Meta-QTL analysis for mining of candidate genes and constitutive gene network development for fungal disease resistance in maize(Zea mays L.)

The development of resistant maize cultivars is the most effective and sustainable approach to combat fungal diseases.Over the last three decades,many quantitative trait loci(QTL)mapping studies reported numerous QTL for fungal disease resistance(FDR)in maize.However,different genetic backgrounds of germplasm and differing QTL analysis algorithms limit the use of identified QTL for comparative studies.The meta-QTL(MQTL)analysis is the meta-analysis of multiple QTL experiments,which entails broader allelic coverage and helps in the combined analysis of diverse QTL mapping studies revealing common genomic regions for target traits.In the present study,128(33.59%)out of 381 reported QTL(from 82 studies)for FDR could be projected on the maize genome through MQTL analysis.It revealed 38 MQTL for FDR(12 diseases)on all chromosomes except chromosome 10.Five MQTL namely 1_4,2_4,3_2,3_4,and 5_4 were linked with multiple FDR.Total of 1910 candidate genes were identified for all the MQTL regions,with protein kinase gene families,TFs,pathogenesis-related,and disease-responsive proteins directly or indirectly associated with FDR.The comparison of physical positions of marker-traits association(MTAs)from genome-wide association studies with genes underlying MQTL interval verified the presence of QTL/candidate genes for particular diseases.The linked markers to MQTL and putative candidate genes underlying identified MQTL can be further validated in the germplasm through marker screening and expression studies.The study also attempted to unravel the underlying mechanism for FDR resistance by analyzing the constitutive gene network,which will be a useful resource to understand the molecular mechanism of defense-response of a particular disease and multiple FDR in maize.

Identification and expression analysis of sugar transporter family genes reveal the role of ZmSTP2 and ZmSTP20 in maize disease resistance

Sugar is an indispensable source of energy for plant growth and development, and it requires the participation of sugar transporter proteins(STPs) for crossing the hydrophobic barrier in plants. Here, we systematically identified the genes encoding sugar transporters in the genome of maize(Zea mays L.), analyzed their expression patterns under different conditions, and determined their functions in disease resistance. The results showed that the mazie sugar transporter family contained 24 members, all of which were predicted to be distributed on the cell membrane and had a highly conserved transmembrane transport domain. The tissue-specific expression of the maize sugar transporter genes was analyzed, and the expression level of these genes was found to be significantly different in different tissues. The analysis of biotic and abiotic stress data showed that the expression levels of the sugar transporter genes changed significantly under different stress factors. The expression levels of Zm STP2 and Zm STP20 continued to increase following Fusarium graminearum infection. By performing disease resistance analysis of zmstp2 and zmstp20 mutants, we found that after inoculation with Cochliobolus carbonum, Setosphaeria turcica, Cochliobolus heterostrophus, and F. graminearum, the lesion area of the mutants was significantly higher than that of the wild-type B73 plant. In this study, the genes encoding sugar transporters in maize were systematically identified and analyzed at the whole genome level. The expression patterns of the sugar transporter-encoding genes in different tissues of maize and under biotic and abiotic stresses were revealed, which laid an important theoretical foundation for further elucidation of their functions.

Pyramiding the disease resistant genes to southern rust and stalk rot in maize(Zea mays L.) with marker-assisted selection

Southern corn rust(SCR) caused by Puccinia polysora Underw and maize stalk rot caused by Pythium inflatum Matthews(MSR-2) are two destructive diseases of maize(Zea mays L.) in China.Our previous studies indicated that maize inbred line Qi319 is highly resistant to SCR but susceptible to MSR-2,while inbred line 1145 is highly resistant to MSR-2 but susceptible to SCR.The SCR resistant gene(RppQ) in Qi319 and MSR-2 resistant gene(Rpi1) in 1145 have been mapped on chromosome 10 and 4 respectively.In this research,through marker-assisted selection(MAS) with the molecular markers,bnlg1937 tightly linked to Rpi1 and phi041 tightly linked to RppQ,pyramid breeding of the two kinds of disease resistant genes were carried out from the year of 2003 to 2007.Two homozygotic inbred lines of F5 generation,DR94-1-1-1 and DR36-1-1-1 were identified.MAS result suggested DR94-1-1-1 and DR36-1-1-1 contained the two resistance genes RppQ and Rpi1.Field inoculation tests confirmed their high resistance to the two diseases.In addition,field investigation indicated that the two selected inbred lines,particularly DR94-1-1-1,had excellent agronomic traits such as plant height,ear height and yield-relating traits including ear length,ear diameter,ear weight,kernels per ear,kernels per row and kernel weight per ear.The two selected inbred lines DR94-1-1-1 and DR36-1-1-1 can either be directly developed into commercial variety or used as immediate donors of SCR and MSR resistance breeding programs in maize.

