甘藍型油菜種子萌發(fā)期耐苯磺隆種質(zhì)篩選與綜合評價

王 倩 崔 翠 葉 桑 崔明圣 趙愉風 林 吶 唐章林 李加納 周清元

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甘藍型油菜種子萌發(fā)期耐苯磺隆種質(zhì)篩選與綜合評價

王 倩**崔 翠**葉 桑 崔明圣 趙愉風 林 吶 唐章林 李加納 周清元*

西南大學農(nóng)學與生物科技學院, 重慶 400715

本研究旨在探討不同基因型甘藍型油菜種子萌發(fā)期耐除草劑苯磺隆特性, 建立其篩選的評價體系, 選育萌發(fā)期耐苯磺隆的甘藍型油菜品種(系)。采用苯磺隆耐性度量值(T值)、平均隸屬函數(shù)值(ASF值)、加權(quán)耐性系數(shù)(WTC值)等指標及相關(guān)分析、頻數(shù)分析、主成分分析、灰色關(guān)聯(lián)度分析、聚類分析和逐步回歸分析相結(jié)合的方法, 鑒定萌發(fā)期耐性。通過11份供試材料相對根長的方差分析, 確定了油菜萌發(fā)期耐苯磺隆種質(zhì)資源篩選及評價研究的最佳苯磺隆濃度為25 mg kg–1。在此濃度脅迫下, 241份甘藍型油菜的根長、芽長、率、發(fā)芽勢、鮮重5個指標均存在顯著性差異。根據(jù)T值的聚類分析, 將供試種質(zhì)劃分為5個苯磺隆耐性級別, 其中I級3份、II級30份、III級198份、IV級6份、V級4份。對苯磺隆耐性較強的甘藍型油菜品種(系)為希望106、SWU95和WH-33, 可作為甘藍型油菜苯磺隆耐性育種和耐性機制研究的重要資源。根長、鮮重、發(fā)芽率可作為評價油菜種質(zhì)資源萌發(fā)期耐性的指標性狀。

甘藍型油菜; 耐苯磺隆; 種質(zhì)資源; 綜合評價; 萌發(fā)期

自1942年除草劑2,4-D問世以來, 人類使用化學除草劑防除農(nóng)田雜草已有70多年的歷史。西方發(fā)達國家已經(jīng)在85%~100%的作物上使用除草劑[1], 我國除草劑生產(chǎn)和使用量也逐年增加[2]。但是, 在所施用的除草劑中, 絕大部分都殘留于土壤或淋溶于水中, 真正起作用的很少[3], 極容易導致對除草劑敏感的作物死亡[4]。不科學地使用半衰期長的除草劑還會毒害下茬作物[5]。因此, 既要高效地消滅雜草, 又不使作物受到藥害, 可以通過新型除草劑產(chǎn)品的研發(fā)和耐除草劑作物品種的培育兩種途徑實現(xiàn), 其中后者相對比較經(jīng)濟, 費用一般不超過研制新除草劑的5%[6]。發(fā)掘農(nóng)作物除草劑耐性基因、培育耐除草劑農(nóng)作物新品種成為作物遺傳育種的重要方向之一。

油菜是當今世界的主要油料作物之一, 我國油菜的種植面積、總產(chǎn)量均約占世界的1/3[7]。田間雜草危害可使油菜產(chǎn)量下降15%, 更甚者可減產(chǎn)50%以上[8-9]。我國油菜主產(chǎn)區(qū)的雜草類型大致可分為以禾本科雜草為主、以闊葉雜草為主以及禾本科與闊葉草混生3種[10]。油菜為闊葉型植物, 所以闊葉雜草就成為油菜田雜草防除的一個關(guān)鍵瓶頸[11]。苯磺隆作為闊葉類雜草除草劑, 在小麥田除草取得良好效果[12], 若要選育出耐苯磺隆的油菜品種, 將苯磺隆和現(xiàn)有油菜田單子葉除草劑混合使用, 可為油菜田雜草的有效防除提供一條新的途徑[13]。因此, 培育和推廣耐苯磺隆除草劑油菜品種可以有效控制闊葉草害, 減少中耕除草用工,提高油菜產(chǎn)量, 增加效益[14]。另外, 苯磺隆作為化學殺雄劑在油菜雜種優(yōu)勢利用研究中效果良好, 已經(jīng)引起廣泛重視[15-16], 選育具有苯磺隆除草劑耐性的甘藍型油菜作為父本,有利于簡化制種程序, 降低制種成本[16-17]。信曉陽等[14]使用苯磺隆溶液處理49個不同基因型油菜幼苗, 篩選出2個在苗期低敏感材料。曲高平等[18]用甲基磺酸乙酯(EMS)溶液對甘藍型油菜中雙9號種子誘變處理, 在M2突變體庫中篩選到3株苯磺隆耐性突變體。華中農(nóng)業(yè)大學報道從華雙5號油菜EMS突變后代群體中鑒定出幾株苯磺隆耐性突變體, 測序表明它們的突變位點均為的Pro197Ser/ Leu突變[16]。吳學莉等[17]從一個耐苯磺隆除草劑的播娘蒿天然突變體中克隆了乙酰乳酸合成酶(ALS)基因, 并通過轉(zhuǎn)基因發(fā)現(xiàn)其表達使甘藍型油菜對苯磺隆的耐性提高至野生型致死濃度的3倍。汪亞琴[19]以甘藍型油菜品系甲572為受體, 轉(zhuǎn)化水稻細胞色素P450基因家族成員基因, 獲得了抗苯磺隆的油菜株系。有研究表明植物耐受苯磺隆除草劑主要與基因突變和非靶標酶代謝解毒能力有關(guān)[1,20-24]。同時, 田間雜草對苯磺隆耐性突變體的機制研究也相繼報道, 其中黑草和看麥娘的基因在擬南芥中表達, 獲得的轉(zhuǎn)基因植物可表現(xiàn)抗多種除草劑[25-28]。在耐除草劑作物的培育中, 鑒定并獲得具有天然耐藥性的植物是研究的關(guān)鍵[29], 因此, 從現(xiàn)有油菜中篩選耐苯磺隆種質(zhì)資源, 并挖掘內(nèi)源耐苯磺隆基因源對于油菜育種具有重要的理論和應(yīng)用價值。

