楊太樺 楊福權(quán) 郜耿東 殷 帥 金慶東 徐林珊 蒯 婕 汪 波 徐正華 葛賢宏 王 晶 周廣生

初步探究LncRNA在甘藍(lán)型油菜生態(tài)型分化中的作用

楊太樺 楊福權(quán) 郜耿東 殷 帥 金慶東 徐林珊 蒯 婕 汪 波 徐正華 葛賢宏 王 晶*周廣生

華中農(nóng)業(yè)大學(xué)植物科學(xué)技術(shù)學(xué)院, 湖北武漢 430070

甘藍(lán)型油菜是我國(guó)重要的油料作物之一, 根據(jù)其成花轉(zhuǎn)變過程中對(duì)低溫春化時(shí)間需求的不同分為3種生態(tài)型: 春性、半冬性和冬性。前人研究發(fā)現(xiàn), 長(zhǎng)鏈非編碼RNA (LncRNA)可在多層面上調(diào)控基因的表達(dá), 參與對(duì)植物生長(zhǎng)發(fā)育的調(diào)控。在擬南芥中, LncRNA可以通過調(diào)控春化途徑相關(guān)基因的表達(dá)來影響開花。本研究以3種生態(tài)型甘藍(lán)型油菜為材料, 利用高通量測(cè)序技術(shù)進(jìn)行苗期葉片的mRNA和LncRNA測(cè)序, 初步探究LncRNA在油菜生態(tài)型分化及適應(yīng)性形成中的作用。3種生態(tài)型甘藍(lán)型油菜共差異表達(dá)基因的GO及KEGG富集分析表明, 不同生態(tài)型甘藍(lán)型油菜之間存在大量基礎(chǔ)化合物合成代謝差異, 特別是脂質(zhì)類化合物。3種生態(tài)型甘藍(lán)型油菜中共鑒定獲得3775個(gè)LncRNA, 其中285個(gè)在2個(gè)及以上生態(tài)型組合中存在差異表達(dá), 涉及到1517個(gè)候選靶基因。這些差異表達(dá)LncRNA涉及到的靶基因也富集到大量基礎(chǔ)化合物合成代謝途徑。通過mRNA-LncRNA聯(lián)合分析, 我們預(yù)測(cè)到了一個(gè)開花基因的調(diào)控網(wǎng)絡(luò), 包含8個(gè)開花基因和23個(gè)LncRNA, 涉及到溫度和光信號(hào)調(diào)控通路。通過比較鑒定得到的LncRNA和972個(gè)甘藍(lán)型油菜重要農(nóng)藝性狀QTL位置信息發(fā)現(xiàn)約90%的LncRNA位點(diǎn)和QTL區(qū)間存在重疊, 且差異表達(dá)LncRNA和QTL的重疊位點(diǎn)在不同生態(tài)型油菜中的分布具有差異。結(jié)果說明LncRNA在油菜生態(tài)型分化及重要農(nóng)藝性狀形成中具有重要作用。

甘藍(lán)型油菜; 生態(tài)型; 長(zhǎng)鏈非編碼RNA; 開花基因; 農(nóng)藝性狀位點(diǎn)

甘藍(lán)型油菜(L., 2= 38)是我國(guó)及世界上重要的油料作物之一, 菜籽油約占我國(guó)國(guó)產(chǎn)食用植物油的1/3[1]。由于自然選擇和人工選擇馴化, 甘藍(lán)型油菜形成了適應(yīng)不同氣候和緯度區(qū)域種植的生態(tài)類型。根據(jù)其成花轉(zhuǎn)化中對(duì)低溫春化需求的不同分為冬油菜(winter oilseed rape, WOSR)、半冬性油菜(semi-winter oilseed rape, SWOSR)和春油菜(spring oilseed rape, SOSR)。冬油菜是最原始的類型, 于歐洲中世紀(jì)晚期開始種植, 開花晚, 需要嚴(yán)格的春化[2]; 春油菜在1700年左右形成, 其開花早, 無需春化[3]; 此外, 甘藍(lán)型油菜于1930年前后傳入中國(guó), 經(jīng)過選擇后產(chǎn)生了新的生態(tài)類型, 即為半冬性油菜, 需要適當(dāng)?shù)拇夯痆3-4]。

作物的開花時(shí)間是重要的農(nóng)藝性狀之一, 受外界環(huán)境因子和自身發(fā)育信號(hào)共同調(diào)控。在擬南芥中有超過300個(gè)開花相關(guān)的基因被鑒定[5]。隨著測(cè)序技術(shù)的發(fā)展及多個(gè)高質(zhì)量甘藍(lán)型油菜參考基因組的公布(Darmor-bzh、寧油7號(hào)、中雙11號(hào)), 研究人員發(fā)現(xiàn)基因是影響甘藍(lán)型油菜春化需求和開花時(shí)間的重要位點(diǎn)[6-9]?；谧匀蝗后w的重測(cè)序及關(guān)聯(lián)分析研究中, 研究人員鑒定到大量的與生態(tài)型分化/開花時(shí)間顯著關(guān)聯(lián)的位點(diǎn), 也發(fā)現(xiàn)基因?qū)τ谏鷳B(tài)型分化起到了重要作用[2,10-13]。An等[14]通過群體轉(zhuǎn)錄組學(xué)數(shù)據(jù)也挖掘到了一些開花或調(diào)節(jié)春化的基因。Song等[15]完成了甘藍(lán)型油菜泛基因組構(gòu)建, 并且通過巢式關(guān)聯(lián)群體進(jìn)行了全基因組關(guān)聯(lián)分析, 鑒定到67個(gè)開花候選基因, 進(jìn)一步證實(shí)基因等位變異在一定程度上決定了油菜對(duì)春化的需求及開花時(shí)間。Yin等[11]則通過等位基因序列分離及轉(zhuǎn)基因驗(yàn)證, 進(jìn)一步發(fā)現(xiàn)與共同調(diào)控不同類型甘藍(lán)型油菜對(duì)春化的需求。與上述研究相一致, 有研究認(rèn)為甘藍(lán)型油菜9個(gè)基因可能均參與了油菜春化需求的調(diào)節(jié)[16-18]。

