食用豆抗豆象种质创新与遗传改良研究进展

杨晓明 程须珍 朱振东 刘昌燕 陈 新

综述

食用豆抗豆象种质创新与遗传改良研究进展

杨晓明1程须珍2,*朱振东2刘昌燕3陈 新4,*

1甘肃省农业科学院作物研究所, 甘肃兰州 730070;2中国农业科学院作物科学研究所, 北京 100081;3湖北省农业科学院粮食作物研究所, 湖北武汉 430064;4江苏省农业科学院经济作物研究所, 江苏南京 210014

食用豆在耕地质量提升、优化农业生态体系及人类膳食结构改善中发挥着重要作用。豆象是世界性仓储性害虫, 严重影响着食用豆产业的健康发展。国内外在食用豆抗豆象种质鉴定评价和利用方面, 从各种野生资源中鉴定出抗不同豆象的种质材料, 并在豆象抗性遗传学分析、QTL定位和基因挖掘等方面取得显著进展。本文通过对豆象发生和危害规律、抗性鉴定方法、抗性机理研究、抗性种质鉴定与评价、抗性遗传和分子标记以及抗豆象育种方法和品种改良等方面的梳理和总结, 对抗豆象研究存在问题进行讨论和展望, 以期为我国抗豆象种质创新和品种改良提供参考价值。

食用豆; 种质创新; 豆象; 抗性育种; 分子标记

食用豆是指除大豆以外, 以收获籽粒为主, 兼做蔬菜, 供人类食用的豆类作物总称[1]。食用豆在耕地质量提升、农业生态系统优化和人类膳食结构改善中发挥着重要作用[2]。食用豆作物有20多种[3], 包括: 蚕豆(Vicia faba L.)、豌豆(Pisum sativum L.)、鹰嘴豆(Cicer arietinum L.)、小扁豆(Lens culinarisMedic)等冷季豆; 绿豆(Vigna radiata L.)、小豆(V. angularis)、黑吉豆(V. mungoL.)、豇豆(V. unguiculata L.)、饭豆(V. umbellate L.)等热季豆; 普通菜豆(Phaseolus vulgaris L.)、多花菜豆(P. coccineus L.)和利马豆(P. lunatus L.)等暖季豆。全球食用豆种植面积约7100万公顷, 产量6770万吨[4]。我国食用豆常年播种约300万公顷, 产量500万吨[1], 主要为蚕豆、豌豆、绿豆、小豆、豇豆和普通菜豆。

随着气候变暖和异地调种频繁, 豆象(Coleoptera: Bruchidae)已成为危害食用豆最为严重的害虫, 其主要分布于亚洲、非洲热带地区以及中美和南美等地[5-6]。我国危害热季豆的豆象主要是绿豆象(Callosobruchus chinensis)和四纹豆象(Callosobruchus maculatus); 危害蚕豆和豌豆的分别是蚕豆象(Bruchus rufimanus)和豌豆象(Bruchus pisorum) (图1), 菜豆象()为检疫性害虫, 但在局部地区也发生。我国河北、安徽、陕西、河南、湖北等产区绿豆象危害达68%; 云南、四川等产区蚕豆象和豌豆象危害高达76%[7]; 豆象危害从疫区向非疫区蔓延, 由低海拔产区向高海拔产区扩张。危害程度总体表现南方产区重于北方产区, 热季豆重于冷季豆, 豆象危害严重制约着食用豆产业的健康发展。

图1 食用豆类作物豆象

A: 绿豆象(♀); B: 绿豆象(♂); C: 豌豆象(♂); D: 豌豆象(♀)。

A: bean weevil (♀); B: bean weevil (♂); C: pea weevil (♂); D: pea weevil (♀).

抗虫品种培育和应用是解决豆象危害最经济、有效和环境友好的途径。食用豆抗虫种质创新和品种改良已成为育种家主要研究目标[8]。迄今, 国内外已鉴定出抗不同豆象的食用豆种质资源, 并在抗性遗传分析、基因发掘、遗传图谱构建、分子标记等方面取得一定成效[9]。目前, 我国培育的抗豆象品种主要是绿豆[10], 其他豆种尚未有育成的抗豆象品种在生产上应用。利用远缘杂交和现代分子育种技术, 培育抗性、品质和产量协同提高的新品种是推动产业发展的有效手段。本文通过国内外主要食用豆类作物抗豆象机理研究、抗性种质鉴定和遗传育种等研究进行系统梳理和总结, 旨在提出当前我国豆象防控的有效措施, 为进一步豆象综合治理和研究提供参考。

1 豆象种类、危害与防治

1.1 豆象种类与危害特征

豆象(Bruchidae)为鞘翅目(Coleoptera)叶甲总科(Chrysomeloidea)豆象亚科(Bruchinae)的通称。全世界已鉴定并记载豆象约102个属1300个种[11]。对食用豆产业造成严重危害的约20多种[12], 包括豆象属(Bruchus)、瘤背豆象属(Callosobruchus)、三齿豆象属(Acanthoscelides)、锥胸豆象属(Bruchidius)。随着全球气候变暖和农业种植结构变化, 豆象发生和危害不断加剧[13]。我国豆象主要有8属25个种。其中四纹豆象(C maculatus)、鹰嘴豆象(C. analis)、西非花生豆象(C. subinnotatus)、菜豆象(A. obtectus)、巴西豆象(Zabrotes subfasciatus)被列为我国重要的植物检疫性有害生物[14]。

