小麦抗根腐病突变体的抗病性研究

 将通过细胞工程技术获得的小麦抗根腐病突变体后代M₁、M₂和M₃进行抗病性鉴定。结果表明,其突变体后代M₁、M₂和M₃都能保持抗病性,同时,突变体的过氧化物酶的活性较标准型对照品种明显增强,在同工酶谱中出现2条新的酶带。     

大白菜抗芜菁花叶病毒基因EST-PCR-RFLP分子标记的研究

 本试验以高抗芜菁花叶病毒C₃株系(TuMV-C₃)的高代自交系A₁56-2和感病自交系P9805杂交后代的F₂代为群体,根据大白菜的抗性相关的表达序列标签(EST)设计引物,利用分离群体分群分析法(BSA),筛选出2个与TuMV-C₃株系抗病基因紧密连锁的EST-PCR-RFLP分子标记BS300及BS160,遗传距离均为6.5 cM,为大白菜分子辅助育种、抗病基因克隆以及研究抗病基因编码特性等奠定基础。     

小麦抗白粉病基因Pm21 的抑制基因

 小麦-簇毛麦6VS. 6AL 易位染色体含有抗白粉病基因Pm21,在我国的小麦育种中被广泛应用。近年来,一些含有Pm21 基因的小麦品种(系)开始感染白粉病。为探索含Pm21 的品种(系)感染白粉病的原因,本研究在6VS. 6AL 易位系与小麦品系(种)R14 和川农12 的杂交后代中利用分子标记CINAU17-1086 和CINAU18-723 辅助选择的遗传背景相对简单的F7 和F8 近等基因系为材料,研究了小麦抗白粉病基因Pm21 的抗病性表达。结果发现,在3 个含有6VS. 6AL 易位染色体的感病F6 植株繁殖的F7 近等基因系中发生了白粉病抗性的分离,分离比率符合13 感病︰ 3 抗病的理论值。在随机选取的F7 感病小麦单株所繁殖的F8 近等基因系中,有7 / 13 的株系一致地重感白粉病,有6 / 13 的株系发生了抗白粉病的分离,其中2 / 13 的株系分离比符合3 感病︰ 1 抗病、4 / 13 的株系分离比符合13 感病︰ 3 抗病的分离模式。这一结果指出,小麦株系中的抗白粉病基因Pm21 的抗性表达受小麦基因组中的一对显性抑制基因所控制,该基因来源于小麦品种(系)川农12或R14,建议命名为SuPm21。本研究指出,在把外源基因引入小麦的研究中,有利的外源基因与不含抑制基因的受体遗传资源同等重要。     

来自簇毛麦抗条锈病新基因的SSR标记

 用小麦条锈菌条中30号生理小种,对小麦抗病种质小麦-簇毛麦易位系V9128-1和铭贤169的杂交后代进行抗条锈性遗传分析,小麦-簇毛麦易位系V9128-1的抗病性符合1对显性抗条锈病基因控制。并根据F₂抗、感病单株分离比例组建抗感池,用SSR技术寻找与抗病基因连锁的分子标记。从121个SSR引物组合中筛选到2个与抗病基因YrV1(暂命名)紧密连锁的微卫星标记Xgwm566和Xgwm376,遗传距离分别为3.6和5.5cM;因此,该抗条锈病基因位于小麦3B染色体短臂上。这2个标记不仅能在小麦-簇毛麦易位系V9128-1中检测到,而且在抗病基因供体亲本簇毛麦中也能检测到。综合抗病基因来源和分子生物学试验结果,可以推断,YrV1很可能是1个来自簇毛麦并与已知抗条锈病基因不同的新基因。     

应用DNA标记定位水稻的抗稻瘟病基因

 从水稻分子遗传图谱选取177个RFLP标记,比较以红脚占为抗源,感病品种IR24为轮回亲本所构建的近等基因对K80R和K79S之间的多态性表现。发现了一些可能与稻瘟病抗性基因连锁的阳性标记。在(K80R×K79S)的F₂群体中,经稻瘟病菌ZB₁小种接种,110株为抗病,33株为感病,用总共10个阳性标记与F₂群体中每个单株的DNA杂交,发现抗病基因与第12染色体上的标记RG81、RG869和RZ397共分离;检测F₃株系的抗病性分离情况,确定F₂植株的抗病性基因型,计算出抗病基因与分子标记的遗传距离,将该基因定位在第12连锁群上。应用近等基因池DNA和随机引物,经PCR扩增和共分离分析,建立了二个RAPD片段与抗病基因紧密连锁。     