非編碼RNA是一種不具有編碼蛋白質(zhì)能力的RNA, 長(zhǎng)期以來被認(rèn)為是無用的轉(zhuǎn)錄“噪音”[19]。長(zhǎng)鏈非編碼RNA (LncRNA)作為非編碼RNA的一種, 是長(zhǎng)度大于200個(gè)核苷酸的RNA, 普遍存在于動(dòng)植物基因組中, 參與細(xì)胞分化及個(gè)體發(fā)育等一系列生物學(xué)過程。前人研究發(fā)現(xiàn), 在擬南芥中LncRNA作為重要的轉(zhuǎn)錄調(diào)控因子調(diào)控基因, 從而調(diào)控開花進(jìn)程[20]。目前為止, 甘藍(lán)型油菜中有關(guān)LncRNA的研究還不多見。Song等[21-22]最早鑒定到了一條花粉特異性的非編碼RNA, 其參與甘藍(lán)型油菜花粉發(fā)育的調(diào)控。Joshi等[23]在甘藍(lán)型油菜中鑒定到3181個(gè)LncRNA, 發(fā)現(xiàn)一個(gè)LncRNA可能參與了核盤菌防御響應(yīng)。Zhang等[24]分別在甘藍(lán)型油菜中鑒定到1885個(gè)LncRNA, 發(fā)現(xiàn)LncRNA表現(xiàn)出了A亞基因組傾向性的表達(dá)。Shen等[25]發(fā)現(xiàn)LncRNA可能在脂質(zhì)代謝中發(fā)揮作用, 可能參與了對(duì)編碼oleosin1蛋白基因的調(diào)控, 進(jìn)而參與種子脂質(zhì)的積累過程。Tan等[26]的研究發(fā)現(xiàn), 干旱脅迫和復(fù)水處理影響了部分LncRNA的表達(dá), 表明LncRNA存在響應(yīng)應(yīng)激的功能。

本研究基于第二代高通量測(cè)序技術(shù), 對(duì)3種生態(tài)型甘藍(lán)型油菜進(jìn)行了LncRNA-seq和mRNA-seq分析, 并結(jié)合已報(bào)道的重要農(nóng)藝性狀的QTL信息, 初步探究LncRNA在甘藍(lán)型油菜生態(tài)分化及適應(yīng)性形成中的作用, 為油菜遺傳育種以及適應(yīng)性改良提供理論依據(jù)。

1 材料與方法

1.1 試驗(yàn)材料與測(cè)序數(shù)據(jù)

本研究中用到的甘藍(lán)型油菜品種為Westar (春油菜)、Dunkeld (春油菜)、Tapidor (冬油菜)、Darmor (冬油菜)、中雙11號(hào)(半冬性油菜)和寧油7號(hào)(半冬性油菜), 材料于2017—2018年種植于華中農(nóng)業(yè)大學(xué)試驗(yàn)田。待6種甘藍(lán)型油菜都生長(zhǎng)至五片真葉期時(shí), 同時(shí)取第3片幼嫩葉片用于總RNA的提取用于建庫測(cè)序, 每個(gè)材料各2個(gè)生物學(xué)重復(fù),-80℃保存?zhèn)溆谩?/p>

使用高純總RNA快速提取試劑盒(北京百泰克生物技術(shù)有限公司)進(jìn)行RNA的提取, cDNA反轉(zhuǎn)引物為隨機(jī)六引物, 建庫試劑盒為TruSeq Stranded Total RNA with Ribo-Zero Plant (Illumina)。LncRNA、mRNA建庫和測(cè)序均由華中農(nóng)業(yè)大學(xué)作物遺傳改良國(guó)家重點(diǎn)實(shí)驗(yàn)室高通量測(cè)序平臺(tái)完成。為更好地鑒定LncRNA, 本研究選用組裝質(zhì)量較好的v10 (contig N50 = 11.49 Mb)[27]為參考基因組進(jìn)行后續(xù)的分析。

1.2 轉(zhuǎn)錄組數(shù)據(jù)分析

原始下機(jī)數(shù)據(jù)通過FastQC (version = 0.11.5)[28]軟件進(jìn)行質(zhì)量檢測(cè), 并使用Trimmomatic (version = 0.39)[29]軟件去接頭序列和除低質(zhì)量reads。使用Hisat2 (version = 2.1.0)[30]軟件將質(zhì)控后的clean reads比對(duì)到參考基因組上, 使用StringTie (version = 2.1.4)[31]獲取每個(gè)基因上的reads數(shù)量并用TPM (transcripts per kilobase of exon model per million mapped reads, 每千個(gè)堿基的轉(zhuǎn)錄每百萬映射讀取的轉(zhuǎn)錄本數(shù))表示基因的表達(dá)量。使用Deseq2 (version = 3.3.3)[32]軟件進(jìn)行基因差異表達(dá)分析, 篩選標(biāo)準(zhǔn)為 |log2(Fold Change) | ≥ 2且adj< 0.01。

1.3 LncRNA數(shù)據(jù)分析

原始下機(jī)數(shù)據(jù)通過FastQC軟件進(jìn)行質(zhì)量檢測(cè), 并使用Trimmomati軟件去除低質(zhì)量的reads, 然后使用Hisat2軟件將質(zhì)控后的clean reads比對(duì)到參考基因組上。使用StringTie軟件進(jìn)行有參組裝, 得到組裝的gtf文件。使用gffcompare (version = v0.12.2)[33]將得到的gtf文件與參考基因組gff注釋文件進(jìn)行比較, 提取標(biāo)記為i、j、o、u和x的轉(zhuǎn)錄本序列。對(duì)得到的轉(zhuǎn)錄本進(jìn)行過濾: (1) 保留轉(zhuǎn)錄本長(zhǎng)度≥200 bp、表達(dá)量TPM≥1以及外顯子數(shù)目 > 1的轉(zhuǎn)錄本。(2)使用CPC2[34]、CPAT[35]和PLEK[36]軟件預(yù)測(cè)轉(zhuǎn)錄本編碼能力, 取交集保留不具有編碼能力的轉(zhuǎn)錄本。將最終得到的LncRNA轉(zhuǎn)錄本比對(duì)到LncRNA數(shù)據(jù)庫-CANTATAdb (http://yeti.amu.edu.pl/CANTATA)[37], 沒比對(duì)上的視為新鑒定的LncRNA。使用Deseq2軟件進(jìn)行基因差異表達(dá)分析, 篩選標(biāo)準(zhǔn)為 |log2(Fold Change)|≥2且adj< 0.01。