不同豆象生活史、发生规律和危害特征不同[15]。其显著特征是绿豆象等多寄主豆象可多次侵染和危害; 而豌豆象、蚕豆象等单寄主豆象只有在田间采食花粉花蜜, 完成交配产卵后, 并在特定寄主上危害[16]。多寄主豆象可为害多种豆类, 如绿豆象、四纹豆象、菜豆象等在一年可繁殖多代, 并危害绿豆、小豆等多种豆种[17]。豌豆象、蚕豆象等单寄主豆象, 每年发生1代, 分别只危害蚕豆和豌豆[16]。蚕豆象和豌豆象首次危害均发生在田间, 于花荚期采食花粉花蜜, 交配产卵在嫩荚上, 随着种子成熟, 卵孵化为幼虫蛀入种子进行危害, 4龄幼虫危害最大[18]。绿豆象、菜豆象、四纹豆象等杂食性豆象在热带地区全年繁殖为害, 成虫不通过采食花粉花蜜, 可完成交配产卵, 幼虫蛀入种子内部进行危害[19]。受豆象危害种子芽率和商品性显著下降[20], 严重影响种子质量和食用品质[21](图2)。

1.2 豆象防控技术研究

基于豆象生长发育和危害规律研究, 田间防治主要是在开花期进行化学药剂防治[22], 也有研究认为将携带Np基因的抗豆象豌豆品种和高粱间作, 可有效控制豆象发生和危害[23]; 还有学者开展了利用屏腹茧蜂(Sigalphus thoracicus)和叶蜂(Triaspis luteipes)等自然天敌[24-25]以及白僵菌(Beauveria bassiana)等病原微生物进行生物防治[26]。储藏期间主要是通过温湿度调控[27]、微波处理[28]、聚乙烯膜密封处理[29], 植物精油熏蒸[30]等方法通过微环境调控影响豆象的生长发育和繁育, 从而达到预防豆象危害的目的, 这些方法由于种种原因尚未规模化应用。目前, 最为普遍的方法是采用磷化铝等化学物质熏蒸, 但该方法存在污染环境和食品安全等问题[31]。为根除豆象对豌豆产业的影响, 2016—2017年新西兰开展了种子检疫、种植结构调整、田间化学杀虫等各个防控环节联动的豌豆象综合治理, 使豌豆象种群降低了99.1%, 成为世界有效综合治理豆象的典范[32]。

图2 绿豆象危害各种食用豆

2 豆象抗性鉴定方法及分级标准

2.1 豆象鉴定方法

抗虫性鉴定是作物抗虫研究的基础, 其一致性和标准性将直接影响作物抗性种质筛选、品种选育和遗传研究等学科的发展。快速、准确、有效的鉴定方法和分级标准对大量抗性种质的科学评价具有重要的意义。田间自然感虫鉴定[33]和人工接虫鉴定[34-35]是豆象抗性鉴定的主要方法。田间自然感虫鉴定受气候环境、豆象种群大小等多种因素, 豆象鉴定的一致性和准确性很难有效控制。人工接虫鉴定包括智能温室鉴定[36]和人工气候箱接虫鉴定[34]。人工气候箱接虫鉴定可有效调控豆象的生活环境、种群大小和雌雄虫比例, 豆象饲养简单、检测周期短、检测结果一致性高, 现已广泛应用于绿豆象、四纹豆象等多寄主豆象的抗虫鉴定。智能温室接虫鉴定主要用于蚕豆象和豌豆象等单寄主豆象的抗虫鉴定,豆象饲养必须以特定寄主的花粉或花蜜为食源进行饲养[36], 豆象种群培养费事费工。我国学者利用绿豆象多寄主生活习性和危害规律, 采用绿豆象替代蚕豆象和豌豆象, 进行人工接虫鉴定蚕豆和豌豆抗虫种质[37-38], 有效的提高了鉴定效率。

2.2 豆象抗性分级标准

在豌豆抗虫资源鉴定中, 美国学者基于豌豆象幼虫在豆荚和种子中的死亡率、以及成虫在豆粒中的出现率和种子危害等指标, 制定了一套豌豆田间自然感虫抗性分级标准和方法[39]。豌豆成熟期采收荚果, 检测种皮上的幼虫取食刺孔, 然后将种子破开, 按1～5个级别进行损伤评分。1级: 种皮有幼虫取食刺孔, 0～1%子叶组织被食或损伤, 幼虫死亡或未发现损伤; 2级: 2%～5%的子叶组织被豆象危害, 1龄幼虫死亡; 3级: ≥5%的子叶被豆象危害, 2～4龄幼虫死亡; 4级: 子叶损伤较重, 蛹和成虫死亡; 5级: 子叶大部分被豆象危害, 有活成虫或尚未羽化的蛹。

在人工气候箱接虫鉴定中, 国内外学者基于豆象种子危害率, 制定了一套绿豆抗豆象分级标准[34-35,40]。在相同的接虫种群压力和豆象饲养条件下, 即温度(27±1)℃、相对湿度(65±5)%[41], 每5～20粒种子接入7对成虫, 接虫28～45 d[41]或50 d[42]后, 统计受检种子危害粒数, 用危害种子粒数除以受检种子总粒数, 即为种子受害率。并根据受害率来评价抗性等级。1级(高抗, HR): 种子受害率0～10%; 3级(抗, R): 10.1%～35.0%; 5级(中抗, MR): 35.1%～ 65.0%; 7级(中感, S): 65.1%～90.0%; 9级(高感, HS): >90%。这套分级标准也有效的用于蚕豆[37]和豌豆[38]等食用豆作物抗绿豆象种质评价研究[43]。但也有一些学者并不完全采用该分级标准,而将豆象抗性划分为5级: 高抗(HR, 0～10%), 抗(R, 10.1%～20.0%), 中抗(MR, 20.1%～40.0%), 感(S, 40.1%～80.0%), 高感(HS, 80.1%～100.0%)[42,44]。