雖然在4種磺酰脲類除草劑中, 苯磺隆的植物毒性最低[30], 且苯磺隆為短殘留除草劑[31], 但是也有報道指出小麥地土壤中苯磺隆降解半衰期為5~12 d[32-33], 因此, 苯磺隆仍存在短期土壤殘留現(xiàn)象。目前, 關(guān)于苯磺隆土壤殘留對作物種子, 尤其是對油菜種子萌發(fā)的影響未見報道。本研究擬綜合鑒定與評價供試品種(系)的發(fā)芽率、發(fā)芽勢、鮮重、干重、根長和芽長, 篩選出萌發(fā)期耐性較強的油菜種質(zhì)及相關(guān)性較強的測定指標, 研究播種期苯磺隆殘留對油菜種子萌發(fā)的影響, 為苗期、蕾薹期、花期篩選耐苯磺隆油菜品種(系)提供基礎(chǔ), 也為進一步在遺傳和育種研究中利用這些優(yōu)異種質(zhì)提供理論依據(jù)。

1 材料與方法

1.1 供試油菜種質(zhì)資源

試驗材料為國內(nèi)外各農(nóng)業(yè)高校、科研院所選育或收集的, 具有不同遺傳背景和廣泛地理來源的241份甘藍型油菜種質(zhì)(附表1), 該群體包含部分自交系和常規(guī)品種, 種子均由重慶市油菜工程技術(shù)研究中心提供。

處理藥劑為10%苯磺隆除草劑(江蘇瑞東農(nóng)藥有限公司), 有效成分含量100 g L–1, 生產(chǎn)用量每公頃150 g兌水600 kg (250 mg kg–1)。

1.2 方法

1.2.1 萌發(fā)期最適苯磺隆處理濃度篩選 參照陳新等[34]的培養(yǎng)皿紙上發(fā)芽法并略有改動。以遺傳背景不同的11份甘藍型油菜品種(系)為材料(附表1), 對每個品種設(shè)置蒸餾水對照和不同濃度(250、25、2.5、0.25 mg kg–1)苯磺隆脅迫共5種處理, 各處理3次重復。在鋪有2層濾紙的培養(yǎng)皿中加入3?mL不同濃度的苯磺隆溶液, 對照中加入相同體積蒸餾水, 將已清洗的飽滿種子均勻放在濾紙上, 每皿20粒, 蓋上皿蓋, 于人工培養(yǎng)箱中培養(yǎng), 設(shè)置溫度為25℃, 相對濕度為85%, 光照和黑暗時間為16 h/8 h。參照汪夢竹等[35]的研究, 于第7天測定油菜主根根長, 分析并確定苯磺隆最佳脅迫濃度。

1.2.2 萌發(fā)期耐苯磺隆鑒定 用1.2.1中確定的最佳處理濃度, 以蒸餾水為對照, 依照上述方法培養(yǎng)241份甘藍型油菜(附表1), 3次重復。于第3天統(tǒng)計種子發(fā)芽勢(germination vigor, GV), 第7天統(tǒng)計種子發(fā)芽率(germination rate, GR), 隨機選取每個重復10株幼苗分別測定其莖長(shoot length, SL)、根長(root length, RL)、鮮重(fresh weight, FW)和干重(dry weight, DW)形態(tài)指標。

