦胄投i稻品種R99和直立穗型粳稻品種SN265構建的151個重組自交系為試材，應用Illumina測序平臺對重組自交系和雙親進行全基因組重測序。結合表型數據與遺傳圖譜，對每穗粒數、一次枝梗著粒數、二次枝梗著粒數和粒型進行QTL分析，篩選QTL區(qū)間內的候選基因，應用基于三代測序組裝的SN265和R99高質量基因組進行候選基因預測和序列比對，在重組自交系中篩選產量性狀表現最好的基因組合，并在SN265遺傳背景下應用CRISPR基因編輯技術對目標位點進行基因編輯?！尽縍99每穗粒數和二次枝梗著粒數顯著多于SN265，SN265的一次枝梗著粒數顯著高于R99，R99粒型細長，SN265粒型短圓。每個重組自交系平均測序深度為6.25×，R99和SN265的測序深度分別為30×和32×。獲得1 456 445個高質量的SNP，利用劃bin策略進行圖譜構建，得到一個包含3 569個bins，平均長度為58.17 kb的遺傳圖。QTL分析在第9染色體檢測到一個同時調控每穗粒數、一次枝梗著粒數和二次枝梗著粒數的QTL，在第1染色體鑒定到一個調控每穗粒數和二次枝梗著粒數的QTL，在第5染色體鑒定到一個調控粒型的QTL。候選基因預測和序列比對發(fā)現第9染色體的同時調控水稻一次和二次枝梗著粒數，第1染色體的主要調控水稻二次枝梗著粒數，第5染色體的主要調控粒型。在151個重組自交系中，對、和的不同組合進行分類并調查產量構成因素，發(fā)現Gn1a/DEP1/qSW5等位基因組合產量表現最好，Gn1a/DEP1/qSW5產量表現最差。對SN265的位點進行分子設計育種，獲得2個獨立的CRISPR基因編輯株系，通過調查其產量構成因素，發(fā)現基因編輯植株穗長顯著變長，每穗粒數顯著增加，進而顯著增加單株產量?！尽拷沂玖?、和對每穗粒數和粒型的影響，明確了Gn1a/DEP1/qSW5為重組自交系中最佳基因組合，通過改良SN265的位點進一步提高了其單株產量。

水稻；高密度遺傳圖譜；每穗粒數；粒型；基因編輯

0 引言

【研究意義】水稻（L.）是重要的糧食作物，約為世界一半人口提供主糧，在全球糧食安全中也發(fā)揮重要作用[1]。水稻產量是一個復雜性狀，主要由穗部性狀控制。在過去的40年中，中國水稻的增產主要通過提高每穗粒數來實現[1]，因此，剖析調控穗部性狀的遺傳機制對育種家提高水稻產量具有重要的意義。【前人研究進展】水稻的圓錐狀花序由穗軸、枝梗、籽粒組成，且籽粒著生在枝梗上，枝梗著生于穗軸上。水稻穗的發(fā)育過程是復雜的，包括小穗分化、發(fā)育和退化等一系列生理過程。在生殖生長期，水稻莖尖分生組織轉變?yōu)榛ㄐ蚍稚M織，分化為一次枝梗分生組織，二次枝梗分生組織在一次枝梗上相繼產生，進一步分化為小穗分枝分生組織和側穗分生組織。在同一時期，一次枝梗分生組織頂部分化為末端小穗分生組織[2-3]。這些分枝及其分化的穗狀分生組織最終形成水稻穗的基本結構，決定每穗粒數。許多參與穗分枝形成的基因已被克隆，如、、/（/）、（）、（）、（）、等參與頂端分生組織分化形成花序分生組織，進而形成一次枝梗和二次枝梗分生組織的分化過程[4-7]；/（/）、（）、（）等參與枝梗分生組織到小穗分生組織的分化過程[8-11]；/與（）調控小穗分生組織到穎花分生組織的分化[12-16]。水稻粒形是穗部性狀的重要組成部分，屬于受多基因控制的數量性狀，單個基因的效應值通常較小，受環(huán)境影響較大[17]。目前，已報道的粒形相關QTL位點約600個，隨著水稻基因組測序和功能基因組研究的深入，已成功克隆近百個粒形相關基因[18-21]。這些基因通過多種途徑調控水稻粒形，主要包括轉錄因子調控（如、、和[16, 22-31]）、泛素途徑（如、和[27-28, 32-33]）、G蛋白途徑（如、和[34-36]），以及激素水平控制粒形途徑（如、、、和）等?！颈狙芯壳腥朦c】數十年來，中國科學家在水稻穗部性狀遺傳基礎、調控基因的定位與功能研究等方面取得了卓越成績，但由于材料背景的限制，單一的雜交組合僅能鑒定到少數穗部性狀相關QTL，單個QTL貢獻率因試驗材料和研究方法的不同往往表現出巨大差異。目前對穗部性狀的研究還主要集中于單個功能基因的解析和應用，對每穗粒數和粒形調控基因的組合應用研究較少?！緮M解決的關鍵問題】本研究以穗型和粒型差異顯著的雙親所構建的重組自交系為試材，對每穗粒數、一次枝梗著粒數、二次枝梗著粒數和粒型進行QTL分析和候選基因功能鑒定，評價不同基因組合的產量構成因素，并對超級稻品種SN265進行分子設計基因編輯育種，為水稻優(yōu)勢等位基因聚合育種提供重要種質和基因資源。