2 結(jié)果與分析

2.1 3種生態(tài)型油菜的開花時(shí)間具有差異

在半冬性環(huán)境條件下, 春油菜開花所需時(shí)間最短, 其次是半冬性油菜, 冬油菜開花所需的時(shí)間最長(zhǎng)(2014年武漢: Westar: 146 d、Dunkeld: 157 d、中雙11號(hào): 158 d、寧油7號(hào): 152 d、Tapidor: 179 d、Darmor: 181 d); 在春環(huán)境條件下, 冬油菜基本無法開花, 春油菜開花所需時(shí)間短于半冬性油菜, 并且他們的開花所需時(shí)間為在半冬性環(huán)境下的一半(2013年青海: Westar: 56 d、Dunkeld: 61 d、中雙11號(hào): 78 d、寧油7號(hào): 70 d、Tapidor和Darmor不開花; 2014年甘肅: Westar: 51 d、Dunkeld: 67 d、中雙11號(hào): 70 d、寧油7號(hào): 65 d、Tapidor和Darmor不開花)[4,10,15]。在武漢半冬性環(huán)境條件下, 2021—2022年度, Westar (春油菜)、中雙11號(hào)(半冬性油菜)和Tapidor (冬油菜)的開花時(shí)間分別為107、162和189 d。播種后110 d, 3種油菜外觀形態(tài)也存在較大差別(圖1)。

圖1 3種生態(tài)型甘藍(lán)型油菜同時(shí)期形態(tài)

在武漢半冬性環(huán)境下, 2021-2022年度, 播種約110 d時(shí), 3種生態(tài)型甘藍(lán)型油菜形態(tài)差異明顯。A: Westar; B: 中雙11號(hào); C: Tapidor。標(biāo)尺為20 cm。

Morphology of three ecotypes of, about 110 days after sowing in semi-winter environment of Wuhan, in 2021-2022. A: Westar; B: Zhongshuang 11; C: Tapidor. Bar: 20 cm。

2.2 甘藍(lán)型油菜全基因表達(dá)譜

為探究不同生態(tài)型甘藍(lán)型油菜之間的基因表達(dá)情況, 本研究對(duì)3種生態(tài)型共6種甘藍(lán)型油菜品種進(jìn)行轉(zhuǎn)錄組測(cè)序, 按照3種生態(tài)型進(jìn)行分組分析。將表達(dá)量TPM≥1的基因視為存在表達(dá)的基因, 經(jīng)過過濾, 每個(gè)樣本mRNA-seq產(chǎn)生了168.42～256.30百萬條clean reads。12個(gè)甘藍(lán)型油菜mRNA-seq數(shù)據(jù)比對(duì)率為92.96%～98.00%。主成分分析表明同組樣本之間具有很好的一致性(圖2-A)。總體而言, 甘藍(lán)型油菜中, 40.45%～44.75%的基因存在表達(dá), 37.26% (40,308)的基因在春油菜中表達(dá), 39.22% (42,429)的基因在冬油菜中表達(dá), 38.56% (41,713)的基因在半冬性油菜中表達(dá)(圖2-B)。32.52% (35,184)的基因在3種生態(tài)型甘藍(lán)型油菜中均表達(dá), 51.82% (56,066)的基因至少在一種甘藍(lán)型油菜中表達(dá)(圖2-B)。2843、2629和2225個(gè)差異表達(dá)基因分別在春油菜與冬油菜、春油菜與半冬性油菜和半冬性油菜與冬油菜之間檢測(cè)到(圖2)。其中27個(gè)基因在3種生態(tài)型組合中均鑒定為差異表達(dá)基因(圖2-D)。

圖2 3種生態(tài)型6個(gè)甘藍(lán)型油菜品種mRNA-seq分析相關(guān)結(jié)果

A: 6種甘藍(lán)型油菜品種12個(gè)樣本mRNA-seq主成分分析; B: 在6個(gè)甘藍(lán)型油菜品種和3種生態(tài)型中甘藍(lán)型油菜全部基因表達(dá)情況; C: 甘藍(lán)型油菜3種生態(tài)型兩兩之間差異表達(dá)基因數(shù)量; D: 甘藍(lán)型油菜3種生態(tài)型兩兩之間差異表達(dá)基因韋恩圖。SOR: 春油菜; SWOR: 半冬性油菜; WOR: 冬油菜。

A: mRNA-seq PCA analysis of 12 samples from sixcultivars; B: the relative expression level of all genes in sixcultivars and three ecotypes; C: the number of differentially expressed genes among three ecotypes; D: Venn diagram of differentially expressed genes among three ecotypes of. SOR: spring type rapeseed; SWOR: semi-winter type rapeseed; WOR: winter-type rapeseed.

2.3 不同生態(tài)型甘藍(lán)型油菜差異表達(dá)基因分析

為探究這些差異基因在不同生態(tài)型中的參與的功能, 本研究對(duì)在2組及以上生態(tài)型中出現(xiàn)差異表達(dá)的基因(mRNA-DEG, 共2300個(gè)基因)進(jìn)行GO和KEGG富集分析。GO富集分析表明, 這些基因顯著富集在脂質(zhì)定位(GO:0010876)、脂質(zhì)轉(zhuǎn)運(yùn)(GO:0006869)、磷脂酶C活性(GO:0004629)、磷脂酰肌醇磷脂酶C活性(GO:0004435)、磷酸二酯水解酶活性(GO:0008081)、磷脂酶活性(GO:0004620)等通路(圖3-A)。KEGG富集分析表明, 這些基因顯著富集在黃酮類物質(zhì)生物合成、苯丙烷類物質(zhì)生物合成、異黃酮類生物合成、α-亞麻酸代謝等通路(圖3-B)。表明不同生態(tài)型甘藍(lán)型油菜的分化形成可能涉及到了大量基礎(chǔ)化合物的合成代謝, 并且脂質(zhì)類相關(guān)化合物可能發(fā)揮了重要的作用。

圖3 3種生態(tài)型甘藍(lán)型油菜差異表達(dá)基因GO功能和KEGG通路富集分析

A: 3種生態(tài)型甘藍(lán)型油菜差異表達(dá)基因GO功能富集分析; B: 3種生態(tài)型甘藍(lán)型油菜差異表達(dá)基因KEGG通路富集分析。

A: GO function enrichment of differentially expressed genes in three ecotypes of; B: KEGG pathway enrichment of differentially expressed genes in three ecotypes of.

