【作者】 李得孝；

【导师】 陈耀锋； 陈鹏印；

【作者基本信息】 西北农林科技大学 ， 作物遗传育种， 2009， 博士

【摘要】 大豆花叶病（SMV）是全球主要大豆产区分布最广的病毒性病害之一。全球范围内大豆种质交流和新的强毒力病毒株系的不断出现,使大豆安全生产面临挑战。大豆花叶病毒归马铃薯Y病毒科（Potyviridae）、马铃薯Y病毒属（Potyvirus）,寄主范围狭窄,主要局限于豆科植物。可通过受侵染的种子,蚜虫和机械接种传播。植株受SMV侵染后,通常出现三种反应:无症状或仅在接种叶有局部致死斑（抗病类）,致死（局部致死或整株致死）及花叶症状（感病类）。大豆对SMV的侵染反应受寄主基因型、病毒株系、侵染时期和环境条件（特别是温度）的影响。依据一套大豆鉴别材料的表型反应,美国已鉴定出7个SMV病毒株系（G1-G7）,而在中国和日本,利用不同的大豆材料鉴定出不同的病毒株系,但各地鉴定的株系之间的关系尚不清楚,有待建立统一的病毒株系鉴别体系。美国利用Cho & Goodman（1979）的鉴别体系已经鉴定出位于大豆不同连锁群（MLG-F,-B2,-D1b）上的3个抗性基因位点（Rsv1,Rsv3,Rsv4）,并在Rsv1位点发现9个等位基因。对SMV和大豆互作中是否存在“一因多效”现象,国内外专家的认识存在分歧。中国学者认为大豆对不同病毒株系的抗性受不同基因控制,不存在“一因多效”现象,而美国专家认为,SMV抗性基因的作用是“多效的”。分子标记作图的结果表明,中国报道的抗性基因位点与美国鉴定的基因基本重合。本研究在美国大豆花叶病鉴别体系的基础之上,进行了如下研究:（1）发掘大豆种质中新的SMV抗病基因,为大豆抗SMV育种提供多样化抗源,提高大豆抵抗病毒侵袭的能力;（2）结合经典遗传研究,探讨利用与抗性位点紧密连锁的分子标记确定抗性基因的可能性;（3）利用分子标记辅助选择育种技术聚合多个抗性基因,创制大豆新抗源,为广谱持久抗性材料的选育提供有用的育种种质;（4）研究抗性基因杂合状态下,温度对致死反应的影响,深化对大豆抗病基因与病毒互作机制的认识。研究用6个株系（G1～G3,G5～G7）鉴定了127份大豆材料的抗性反应、甄别这些材料中携带的可能抗性基因。结果显示:84份材料携带Rsv1位点上的抗性基因,分别是Rsv1-y 42份, Rsv15份,Rsv1-k 19份,8份Rsv1-t, 8份Rsv1-r, 1份Rsv1-n, 1份Rsv1-h;Rsv3位点材料各3份;16份材料可能携带Rsv1, Rsv3,或Rsv4位点上的新基因,比较其与已报道的9个抗性基因对不同病毒株系的反应模式,发现其中9份材料（Kosuzu, Suzumaru, PI 398877, Jitsuka, Clifford, and Tousan 65, Cosica, PI 61944,和PI 61947）可能携带Rsv1位点的新基因,2份（PI 339870 and PI 399091）可能携带Rsv3位点的新抗性基因,5份（KAERI-GNT-220-7, PI 398593, PI 438307, Rhosa和Beeson）可能携带Rsv4位点的新抗性基因。另外,18份材料全抗6个株系,可能携带Rsv1-h, Rsv4, Rsv1Rsv3, Rsv1Rsv4,或Rsv3Rsv4基因。遗传等位测验显示中国大豆品种J05携带两个独立的大豆花叶病抗性基因Rsv1和Rsv3。研究利用分子标记证实J05携带两个抗性基因。用SMV-G1接种的F（2J05 x Essex）单株,结果很好地符合3:1的分离比例,Rsv1位点附近的三个SSR标记,Satt114, Satt510和Sat₁54,可在亲本J05和Essex中扩增出多态性条带,并与F连锁群的基因紧密连锁,证实J05中含有SMV G1的抗性基因Rsv1;选感SMV-G1的F₂单株的F_2:3家系接种SMV-G7株系,表现型出现1:2:1的分离比例,证实J05中存在Rsv3并与B2连锁群的两个SSR标记Satt726和Sat₄24紧密连锁;但与基因Rsv4紧密连锁的两个标记Satt296和Satt542与SMV的抗性分离表现相互独立,表明J05中不含Rsv4基因。Rsv1和Rsv3位点的这些标记可用于J05中SMV抗性基因选择和聚合的有效分子工具。试验以J05（Rsv1Rsv3）和V94-5152（Rsv4）做抗性基因供体,利用分子标记聚合Rsv1, Rsv3和Rsv4三个SMV抗性基因。用J05 x V94-5152的F₂:3, F3: 4,和F4:5家系筛选携带三个抗性基因的聚合个体。8个与三个抗性基因连锁的PCR标记被用于分子辅助选择。两个SSR标记（Sat₁54和Satt5 10）和一个基因特异性标记（Rsv1-f/r）被用于筛选携带Rsv1的单株; Satt560和Satt063用于筛选Rsv3;Satt266, AI856415和AI856415-g用于筛选Rsv4。有五个F4:5家系在所有8个标记位点上都重合,推测可能已经聚合了3个SMV抗病基因。迄今在大豆种质中已鉴定出了3个SMV抗性基因位点,Rsv1, Rsv3和Rsv4。Rsv1位点的大多数基因对一些SMV株系（而非全部）表现抗病;Rsv3对G5- G7表现抗病,对G1- G4表现感病;Rsv4对G1-G7表现在幼苗早期抗病。Rsv1位点的抗性基因常表现剂量效应,出现杂合致死;而当与特定的SMV株系互作时,可出现纯合体的致死反应。研究利用一套Essex等基因系和F₁杂种,探究了在不同温度条件下SMV的致死反应与抗性基因的剂量效应（基因型纯合对基因型杂合）之间的关系。结果表明,SMV侵染导致的致死症状受外界温度的影响。SMV接种携带Rsv3和Rsv4的植株,不论基因纯合或杂合,在所有温度下皆表现无症状。在纯合状态下,V94-3971（Rsv1）、PI 96983 （Rsv1）和V262 （Rsv1-n）植株接种G7或G1后,茎尖致死转化成花叶的温度阈值中分别为30oC, 33oC和33oC。但在杂合状态下,G7接种的F₁（V94-3973 x Essex）的温度阈值为29oC;G7接种的F₁（Essex x PI 96983）为30oC;G1接种的F₁（V262 x Essex）为31oC。而且,在G1接种的F₁（V262 x Essex）和G7接种的F₁（V94-3973 x Essex）杂合体中,还观察到不完全致死症状,即致死和花叶症状是混合出现的。茎尖致死对温度的反应受抗性基因、基因剂量效应、和细胞核背景的影响,细胞质效应可能存在但很有限。更多还原

【Abstract】 Soybean mosaic virus （SMV） is one of the most prevalent viral diseases in all major soybean-growing areas worldwide. Germplasm exchanges and continual appearances of new virulent SMV strains have caused significant challenges to security of soybean production worldwide. Soybean mosaic virus, classified as the genus potyvirus in the family potyviridae with narrow hosts, limited primarily to the leguminosea, was transmitted by infected seeds, aphids, and mechanical inoculation. A plant infected by SMV would show reaction of symptomless or local necrotic spots on inoculated leaves （resistant type）, systematic necrosis （local necrosis or stem top necrosis）, or mosaic symptom （susceptible type）. Symptoms of plant infected by SMV are alternated by host genotype, virus strain, development stage of infected