1 材料與方法

1.1 供試水稻材料

R99為典型的彎穗型秈稻品種，SN265為中國第一個直立大穗型粳型超級稻品種。以SN265和R99為雙親配制雜交組合，采用單粒傳法套袋自交12代，獲得包含151個株系的穩(wěn)定遺傳重組自交系。親本和重組自交系于2021年春季種植于沈陽農業(yè)大學水稻所試驗田（123°E，41°N），每個株系按照3行×6株規(guī)模種植一小區(qū)，株行距均為20 cm，單苗插植，常規(guī)栽培管理。

1.2 表型考察

抽穗后45 d，每小區(qū)取中部5株收獲，充分曬干后，考察單株穗數。取長勢均勻的5穗，統(tǒng)計一次枝梗著粒數、二次枝梗著粒數、每穗粒數和結實率。隨機取500粒飽滿籽粒稱重統(tǒng)計千粒重。最后以Microsoft Excel 2016計算各材料每株的各性狀平均值、標準差，并對各性狀進行兩尾等方差檢驗，使用GraphPad Prism 8進行作圖。

1.3 全基因組測序和遺傳圖譜構建

選取插秧后3周齡植株的幼嫩葉片，采用CTAB法提取DNA，送北京百邁克生物科技有限公司進行高通量測序分析。參照Li等[37]報道的重測序手段，采用“滑動窗口”法構建R99和SN265的重組自交系群體的遺傳圖譜。利用劃bin策略得到3 569個bins，平均長度為58.17 kb的遺傳圖譜。采用R/qtl的CIM方法進行QTL定位，采用mqmpermutation命令進行排列組合1 000次的LOD閾值（=0.05）確定，當實際求得的LOD值大于LOD閾值時，就認為該區(qū)段存在1個QTL，其置信區(qū)間為LOD峰值向下1個LOD值單位的區(qū)間[38]。

1.4 CRISPR/Cas9基因編輯

以超級稻品種SN265為遺傳背景材料進行CRISPR基因編輯。通過華南農業(yè)大學亞熱帶農業(yè)生物資源保護與利用國家重點實驗室劉耀光院士團隊開發(fā)的基因編輯工具包CRISPR-GE（http://skl.scau.edu. cn/）進行靶位點的設計，應用BLAST比對日本晴參考基因組確認靶點的特異性（https://rapdb.dna. affrc.go.jp/tools/blast）?；蚓庉嫲悬c序列和引物合成，以及測序服務均由華大基因完成。參照Li等[37]方法構建基因編輯載體以及基因編輯植株的遺傳轉化和篩選。