3 豆象抗性机理研究

解析作物抗虫性机制是有效开展作物抗虫种质创新和品种改良的基础[45]。作物抗虫性是指作物利用形态特征、生理特性或以自身所特有的物质等来阻碍昆虫侵害的自我防护能力[46], 具有可遗传的生物学特性。作物抗虫性机制复杂多样[47], 除与作物遗传特性有关外, 还与作物生长发育相协同。在作物抗虫性机制研究方面, 近年来我国科学家基于作物和昆虫互作机制创新研究, 在作物免疫受体转录、抗性信号转导、效应蛋白表达、抗虫基因分离、小RNA干扰以及利用基因编辑技术创制抗虫新种质等方面取得了显著进展[48]。相对玉米、大豆、水稻等大宗作物抗虫研究, 食用豆类作物抗虫机制研究较为落后[49], 而且多数集中在绿豆抗豆象机理研究方面。国内外研究较多的植物抗虫机制主要有组成型抗性(constitutive defenses)和诱导型抗性(induced defenses)。组成抗性是植物的一种固有特性, 取决于不同基因型, 抗性程度受环境影响较小, 植物在被侵害之前通常以特定的形态结构、特异的组织成分或气味来预防虫害危害[50]。食用豆类作物豆象抗性主要是特有的组织结构和形态特征以及所具有的化学物质介导的组成抗性, 诱导抗性在食用豆抗豆象研究方面鲜有报道。

3.1 组成型抗性

物理抗性(morphological defense)是组成抗性的一种主要形式, 是作物具备特有的形态特征或组织结构而对害虫产生的抗虫性[50]。亚洲蔬菜研究发展中心在对绿豆抗豆象机理研究中发现, 绿豆抗豆象性与种子大小、种皮特性等性状有关[51]。种子较小和种皮有毛层的品种对绿豆象具有抗性[52]; Lambrides等[53]通过对TC1966、ACC23、ACC41等抗性特性研究发现, 抗性水平与绿豆种皮薄厚和种子大小有关。在红小豆抗豆象研究中发现, 种皮光滑、籽粒饱满的品种很容易受到豆象危害[54]。

生化抗性(biochemical defense)是组成抗性的另外一种表现形式, 是植物所特异化学物质介导的抗性[55]。特异抗虫性物质主要有两大类。一类是生物碱、凝集素、蛋白酶抑制剂等生化物质; 另一类是吲哚、茉莉酸、水杨酸等植物激素或抗生素[50]。目前分离出的抗豆象物质主要是抑制或阻遏昆虫对食物进行消化和利用的化学物质。这些物质包括植物凝集素[56]、蛋白酶抑制剂[57]和α-淀粉酶抑制剂[58], 以及从绿豆中分离出来的豇豆酸A (Vignatic acid A)[59], 从豇豆中分离出抗性糖蛋白(Vivilins)[60], 但后来发现Vivilins对绿豆象没有抗虫性[61]。Chen等[62]和Lin等[63]从绿豆抗豆象种质TC1966与感豆象品系VC1973A杂交的后代材料VC6089A中发现了一种豆象抗性蛋白VrCRP; 其生物活性和α-淀粉酶抑制剂相当, 能够有效抑制豆象幼虫发育[64]。在对豇豆属野生种豆象抗性研究中发现, 一种非蛋白质芳香族氨基酸对氨基苯甲酸(PAPA)与绿豆象和菜豆象抗性有关[65]。Zhang等[66]发现多聚半乳糖醛酸酶抑制蛋白(VrPGIP)介导绿豆抗虫性, 但尚未对基因功能进一步验证[67]。早在1988年, Osborn等[68]在对菜豆种子生物活性鉴定中, 从野生菜豆中发现了一种种子储藏蛋白安赛林(Arcelins), 该物质区别于植物凝集素和植物血凝素(PHA)[69], 为60 kD二聚糖蛋白[70], 对菜豆象具有显著抗性。食用豆抗豆象机理的深度解析对抗性种质创制和品种改良具有重要指导作用。

3.2 诱导抗性

植物诱导抗虫性是指在遭受植食性害虫为害后,植物能产生各种诱导防卫反应, 进而通过生理、生化及形态特征等生理变化而形成的抗虫特性[71]。植食害虫取食植物后引起植物体内产生的生理生化反应能够影响植食者的产卵选择、幼虫发育等危害特性[72], 从而降低植食虫害危害。近年来, 在植物虫害诱导抗性研究方面, 基于植物化学防御作用机制创新研究, 在虫害信号识别和传导、防御基因调控和表达、生理代谢或变化等方面对植食者和宿主互作开展了大量研究[73]。在对豌豆抗豆象诱导抗性机制研究中发现, 携带Np基因豌豆, 当受到豆象危害后, 可激发豆荚产生瘤状愈伤组织, 通过瘤状愈伤组织防御体系降低豆象产卵, 从而降低豆象危害[74]。而在随后的研究中发现, 结瘤基因Np提供的抗性是有限的[75]。Aznar-Fernández等[76]通过豌豆象在不同寄主作物上的发育研究表明, 非寄主作物山黧豆(Lathyrus sativus)花粉和荚果可显著抑制豆象卵和幼虫正常发育。Underwood等[77]在用墨西哥豆象(Epilachna varivestis)诱导大豆性抗性研究中发现, 不同大豆品种间诱导抗性有显著差异, 但同一品种间诱导抗性和组成抗性没有显著差异。诱导抗性表达受昆虫种群基数和分布以及植物生长等因素的影响。

4 抗豆象种质资源鉴定与评价

抗性种质资源是品种创新的基础。从不同豆类作物豆象发生和危害规律分析, 鉴定评价冷季豆类作物豆荚和种子抗性具有应用价值; 而热季豆类作物鉴定评价种子抗性更具有重要应用价值。不同的研究者从豆荚抗性和种子抗性等不同方面开展了抗豆象种质资源鉴定和评价[78]。