2.4 LncRNA鑒定和靶基因鑒定分析

在之前的研究中, 報(bào)道了lncRNA作為重要的轉(zhuǎn)錄調(diào)控因子調(diào)控植物的開花[20]。本研究對(duì)3種生態(tài)型6種甘藍(lán)型油菜品種進(jìn)行LncRNA測(cè)序分析。每個(gè)樣本LncRNA-seq產(chǎn)生了157.31～537.70百萬條clean reads。12組甘藍(lán)型油菜LncRNA-seq數(shù)據(jù)比對(duì)率為93.30%～95.67%。用Stringtie軟件對(duì)LncRNA測(cè)序數(shù)據(jù)進(jìn)行組裝, 12組樣本中共得到了146,188條不同的轉(zhuǎn)錄本, 過濾后共鑒定得到3775個(gè)LncRNA序列, 其中97.91% (3696)的LncRNA可以定位在甘藍(lán)型油菜19條染色體上。主成分分析表明同組樣本之間的LncRNA表達(dá)量具有很好的一致性(圖4-A)。將鑒定到的甘藍(lán)型油菜LncRNA序列與白菜、甘藍(lán)和甘藍(lán)型油菜LncRNA數(shù)據(jù)庫進(jìn)行比對(duì)分析, 分別比對(duì)上了1190、1418和2176個(gè)LncRNA, 另外鑒定到1147個(gè)新LncRNA序列。28.90% (1091), 26.86% (1014)和28.32% (1069)的LncRNA分別在春油菜、半冬性油菜和冬油菜中表達(dá)(圖4-B)。其中, 688個(gè)LncRNA在春油菜與冬油菜中都表達(dá); 645個(gè)在半冬性油菜與春油菜中表達(dá); 640個(gè)在冬油菜與半冬性油菜中表達(dá)。522 (13.83%)個(gè)LncRNA在3種生態(tài)型甘藍(lán)型油菜中都表達(dá)(圖4-C)。在油菜19條染色體上, A10染色體上鑒定到的LncRNA數(shù)量最少(79), C03上數(shù)量最多(307)。C亞基因組上LncRNA數(shù)量要遠(yuǎn)多于A亞基因組(A亞基因組: 1486, C亞基因組: 2210), 這可能與甘藍(lán)型油菜參考基因組A、C 亞基因組實(shí)際大小和基因數(shù)量有關(guān)(A亞基因組大小: 346M, 基因數(shù)量: 47,193; C亞基因組大小: 520M, 基因數(shù)量: 59,692)(圖4-D)。

圖4 3種生態(tài)型6個(gè)甘藍(lán)型油菜品種LncRNA-seq分析相關(guān)結(jié)果

A: 6種甘藍(lán)型油菜品種12個(gè)樣本LncRNA-seq主成分分析; B: 在6個(gè)甘藍(lán)型油菜品種和3種生態(tài)型中存在表達(dá)的LncRNA數(shù)量; C: 甘藍(lán)型油菜3種生態(tài)型之間LncRNA表達(dá)情況韋恩圖; D: 在3種生態(tài)型甘藍(lán)型油菜中共鑒定到了LncRNA在19條染色體以及An/Cn亞基因組上的分布。SOR: 春油菜; SWOR: 半冬性油菜; WOR: 冬油菜。

A: LncRNA-seq PCA analysis of 12 samples from sixcultivars; B: the number of expressed LncRNA in sixcultivars and three ecotypes; C: Venn diagram of expressed LncRNA among three ecotypes; D: the distribution of LncRNA on 19 chromosomes and An, Cn subgenomes in three ecotypes of. SOR: spring type rapeseed; SWOR: semi-winter type rapeseed; WOR: winter-type rapeseed.

2.5 不同生態(tài)型甘藍(lán)型油菜LncRNA差異表達(dá)分析

按照生態(tài)型, 本研究對(duì)LncRNA進(jìn)行了差異表達(dá)分析。在春油菜和冬油菜、春油菜和半冬性油菜、半冬性油菜和冬油菜中分別檢測(cè)到291、352和285個(gè)差異表達(dá)LncRNA序列。其中有283種LncRNA序列在多個(gè)組合中存在表達(dá)差異。LncRNA可以通過調(diào)控mRNA的表達(dá)來實(shí)現(xiàn)功能, 本研究通過其與蛋白質(zhì)編碼基因的位置關(guān)系(順式)和表達(dá)相關(guān)性(反式)預(yù)測(cè)LncRNA可能參與的生物學(xué)功能。預(yù)測(cè)得到1517個(gè)預(yù)測(cè)靶基因(順式: 617, 反式: 911, 11個(gè)預(yù)測(cè)靶基因在順式和反式預(yù)測(cè)中均檢測(cè)到), 并對(duì)這些表達(dá)差異LncRNA的預(yù)測(cè)靶基因(LncRNA-DEG)進(jìn)行GO和KEGG分析。對(duì)于GO功能富集分析表明, 差異表達(dá)LncRNA預(yù)測(cè)候選靶基因富集在眾多跨膜轉(zhuǎn)運(yùn)蛋白活性(如: 含核堿基的化合物, GO:0015932、碳水化合物衍生物, GO:1901505、有機(jī)磷酸酯: GO:0015605等),同樣也顯著富集在脂質(zhì)定位(GO:0010876)、脂質(zhì)轉(zhuǎn)運(yùn)(GO:0006869)通路(圖5-A)。KEGG通路分析富集到DNA復(fù)制, 花生四烯酸代謝,錯(cuò)配修復(fù), 氮素代謝以及一些氨基酸萜類物質(zhì)的合成代謝(圖5-B)。

圖5 3種生態(tài)型甘藍(lán)型油菜差異表達(dá)LncRNA候選靶基因GO功能和KEGG通路富集分析

A: 3種生態(tài)型甘藍(lán)型油菜差異表達(dá)LncRNA候選靶基因GO功能富集分析; B: 3種生態(tài)型甘藍(lán)型油菜差異表達(dá)LncRNA候選靶基因KEGG通路富集分析。

A: GO function enrichment of candidate target gene of differentially expressed LncRNA in three ecotypes of; B: KEGG pathway enrichment of candidate target gene of differentially expressed LncRNA in three ecotypes of.