小麦品种对条绣病抗病性遗传规律研究初报

对133个小麦品种间杂交组合的观察结果指出,凡有免疫品种为亲本的组合,其 F_1抗病性表现显性的机率都很高,而其机率的高低又与另一亲本抗病性的强弱呈正相关。对24对正反交组合的 F_1代,8对的 F_2代和2对的 F_3代作比较观察的结果表明用免疫品种作父本与母本,对后代抗病性的表现和分离并无不同影响。根据田间调查结果,看出杂种后代中底部叶片轻微发病的植株其抗病性稳定程度和遗传力低于“全株免疫株”;用底部叶片发病,中上部叶片表现免疫的品种作抗病亲本,F_1代的中下部叶片发病比较普遍;而用全株免疫的品种作抗病亲本,F_1代一般没有发病现象。试验结果证明 F_1代和 F_2代中的“少茎病株”(同一植株中多数分蘖免疫,个别或少数分蘖感病),其后代(F_2和 F_3)分离出的免疫株率与全株免疫株差别不明显。感病分蘖与同株免疫分蘖的后代在抗病性的分离上也无显著差别。石特14×胜利组合 F_2免疫株的后代(F_3)中有15.8%的株系全部植株免疫;“0;”型和“1”型 F_3植株分离出的免疫株率各达到80.3%和55.5%,但汉有出现免疫的株系。     

利用分子标记提高筛选普感水稻抗病突变体的准确性

 水稻丽江新团黑谷(LTH)是一个云南地方粳稻普感品种,对全球近2 800多个稻瘟病菌生理小种均表现为感病表型。本研究为利用⁶⁰Co-γ射线辐照处理LTH种子,分单株收获M₁代种子。对M₂和M₃代植株分别进行稻瘟病的自然诱发筛选和人工喷雾接种筛选,以期从LTH中筛选获得抗性提高的突变体。在突变体筛选过程中,我们发现突变体的遗传背景受到零星异交或混杂干扰,这种情况在其他本底抗性水平较高的抗性突变体筛选中往往容易被忽略。为了确保突变体遗传背景的真实性,我们利用分布在水稻12条染色体上的在粳稻LTH与籼稻品种间存在多态性的In/Del标记,分析候选抗性植株是否为LTH纯合背景,以排除杂合假抗性个体。通过两种不同的筛选策略,最终从M₃代植株中分别鉴定出1份和4份抗性突变体,为进一步研究LTH普感特性奠定基础。将其中一个LTH抗病突变体分别与野生型LTH和籼稻普感品种CO39进行杂交,获得的F₂群体人工喷雾接种稻瘟病菌后调查抗、感植株的分离情况,分析结果显示,该突变体性状符合单个显性基因控制的遗传规律。另外,本研究结果表明:在水稻诱变育种工作中,不仅要做到充分隔离,还需借用分子标记辅助剔除零星意外串粉造成的杂合假突变体,以提高水稻突变体筛选的准确率。     

水稻恢复系华占抗稻瘟病遗传分析及基因鉴定

 华占是近年来我国新选育的恢复系,广泛应用于杂交稻育种中,其配组的杂交稻组合对稻瘟病均表现出良好的抗性。本研究利用来源于华南稻区的62个稻瘟病菌株对恢复系华占、明恢63和广恢998进行抗病性鉴定,结果表明:华占对接种菌株的抗谱达到95.2%,对稻瘟病表现出广谱抗性。为进一步了解该恢复系所含的抗稻瘟病基因,以高感稻瘟病品种丽江新团黑谷为母本,以华占为父本,构建了丽江新团黑谷(LTH)/华占的F₂抗病遗传分析群体。利用对华南水稻品种具有致病谱较广的稻瘟病代表菌株GD00-193a对随机选择的1 000个来源于LTH/华占的F₂个体及其F₁个体进行抗病遗传分析,结果发现F₁个体表型全为抗病,1 000个F₂个体的抗/感比率符合3∶1的显性单基因分离比,说明华占至少含有一个显性抗稻瘟病基因。利用均匀分布于水稻12条染色体的250对SSR引物,通过分离群体分析结合隐性群体分析法将华占的一个抗稻瘟病基因定位于水稻第6染色体的Pi2/Pi9/Pi50区域。通过Pi2等位基因的测序分析,结果表明华占含有Pi2抗病基因。本试验结果为华占在杂交稻上的应用及其配组组合的品种布局提供重要依据。     