4.1 绿豆抗豆象种质资源鉴定

在抗绿豆象方面, 早在1973年Doria等[79]通过绿豆不同生长阶段豆荚上产卵特性和幼虫发育的研究, 从66份材料中鉴定出EG Glabrous、EG-MG- 4和EG-MG-7抗性种质, 其豆荚能够明显抑制绿豆象幼虫正常发育。在对豇豆属豆象抗性鉴定中发现野生绿豆TC1966具较高抗性[80]。室内接虫鉴定表明, TC1966对绿豆象和四纹豆象具有抗虫性[81]。Lambrides等[53]鉴定出抗绿豆象野生绿豆资源ACC41和ACC23。但后来也有研究发现, TC1966和ACC41对四纹豆象表现易感[82]。在栽培种抗绿豆象种质鉴定方面, 有学者从绿豆栽培种中鉴定出中抗种质V2709和V2802[83], 以及高抗种质V1128和V2817[84]。我国学者通过对国内外绿豆抗虫鉴定, 筛选出高抗绿豆象品系98-15[85]以及C05199、C05202和C05528等高抗种质[86], 并在对绿豆胰蛋白酶抑制剂稳定性研究中, 鉴定出抑制剂活性较高的抗豆象种质C5200和C5193[87]。

4.2 豇豆属抗豆象种质鉴定

小豆是绿豆象主要寄主, 段灿星等[7]对国家保存的小豆资源进行了抗绿豆象鉴定, 筛选出优异抗性种质。Tomooka等[88]在对豇豆属的7个豆种抗绿豆象种质鉴定中发现, 黑吉豆(V. mungo var.)祖先种、饭豆(V. umbellata)野生种及硬毛豇豆(V. hirtella)等种质资源对绿豆象和四纹豆象具有抗虫性。Dongre等[89]从黑吉豆种质资源中鉴定出抗绿豆象种质Silvestris; Kashiwaba等[90]从饭豆种质中鉴定出高抗绿豆象和四纹豆象种质JP99485、JP100304、JP100311。Seram等[91]从饭豆地方品种中鉴定出具完全抗豆象资源LR(M) 3、LR(M) 4和TNAU Red; Mariyammal等[42]从饭豆品种Tnau Red和绿豆品种VRM(Gg) 1远缘杂交构建的抗豆象近等基因系中鉴定出RIL 165高抗绿豆象种质。

在豇豆抗豆象种质鉴定方面, 早在1985年国际热带农业研究中心(IITA)开展了大量的抗性评价研究, 鉴定出抗四纹豆象种质TVu11952、TVu11953和TVu2027[92]。随后相继从豇豆中鉴定出IT81D-994、Adom (CR-06-07)[90], 以及Pajeu、Guariba、Tucumaque、Xiquexique BRS等抗性种 质[93]。Tripathi等[94]基于种子抗性特性的研究, 从豇豆中鉴定出EC528425和EC5283872等抗豆象种质。

4.3 冷季豆类抗豆象种质鉴定

在豌豆抗豆象种质鉴定方面, 基于豆荚抗性和种子抗性, 从豌豆野生种(Pisum fulvum)中鉴定出12份抗豆象种质[39]; Hardie等[95]通过1900份野生种和1745份栽培种两大基因库种质鉴定, 从野生种中鉴定出ATC113等18份抗豆象种质; 仲伟文等[96]采用室内绿豆象接虫鉴定和田间抗性评价, 筛选出8份对绿豆象和豌豆象具有抗性的豌豆种质资源, 但这些抗虫资源均为豌豆近缘种。为从栽培种中发掘抗豆象资源, Aznar-Fernandez等[33]通过多环境条件下对野生种和栽培种鉴定, 基于豆荚和子叶抗性鉴定出栽培种(P. sativum ssp. syriacum)抗豆象种质P665。康永生等[43]通过2年的室内接虫鉴定, 从49份栽培豌豆种中筛选出重庆黄豌豆、安徽涡药豌豆、梭沙大白豌等抗豆象地方资源, 这些优异的抗虫材料目前用于豌豆象抗虫育种。

在抗蚕豆象种质鉴定方面, 国际干旱地区农业研究中心(ICARDA)对1000余份蚕豆种质进行了4年田间抗性评价, 仅鉴定出ILB1814和BPL33抗性材料[97]。法国第戎大学通过对1858份蚕豆资源抗豆象田间鉴定, 筛选出2套抗性机制不同的抗性种质。QUASAR和223303等抗虫种质可延缓豆象幼虫发育; 而BOBICK ROD115和NOVA GRADISKA等抗虫种质延缓豆象幼虫发育的同时, 可有效阻遏豆象幼虫蛀入子叶[98]。Duan等[99]通过室内接绿豆象鉴定出通蚕5号、云蚕82以及H5067等抗性种质。杨新等[100]通过对874份不同地理来源蚕豆种质对绿豆象抗性特性研究, 鉴定出27份高抗种质, 抗性表现与籽粒大小和种皮颜色有关。Shaheen等[101]从鹰嘴豆(C. arietinum)中鉴定出抗鹰嘴豆象和四纹豆象Punjab-91、Dasht、Bittle-98、Parbat等抗性材料, Swamy等[102]则鉴定出高抗四纹豆象鹰嘴豆NBeG 511、JAKI、9218、JG 11。

4.4 菜豆抗豆象种质鉴定

在菜豆抗豆象种质鉴定方面, 从墨西哥地方资源中鉴定出凝集素家族蛋白Arcelin介导的抗菜豆象野生菜豆种质资源G24582[103], 随后相继鉴定出G02771 (Arcelia 5)、G12952 (Arcelia 4)、G12882 (Arcelia 1)、G12866 (Arcelia 2)[104]以及QUES (Arcelin-8)[105]等不同抗性水平的菜豆资源, 这些资源均为菜豆野生种。

5 豆象抗性遗传和分子标记

发掘野生种质资源和培育抗性品种是解决豆象危害最为经济有效的途径[106]。作物抗性遗传解析是抗性育种的理论基础, 而抗性基因发掘和利用是培育抗性品种的前提。国内外在抗豆象遗传解析、基因发掘和分子定位等方面取得显著进展。