2.6 甘藍(lán)型油菜開花基因與LncRNA、mRNA之間的關(guān)系

根據(jù)擬南芥中鑒定獲得的300多個(gè)開花時(shí)間相關(guān)基因[5], 使用雙向BLASTP方法在甘藍(lán)型油菜_v10基因組中得到同源基因1165個(gè)。將上述mRNA-seq與LncRNA-seq數(shù)據(jù)進(jìn)行整合分析, 結(jié)果發(fā)現(xiàn)開花基因中有39個(gè)基因?yàn)椴町惐磉_(dá)基因, 包括、、等基因(附表1)。其中, 有16個(gè)基因?yàn)長(zhǎng)ncRNA差異表達(dá)預(yù)測(cè)的候選靶基因, 包括、、和(附表2)。特別是, 11個(gè)開花相關(guān)基因同時(shí)為差異表達(dá)基因和LncRNA差異表達(dá)預(yù)測(cè)候選靶基因,、、、、和(附表3)。這11個(gè)共差異基因聚類到4個(gè)mRNA-LncRNA互作模塊, 其中最大的一個(gè)大模塊包含8個(gè)基因和23個(gè)LncRNA序列(圖6)。其中包含基因的4個(gè)拷貝(A03p23680.1_BnaDAR、A03p23700.1_BnaDAR、C03p28240.1_BnaDAR、A03p28260.1_BnaDAR)、基因的2個(gè)拷貝(A02p03320.1_BnaDAR、C02p08090.1_BnaDAR)、1個(gè)基因(A05p05620.1_ BnaDar)和1個(gè)基因(A01p22980.1_BnaDAR)。此外, 本研究用已經(jīng)發(fā)表的甘藍(lán)型油菜群體轉(zhuǎn)錄組學(xué)數(shù)據(jù)[38]進(jìn)行了驗(yàn)證, 該模塊中的絕大多數(shù)基因在3種生態(tài)型油菜群體中存在表達(dá)差異。

2.7 LncRNA與甘藍(lán)型油菜農(nóng)藝性狀QTL的位置關(guān)系

本研究收集了972個(gè)甘藍(lán)型油菜農(nóng)藝性狀QTL[39](附表4), 利用BEDTools軟件將鑒定到的LncRNA與972 (A亞基因組: 624; C亞基因組: 348)個(gè)QTL的物理區(qū)間進(jìn)行比較。發(fā)現(xiàn)在972個(gè)QTL位點(diǎn)置信區(qū)間內(nèi), 其中的844個(gè)QTL區(qū)間中共有1068個(gè)LncRNA位點(diǎn)存在(28.29%, A亞基因: 572; C亞基因: 496) (圖7)。結(jié)合前面的差異表達(dá)LncRNA分析, 我們發(fā)現(xiàn)分別有31.96% (93)、30.97% (109)和30.18% (86)與農(nóng)業(yè)性狀QTL位點(diǎn)重合的LncRNA分別為春油菜-冬油菜、春油菜-半冬性油菜和半冬性油菜-冬性油菜差異表達(dá)LncRNA。與性狀QTL位點(diǎn)重合的LncRNA, 更多的分布在甘藍(lán)型油菜的A亞基因組上, 這可能是由于更多的農(nóng)藝性狀相關(guān)的QTL在A亞基因組上被檢測(cè)到。但有趣的是, 在與性狀QTL位點(diǎn)重合的LncRNA中, A亞基因組中檢測(cè)到了更高比例的差異表達(dá)LncRNA (春油菜-冬油菜、春油菜-半冬性油菜和半冬性油菜-冬油菜A亞基因組中分別為8.92%、12.76%和8.39%, 對(duì)應(yīng)的C亞基因組為: 8.47%、7.26%和7.66%)。這意味著A亞基因組在甘藍(lán)型油菜生態(tài)型分化上起到了很大部分的作用。

3 討論

目前為止, 3種生態(tài)型甘藍(lán)型油菜都有至少2個(gè)品種完成了高質(zhì)量參考基因組的構(gòu)建, 這些基因組數(shù)據(jù)的釋放為重要農(nóng)藝性狀的基因定位及其功能研究提供了極大的便利[6-9,15]。甘藍(lán)油菜不同生態(tài)類型, 不僅開花時(shí)間的早晚差別顯著, 在形態(tài)及重要農(nóng)藝性狀上也有顯著不同。盡管不同調(diào)控途徑的開花基因在擬南芥中都已經(jīng)研究的很透徹, 但對(duì)于經(jīng)歷了多次的基因組多倍化事件的甘藍(lán)型油菜而言, 由于基因拷貝數(shù)的增加及功能分化, 鑒定決定其生態(tài)型分化、開花時(shí)間及其他農(nóng)藝性狀的關(guān)鍵基因仍然具有挑戰(zhàn)性。通過雙向BLASTP方法和嚴(yán)格的篩選過濾, 本研究在甘藍(lán)型油菜Darmor-bzh v10參考基因組中得到1165個(gè)甘藍(lán)型油菜開花基因, 這與之前的報(bào)導(dǎo)的1173個(gè)基因數(shù)量較為接近[40]。

基于mRNA-seq分析, 本研究在不同生態(tài)型甘藍(lán)型油菜中檢測(cè)到了5個(gè)同源基因(A02p00340.1_ BnaDAR、A03p16730.1_BnaDAR、C03p04920.1_ BnaDAR、C09p67350.1_BnaDAR、C09p67380.1_ BnaDAR)和2個(gè)(A05p05620.1_BnaDAR、C03p30160.1_BnaDAR)出現(xiàn)了差異表達(dá)(附表1), 但是沒有鑒定到成花素基因, 這可能是因?yàn)榛蛟陂_花之前表達(dá)量極低。在模式植物擬南芥中, 多個(gè)開花途徑, 包括環(huán)境響應(yīng)和發(fā)育信號(hào), 都匯集于對(duì)基因的轉(zhuǎn)錄調(diào)控。基因的表達(dá)由光周期途徑中基因激活, 并且受到基因的抑制,表達(dá)的產(chǎn)物同時(shí)受到春化途徑和自主途徑的抑制[41-42]。

圖6 差異表達(dá)mRNA-LncRNA互作模塊及基因在甘藍(lán)型群體中的達(dá)量

A: 差異表達(dá)mRNA-LncRNA互作模塊, 包含8個(gè)基因和23個(gè)LncRNA序列; B: 差異表達(dá)mRNA-LncRNA互作模塊中,、和基因(多個(gè)拷貝)在甘藍(lán)型油菜不同生態(tài)型群體中的表達(dá)量。FPKM (Fragments Per Kilobase of exon model per Million mapped fragments): 每千個(gè)堿基的轉(zhuǎn)錄每百萬映射讀取的片段數(shù), *表示在< 0.05水平差異顯著, **表示在< 0.01水平差異顯著, ns表示差異不顯著> 0.05。SOR: 春油菜, SWOR: 半冬性油菜, WOR: 冬油菜。

A: the differentially expressed mRNA-LncRNA interaction module, including 8 genes and 23 LncRNA sequences; B: the relative expression level of COR15b, KIN2, and SOC1 genes (multiple copies) in different ecotypes ofpopulation. FPKM: the fragments per kilobase of exon model per million mapped fragments; * indicates significant differences at the 0.05 probability level; ** indicates significant differences at the 0.01 probability level; ns: no significant difference (> 0.05). SOR: spring type rapeseed; SWOR: semi-winter type rapeseed; WOR: winter-type rapeseed.