西瓜抗小西葫芦黄花叶病毒基因的连锁分子标记研究

 小西葫芦黄花叶病毒中国株系(Zucchini yellow mosaic virus Chinese strain,ZYMV-CH)是危害我国西瓜的主要病毒。本实验以抗病毒病西瓜野生种质P.I.595203与感病的普通西瓜自交系98R为亲本,采用单粒传方式得到109个E代株系,分别对亲本、F₁及109个F₃代株系群体进行了苗期抗ZYMV-CH接种鉴定,通过F₃代群体的抗感分离情况,推测得到F₂代各单株的基因型,采用集团分离分析法(bulked segregant analysis,BSA)在F₂代建立抗感基因池,以亲本、F₁和抗感基因池为模板,对640条RAPD引物进行PCR扩增筛选,其中引物AK13在亲本、F₁和抗感基因池之间扩增出一条多态性片段(644bp),在F₂代群体上验证该多态性条带与ZYMV-CH的抗性基因呈现连锁关系,遗传连锁距离为8cM,定名为AK13_-644,该连锁标记在ZYMV-CH抗性转育后代自交系上得到了验证。最终将此RAPD标记成功转化成SCAR标记SCAK13_-644,该标记可以作为西瓜抗病毒病辅助选择的分子标记。     

小麦秆锈菌鉴别寄主品种Orofen对小种21C3和34C2抗病基因的单体分析

 用中国春单体系列和二体中国春作为母本与Orofen杂交。选择出所有类型的单体杂种F1植株,令其自交结实。在温室内(10-25℃)用秆锈菌小种21C3和34C2的单孢菌系分别接种鉴定各杂交组合的F₂代苗期的抗性分离表现。对小种21C3,除2D和6D之外,其它单体类型和二体对照的F₂代都符合抗病15:1感病的分离比例;对小种34C2,除2D之外,其它单体类型和二体对照的F₂代都符合抗病3:1感病的分离比例。用Orofen与含有国际上已定位于2D和6D染色体上的已知Sr基因的品系(或品种)杂交。对小种21C3,Orofen与含有Sr5和Sr6的单基因系的杂交F₂未分离出感病的植株;对小种34C2,只有与含有Sr6的单基因系的杂交F₂代未分离出感病的植株。这表明,Orofen在2D染色体上含有Sr6,它兼抗小种21C3和34C2,分别提供0-1;1⁺⁺x^-和;1-;1⁺⁺x^-的抗性效应;而在6D染色体上携带抗病基因Sr5,它只抗小种21C3.控制0-;1^-的侵染型。对无毒性的小种,Sr5对Sr6的抗病效应是上位的。     

番木瓜环斑病毒株系的分子生物学方法鉴定

 以PRSV株系特异性引物对PRSV的PRSV126(PRSV日本分离物)、Ys、Vb和Sm等株系进行RT-PCR方法鉴定,引物PR21/PR22能把Ys从Vb和Sm中鉴定出来,PR300/PR301则能把Vb从Ys、Sm和PRSV126中鉴定出来;用限制性内切酶Hae Ⅱ、Sau3A I和Hinf I对PRSV的PRSV126、Ys、Vb和Sm等株系进行单酶切RT-PCR-RFLP分析,Hinf I能把PRSV126与Ys、Vb和Sm鉴别开来,Sau3A I能把Ys与Vb和Sm鉴别开来,Hae Ⅱ则能把Ys与PRSV126、Vb和Sm鉴别开来;以P1/P2为引物,对Vb、Ys和Sm株系进行RT-PCR-RFLP-SSCP分析,结果能一次把三者较好地区别开来。     

Broad-Spectrum Resistance to Different Geographic Strains of Papaya ringspot virus in Coat Protein Gene Transgenic Papaya

ABSTRACT Papaya ringspot virus (PRSV) is a major limiting factor for cultivation of papaya (Carica papaya) in tropical and subtropical areas throughout the world. Although the coat protein (CP) gene of PRSV has been transferred into papaya by particle bombardment and transgenic lines with high resistance to Hawaii strains have been obtained, they are susceptible to PRSV isolates outside of Hawaii. This strain-specific resistance limits the application of the transgenic lines in other areas of the world. In this investigation, the CP gene of a local strain isolated from Taiwan, designated PRSV YK, was transferred into papaya via Agrobacterium-mediated transformation. A total of 45 putative transgenic lines were obtained and the presence of the transgene in papaya was confirmed by polymerase chain reaction amplification. When the plants of transgenic lines were challenged with PRSV YK by mechanical inoculation, they showed different levels of resistance ranging from delay of symptom development to complete immunity. Molecular analysis of nine selected lines that exhibited different levels of resistance revealed that the expression level of the transgene is negatively correlated with the degree of resistance, suggesting that the resistance is manifested by a RNA-mediated mechanism. The segregation analysis showed that the transgene in the immune line 18-0-9 has an inheritance of two dominant loci and the other four highly resistant lines have a single dominant locus. Seven selected lines were tested further for resistance to three PRSV heterologous strains that originated in Hawaii, Thailand, and Mexico. Six of the seven lines showed varying degrees of resistance to the heterologous strains, and one line, 19-0-1, was immune not only to the homologous YK strain but also to the three heterologous strains. Thus, these CP-transgenic papaya lines with broad-spectrum resistance have great potential for use in Taiwan and other geographic areas to control PRSV.     