5.1 绿豆抗豆象遗传和分子定位

Kitamura等[107]对抗豆象绿豆种质资源TC1966遗传研究表明, 其抗性由单显性基因控制。程须珍等[108]通过对中绿1号和TC1966杂交对其F2代的遗传研究进一步证实TC1966抗豆象遗传学特性。Lambrides等[109]通过遗传等位性研究, 进一步发现野生种TC1966和栽培种ACC41含有相同的抗豆象基因。Young等[110]采用RFLP分子标记方法, 将TC1966携带的抗豆象基因定位在LG VIII上(后来将含有Br基因的LG VIII命名为LG9), 两侧标记分别是pA882和pM151。Kaga等[111]利用RFLP分子标记进行了Br抗豆象基因定位。而对抗绿豆象和四纹豆象种质V2709BG和V2802BG抗性遗传分析表明, 其抗性也是由单显性基因(Br)控制, 但不同于TC1966和ACC41遗传特性, 其遗传受母性遗传和修饰基因影响[112]。王丽侠等[113]利用SSR、RFLP和STS标记, 基于澳大利亚高感豆象绿豆栽培种(Berken)和高抗豆象绿豆野生种(ACC41)杂交构建的RILs群体, 将豆象抗性基因(Br1)定位在LG9连锁群, 距两侧SSR标记BM202、Vr2-627的距离分别为0.7 cM和1.7 cM。吴传书等[114]构建了我国第1张图谱总长732.9 cM、包括11个连锁群的绿豆高密遗传图谱, 为绿豆重要性状相关基因定位、克隆及分子标记辅助育种提供了技术平台。

亚洲蔬菜研究发展中心(AVRDC)采用TC1966× NM92和V2802×NM94构建的2套RILs, 通过基因测序分型(GBS)技术研究表明绿豆抗性种质TC1966和V2802具有相同抗性位点, 并从获得的6000多个SNP中, 将抗豆象主效QTL定位在5号染色体。根据标记的物理图谱位置进行QTL分析表明, 在不同染色体上存在多个抗豆象QTL, 其分子标记抗性预测准确率100%[115]。刘长友等[116]在对抗豆象种质V1128遗传解析方面, 采用分离集团分析法(BSA)将抗性基因(Br3)定位在5号染色体; Chotechung等[117]通过418个抗(V2802)感(Kamphaeng Saen 1)绿豆品种杂交获得的BC11F2回交群体, 采用SSR标记发现编码多聚半乳糖醛酸酶抑制剂基因(VrPGIP2)与绿豆象抗性基因(Br)位于5号染色体相同基因座位。

Zhang等[66]对V2802种质抗绿豆象遗传解析和基因定位研究发现, 调控多聚半乳糖醛酸酶合成的2个基因(VrPGIP1、VrPGIP2)共同参与了绿豆抗豆象遗传调控。Kaewwongwal等[118]进一步利用SNP标记和等位基因测序, 在Br基因座位发现了2个新的调控豆象抗性等位基因(VrPGIP1-1、VrPGIP2-2)。Rathnayaka-Gamage等[119]采用抗感乌头叶菜豆杂交构建的2个不同F2作图群体(F2OA、F2NB), 对调控豆象抗性基因进行精细定位和测序进一步验证, 高抗豆象野生乌头叶菜豆TN67中编码多聚半乳糖醛酸酶抑制剂的2个基因(VacPGIP1、VacPGIP2)协同调控豆象抗性。Lin等[120]和Liu等[121]利用感豆象品种VC1973A和抗豆象品种VC6089A构建的绿豆近等基因系NILs, 在转录组和蛋白组水平上, 通过对差异表达基因分析对抗豆象基因进行发掘, 鉴定出2个分别位于5号和1号染色体上编码含有BURP结构域的蛋白基因(g39185、g34458), 其中g39185被定位到与抗豆象主效基因座位标记最近的区域。

5.2 豇豆属抗豆象遗传和分子定位

在豇豆属抗豆象遗传研究方面, Gupta等[122]通过对豇豆种质TVu11952、TVu11953和TVu2027遗传分析, 发现其抗性由2个隐性基因(rm1, rm2)控制。Redden等[123]以抗虫品种TVu2027为母本, 通过5个不同抗感杂交后代苗期胰蛋白酶抑制剂的研究, 发现其抗虫性表达受母系基因型决定, 为主效隐性基因遗传, 且在不同杂交组合中存在不同效应修饰基因影响; Thandar等[124]进一步证实豇豆抗性种质TVu2027对绿豆象抗性由2个基因调控, 而对四纹豆象抗性由单基因调控, 其抗性遗传力与加性效应以及加性和显性基因互作有关, 而且不同的抗性基因之间没有连锁关系。Somta等[35]通过对饭豆与小豆近缘野生种(V. nakashimae)杂交遗传群体研究, 饭豆抗性由4个QTL控制。但后来采用序列相关扩增多态性(SRAP)分子标记, 通过抗(LRB238)感(LRB26)杂交构建的F2作图群体, 从检测到的11个QTL位点发掘出2个调控豆象抗性的主效基因和, 解释表现变异率达67.3%和77.4%[125]。Seram等[126]以抗豆象饭豆地方品种TNAU Red和高感品种VRMGg 1杂交构建的RILs为研究对象, 采用分离集团分析法(BSA)找到与豆象抗性关联的RAPD标记(OPB08, OPX04)和ISSR标记(UBC810)。

Belay等[127]对217份豇豆微核心种质进行表型和基因型分析, 基于全基因组关联分析, 利用41,948个SNP标记, 鉴定出11个与豆象抗性高度相关的SNP标记, 并筛选6个与抗性相关的候选基因(Vigun08g132300、Vigun08g158000、Vigun06g053700、Vigun02g131000、Vigun01g234900和Vigun01g201900), 分别位于豇豆1号、2号、6号和8号染色体。王彦等[128]利用中豇1号(感)和Pant-lobia-1 (抗)为亲本构建的RILs群体, 结合豆象(绿豆象和四纹豆象)抗性表型鉴定和基因型分型, 采用完备区间作图法(ICIM- ADD), 检测到2个与抗豆象相关的QTL位点, 解释表现变异7.16%和6.92%; 并构建了包含182个多态性标记, 平均遗传距离5.85 cM, 图谱总长1065.23 cM, 包含11个连锁群的豇豆遗传连锁图谱。