圖7 甘藍(lán)型油菜QTL和鑒定到的LncRNA共線性分布

植物中第一個(gè)被鑒定的LncRNA是黃瓜中的CR20[43], 它是一個(gè)細(xì)胞分裂素抑制基因。在擬南芥中具有代表性的且通過功能驗(yàn)證的是名為COOLAIR的反義轉(zhuǎn)錄本(NAT), 其在低溫處理14 d的條件下高表達(dá), 通過抑制基因表達(dá)從而促進(jìn)開花[17,41]。在白菜中經(jīng)過低溫處理后在基因也鑒定到反義轉(zhuǎn)錄本的存在, 并且同樣能抑制基因的表達(dá)[44]。本研究中, 并沒有鑒定到以為靶基因的LncRNA序列, 但是檢測(cè)到了19個(gè)LncRNA序列涉及到3個(gè)靶基因。本研究鑒定得到了一個(gè)包含8個(gè)開花相關(guān)基因和23個(gè)LncRNA的互作表達(dá)模塊, 其中包含4個(gè)基因、2個(gè)基因、1個(gè)基因和1個(gè)基因(圖6)。其中編碼一種在冷脅迫過程中保護(hù)葉綠體膜的蛋白質(zhì)[45],編碼一個(gè)可被低溫和脫落酸誘導(dǎo)的蛋白[46], 可能與低溫馴化有關(guān)。編碼一種光誘導(dǎo)蛋白, 響應(yīng)光信號(hào)[47],則編碼開花途徑中的整合因子, 調(diào)控及其他基因表達(dá)[48]。因此, 該模塊可能參與通過響應(yīng)光信號(hào)和溫度信號(hào)調(diào)控甘藍(lán)型油菜的開花時(shí)間, 但具體的功能還待進(jìn)一步探究。目前為止有關(guān)LncRNA的功能研究還較少。有研究表明, LncRNA可能通過調(diào)節(jié)開花相關(guān)基因在花發(fā)育過程中的表達(dá)從而影響鷹嘴豆的產(chǎn)量[50]; LncRNA可能介導(dǎo)植物體對(duì)營(yíng)養(yǎng)元素脅迫響應(yīng)[51-52]; LncRNA可能參與對(duì)病原體以及干旱等逆境脅迫的調(diào)控[53-54]。本研究的相關(guān)結(jié)果為解析LncRNA油菜生態(tài)型分化、適應(yīng)性及、農(nóng)藝性狀的形成中的作用奠定了基礎(chǔ)。

多項(xiàng)研究表明, 甘藍(lán)型油菜A亞基因組變異程度、核苷酸多樣性、基因組互作程度和連鎖不平衡衰退都要強(qiáng)于C亞基因組[2-3,14,49]。大量的GWAS分析也表明, 甘藍(lán)型油菜重要農(nóng)藝性狀QTL多在A亞基因組中, A亞基因組在甘藍(lán)型油菜形成、進(jìn)化和馴化中起到了關(guān)鍵作用。本研究也發(fā)現(xiàn)與性狀QTL位點(diǎn)重合的LncRNA, 更多的分布在甘藍(lán)型油菜的A亞基因組上, 且A亞基因組中也檢測(cè)到了更高比例的差異表達(dá)LncRNA。這些結(jié)果預(yù)示LncRNA可能參與了甘藍(lán)型油菜的生態(tài)型分化及與之相適應(yīng)的農(nóng)藝性狀形成的調(diào)控, 且與C基因組相比, A亞基因組在其中扮演了更為重要的角色。

必須指出, 為兼顧結(jié)果的準(zhǔn)確可靠及研究的經(jīng)濟(jì)性, 本研究中每種甘藍(lán)型油菜的轉(zhuǎn)錄組測(cè)序只進(jìn)行了2次生物學(xué)重復(fù)。盡管差異表達(dá)的基因及差異表達(dá)的LncRNA均是按照不同生態(tài)型分組進(jìn)行的比較, 即每種生態(tài)型包含2個(gè)品種、4個(gè)生物學(xué)重復(fù), 但由于同一個(gè)生態(tài)型的2個(gè)品種遺傳背景差異較大, 可能會(huì)縮小不同生態(tài)型間的共差異基因及生態(tài)型保守LncRNA數(shù)量。

4 結(jié)論

本研究通過高通量測(cè)序技術(shù), 在3種生態(tài)型6個(gè)甘藍(lán)型油菜品種中進(jìn)行mRNA-seq和LncRNA-seq,探究LncRNA在甘藍(lán)型油菜生態(tài)型分化中的潛在作用。本研究鑒定到了3775條LncRNA序列, 其中新鑒定到1147條LncRNA序列, 預(yù)測(cè)到了8575個(gè)LncRNA候選靶基因?；诓町惐磉_(dá)基因及LncRNA靶基因的GO和KEGG富集分析表明, LncRNA可能參與到氨基酸、脂質(zhì)和酶等相關(guān)基礎(chǔ)物質(zhì)的合成代謝。特別是, 還鑒定到一個(gè)包含開花相關(guān)基因和LncRNA的互作模塊, 可能參與光信號(hào)及和溫度響應(yīng)過程。結(jié)合與QTL的共定位分析, 進(jìn)一步發(fā)現(xiàn)LncRNA可能在甘藍(lán)型油菜生態(tài)型分化、開花期調(diào)控及農(nóng)藝性狀形成中發(fā)揮重要作用。