1.3 數(shù)據(jù)處理與分析

利用Microsoft Excel 2013、IBM SPSS19.0[36]和DPS 2006[37]統(tǒng)計軟件整理分析數(shù)據(jù)。

參考閆峰等[38]、Upadhyaya[39]、汪燦等[40]的方法, 以各品種6個性狀測定值作為基礎(chǔ)數(shù)據(jù), 分析其平均數(shù)差異顯著性, 按公式(1)計算對苯磺隆單項耐性系數(shù)(tribenuron-methyl tolerance coefficient, TC), 式中XCK分別表示第個指標下第個品種(系)苯磺隆脅迫和對照處理的指標測定值。針對各指標TC值, 進行簡單相關(guān)分析、連續(xù)次數(shù)分布統(tǒng)計分析和主成分分析。

按公式(2)、(3)和(4)計算取各種質(zhì)各指標的隸屬函數(shù)值[(X)]和平均隸屬函數(shù)值(ASF, average subordinative function value)。

(X)(X– Xmin) /( Xmax – Xmin)= 1, 2, 3…,;1, 2, 3…,(3)

式中,P為第個綜合指標貢獻率, 表示第個指標在所有指標中的重要程度。X、Xmin、Xmax分別表示第個指標下第個品種的TC值及第個指標下的最小值和最大值。其中為選取的主成分個數(shù)。

根據(jù)因子權(quán)重系數(shù)(ω)和各主成分得分值[(X)], 按公式(5)計算苯磺隆耐性度量值(T, tribenuron-methyl tolerance comprehensive evaluation value)。

= 1, 2, 3, …,(5)

按公式(6)、(7)計算關(guān)聯(lián)系數(shù)(ξ)及等權(quán)關(guān)聯(lián)度(γ)。

ξ=,= 1, 2, 3, …,(6)

式中,ξ為關(guān)聯(lián)系數(shù),Δ為品種最優(yōu)性狀與第個品種(系)第個性狀的絕對差值。minΔ為最小二級絕對差值, maxΔ為最大二級絕對差值,為分辨系數(shù)(取0.5)。

分別按公式(8)和(9)計算各指標權(quán)重系數(shù)[ω(γ)]和加權(quán)關(guān)聯(lián)度(weight tribenuron-methyl tolerance coefficient, WTC)。

式中,γ為各指標關(guān)聯(lián)度, TC為對苯磺隆單項耐性系數(shù)。

以各指標TC值為比較序列, 分別以T值和WTC值為參考序列進行灰色關(guān)聯(lián)度分析, 獲得各指標TC值與T值間的關(guān)聯(lián)度(T)和TC值與WTC值間的關(guān)聯(lián)度(WTC)。

最后基于供試甘藍型油菜種質(zhì)T值, 采用加權(quán)配對算術(shù)平均法(weighted pair group method average, WPGMA)和歐氏距離進行聚類分析, 劃分苯磺隆耐性等級, 并分別以T值、ASF值和WTC值為參考序列, 對各指標TC值進行逐步回歸分析, 獲得回歸方程。

2 結(jié)果與分析

2.1 苯磺隆脅迫濃度篩選

11份甘藍型油菜品種在不同苯磺隆濃度處理條件下, 根長均受到不同程度的抑制(表1)。在0、0.25、2.5、25和250 mg kg–1濃度下, 根長均值分別為7.73、4.44、1.62、1.26和1.15 cm, 處理較對照分別下降36.12%、79.01%、83.64%和85.15%, 根長在2.5 mg kg–1處理時下降接近最大值, 到25 mg kg–1和250 mg kg–1處理時下降趨勢變緩。11份品種在5個處理中差異均具有統(tǒng)計學意義, 但是25 mg kg–1脅迫處理時, 更能夠區(qū)分材料本身的差異; 在25 mg kg–1具有差異的材料, 在0.25、2.5和250 mg kg–1脅迫處理均沒有差異(表1)。如25 mg kg–1脅迫下, P18與所有品種(系)間差異均顯著, 甲預31棚與中雙12等5個品種差異顯著。2.5 mg kg–1脅迫下, P18與至尊和B265品種(系)間差異不顯著, 甲預31棚與6個品種(系)差異不顯著。250 mg kg–1脅迫下, P18與至尊品種(系)間差異不顯著, 甲預31棚與9個品種差異不顯著。另外, 250 mg kg–1脅迫處理7 d后, 各品種根部萎縮生長普遍變差。因此, 25 mg kg–1濃度可作為鑒定甘藍型油菜萌發(fā)期耐苯磺隆的適宜濃度。

表1 不同濃度下11份品種根長相對值的顯著性分析

同列標有不同小寫字母者組間差異顯著(<0.05)。

Values within a column followed by different letters are significant by different between groups (<0.05).