2 結果

2.1 雙親穗部性狀差異顯著

R99穗型松散，SN265穗型緊湊。R99每穗粒數顯著多于SN265。進一步調查一次枝梗和二次枝梗，發(fā)現SN265的一次枝梗著粒數顯著多于R99，而R99的二次枝梗著粒數顯著多于SN265。此外，R99和SN265的粒型也存在顯著差異，R99籽粒細長，籽粒長寬比超過2.5，而SN265的籽粒較為短圓，籽粒長寬比約為2（圖1）。

2.2 遺傳圖譜構建與QTL分析

應用Illumina測序平臺對R99和SN265為親本構建的重組自交系和雙親進行全基因組重測序，每個重組自交系平均測序深度為6.25×，R99和SN265的測序深度為30×和32×。得到1 456 445個高質量的SNP，利用劃bin策略進行圖譜構建，得到3 569個bins，平均長度為58.17 kb的遺傳圖譜[37]。利用R/qtl軟件對重組自交系群體的每穗粒數、一次枝梗著粒數、二次枝梗著粒數和粒型進行QTL分析。獲得2個控制每穗粒數的QTL，分布在第1和第9染色體，其LOD值分別為7.8和12.6，表型貢獻率分別為16.8%和28.1%。隨后，把每穗粒數拆分成一次枝梗著粒數和二次枝梗著粒數分別進行QTL分析。結果顯示，第9染色體的QTL同時控制一次枝梗著粒數和二次枝梗著粒數，而第1染色體的QTL只調控二次枝梗著粒數。在第5染色體檢測到一個LOD值為18.5，貢獻率為42.1%的主效粒型QTL（圖2）。

2.3 候選基因序列比對

通過數據庫比對發(fā)現第1染色體上調控每穗粒數和二次枝梗著粒數的QTL與已報道的編碼細胞分裂素降解酶位置重合[6]，第9染色體控制每穗粒數、一次枝梗著粒數和二次枝梗著粒數的QTL與已經報道的G蛋白伽馬亞基位置重合[7]，第5染色體控制粒型的QTL與已報道的油菜素內酯信號傳導的新型正調因子位置重合[33, 39-40]。應用基于三代測序組裝的SN265和R99高質量基因組[37, 41]，比對雙親、和的基因序列發(fā)現，與R99相比，SN265在位點上游5 kb存在一個1 212 bp的缺失，該1 212 bp缺失通過調控的表達量進而調控籽粒大小[33]。SN265在的3′端有一段637 bp的序列被12 bp序列所替換，使蛋白缺失了C端的Cys富集區(qū)域，該突變能促進細胞分裂，降低穗頸節(jié)長度并使稻穗變密、枝梗數增加、每穗籽粒數增多，從而促進水稻增產。SN265在和位點均為優(yōu)勢等位基因，而R99在位點的第1個外顯子處6 bp的插入和2個SNP（C/G和G/A），以及第4個外顯子處的1個SNP（G/T），導致其蛋白產物與粳稻品種產生差異，引起花序分裂組織中細胞分裂素的積累，因而增加每穗粒數，最終導致產量提高（圖3）。

A：株型；B：穗型；C：粒型；D：每穗粒數；E：一次枝梗著粒數；F：二次枝梗著粒數；G：籽粒長寬比。*：P＜0.05

2.4 不同DEP1、Gn1a和qSW5組合的產量表現

為了闡明、和的不同基因組合對水稻產量表現的影響，根據3個基因的等位基因型，將151個重組自交系分為8個類型（圖4）。通過比對其每穗粒數、千粒重和單株產量，發(fā)現Gn1a/DEP1等位基因的組合每穗粒數表現最佳，Gn1a/DEP1和Gn1a/DEP1次之，Gn1a/DEP1組合的每穗粒數最少。千粒重主要受基因型調控，含有qSW5的4種類型千粒重普遍顯著高于含有qSW5等位基因4種類型?？傊?，Gn1a/DEP1/qSW5因每穗粒數和千粒重的優(yōu)勢體現出最好的產量表現，Gn1a/DEP1/qSW5則因為每穗粒數和千粒重的劣勢產量表現最差（圖4）。