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附表1 差異表達(dá)開花基因

Table S1 Differentially expressed genes related flowering time

擬南芥基因Arabidopsis thaliana gene-ID基因縮寫Symbol甘藍(lán)型油菜基因IDBrassica napus gene-ID開花調(diào)控途徑Flowering regulation pathway AT4G11880AGL14A09p27210.1_BnaDAR其他途徑 Other AT5G13790AGL15C09p63310.1_BnaDAR開花整合基因 Floral integrator AT1G69120AP1A02p15620.1_BnaDAR花分化和發(fā)育途徑 Floral meristems and development AT1G69120AP1C02p17480.1_BnaDAR花分化和發(fā)育途徑 Floral meristems and development AT3G21320AT3G21320A03p42190.1_BnaDAR其他途徑 Other AT5G37780CAM1C09p30310.1_BnaDAR其他途徑 Other AT4G17640CKB2C01p11310.1_BnaDAR光信號(hào)途徑 Light singnalling AT2G42530COR15bA03p23680.1_BnaDAR春化途徑 Vernalization AT2G42530COR15bA03p23700.1_BnaDAR春化途徑 Vernalization AT2G42530COR15bC03p28240.1_BnaDAR春化途徑 Vernalization AT2G42530COR15bC03p28260.1_BnaDAR春化途徑 Vernalization AT1G12610DDF1A06p09040.1_BnaDAR赤霉素途徑 Gibberellin AT1G12610DDF1A08p32730.1_BnaDAR赤霉素途徑 Gibberellin AT1G12610DDF1C05p10640.1_BnaDAR赤霉素途徑 Gibberellin AT1G12610DDF1C08p15540.1_BnaDAR赤霉素途徑 Gibberellin AT1G12610DDF1C08p47250.1_BnaDAR赤霉素途徑 Gibberellin AT5G54510DFL1A02p10230.1_BnaDAR其他途徑 Other AT4G14690ELIP2A01p22980.1_BnaDAR光信號(hào)途徑 Light singnalling

(續(xù)附表1)

擬南芥基因Arabidopsis thaliana gene-ID基因縮寫Symbol甘藍(lán)型油菜基因IDBrassica napus gene-ID開花調(diào)控途徑Flowering regulation pathway AT4G14690ELIP2C01p28420.1_BnaDAR光信號(hào)途徑 Light singnalling AT5G10140FLCA02p00340.1_BnaDAR春化途徑 Vernalization AT5G10140FLCA03p16730.1_BnaDAR春化途徑 Vernalization AT5G10140FLCC03p04920.1_BnaDAR春化途徑 Vernalization AT5G10140FLCC09p67350.1_BnaDAR春化途徑 Vernalization AT5G10140FLCC09p67380.1_BnaDAR春化途徑 Vernalization AT1G30040GA2OX2C03p76330.1_BnaDAR赤霉素途徑 Gibberellin AT5G67100ICU2A02p33280.1_BnaDAR花分化和發(fā)育途徑 Floral meristems and development AT5G15970KIN2A02p03320.1_BnaDAR春化途徑 Vernalization AT5G15970KIN2C02p08090.1_BnaDAR春化途徑 Vernalization AT4G32040KNAT5C01p06890.1_BnaDAR赤霉素途徑 Gibberellin AT5G65060MAF3A02p43650.1_BnaDAR春化途徑 Vernalization AT5G65060MAF3C02p63700.1_BnaDAR春化途徑 Vernalization AT3G01460MBD9C09p75140.1_BnaDAR光周期和生物鐘途徑Photoperiod and circadian clock AT3G62090PIL2C08p37050.1_BnaDAR光信號(hào)途徑 Light singnalling AT1G13260RAV1A09p63190.1_BnaDAR光周期和生物鐘途徑Photoperiod and circadian clock AT2G03710SEPALLATA4A02p33530.1_BnaDAR花分化和發(fā)育途徑 Floral meristems and development AT2G45660SOC1A05p05620.1_BnaDAR開花整合基因 Floral integrator AT2G45660SOC1C03p30160.1_BnaDAR開花整合基因 Floral integrator AT2G33810SPL3C04p15220.1_BnaDAR花分化和發(fā)育途徑 Floral meristems and development AT3G16640TCTPC01p47690.1_BnaDAR花分化和發(fā)育途徑 Floral meristems and development

附表2 差異表達(dá)LncRNA涉及到的開花基因

Table S2 Differentially expressed LncRNA related flowering time gene

擬南芥基因Arabidopsis thaliana gene-ID基因縮寫Symbol甘藍(lán)型油菜基因IDBrassica napus gene-ID開花調(diào)控途徑Flowering regulation pathway AT4G14690ELIP2A01p22980.1_BnaDAR光信號(hào)途徑 Light singnalling AT5G15970KIN2A02p03320.1_BnaDAR春化途徑 Vernalization AT1G69120AP1A02p15620.1_BnaDAR花分化和發(fā)育途徑 Floral meristems and development AT1G78440GA2OX1A02p22190.1_BnaDAR赤霉素途徑 Gibberellin AT2G42530COR15bA03p23680.1_BnaDAR春化途徑 Vernalization AT2G42530COR15bA03p23700.1_BnaDAR春化途徑 Vernalization AT2G45660SOC1A03p25280.1_BnaDAR開花整合基因 Floral integrator AT2G45660SOC1A05p05620.1_BnaDAR開花整合基因 Floral integrator AT5G11260HY5A10p26460.1_BnaDAR光信號(hào)途徑 Light singnalling AT5G15970KIN2C02p08090.1_BnaDAR春化途徑 Vernalization AT1G69120AP1C02p17480.1_BnaDAR花分化和發(fā)育途徑 Floral meristems and development AT4G25470CBF2C03p19680.1_BnaDAR春化途徑 Vernalization AT2G42530COR15bC03p28240.1_BnaDAR春化途徑 Vernalization AT2G42530COR15bC03p28260.1_BnaDAR春化途徑 Vernalization AT1G12610DDF1C08p47250.1_BnaDAR赤霉素途徑 Gibberellin AT5G20730NPH4C09p55450.1_BnaDAR光信號(hào)途徑 Light singnalling

附表3 差異表達(dá)基因與差異表達(dá)LncRNA候選靶基因

Table S3 Differentially expressed genes and differentially expressed candidate target genes of LncRNA