5.3 菜豆抗豆象遗传和分子标记

在对菜豆抗豆象遗传和分子研究方面, 主要集中在植物凝集素、α-淀粉酶抑制剂和Arcelin等种子储藏蛋白家族(APA)的研究上[129-130]。Blair等[131]通过感豆象品种SEQ1006和抗豆象品种RAZ106杂交构建的F2群体, 利用SSR分子标记从野生普通豆种RAZ106中鉴定了一个等位基因的连锁位点。Kamfwa等[132]通过地方品种Solwezi和抗豆象品系AO-1012-29-3-3A构建的RILs群体, 采用SNP分子标记鉴定出3个抗豆象QTL, 分别位于Pv04和Pv06染色体上, 其中Pv04染色体上的1个QTL (AO4.1SA)和APA调控的抗性位于同一基因座位。Li等[133]为从菜豆栽培种中发掘抗性基因, 通过高抗菜豆象地方品种黑芸豆和高感品种龙芸豆3号杂交构建的RILs群体, 基于全基因组测序和高密度遗传图谱构建, 在Pv06染色体上找到一个调控脂质转运蛋白和种子储藏蛋白有关的抗菜豆象候选基因。Somta等[134]通过对豇豆属中乌头叶菜豆(V. aconitifolia)抗性基因定位和测序, 鉴定出豆象抗性调控主效基因(qVacBrc2.1)和修饰基因(qVacBrc5.1), 其中主效基因qVacBrc2.1位于2号连锁群, 解释表现变异率50.41%～64.23%, 两侧标记分别为CEDG261和DMB-SSR160, 该基因和野生小豆(V. nepalensis)中定位到的抗豆象基因QTL Brc2.1具有高度同源性。

5.4 豌豆抗豆象遗传和分子定位

抗豆象遗传学研究表明, 豌豆象抗性是由多基因控制[135]。Aryamanesh等[136]首次在豌豆属(Pisum)种质资源中用QTL标记检测到抗豆象基因片段。通过抗豆象野生种ATC113 (PI 595933)和感豆象品种Pennant杂交构建的F2群体, 结合豆荚、种皮和子叶抗性表型, 采用AFLP和SSR分子标记, 利用MultiQTL共检测到8个调控子叶抗性的QTL。其中3个主效QTL (COR2、COR4b、COR5a)分别定位在LG2、LG4和LG5连锁群, 解释表现变异率80%。对豆荚和子叶抗性的QTL共线性分析表明, 这2个性状的抗性机制可能存在共同的信号调控通路和途径[136]。

6 抗豆象育种方法和进展

6.1 抗豆象育种方法

野生种或近缘野生种和栽培种种间远缘杂交以及利用转基因技术进行目标基因导入等育种方法, 是抗豆象种质创制和品种抗性改良的重要手段。远缘杂交抗豆象育种研究主要集中在豇豆属不同食用豆种间杂交育种上, 转基因抗豆象种质改良研究主要集中在α-淀粉酶抑制剂基因()转基因研究上。

6.1.1 远缘杂交抗豆象育种 野生种或近缘野生种与栽培种种间远缘杂交, 采用多代回交导入野生种优异性状是抗豆象种质创制和品种抗性改良的重要手段。豇豆属中饭豆(V. umbellata)、野生绿豆(V. radiata var. sublobata)、野生黑吉豆(V. mungo var. silvestris)和野生小豆(V. nepalensis)均对豆象具有很好的抗性[42]。早在20世纪90年代Sugawara等[59]利用种间杂交手段, 将野生绿豆抗豆象物质豇豆酸A成功转育到栽培绿豆中。2006年Somta等[35]通过饭豆和小豆近缘野生种远缘杂交, 成功将饭豆抗豆象特性转育到小豆中。2019年Manyammal等[42]则首次通过饭豆和绿豆远缘杂交构建了抗豆象RFLs, 成功将饭豆抗豆象特性转育到绿豆中。为有效开展抗豆象野生种或近缘野生种利用研究, 刘长友等[137]通过远缘杂交亲和性研究, 明晰了豇豆属中不同豆种亲缘关系和远缘杂交育种技术, 戴希刚等[138]并以饭豆作为桥梁亲本, 在豇豆抗性种质创新方面取得阶段性成效。Samyuktha等[41]通过6个高感绿豆品种分别和2个高抗品种种间杂交, 构建了12个不同杂交群体, 通过后代株系评价鉴定, 筛选出优异杂交组合(CO6×V2802BG), 并在F4～F5代中鉴定筛选出高抗豆象优异株系5株。在利用远缘杂交豌豆抗豆象育种方面, Clement等[139]利用野生种资源PI 595946为母本, 白花栽培种豌豆Alaska 81为父本, 通过种间杂交将抗豆象基因转入到栽培种豌豆。Aryamanesh等[140]则利用近缘野生种ATC113和感豆象栽培种Pennant杂交, 采用30% CsCl溶液对抗感株系的分离, 经多年回交也成功将抗豆象基因转入到栽培种豌豆。