soybean plant, and environmental conditions, especially temperature. Based on the phenotypic reactions to a set of differential soybean genotypes, seven strains of SMV（G1-G7） were identified in the U.S. However, in China and Japan, different soybean genotypes were used for identification of SMV strains. But the relationship of SMV strains identified in different countries was unknown and uniformity of SMV differential system was expected. Three resistance genes, Rsv1, Rsv3, and Rsv4 located on the molecular linkage group（MLG） F, B2, and D1b, respectively, were identified in the U.S. on the base of SMV identification system of Cho and Goodman（1979）, and nine alleles were reported at the Rsv1 locus. There are different perception on the interaction of SMV and soybean. In China, resistance to different SMV strains was presumed to be conditioned by different genes and there were no pleiotrophic effect on a resistance gene. However, in the U.S., SMV resistance genes were shown to be pleiotrophic,that is, two different reactions to separate SMV strains may be controlled by the same host gene. But mapping results of resistance genes with molecular markers indicated that resistance genes named differently in different countries maybe the same or closely linked in MLG.This dissertation was developed on the base of SMV identification system in the U.S. and four items were conducted, including （1） searching for the new resources of SMV resistance from the differential soybean germplasm, which can be used in the soybean breeding program as a resistance parent of SMV, and strengthening ability resistant to SMV infection; （2） exploring the possibility of conforming appearance of specific SMV resistance gene in a giving genotype combining molecular marker information and classical genetic procedure; （3） pyramiding three SMV resistance gene into a soybean plant through the procedure of marker-assisted selection （MAS）, and created a new resistance resource unreported in nature, and used in the development of new soybean varieties with broader resistance to ever-changing SMV strains; （4） studying effect of temperature under the heterozygous state of resistance gene and extending the knowledge of soybean resistance gene and virus interaction.127 genotypes were screened with six SMV strains and specific alleles for resistance in these genotypes were differentiated in this research. The results demonstrated that 84 genotypes carry alleles at the Rsv1 locus: 42 with Rsv1-y, five with Rsv1, 19 with Rsv1-k, eight with Rsv1-t, eight with Rsv1-r, one with Rsv1-n, and one with Rsv1-h. Sixteen genotypes were identified to presumably carry new alleles for SMV resistance at Rsv1, Rsv3, or Rsv4. Nine of these 16 genotypes （‘Kosuzu’,‘Suzumaru’, PI 398877,‘Jitsuka’,‘Clifford’, and‘Tousan 65’,‘Corsica’, PI 61944, and PI 61947） may carry new alleles at the Rsv1 locus based on the comparison of their differential reaction patterns against the nine reported Rsv1 alleles. Two genotypes （PI 339870 and PI 399091） may carry new resistance alleles at the Rsv3 locus. Five genotypes （KAERI-GNT-220-7, PI 398593, PI 438307,‘Rhosa’, and‘Beeson’） may carry new alleles at the Rsv4 locus. In addition, 18 genotypes were resistant to all six strains tested and may carry Rsv1-h, Rsv4, Rsv1Rsv3, Rsv1Rsv4, or Rsv3Rsv4. Research is underway to confirm the new SMV resistance alleles through genetic and molecular approaches. J05 Soybean was previously identified to carry two independent genes, Rsv1 and Rsv3, for Soybean mosaic virus （SMV） resistance by inheritance and allelism studies. This