+：R99基因型；-：SN265基因型。不同字母表示差異顯著（P＜0.05）。下同

2.5 CRISPR/Cas9基因編輯聚合優(yōu)勢基因型

基于2.4結果，Gn1a/DEP1/qSW5有最好的產量表現，而超級稻品種SN265的基因型僅與Gn1a/DEP1/qSW5在位點上存在差別，在粳稻中花11遺傳背景下對進行Knock-out突變，可以顯著增加每穗粒數[42-43]。因此，對SN265進行基因設計育種，在的第一個外顯子設計PAM序列，進行基因編輯，在T0篩選陽性植株，在T1進行測序，鑒定到CR-1和CR-2 2個純合突變株系，分別缺失了1和2 bp，造成移碼突變（圖5）。2個基因編輯突變體穗長較SN265穗長顯著增長，每穗粒數顯著增加（圖5）。基因編輯植株與SN265的結實率、穗數和千粒重差異不顯著，因為每穗粒數的增加，基因編輯植株的單株產量顯著高于SN265（圖5）。綜上，Gn1a/DEP1/qSW5基因編輯植株較SN265體現出更好的單株產量表現。

A：CRISPR基因編輯的PAM序列和突變體序列；B：SN265和基因編輯植株的穗型；C：SN265和基因編輯植株的穗長；D：SN265和基因編輯植株的每穗粒數；E：SN265和基因編輯植株的結實率；F：SN265和基因編輯植株的穗數；G：SN265和基因編輯植株的千粒重；H：SN265和基因編輯植株的單株產量

3 討論

3.1 優(yōu)異等位基因聚合

水稻育種實踐表明增加每穗粒數是提高水稻產量的最有效途徑之一。近幾十年來，水稻每穗粒數的研究取得了很大進展，已成功克隆了多個影響每穗粒數的基因，這些影響每穗粒數的基因可以作為育種家的潛在種質資源。分子標記輔助選擇是基因聚合的有效工具[44]，如的3型等位基因和OsSPL14等位基因通過重復回交聚合，顯著增加每穗粒數[45]。和的聚合可以通過增加每穗粒數來提高水稻產量[46]。因為一些控制每穗粒數的基因在水稻中產生其他不良影響，例如延遲水稻抽穗[47]，使得水稻籽粒變小[9]，減少分蘗數[31, 48]，直立穗型等位基因有降低千粒重的趨勢[49-50]。如何平衡這些性狀與每穗粒數之間的關系仍然是育種中亟待解決的問題。本研究發(fā)現同時調控水稻一次枝梗著粒數和二次枝梗著粒數，主要調控二次枝梗著粒數，qSW5等位基因通過增加粒寬提高千粒重，可減輕所引起的千粒重降低，、和優(yōu)勢等位基因的組合應用可以獲得最優(yōu)的產量表現。