2.2 耐苯磺隆油菜種質(zhì)資源篩選

2.2.1 241份甘藍型油菜萌發(fā)期指標 對照和處理(25 mg kg–1苯磺隆脅迫)的6個性狀在品種間差異均具有統(tǒng)計學意義, 各性狀的變異系數(shù)分別為16.45%~54.56%和20.01%~54.83%, 說明試驗中的241份種質(zhì)在兩個條件下均表現(xiàn)出廣泛的遺傳變異。在苯磺隆脅迫下, 各性狀受到影響程度不同。脅迫處理下根長均值僅為0.94 cm, 較對照下降87.99%, 受抑制最嚴重。脅迫處理下的發(fā)芽勢、發(fā)芽率、鮮重均值分別為75.77%、83.22%、0.36 g, 較對照分別下降6.81%、5.37%、5.26%。脅迫處理下芽長均值為2.37 cm, 較對照增加4.61%。在脅迫條件下, 根長、芽長、發(fā)芽勢、發(fā)芽率的變異系數(shù)均增加, 其中根長的變異系數(shù)較對照增加最明顯(表2)。

2.2.2 單項指標分析 供試種質(zhì)在苯磺隆脅迫處理后, 與對照相比各指標均發(fā)生不同程度的變化(表3)。同一指標下不同品種對苯磺隆單項耐性系數(shù)(TC)的最大值和最小值之間差異較大, 說明不同品種間對苯磺隆的耐性不同。不同指標下TC值存在明顯差異, 變異系數(shù)介于10.3%~49.0%之間, 表明各指標對苯磺隆脅迫反應(yīng)的敏感性不同。此外對各單項指標進行單因素方差分析, 表明不同種質(zhì)間在根長、芽長、發(fā)芽率、發(fā)芽勢、鮮重5個指標下均存在具有統(tǒng)計學意義的差異。相關(guān)分析表明(表4), 各指標間存在一定程度的相關(guān)性。其中干重與根長呈顯著負相關(guān), 與發(fā)芽率呈顯著正相關(guān), 而與其余指標相關(guān)不顯著。發(fā)芽勢與發(fā)芽率呈極顯著正相關(guān), 與根長呈極顯著負相關(guān), 相關(guān)系數(shù)分別為0.687和–0.150。發(fā)芽率與根長呈顯著負相關(guān), 鮮重與芽長呈極顯著正相關(guān), 其余指標間無顯著相關(guān)性。除發(fā)芽勢與發(fā)芽率相關(guān)系數(shù)較高, 其余相關(guān)系數(shù)絕對值均在0.1~0.3之間。此外, 各指標TC值分布次數(shù)相差較大(圖1)。根長的TC值在0.095~0.215之間的品種最多。TC>0.90的鮮重、發(fā)芽勢、發(fā)芽率、芽長、干重的分布頻率分別為68.0%、70.5%、80.5%、81.3%和96.3%, 各指標對苯磺隆脅迫的敏感性由強到弱依次是根長、鮮重、發(fā)芽勢、發(fā)芽率、芽長、干重。因此, 如果直接采用各個指標, 由于指標間信息的疊加重合, 很難準確客觀地評價各種質(zhì)的耐性, 從而影響對苯磺隆耐性鑒定結(jié)果。

表2 苯磺隆脅迫下油菜萌發(fā)期各指標的變化

**表示在0.01水平上差異顯著。

**Significant difference at the 0.01 probability level. RL: root length; SL: shoot length; GV: germination vigor; GR: germination rate; FW: fresh weight; DW: dry weight.

表3 供試油菜種質(zhì)各指標的苯磺隆耐性系數(shù)

**表示在0.01水平上差異顯著。

**Significant difference at the 0.01 probability level. RL: root length; SL: shoot length; GV: germination vigor; GR: germination rate; FW: fresh weight; DW: dry weight.

表4 供試油菜種質(zhì)各指標耐性系數(shù)的相關(guān)性

*和**分別表示在0.05和0.01水平上相關(guān)顯著。

*,**Significant correlations at the 0.05 and 0.01 probability levels, respectively. RL: root length; SL: shoot length; GV: germination vigor; GR: germination rate; FW: fresh weight; DW: dry weight.

圖1 241份甘藍型油菜不同指標耐性系數(shù)(TC)的頻次分布

RL: 根長; FW: 鮮重; GV: 發(fā)芽勢; GR: 發(fā)芽率; SL: 芽長; DW: 干重。

RL: root length; SL: shoot length; GV: germination vigor; GR: germination rate; FW: fresh weight; DW: dry weight.

2.2.3 萌發(fā)期主成分分析 由表5可知, 第1、第2、第3和第4主成分的貢獻率分別為30.17%、21.83%、18.27%和13.16%, 累計貢獻率達到83.42%, 已基本代表了所測指標的信息, 可反映影響萌發(fā)期油菜耐苯磺隆性主導因素。從各個指標在綜合指標的貢獻率及主成分特征向量值可以看出, 第1主成分中發(fā)芽率、發(fā)芽勢所占的比率較大, 第2主成分中鮮重所占的比率較大, 第3主成分中根長所占的比率較大, 第4主成分中芽長所占比率較大。

表5 各綜合指標的特征值、貢獻率和主成分特征向量值

GR: germination rate; GV: germination vigor; FW: fresh weight; RL: root length; SL: shoot length; DW: dry weight.