research has confirmed that the two genes in J05 using molecular markers and a marker-assisted selection can be implemented. The segregation of F₂ plants from J05 x Essex exhibited a good fit to a 3:1 ratio when inoculated with SMV G1. Three simple sequence repeat （SSR） markers near Rsv1, Satt114, Satt510, and Sat₁54, amplified polymorphic DNA fragments between J05 and Essex, and were closely linked to the gene on soybean molecular linkage group （MLG） F, thus verifying the presence of Rsv1 in J05 for resistance to SMV G1. The presence of Rsv3 in J05 was confirmed by two closely linked SSR markers on MLG B2, Satt726 and Sat₄24, in F₂:3 lines that were derived from the SMV G1-susceptible F₂ plants and segregated in a 1:2:1 ratio for reaction to SMV G7. Two closely linked markers for Rsv4, Satt296 and Satt542, segregated independently of SMV resistance, indicating the absence of Rsv4 in J05. These SSR markers for Rsv1 and Rsv3 can serve as a useful molecular tool for selection and pyramiding of genes in J05 for SMV resistance.This research was to pyramid Rsv1, Rsv3, and Rsv4 for SMV resistance using molecular markers. J05 carrying Rsv1 and Rsv3 and V94-5152 carrying Rsv4 were used as the donor parents for gene pyramiding. A series of F_2:3, F_{3: 4}, and F_4:5 lines derived from J05 x V94-5152 were developed for selecting individuals carrying all three genes. Eight PCR-based markers linked to the three SMV resistance genes were used for marker-assisted selection. Two SSR markers （Sat₁54 and Satt510） and one gene-specific marker （Rsv1-f/r） were used for selecting plants containing Rsv1; Satt560 and Satt063 for Rsv3; and Satt266, AI856415, and AI856415-g for Rsv4. Five F_4:5 lines were homozygous for all eight marker alleles and presumably carry all three SMV resistance genes for multiple and durable resistance to SMV. Three resistance loci, Rsv1, Rsv3, and Rsv4, have been identified in soybean germplasm by far. Most alleles at the Rsv1 locus exhibit resistance to some, but not all, strains of SMV, Rsv3 confers resistance to G5 through G7, and susceptibility to G1 to G4, and Rsv4 alleles provide genetic resistance to G1-G7 at the early seedling stage. Resistance genes at the Rsv1 locus often have a dosage effect resulting in necrosis in the heterozygous state and may confer necrotic reaction in the homozygous state when interacting with specific SMV strains. Necrotic symptom induced by SMV infection is affected by environmental factors such as temperature. Using a set of Essex isolines and F₁ hybrids, this study has explored relationship of SMV-induced necrosis expression and resistance gene dosage （homozygous vs heterozygous alleles） at different temperature regimes. The results showed that SMV-inoculated plants carrying Rsv3 and Rsv4 showed symptomless at homozygous and heterozygous state at all temperature regimes; Threshold temperature of symptom shifting from stem tip necrosis （STN） into mosaic were 30oC, 33oC, and 33oC in G7-inoculated homozygous genotypes V94-3971（Rsv1）, PI 96983 （Rsv1）, and G1-inoculated V262 （Rsv1-n）, respectively. But at heterozygous state, threshold temperature was 29℃in G7-inoculated F₁（V94-3973 x Essex）, 30℃in G7-inoculated F₁（Essex x PI 96983）, and 31℃in G1-inoculated F₁（V262 x Essex）; In addition, incomplete necrosis was observed in heterozygous state in G1-inoculated F₁（V262 x Essex） and G7-inoculated F₁（V94-3973 x Essex） where necrotic and mosaic symptoms were mixed. STN expression to temperature was affected by resistance gene, gene dosage, and nuclear background. Cytoplasm effect may exist but very limited.更多还原