6.1.2 转基因抗豆象育种 随着作物高效再生体系的建立和优异抗虫基因的克隆, 转基因抗虫育种取得了显著进展。利用农杆菌介导等方法的转基因品种改良成为现代作物品种育种的有效手段。但是相对玉米、水稻等禾本科作物, 食用豆作物转基因育种较为落后[141]。特别是蚕豆[142]、豌豆[143-144]等豆类作物离体培养再生体系和遗传转化体系建立相对困难, 可供利用的有效抗虫基因缺乏[145], 这些因素严重制约着转基因技术在食用豆作物遗传改良中的应用。转基因抗豆象品种改良是食用豆作物抗虫研究的主要方向。已报道的研究重点是针对从普通菜豆中克隆到的α-淀粉酶抑制剂基因()进行食用豆品种抗虫性改良。通过农杆菌介导的转基因技术, 已成功将α-淀粉酶抑制剂基因导入绿豆[146]、豇豆[147]、小豆[148]、鹰嘴豆[149]和豌豆[150-151]等食用豆中。值得一提的是, 转基因小豆对绿豆象、四纹豆象、鹰嘴豆象均具有很高的抗性[148]。早在1994年Shade等[153]将α-AI-Pv基因导入到栽培豌豆中, 可显著提高豌豆对绿豆象和四纹豆象的抗性水平[152]。但后来临床试验表明, 含有α-AI基因的转基因豌豆可使小白鼠肺部产生炎症, 主要原因是转基因豌豆蛋白质结构发生改变[154]。Negawo等[155]将苏云金芽胞杆菌(Bacillus thuringiensis, Bt)中对鳞翅目具有杀虫活性的特异晶体蛋白基因(cry1Ac), 通过农杆菌介导转入到豌豆中, 可提高对烟芽夜蛾(Heliothis virescens)的抗性, 对豌豆象是否具有抗性尚未做进一步研究。

6.1.3 分子标记辅助选择抗豆象育种 抗豆象基因定位和连锁分子标记发掘对分子标记辅助育种具有重要意义。豇豆属中绿豆、小豆、饭豆、黑吉豆等抗豆象分子标记鉴定取得显著进展, 但这些标记常常与子粒小、品质差等不利性状紧密连锁, 在一定程度上限制了分子标记开发和应用, 相对大宗作物食用豆抗虫分子标记育种仍处于起步阶段[156]。在绿豆抗豆象种质鉴定和分子标记辅助选择育种上, Sarkar等[157]和Majhi等[158]利用与绿豆野生种ACC41抗性基因Br1紧密连锁的STS标记STSbr1进行分子标记辅助选择; Wu等[159]以携带豆象抗性基因V2802绿豆品种为供体, 利用VrBR-SSR013和DMB-SSR158两个SSR标记作为前景选择, 豆象抗性表型作为背景选择, 经3代回交分子标记辅助鉴定(MAB, marker-assisted backcrossing), 成功将多聚半乳糖醛酸酶抑制剂抗性基因(VrPGIP2)导入到泰国主栽绿豆品种Kamphaeng Saen 1中, 并创制出抗绿豆象种质R67-22。

6.2 抗豆象育种进展

抗性资源鉴定以及抗性基因发掘和利用最终目标是抗性品种的选育和应用。基于食用豆抗豆象种质鉴定和分子标记开发, 国内外在抗豆象育种方面取得了显著成效, 但主要集中在绿豆抗虫品种改良上。在绿豆抗豆象育种方面, 由于抗性种质发掘和利用, 泰国和韩国科学家利用TC1966、ACC41、V2709、V2802等野生种抗性种质, 开展了绿豆抗豆象品种育种研究。亚洲蔬菜研究发展中心以高抗豆象野生种TC1966和高感栽培种VC1973A杂交, 通过高抗亲本回交, 选育出近等基因系VC6089A[83]; Cisse等[160]采用杂交手段培育出极早熟抗绿豆象的豇豆品种Mouride; 2000年Lee等[161]从TC1966和V2709杂交后代中选育出Jangannogdu抗豆象品种。我国在抗豆象绿豆品种改良方面, 通过引进亚洲蔬菜研究发展中心抗豆象野生资源, 采用远缘杂交在抗豆象品种改良方面也取得显著成效。中国农业科学院作物科学研究所利用TC1966、V2709等抗性种质, 先后培育出中绿3号、中绿4号、中绿6号、中绿7号等抗豆象绿豆新品种[162]。山西省农业科学院以TC1966为抗性亲本, 通过有性杂交和定向选择, 培育出晋绿豆7号[163], 并采用诱变和杂交技术选育出高抗绿豆象品种晋中10号[164]; 江苏省农业科学院以从泰国引进的抗豆象资源1号和地方资源苏资8号杂交, 选育出苏绿5号和6号抗豆象品种[165-166]。河北省农林科学院以抗豆象材料抗豆象4号(V1128×中绿1号)为母本, 以冀绿7号为父本, 通过杂交、回交、室内抗豆象鉴定及定向选择, 培育出抗豆象绿豆品种冀绿17号[167]。这些品种的研究和应用对有效解决豆象危害的产业瓶颈问题提供了技术支撑。

7 问题与展望

7.1 抗豆象种质创新和品种改良存在的问题

食用豆在农业可持续发展及人类膳食结构改善中发挥着重要作用, 而豆象严重制约着产业的健康发展, 有效解决豆象最为安全有效的措施是发掘优异种质资源并利用现代生物技术培育抗豆象品种。世界各国现保存食用豆种质资源56.1万份[168], 我国拥有丰富的食用豆种质资源[169], 但目前鉴定出的抗豆象资源还很有限。在对种质抗豆象基因鉴定上, 主要是基于传统分子遗传学上的单基因鉴定和利用,缺乏全基因组水平上多基因发掘和多组学(基因组学、表观基因组学、转录组学、蛋白质组学、代谢组学等)水平上抗性机理解析, 缺乏抗性基因表达和信号网络调控方面的研究。在抗豆象品种改良上, 主要利用远缘杂交和常规育种进行抗性选择, 有效针对目标基因进行的转基因育种和分子辅助育种尚处于起步阶段。在优质多抗多亲本聚合育种上相对大宗作物研究还存在差距。目前培育推广的绿豆抗豆象品种, 由于豆象不同地理种群间遗传结构变异[170], 抗虫品种存在抗性丧失风险。发掘新的抗虫基因和利用现代育种技术培育抗虫品种任务重大。