2.2.4 甘藍型油菜品種資源耐苯磺隆性綜合評價

供試種質(zhì)平均隸屬函數(shù)值(ASF值)介于0.155~ 0.687之間, 平均值均為0.485, 變異系數(shù)為15.3%, 根據(jù)ASF值大小對供試種質(zhì)進行耐性排序, 其結(jié)果見表6。其中, 對苯磺隆耐性強的品種(系)有SWU95、SWU113、希望106和WH-33, 耐性弱的品種(系)有10-804、中雙7號、中油589和10-1043, 其余種質(zhì)介于兩者之間。供試種質(zhì)加權(quán)關(guān)聯(lián)系數(shù)(WTC值)介于0.360~0.615之間, 平均值均為0.508, 變異系數(shù)為8.14%。根據(jù)WTC值大小對供試種質(zhì)進行耐性排序, 對苯磺隆耐性強的品種(系)有希望106、WH-33、SWU113和SWU95, 耐性弱的品種(系)有中雙7號、中油589和10-1043, 其余種質(zhì)介于兩者之間(表6)。供試種質(zhì)苯磺隆耐性度量值(T值)介于–2.58~ +2.22之間, 根據(jù)T值大小對供試種質(zhì)進行耐性排序, 對苯磺隆耐性強的品種(系)有希望106、SWU95和WH-33, 耐性弱的品種(系)有中油589和10-1043, 其余種質(zhì)介于兩者之間(表6)。三者之間的鑒定結(jié)果有所區(qū)別, 但供試種質(zhì)耐性較強較弱的鑒定結(jié)果基本一致。為方便比較, 將ASF值、WTC值和T值的排序數(shù)值相加, 再根據(jù)累加值從小到大進行排序, 從241份種質(zhì)資源的綜合排序看, 其中排名前五的依次為希望106、SWU95、WH-33、SWU113和97097。從4種排序方法看, 希望106、SWU95、WH-33均表現(xiàn)為對苯磺隆耐性強。