玉米抗粗缩病遗传及分子标记研究

Maize rough dwarf disease caused by Rice black-streaked dwarf virus (RBSDV) is transmitted by planthopper in China. Identification and development of resistant hybrids are complicated because of the inconsistencies in viral disease pressure every year. Marker-assisted selection can provide means for main-taining virus resistance alleles even in the absence of disease. In this paper a F₂ segregation population was constructed to identity the molecular markers linked to the resistance gene using a cross between a resistant and a susceptible parents (Qi319×Ye107). Fifteen-day-old seedlings of F₂ population were exposed to small brown planthoppers carrying RBSDV for 3 days in specific inoculation chamber. The inoculated plants were transplanted to screenhouse after removing the insects completely. In plant maturity stage the disease resistance of all the individuals were visually assessed. The results showed that 17, 8, 11, 51 and 122 plants were scaled from 0-4 respectively, in which 0 means no symptoms and 4 represents highly susceptible. Chi-square test demonstrated that the segregation ratio of phenotype was 1∶15 (resistant: susceptible) or 1∶6∶9 (resistant∶moderate∶susceptible) in the F₂ population, indicating RBSDV resistance of maize was controlled by two recessive genes. The F₂ individuals DNA were extracted and 261 SSR (simple sequence repeat) primers derived from maize genome ten chromosomes were selected from maize GDB database to construct genetic linkage map. The linkage map consisted of 71 polymorphic SSR markers, spanning a genetic distance of 996.6 cM with an average interval of 14.0 cM between adjacent markers. The resistant and susceptible gene pools were set up for BSA (bulked segregant analysis) and 6 polymorphism markers were obtained with BSA-SSR method between the two pools. The F₂individuals were further analyzed with 6 polymorphism markers. Chi-square test showed that phi 051, umc1407 and umc1432, mapped on chromosome 7 and 10, exhibited segregation distortion significantly and very significantly in susceptible individuals. These three SSR markers were identified as potential markers linked to the resistant loci.     

The ability of <Emphasis Type="Italic">Papaya ringspot virus</Emphasis>strains overcoming the transgenic resistance of papaya conferred by the coat protein gene is not correlated with higher degrees of sequence divergence from the transgene

The coat protein (CP) gene mediated transgenic resistance is found to be the best approach for protecting papaya plants against the destructive disease caused by Papaya ringspot viruses(PRSV). In order to study the variability of PRSV and the potential threat to the CP-transgenic resistance, five virus isolates were collected from transgenic plants of papaya line 16-0-1, which carry the CP gene of the typical mosaic strain of Taiwan PRSV YK, in an approved test field and fourteen from untransformed papaya plants in different areas of Taiwan. The results of biological, serological, and molecular characterization indicated that all isolates are related to PRSV YK. Among them, the isolate 5--19 from the transgenic line and the isolates CS and TD2 from untransformed papaya were able to overcome the YK CP gene-mediated resistance of papaya lines 18--2--4, 17-0-5, and 16-0-1, which provide high degrees of resistance to different geographic PRSV strains of Hawaii (HA), Mexico (MX), and Thailand (TH). These three isolates were also able to cause symptoms on untransformed papaya plants more severe than those induced by YK. In addition to the host reactions, the variability of the collected 19 isolates was also analyzed and compared with YK and other geographic strains by heteroduplex mobility assay (HMA) and sequence analyses. The results of HMA indicated that the CP genes of isolates 5--19 and TD2 are more divergent than those of other isolates when compared with YK. However, sequence analyses of the transgenic-resistance overcoming isolates 5-19, CS, and TD2 revealed that their CP coding regions and the 3 untranslated regions (UTRs) share nucleotide identities of 93.9–96.6% and 94.2–97.9% with those of YK, respectively; whereas the other geographic strains of HA, MX, and TH that could not overcome the transgenic resistance share lower nucleotide identities of 89.8–92.6% and 92.3–95.3% with those of YK, respectively. Our results indicate that the ability for overcoming the transgenic resistance is not solely correlated with higher degrees of sequence divergence from the transgene. The possible mechanism for overcoming the transgenic resistance and the potential threat of these PRSV strains to the application of the transgenic papaya lines carrying PRSV YK CP gene are discussed.     