7.2 加快我国食用豆抗豆象育种的展望

7.2.1 全面解析抗虫性与重要农艺性状协同调控机制 食用豆作物豆象抗虫性和其他植物抗虫性具有相似的遗传机制, 多由多基因或数量位点控制, 并且和重要农艺性状存在高度相关性。抗虫性和农艺性状的协同调控十分复杂, 除基因与基因互作外, 还与基因和环境互作有关。结合传统基因鉴定方法与现代分子生物学技术, 挖掘协同调控抗虫与重要农艺性状的功能基因, 深度解析作物抗虫性调控与形态建成和抗性物质形成的分子机制, 构建协同调控抗性与生长发育的遗传表达和信号转导网络, 为抗虫基因聚合育种提供理论基础和技术支持。

7.2.2 建立抗豆象种质鉴定和基因发掘技术, 加快抗虫基因克隆与应用 抗虫育种最为关键的是有可利用的抗虫种质或基因资源。针对目前抗虫育种中可用抗虫种质或抗虫基因缺乏的问题, 继续加强抗虫种质鉴定和抗虫基因发掘与应用。利用全基因组关联分析(GWAS)与传统克隆技术可实现抗虫基因克隆; 利用基因编辑技术可提高抗性基因功能验证[171]。随着高通量基因测序和生物信息技术的发展, 食用豆作物基因组信息解析和功能注释取得显著进展。近年来国内外先后完成绿豆[172]、小豆[173-174]、普通菜豆[175]、鹰嘴豆[176]、豇豆[177]和豌豆[178]基因组测序。Wu等[179]基于普通菜豆核心种质, 采用全基因组关联分析, 率先构建了世界首张精细普通菜豆单倍型图谱, 明晰了重要农艺性状基因位点, 为有效开展全基因组水平上抗豆象基因发掘和利用奠定了基础。基于我国绿豆核心种质评价和关键性状调控基因发掘, Liu等[180]开展了绿豆全基因组重测序, 率先构建了我国绿豆泛基因组遗传图谱, 实现了图形结构基因组构建。食用豆大量表型数据和基因型数据的发掘和应用, 必将对豆象抗虫种质表型精准鉴定、基因精细定位以及精准设计抗虫育种提供技术支撑。

7.2.3 构建分子设计和全基因组选择的抗虫智能化育种技术体系 基于传统远缘杂交在抗虫育种中存在优异性状重组交换低、重组位点分布不均、有害等位基因连锁、表型选择易受环境影响, 传统育种技术已很难有效实现优异多基因聚合育种目标。随着基因组学、系统生物学、大数据科学和计算生物学等新兴学科的快速发展, 以及大量分子标记技术的开发与应用, 基于对关键基因或QTL功能的认识, 利用TILLING技术、基因组编辑技术和转基因技术构建新型智能化抗虫育种体系, 使现代新型无重组育种技术成为可能。根据预先设定的育种目标, 选择合适的设计元件, 实现分子设计和多基因组装的全基因组选择育种, 已经成为引领作物遗传改良的前沿技术[181]。为解决转基因豌豆存在的科学问题, 我国学者率先在豌豆上通过农杆菌介导的遗传转化,利用CRISPR/Cas9系统定向编辑了八氢番茄红素脱氢酶基因(PsPDS)[182], 这为我国基因编辑创制豌豆新种质迈出了新步伐。近年来, 多个研究报道采用基因编辑可加快野生种人工驯化, 加快抗性基因原位编辑与聚合, 实现精准设计抗虫育种。充分应用转基因技术、分子标记辅助回交选择、循环集合选择、基因编辑技术及全基因组选择等现代分子设计育种必将成为抗性育种的主要方向[183]。未来育种4.0关键育种方案就是合理设计具有高产、优质、高抗等综合性状优良的作物[184]。

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Advances in germplasm innovation and genetic improvement of food legumes resistant to bruchid

YANG Xiao-Ming1, CHENG Xu-Zhen2,*, ZHU Zhen-Dong2, LIU Chang-Yan3, and CHEN Xin4,*

1Crop Research Institute, Gansu Academy of Agricultural Sciences, Lanzhou 730070, Gansu, China;2Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China;3Institute of Food Crops, Hubei Academy of Agricultural Sciences, Wuhan 430064, Hubei, China;4Institute of Industrial Crops, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, Jiangsu, China

Food legumes play a key role in maintaining soil sustainability, developing agroecosystem diversification, and improving human nutrition. However, bruchid (Coleoptera: Bruchidae) is a notorious pest that can devastate the entire seed and cause severe loss in pulses storage. To explore the potential germplasm resources and breed legume varieties resistant to bruchids, a few elite germplasms and genes resistant to bruchids were identified and finely mapped. Lots of studies have been carried out and made some progress on resistance mechanisms, genetic analysis, genetic mapping, gene cloning, and molecular markers of bruchid resistance in pulses. In this paper, studies on pulses germplasm exploring and evaluating for resistance to bruchids, resistance inheritance, discovery and mapping of resistance genes, and the breeding of resistant cultivars were reviewed. Several important directions for future research have prospected. Here, the main objective is to supply useful information for exploring potential germplasm and promoting the genetic improvement of food legumes with resistance to bruchids in China.

food legumes; germplasm exploitation; bruchid; resistance breeding; molecular markers

10.3724/SP.J.1006.2023.24169

本研究由财政部和农业农村部国家现代农业产业技术体系建设专项(CARS-08)和国家自然科学基金项目(32260483)资助。

This study was supported by the China Agriculture Research System of MOF and MARA (CARS-08) and the National Natural Science Foundation of China (32260483).

程须珍, E-mail: chengxuzhen@caas.cn; 陈新, E-mail: cx@jaas.ac.cn

E-mail: yangxm04@hotmail.com

2022-07-21;

2022-11-03;

2022-11-14.

URL: https://kns.cnki.net/kcms/detail/11.1809.S.20221111.1116.002.html

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