表6 苯磺隆脅迫下油菜品種資源的ASF值、WTC值、T值和綜合排序

(續(xù)表6)編號CodeASF值 ASF valueWTC值 WTC valueT值 T value綜合排序Final rank數(shù)值Value排序Order數(shù)值Value排序Order數(shù)值Value排序Order 440.461620.50147–0.13143156 450.441970.49175–0.39186194 460.491200.5447–0.0212287 470.392230.45223–0.74219223 480.51890.54400.356454 490.54430.53810.356352 500.53520.55250.218246 510.422040.47206–0.36185208 520.6450.5791.1365 530.501060.50156–0.07135134 540.501080.511370.366099 550.441870.48186–0.31181191 560.461630.49167–0.17157164 570.451820.47197–0.24167189 580.501030.5458–0.15152106 590.461680.48185–0.31180183 600.54500.55270.464637 610.471600.5375–0.33182144 620.51830.53860.336766 630.322360.42235–1.41236236 640.53600.54550.208658 650.54460.54520.533939 660.55340.56140.533826 670.491240.5389–0.27172131 680.501040.5373–0.11140107 690.451750.52105–0.39188162 700.52710.521020.188878 710.392220.44224–0.81225225 720.57210.54421.001220 730.292370.41237–1.47237237 740.53510.54380.139953 750.451740.5461–0.26169136 760.52690.501420.04114112 770.51850.511330.12100109 780.451730.49181–0.17155172 790.52720.521180.169284 800.402190.45222–0.77223221 810.372280.42234–0.75221229 820.54420.511240.1110182 830.422110.47198–0.64212213 840.471520.501520.02117147 850.491330.50158–0.08136150 860.451790.45215–0.10137182 870.212390.38239–1.99239239 880.56310.54590.444841 890.53630.52960.524057 900.451720.49169–0.15149166 910.432020.46209–0.22164201 920.501010.53740.326973 930.51900.511390.356189 940.52770.56220.425545 950.471560.49165–0.13144160 960.55330.56160.434931 970.52750.53700.0411577 980.53540.53760.543747 990.501020.54450.159671 1000.382260.45219–0.92231227 1010.451710.50161–0.43192180 1020.57250.53720.901734 1030.451760.51131–0.35183167 1040.53620.501430.159595 1050.422120.47205–0.68216216 1060.55360.521100.762648 1070.59130.56150.971413 1080.50930.5379–0.15150111 1090.54440.54530.435044 1100.51880.511360.2083101 1110.481370.50148–0.04127142 1120.491190.5460–0.04129102 1130.52790.501590.375991 1140.52800.53640.208464 1150.491250.52970.277592 1160.491310.51127–0.19162146 1170.52740.53850.405761 1180.471510.5292–0.16153133 1190.491210.52117–0.01120127 1200.451800.49180–0.51197193 1210.50990.52950.0810796 1220.422070.47201–0.65213214 1220.422070.47201–0.65213214 1230.382250.43227–0.62208219 1240.471590.50155–0.40189171 1250.441900.46207–0.19161195 1260.53650.511400.425475 1270.441890.48190–0.63209207 1280.51870.52109–0.05131114 1290.392210.44225–0.77224224 1300.422080.51135–0.44193185 1310.422100.49176–0.17156188 1320.402170.46210–0.76222217 1330.6170.56210.971310 1340.54470.54510.514242 1350.432000.48195–0.36184203 1360.412140.45220–0.90230222 1370.422050.48192–0.71217211 1380.501110.53880.1010297 1390.50940.5456–0.1113988 1400.51820.55340.179159 1410.491260.521030.1397113 1420.58190.55240.722717 1430.491230.51128–0.04128129 1440.501090.5387–0.04125110 1450.471460.52112–0.17159145 1460.55390.57100.802218 1470.441980.49182–0.55202204 1480.55370.56170.524130 1490.392200.44226–0.81226226 1500.491290.52116–0.25168143 1510.50960.501530.08106125 1520.491320.50162–0.07134151 1530.501130.521200.07109120 1540.491300.50154–0.24166155 1550.432010.45217–0.65214215 1560.491280.521190.1790117 1570.59160.5760.663015 1580.57200.56230.603322 1590.56260.56200.405832 1600.50950.54390.1010368 1610.451830.48184–0.45194198 1620.6360.56111.2556 1630.441950.50149–0.46195187 1640.56280.55290.891821 1650.491170.462110.7128126 1660.352320.42230–1.29235233 1670.461700.51130–0.49196170 1680.491350.5369–0.04126115 1690.59120.5780.83209 1700.53560.54500.307049 1710.501140.501500.2379121 1720.471580.5377–0.26170137 1730.52730.491730.3565104 1740.53640.54430.307150 1750.51810.511290.435179 1760.58180.5750.543616 1770.53550.55360.159451 1780.491150.52930.10104105 1790.441910.52108–0.64211173 1800.6810.5842.1622 1810.52660.521070.415665 1820.57230.55310.792323 1830.52700.511250.326880 1840.441930.47200–0.12142184 1850.6820.6021.1274 1860.53530.511260.931655 1870.481420.49171–0.26171163 1880.54450.491790.861972 1890.481390.48187–0.05130157 1900.372290.45221–1.19233230 1910.172400.37240–2.14240240 1920.53610.521150.464563 1930.382240.43229–0.63210220 1940.412160.45218–0.14148205 1950.481450.53780.287493 1960.441860.47202–0.28173199 1970.51860.481880.5143108 1980.491180.501570.04112132 1990.6730.6212.2111 2000.461650.46213–0.28175190 2010.50970.52910.188983 2020.451780.48193–0.39187196 2030.6090.56181.03108 2040.6080.53681.34424 2050.461610.49177–0.28174176 2060.362300.42233–0.89229232 2070.59150.56121.0497 2080.56300.55370.822127 2090.451850.45216–0.23165200 2100.491160.50146–0.14147139 2110.59170.56191.011114 2120.471490.47203–0.17158174 2130.471530.46214–0.02123168 2140.56290.55280.563428 2150.6740.6031.8533 2160.431990.47199–0.19160197 2170.362310.43228–0.75220228 2180.52670.52900.208569 2190.501050.511220.297294 2200.451810.491680.2676149 2210.54480.54440.435243 2220.501120.491720.2478128 2230.57220.54460.792529 2240.56270.54480.672933 2250.50910.511380.356690 2260.481360.501510.04113135 2270.55400.521110.356260 2280.481440.48189–0.03124159 2290.60100.54541.07819 2300.52760.511340.169398 2310.461660.48196–0.06132169 2320.441880.50160–0.41190186 2330.501000.472040.2577130 2340.53570.491700.633276 2350.372270.42231–0.87228231 2360.55380.55350.484436 2370.55320.521130.425356 2380.54410.54410.444738 2390.53590.53710.198762 2400.332340.5380–0.57205177 2410.342330.42232–1.18232234

2.2.5 灰色關(guān)聯(lián)度分析 各指標TC值與T值間的關(guān)聯(lián)度大小依次為根長、鮮重、發(fā)芽率、發(fā)芽勢、干重和芽長, 反映了各指標TC值與T值的密切程度, 這與各指標對苯磺隆脅迫反應(yīng)的敏感性基本吻合。各指標TC值與WTC值間的關(guān)聯(lián)度大小依次為根長、鮮重、芽長、發(fā)芽勢、發(fā)芽率和干重(表7)。

表7 供試油菜種質(zhì)各指標TC值與T值和WTC值的關(guān)聯(lián)度及各指標權(quán)重

GR: germination rate; GV: germination vigor; FW: fresh weight; DW: dry weight; RL: root length; SL: shoot length.