Inheritance of partial resistance to race 2 of Albugo candida in canola-quality mustard (Brassica juncea) and its role in resistance breeding

The inheritance of partial resistance to race 2 of Albugo candida was studied in a canola-quality line of Brassica juncea . This partially resistant line was crossed with the susceptible B. juncea cultivar Commercial Brown. F₁, F₁(reciprocal), F₂, BC and doubled haploid generations from the cross were inoculated with a zoospore suspension of race 2 to study segregation of partial resistance. The partially resistant phenotype appeared to be controlled by a single dominant gene that has variable expression. This partial resistance can have implications in breeding for disease resistance against white rust, as adult plants did not develop hypertrophic growth or stagheads under greenhouse and field conditions.     

水稻抗病基因Xa21转入3个粳稻品种的抗性初步分析

 通过农杆菌介导转化,将已克隆的水稻白叶枯病抗性基因Xa21转入我国辽宁省3个粳稻品种:沈农606、辽粳454、辽粳294,共获得独立转基因系205个。通过PCR、GUS染色和Southern blot分析,证明Xa21基因已经整合到3个粳稻栽培品种的基因组中。通过温室接种菲律宾白叶枯病小种6的代表菌系PXO99,T₀代转基因水稻除了3-34表现部分抗性外,其它的转基因材料均表现高度抗病,表明Xa21转基因水稻已经获得抗性;温室接种T₁和T₂代试验表明,单拷贝整合的转化体呈现3:1分离,同时单拷贝3-34材料也表现部分抗性和感病3:1分离,证明已整合的Xa21基因能稳定遗传;同时对3-34部分抗性机制进行了分子生物学检测,证明GUS基因全部丢失、Xa21基因部分丢失并导致部分抗性。获得部分抗性材料,对于深入研究Xa21基因的功能区和研究抗病分子机制具有重要的意义。     

Transgene-mediated resistance to <Emphasis Type="Italic">Papaya ringspot virus</Emphasis>: challenges and solutions

Global papaya production is severely affected by papaya ringspot disease caused by Papaya ringspot virus (PRSV). Management of this potyvirus is challenging, due to 1) its non-persistent transmission by numerous aphid species and 2) the diversity of PRSV strains that exists within a country or between different geographical regions. Papaya cultivars with transgenic resistance have reduced the impact of the disease. There are no effective alternatives to transgenic resistance available in areas where disease pressure is high. In Hawaii, transgenic papayas such as “SunUp” and “Rainbow” have remained resistant to PRSV more than two decades saving the commercial papaya industry. Following the success in Hawaii, researchers from other countries have focused on developing PRSV-resistant transgenic papaya. These transgenic cultivars often demonstrated an initial transitory resistance that was ultimately overcome by the virus. For other cases, resistance was inconsistent. That is, some transgenic lines were resistant while others were not. Transgenic cultivars are now losing PRSV-resistance for various reasons in China and Taiwan. In this review, we present an update on work with transgenic papaya with resistance to PRSV. The focus is on factors affecting transgenic resistance in papaya and our attempt to explain why the Hawaiian scenario of complete and durable resistance has not been replicated in other regions. The utilization of more recent technologies to the development of virus resistance in papaya is also discussed.     

Occurrence and inheritance of resistance to powdery mildew (Oidium lycopersici) in Lycopersicon species

Among 146 accessions of Lycopersicon pimpinellifolium , 132 of L. esculentum var. cerasiforme and 53 of L. peruvianum screened for resistance to powdery mildew, caused by Oidium lycopersici , a wide variation in reactions was found. Two plants of L. esculentum var. cerasiforme accession LA-1230 were resistant. One resistant symptomless plant of accession LA-1230, designated LC-95, produced homozygous resistant progenies. LC-95 was crossed with cv. Marmande (susceptible parent) and F₁, F₂ and backcrosses to the resistant and the susceptible parents were derived. These genotypes were grown in glasshouses at 23°C and 95–100% RH and inoculated with O. lycopersici . The F₁ plants were susceptible. F₂ and backcross segregations fitted the hypothesis of a single recessive gene which is here designated ol-2 .