Identification and Evaluation of Insect and Disease Resistance in Transgenic Cry1Ab13-1 and NPR1 Maize

PCR detection,quantitative real-time PCR(q-RTPCR),outdoor insect resistance,and disease resistance identification were carried out for the detection of genetic stability and disease resistance through generations(T2,T3,and T4)in transgenic maize germplasms(S3002 and 349)containing the bivalent genes(insect resistance gene Cry1Ab13-1 and disease resistance gene NPR1)and their corresponding wild type.Results indicated that the target genes Cry1Ab13-1 and NPR1 were successfully transferred into both germplasms through tested generations;q-PCR confirmed the expression of Cry1Ab13-1 and NPR1 genes in roots,stems,and leaves of tested maize plants.In addition,S3002 and 349 bivalent gene-transformed lines exhibited resistance to large leaf spots and corn borer in the field evaluation compared to the wild type.Our study confirmed that Cry1Ab13-1 and NPR1 bivalent genes enhanced the resistance against maize borer and large leaf spot disease and can stably inherit.These findings could be exploited for improving other cultivated maize varieties.

Quantitative Disease Resistance： Dissection and Adoption in Maize

Maize is the world＇s most produced crop, providing food, feed, and biofuel. Maize production is constantly threatened by the presence of devastating pathogens worldwide. Characterization of the genetic compo- nents underlying disease resistance is a major research area in maize which is highly relevant for resistance breeding programs. Quantitative disease resistance （QDR） is the type of resistance most widely used by maize breeders. The past decade has witnessed significant progress in fine-mapping and cloning of genes controlling QDR. The molecular mechanisms underlying QDR remain poorly understood and exploited. In this review we discuss recent advances in maize QDR research and strategy for resistance breeding.

Identification of genes contributing to quantitative disease resistance in rice

Despite the importance of quantitative disease resistance during a plant’s life, little is known about the molecular basis of this type of host-pathogen interaction, because most of the genes underlying resistance quantitative trait loci (QTLs) are unknown. To identify genes contributing to resistance QTLs in rice, we analyzed the colocalization of a set of characterized rice defense-responsive genes and resistance QTLs against different pathogens. We also examined the expression patterns of these genes in response to pathogen infection in the parents of the mapping populations, based on the strategy of validation and functional analysis of the QTLs. The results suggest that defense-responsive genes are important resources of resistance QTLs in rice. OsWRKY45-1 is the gene contributing to a major resistance QTL.NRR,OsGH3-1,and OsGLP members on chromosome 8 contribute alone or collectively to different minor resistance QTLs. These genes function in a basal resistance pathway or in major disease resistance gene-mediated race-specific pathways.

Mapping of Defense Response Gene Homologs and Their Association with Resistance Loci in Maize

Defense response genes in higher plant species are involved in a variety of signal transduction pathways and biochemical reactions to counterattack invading pathogens. In this study, a total of 366 non-redundant defense response gene homologs （DRHs）, including 124 unigenes/expressed sequence tags, 226 tentative consensuses, and 16 DRH contigs have been identified by mining the Maize Genetics and Genomics and The Institute for Genomic Research maize databases using 35 essential defense response genes. Of 366 DRHs, 202 are mapped to 152 loci across ten maize chromosomes via both the genetic and in silico mapping approaches. The mapped DRHs seem to cluster together rather than be evenly distributed along the maize genome. Approximately half of these DHRs are located in regions harboring either major resistance genes or quantitative trait loci （QTL）. Therefore, this comprehensive DRH linkage map will provide reference sequences to identify either positional candidate genes for resistance genes and/or QTLs or to develop makers for fine-mapping and marker-assisted selection of resistance genes and/or QTLs.