3.2 水稻分子設計育種

分子設計育種技術體系不斷實踐與完善對促進作物育種技術發(fā)展有重要作用，近年來越來越多的水稻產量構成因素調控基因被克隆，其分子調控網絡也被深入解析，加上CRISPR基因編輯技術的廣泛應用，分子設計育種開始應用到水稻生產實踐當中[51-52]，以為核心的理想株型設計育種[53]，以為核心的品質改良設計育種都已初見成效[54]。本研究針對中國第一個直立大穗型超級稻品種，根據試驗結果嘗試對其位點進行基因編輯以期進一步增加SN265的每穗粒數。本研究證實了SN265的編輯植株每穗粒數顯著增加，顯著提高了其單株產量。但是水稻作為群體作物，單株產量的提高并不能保證其單位面積產量增產。超級稻SN265的直立穗型有助于改善群體受光結構，適應高密度密植[55]，其編輯植株穗長增加，是否會削弱其對高密度栽培的適應能力還需進一步研究，與其配套的栽培技術也將是今后研究的方向之一。在水稻種質資源中發(fā)現有利基因或等位基因并在不同遺傳背景下對其功能進行評估，以及通過分子標記輔助選擇和基因編輯技術闡明這些有利等位基因之間的遺傳互作及其對產量的聚合效應是今后水稻分子設計育種的兩項主要任務。

4 結論

闡明了、和對一次枝梗著粒數、二次枝梗著粒數和粒型的影響，發(fā)現了Gn1a/DEP1/qSW5為重組自交系中最佳基因組合，通過改良SN265的位點增加每穗粒數，從而進一步提高了其單株產量。

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Combination of,, andregulates the panicle architecture in rice

1College of forestry, Shenyang Agricultural University, Shenyang 110866;2Rice research institute of Shenyang Agricultural University, Shenyang 110866

【】Rice is an important food crop, providing staple food for more than half of the world’s population. Panicle traits are the main factors affecting rice yield. Discover the elite haplotype of the panicle regulation gene, and provide important germplasm and gene resources for pyramiding breeding. 【】In this study, recombinant inbred lines (RILs) derived from a cross between SN265 and R99 were re-sequenced through high-throughput sequencing. QTL analysis and candidate gene identification were conducted on the grain number on the primary branch, the grain number on the secondary branch, and the grain shape. The sequences of candidate genes were compared using the long-read sequence assemblies of SN265 and R99. The combination of candidate genes that can maximize grain yield was selected among RILs. Finally, the super rice variety SN265 was improved using CRISPR/Cas9 gene editing technology. 【】The R99 had significantly more grain number per panicle and grain number on the secondary branch, whereas SN265 had significantly more grain number on the primary branch. The grain of R99 is slender, and the grain of SN265 is short and round. The RILs were sequenced with approximately 6.25-fold depth. For parent lines, 30.0-fold depth and 32.0-fold depth data were generated for R99 and SN265, respectively. Subsequently, a bin map was constructed by 1456445 high-quality SNPs. The genetic map containing 3 569 recombinant blocks, with an average length of 58.17 kb. The QTL analysis detected a QTL on Chr.9 for grain number per panicle and grain number on both primary and secondary branch, a QTL on Chr.1 for grain number per panicle and grain number on the secondary branch, a QTL on Chr.5 for grain shape. The candidate gene prediction and sequence comparison showed thatregulated the grain number on both primary and secondary branches of rice,mainly regulated the grain number on secondary branches of rice, andmainly regulated the grain shape. The yield of the combination ofGn1a/DEP1/qSW5alleles showed an advantage in yield performance among the RILs. We further conducted a molecular design breeding to SN265 by knocking out thelocus using CRISPR/Ca9 gene editing technology, and the grain number per panicle of the transgenic plants increased significantly compared to that of SN265. 【】This study used RILs derived from a XI/GJ cross and high-throughput sequencing technology to conduct QTL analysis of rice panicle traits, revealed the effects of,andon grain number per panicle and grain shape, and clarified thatGn1a/DEP1/qSW5was the best gene combination in RILs. The yield per plant was further improved by knocking out thelocus of SN265. This study provided important germplasm and gene resources for pyramiding breeding with elite alleles.

rice; high density genetic map; grain number; grain shape; gene editing

2022-10-21；

2022-11-14

國家自然科學基金（32071982）

溫一博，E-mail：wenyibo@syau.edu.cn。通信作者徐銓，E-mail：kobexu34@syau.edu.cn。通信作者孫健，E-mail：sunjian811119@syau.edu.cn

（責任編輯 李莉）