2.2.6 聚類分析及耐性級別的劃分 基于T值, 在歐氏距離為0.75處將241份供試種質(zhì)分為5類(附圖1)。其中第I類為高度耐苯磺隆的品種(系), 有SWU95、希望106和WH-33共3份; 第II類為耐性品種(系), 共30份, 占總數(shù)的12.4%; 第III類為耐性一般品種(系), 共198份, 占總數(shù)的82.2%; 第IV類為不耐苯磺隆的品種(系)共6份; 第V類為高度不耐苯磺隆的品種(系), 有10-1043、10-804、中雙7號和中油589共4份。根據(jù)供試材料對苯磺隆的耐性聚類分析及耐性級別劃分結(jié)果, 對其耐性評價指標分級統(tǒng)計表明, 除芽長外, 其余單項指標的隸屬函數(shù)值、ASF值、WTC值和T值均隨對苯磺隆耐性級別的升高而增大(表8)。

2.2.7 回歸方程 分別以ASF值、WTC值和T值為參考序列, 對供試種質(zhì)各指標TC值進行逐步回歸分析, 得到的3個回歸方程的相關(guān)系數(shù)≈ 1,檢驗均達極顯著水平(表9), 說明回歸方程最優(yōu), 模型擬合度好, 用這3個方程進行甘藍型油菜種質(zhì)資源萌發(fā)期對苯磺隆耐性評價的效果較好。根據(jù)T值與各指標TC值的回歸方程可知, 在甘藍型油菜種質(zhì)資源萌發(fā)期耐性鑒定中, 有選擇性地測定與T值密切相關(guān)的指標, 如根長、鮮重、發(fā)芽率可有效鑒定油菜種質(zhì)資源對苯磺隆的耐性。

表8 供試油菜種質(zhì)苯磺隆耐性評價指標的分級

I、II、III、IV、V表示不同耐性級別。

I, II, III, IV, and V represent different tribenuron-methyl tolerance levels. GR: germination rate; GV: germination vigor; FW: fresh weight; DW: dry weight; RL: root length; SL: shoot length.

表9 供試油菜種質(zhì)耐性模型預測

1、2、3、4、5、6分別為發(fā)芽率、發(fā)芽勢、鮮重、干重、根長、芽長的TC值。

1,2,3,4,5, and6are TC values of germination rate, germination vigor, fresh weigh, dry weight, root length, and shoot length respectively.

3 討論

種子萌發(fā)是植物生長發(fā)育的前提, 是對環(huán)境因素最為敏感的階段。苯磺隆作為一種化學除草劑, 在殺死雜草的同時, 對作物也是一種非生物逆境脅迫。隨著化學除草劑在農(nóng)業(yè)生產(chǎn)中的廣泛應(yīng)用, 雜草對化學除草劑的耐性增強, 化學除草劑的使用量大幅度增加, 非合理使用導致化學除草劑在土壤中的殘留越來越多, 對種子的萌發(fā)影響會越來越大。苯磺隆在現(xiàn)階段油菜生產(chǎn)中使用量較少, 但播種前結(jié)合其他化學除草劑防除田間雜草時, 苯磺隆殘留就可能影響油菜種子萌發(fā), 目前未見關(guān)于油菜種子萌發(fā)過程中受苯磺隆影響的公開報道。本研究認為萌發(fā)期最佳苯磺隆脅迫濃度為25 mg kg–1, 為生產(chǎn)上除草劑濃度的1/10, 這一結(jié)果與前人[14]篩選濃度不同, 可能與研究對象、處理方式以及測定指標的不同有關(guān)。

不同作物或同一作物的不同品種對除草劑敏感性不同[14,24,41-46], 植物存在天然耐性基因, 通過除草劑的選擇, 可篩選出耐除草劑作物[47]。本研究中25 mg kg–1苯磺隆脅迫條件下, 所有供試油菜種質(zhì)指標均受到不同程度的影響, 且其影響差異均具統(tǒng)計學意義。牛志峰等[42]研究表明不同小麥品種種芽對苯磺隆的耐藥性明顯較種根強, 婁國強等[43]研究發(fā)現(xiàn)苯磺隆對不同小麥品種安全性存在較大差異, 且對根長的抑制作用明顯大于株高。本研究單項耐性系數(shù)表明, 根長受抑制最嚴重, 這與前人研究結(jié)果基本一致[29,42,48]。另外, 頻次分析表明, 發(fā)芽勢、發(fā)芽率指標在0.90

近年來, 隨著作物耐逆性研究的發(fā)展, 采用多種綜合評價, 已在燕麥[34]、花生[39]、薏苡[40]等作物耐逆性鑒定中得到初步應(yīng)用, 可以避免單一指標的片面性和不穩(wěn)定性, 而目前作物耐除草劑研究中評價方法較單一。本研究采用ASF值、T值和WTC值等綜合評價其對苯磺隆耐性, 確定T值為主要綜合評價指標, 以ASF值和WTC值作為輔助綜合評價指標, 進行較確切的聚類分析和耐藥型劃分, 并結(jié)合灰色關(guān)聯(lián)分析、逐步回歸分析篩選關(guān)鍵性狀指標, 建立了擬合度較好的回歸方程。采用ASF值、T值和WTC值對供試油菜種質(zhì)綜合排序, 可消除因各指標單位不同帶來的影響。據(jù)此綜合評價, 選出耐性強的品種(系) SWU95、希望106和WH-33, 耐性弱的品種(系) 10-1043、10-804、中雙7號和中油589。種質(zhì)資源的鑒定結(jié)果為油菜其他生育時期耐苯磺隆種質(zhì)資源篩選和鑒定奠定了基礎(chǔ), 也為油菜耐苯磺隆遺傳和育種研究提供了理論依據(jù)。