The Auxin-Regulated Protein ZmAuxRPI Coordinates the Balance between Root Growth and Stalk Rot Disease Resistance in Maize

To optimize fitness, plants must efficiently allocate their resources between growth and defense. Although phytohormone crosstalk has emerged as a major player in balancing growth and defense, the genetic basis by which plants man age this balance remai ns elusive. We previously ide ntified a quantitative disease . resistance locus, qRfg2, in maize (Zea mays) that protects against the fungal disease Gibberella stalk rot. Here, through map-based cloning, we demonstrate that the causal gene at qRfg2 is ZmAuxRPI, which encodes a plastid stroma-localized auxin-regulated protein. ZmAuxRPI responded quickly to pathogen challenge with a rapid yet transient reduction in expression that led to arrested root growth but enhanced resista nee to Gibberella stalk rot and Fusarium ear rot. ZmAuxRPI was show n to promote the biosynthesis of indole-3-acetic acid (IAA), while suppressing the formation of benzoxazinoid defense compounds. ZmAuxRPI presumably acts as a resource regulator modulating indole-3-glycerol phosphate and/or indole flux at the branch point between the IAA and benzoxazinoid biosynthetic pathways. The concerted interplay between IAA and benzoxazinoids can regulate the growth-defense balance in a timely and efficient manner to optimize plant fitness.

An Atypical Thioredoxin Imparts Early Resistance to Sugarcane Mosaic Virus in Maize

Sugarcane mosaic virus （SCMV） causes substantial losses of grain yield and forage biomass in susceptible maize worldwide. A major quantitative trait locus, Scmvl, has been identified to impart strong resistance to SCMV at the early infection stage. Here, we demonstrate that ZmTrxh, encoding an atypical h-type thioredoxin, is the causal gene at Scmvl, and that its transcript abundance correlated strongly with maize resistance to SCMV. ZmTrxh alleles, whether they are resistant or susceptible, share the identical coding/proximal promoter regions, but vary in the upstream regulatory regions. ZmTrxh lacks two canon- ical cysteines in the thioredoxin active-site motif and exists uniquely in the maize genome. Because of this, ZmTrxh is unable to reduce disulfide bridges but possesses a strong molecular chaperone-like activity. ZmTrxh is dispersed in maize cytoplasm to suppress SCMV viral RNA accumulation. Moreover, ZmTrxh- mediated maize resistance to SCMV showed no obvious correlation with the salicylic acid- and jasmonic acid-related defense signaling pathways. Taken together, our results indicate that ZmTrxh exhibits a distinct defense profile in maize resistance to SCMV, differing from previously characterized dominant or recessive potyvirus resistance genes.

Detection and clinical significance of multidrug resistance-1 mRNA in bone marrow cells in children with acute lymphoblastic leukemia by real-time fluorescence quantitative RT-PCR