基因縮寫Symbol擬南芥基因Arabidopsis thaliana gene甘藍(lán)型油菜基因Brassica napus gene非編碼RNALncRNA COR15bAT2G42530A03p23680.1_BnaDARBnaLncA02_026, BnaLncA02_041, BnaLncA03_157, BnaLncC02_051, BnaLncC03_115, BnaLncC03_185, BnaLncC03_268, BnaLncC05_059, BnaLncC05_060, BnaLncC06_126, BnaLncC06_146 COR15bAT2G42530A03p23700.1_BnaDARBnaLncA02_026, BnaLncA02_041, BnaLncA03_157, BnaLncC02_051, BnaLncC03_115, BnaLncC03_185, BnaLncC03_268, BnaLncC05_059, BnaLncC05_060, BnaLncC06_126, BnaLncC06_146 COR15bAT2G42530C03p28240.1_BnaDARBnaLncA02_026, BnaLncA02_041, BnaLncA03_109, BnaLncA03_157, BnaLncA03_168, BnaLncC02_040, BnaLncC02_051, BnaLncC03_115, BnaLncC03_185, BnaLncC03_268, BnaLncC05_059, BnaLncC05_060, BnaLncC06_126, BnaLncC06_146, BnaLncC07_243 COR15bAT2G42530C03p28260.1_BnaDARBnaLncA02_026, BnaLncA02_041, BnaLncA03_109, BnaLncA03_157, BnaLncA03_168, BnaLncC02_040, BnaLncC02_051, BnaLncC03_115, BnaLncC03_164, BnaLncC03_185, BnaLncC03_268, BnaLncC05_059, BnaLncC05_060, BnaLncC06_126, BnaLncC06_146, BnaLncC07_243 AP1AT1G69120A02p15620.1_BnaDARBnaLncA02_044, BnaLncA06_211, BnaLncC08_129 AP1AT1G69120C02p17480.1_BnaDARBnaLncA01_118, BnaLncA08_034, BnaLncA08_093, BnaLncA10_078, BnaLncC01_179, BnaLncC03_247, BnaLncC04_029 KIN2AT5G15970A02p03320.1_BnaDARBnaLncA02_027, BnaLncA02_142, BnaLncA03_109, BnaLncA03_168, BnaLncA06_218, BnaLncA09_045, BnaLncA09_161, BnaLncC02_040, BnaLncC03_164, BnaLncC05_060, BnaLncC06_146, BnaLncC07_243, BnaLncC08_166 KIN2AT5G15970C02p08090.1_BnaDARBnaLncA02_027, BnaLncA02_142, BnaLncA03_109, BnaLncA03_168, BnaLncA06_218, BnaLncA09_045, BnaLncA09_161, BnaLncC02_016, BnaLncC02_040, BnaLncC03_164, BnaLncC05_060, BnaLncC07_243, BnaLncC08_166 DDF1AT1G12610C08p47250.1_BnaDARBnaLncC08_081 ELIP2AT4G14690A01p22980.1_BnaDARBnaLncA02_026, BnaLncA02_041, BnaLncA03_109, BnaLncA03_157, BnaLncA03_168, BnaLncC02_040, BnaLncC02_051, BnaLncC03_115, BnaLncC03_164, BnaLncC03_185, BnaLncC03_268, BnaLncC05_059, BnaLncC05_060, BnaLncC06_126, BnaLncC06_146, BnaLncC07_243 SOC1AT2G45660A05p05620.1_BnaDARBnaLncA02_027, BnaLncA03_109, BnaLncA03_168, BnaLncA06_218, BnaLncA09_161, BnaLncC02_040, BnaLncC08_166

附表4 甘藍(lán)型油菜收集的QTL信息匯總

Table S4 Summary of collected QTLs in

QTL性狀 QTL trait數(shù)目Number 開花時(shí)間Flowering times181 千粒重Thousand seed weight160 株高Plant height124 種子產(chǎn)量Seed yield89 成熟時(shí)間Maturity time73 第一分支數(shù)Number of primary branches53 其他Others292 A基因組數(shù)目A genome numbers624 C基因組數(shù)目C genome numbers348 總計(jì)Total972

Preliminary exploration of the role of LncRNA in the ecotype differentiation ofL.

YANG Tai-Hua, YANG Fu-Quan, GAO Geng-Dong, YIN Shuai, JIN Qing-Dong, XU Lin-Shan, KUAI Jie, WANG Bo, XU Zheng-Hua, GE Xian-Hong, WANG Jing*, and ZHOU Guang-Sheng

College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, Hubei, China

L. is one of the important oil crops in China. Based on its different requirements for vernalization temperature during flower transition,can be divided into three ecotypes (spring, semi-winter, and winter ecotype). Previous studies found that long non-coding RNA (LncRNA) can regulate gene expression level in multiple levels and participate in the regulation of plant growth and development. In, LncRNA can regulate flowering time by regulating the expression of genes related to vernalization pathway. In this study, to preliminarily explore the ecotype differentiation and adaptive formation among the three ecotypes of, three different ecotypes ofwere used as the materials and high-throughput sequencing technology was used to sequence mRNA and LncRNA in seeding leaves. The GO and KEGG enrichment analysis of co-differential expression genes of three ecotypes ofshowed that there were a lot of differences in the anabolism of basic compounds, especially lipid compounds. A total of 3775 LncRNA sequences were identified from the three ecotypes of, among which 285 LncRNA were differentially expressed in two or three ecotype combinations, and 1517 candidate target genes were involved. The candidate target genes of these differential expression LncRNA were also enriched in large number of anabolism related pathways of basic compounds. Based on conjoint analysis of mRNA and LncRNA, we predicted a putative regulatory network in the flowering genes, included eight flowering time genes and 23 LncRNA, which were involved in the regulation of temperature and light signal pathways. By analyzing the location of QTLs for important agronomic traits in, we found that about 90% of LncRNA and QTLs intervals were overlapped. The distribution of different expression LncRNA and the location of QTLs were different in three ecotypes of, which suggesting that LncRNA played an important role in ecotype differentiation and formation of important agronomic traits in.

; ecotype; long non-coding RNA; flowering genes; QTLs

10.3724/SP.J.1006.2023.24105

本研究由國(guó)家重點(diǎn)研發(fā)計(jì)劃項(xiàng)目“大田經(jīng)濟(jì)作物優(yōu)質(zhì)豐產(chǎn)的生理基礎(chǔ)與調(diào)控”(2018YFD1000900)資助。

This study was supported by the National Key Research and Development Program of China “Physiological Basis and Agronomic Management for High-quality and High-yield of Field Cash Crops” (2018YFD1000900).

王晶, E-mail: wangjing@mail.hzau.edu.cn

E-mail: taihua1996@gmail.com

2022-04-27;

2022-09-05;

2022-09-15.

URL: https://kns.cnki.net/kcms/detail/11.1809.S.20220914.1812.007.html

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).