4 結(jié)論

采用苯磺隆耐性度量值(T值)、平均隸屬函數(shù)值(ASF值)和加權(quán)耐性系數(shù)(WTC值)為綜合評價指標, 結(jié)合相關(guān)分析、頻數(shù)分析、主成分分析、灰色關(guān)聯(lián)度分析、聚類分析和逐步回歸分析等方法, 對241份甘藍型油菜品種的萌發(fā)期耐性鑒定發(fā)現(xiàn), 根長、鮮重、發(fā)芽率可作為油菜品種的萌發(fā)期耐苯磺隆能力和品種選育時優(yōu)先考慮指標。根據(jù)T值進行聚類分析, 可將供試苯磺隆種質(zhì)劃分為5個耐性級別, 其中I級3份、II級30份、III級198份、IV級6份、V級4份。希望106、SWU95和WH-33為苯磺隆耐性較強的種質(zhì), 可作為甘藍型油菜的苯磺隆耐性育種和耐性機理研究的重要資源。

附圖和附表 請見網(wǎng)絡(luò)版: 1) 本刊網(wǎng)站http://zwxb. chinacrops.org/; 2) 中國知網(wǎng)http://www.cnki.net/; 3) 萬方數(shù)據(jù)http://c.wanfangdata.com.cn/Periodical-zuowxb. aspx。

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Screening and Comprehensive Evaluation of Germplasm Resources with Tribenuron-methyl Tolerance at Germination Stage in Rapeseed (L.)

WANG Qian**, CUI Cui**, YE Sang, CUI Ming-Sheng, ZHAO Yu-Feng, LIN Na, TANG Zhang-Lin, LI Jia-Na, and ZHOU Qing-Yuan*

College of Agronomy and Biotechnology, Southwest University, Chongqing 400715, China

The purpose of this study is to explore the characteristics of tribenuron-methyl tolerance in different genotypes of, and establish the evaluation system for screening and breeding new cultivars with tribenuron-methyl tolerance. The germination vigor was measured on the third day after treatment and the germination rate, shoot length, root length, dry weight, as well as fresh weight were measured on the seventh day. The identification of tolerance at germination stage was performed by tribenuron-methyl tolerance comprehensive evaluation value (T), average subordinative function value (ASF) and weight tribenuron-methyl tolerance coefficient (WTC) in correlation analysis, frequency analysis, principal component analysis, grey analysis, cluster analysis and stepwise regression analysis. Based on the variance analysis of relative root length index in 11 tested materials, we determined the optimum treatment concentration (25 mg kg–1) for selecting and evaluating the tolerant germplasm resources to tribenuron-methyl in napeseed. Under the optimum concentration, the single factor variance analysis showed significant differences in root length, shoot length, germination rate, germination vigor and fresh weight among 241 accessions of. The clustering analysis according to T value exhibited that the tested cultivars were roughly divided into five tribenuron-methyl tolerance grades, including three in grade I, 30 in grade II, 198 in grade III, six in grade IV, and four in grade V. We conclude that the varieties (lines) with strong tribenuron-methyl tolerance at germination stage are Xiwang 106, SWU95, and WH-33, which could be used as the germplasm materials for the study on tolerance breeding and mechanism. Furthermore, the root length, fresh weight and germination rate could be used as index traits to evaluate the tolerance of rapeseed germplasm resources during germination stage.

L; Tribenuron-methyl tolerance; germplasm resources; comprehensive evaluation; germination stage

2018-03-26;

2018-04-18.

10.3724/SP.J.1006.2018.01169

周清元, E-mail: zhouqy2005@163.com

**同等貢獻(Contributed equally to this work)

王倩, E-mail: 734747691@qq.com; 崔翠, E-mail: cuigreeny@163.com

2017-11-20;

本研究由國家現(xiàn)代農(nóng)業(yè)產(chǎn)業(yè)技術(shù)體系建設(shè)專項(CARS-12), 國家科技支撐計劃項目(2013BAD01B03-12), 重慶市社會事業(yè)與民生保障科技創(chuàng)新項目(cstc2016shmszx0756)和西南大學博士啟動基金項目(swu113064)資助。

This study was supported by the China Agriculture Research System (CARS-12), the National Key Technology Support Program of China (2013BAD01B03-12), the Science and Technology Committee of Chongqing (cstc2016shmszx0756), and the Doctoral Start-up Fund of Southwestern University (swu113064).

URL:http://kns.cnki.net/kcms/detail/11.1809.S.20180418.0952.002.html