Objective： Multidrug resistance（MDR） is one of the most important reasons for treatment failure and recurrence of acute leukemia. Its manifestations are different in children with acute lymphoblastic leukemia（ALL） which may be due to different detection methods. This study was to detect the expression of MDR1 mRNA in bone marrow cells of children with ALL by real-time fluorescence- quantitative reverse transcription polymerase-chain reaction（FQ-RT-PCR）, and combine minimal residual desease（MRD） detection by flow cytometry（FCM） and to study their relationship with treatment response and prognosis of ALL. Methods：The MDR1 mRNA levels in bone marrow cells from 67 children with ALL[28 had newly diagnosed disease, 27 had achieved complete remission（CR）, 12 recurrent] and 22 children without leukemia were detected by FQ-RT-PCR. MRD was detected by FCM. The patients were observed for 9-101 months, with a median of 64 months. Results：Standard curves of human MDR1 and GAPDH genes were constructed successfully. MDR1 mRNA was detected in all children with a positive rate of 100%. The mRNA level of MDR1 was similar among the newly diagnosed ALL group, CR group, and control group（P 〉 0.05）, but significantly higher in the recurrence group than that in newly diagnosed disease group and control group（0.50 ± 0.55 vs. 0.09 ± 0.26 and 0.12 ± 0.23, P〈 0.05）. 54 ALL patients were followed up, and it was found that MDR1 mRNA level was significantly higher in ALL patients within 3 years duration than that of ALL patients with 3-6 years and over 6 years duration（0.63 ± 0.56 vs. 0.11 ± 0.12 and 0.04 ± 0.06, P〈 0.01）. For the 28 children with newly diagnosed disease, the MDR1 mRNA level was similar between WBC 〉 50 ~ 109 group and WBC〈50 × 10^9 group（P〉 0.05）. In the 33 CR patients, the MDR1 mRNA level was significantly higher in MRD〉10a group than that in MRD〈10a group（0.39 ± 0.47 vs. 0.03 ± 0.03, P 〈 0.05）. Conclusion：The sensitivity and specificity of FQ-RT-PCR in detecting MDR1 mRNA in bone marrowy cells of children with ALL patients are high. MDR1 mRNA is expressed in children with and without leukemia. MDR1 mRNA is highly expressed in the CR ALL patients with high MRD, recurrence and short duration（within 3 years）. Monitoring MRD and the MDR1 mRNA level might be helpful for individual treatment.

基于分子标记技术玉米抗粗缩病种质资源的筛选与应用

【目的】利用与3个玉米抗粗缩病位点qMrdd2、Rmrdd6和qMrdd8紧密连锁的分子标记筛选高抗玉米粗缩病自交系,并进行杂种优势类群分类和配合力分析,为玉米抗粗缩病品种的快速高效选育提供依据和方法。【方法】将抗病自交系K36与感病自交系S221杂交,从F2开始,采用单粒传的方法构建一个包括263个F9家系的重组自交系(recombinant inbred lines,RILs)群体;在不同种植环境下,对RILs进行抗病性鉴定,同时,采用与3个抗病位点qMrdd2、Rmrdd6和qMrdd8紧密连锁的分子标记5FR、6W53和IDP25K进行基因型分型,筛选出抗病且农艺性状优良家系;利用Maize56K SNP芯片对包括优良家系在内的24份玉米自交系进行基因型分型,根据Roger’s算法计算优良家系与其他骨干自交系之间的遗传距离,并在构建聚类图的基础上进行杂种优势类群划分;同时利用优良家系与不同类群的自交系测交配制杂交组合,在田间进行配合力测定和抗病性鉴定,筛选出抗病且杂种优势强的组合。【结果】自交系K36在qMrdd2、Rmrdd6和qMrdd8等3个抗性位点处均为抗病性纯合基因型,自交系S221均为感病性纯合基因型。RILs群体的263个家系在粗缩病抗性遗传组成上分为21种基因型,3个位点均为抗性纯合基因型家系的病情指数(DSI)最小(0.281),均为感病性纯合基因型家系的DSI最大(0.776),这与抗、感性亲本的DSI表现一致(0.257和0.623);2个位点为感病性纯合基因型时,1个位点为抗病性纯合基因型的DSI由小到大的位点是Rmrdd6(0.396)、qMrdd8(0.478)和qMrdd2(0.654),结果表明,Rmrdd6抗病性最强,qMrdd8次之,qMrdd2最弱。筛选出的3个抗性位点纯合基因型家系JR2136与其他23份自交系的遗传距离变化范围为0.2234—0.2895,平均值为0.2612,与之遗传距离最小的自交系是C413,最大的是Chang7-2。根据聚类分析结果,将JR2136划分在瑞德群,与属于黄改群的自交系H92和H521配制出抗病强的优势组合,分别比郑单958增产7.01%和7.80%,表明JR2136与属于黄改群的自交系之间具有良好的配合力。【结论】玉米自交系K36对粗缩病的抗性由qMrdd2、Rmrdd6和qMrdd8等3个基因控制、呈数量遗传特征且具有基因累加效应,具有3个抗性纯合基因型的玉米品种抗病性最强。开发的与3个位点紧密连锁的分子标记在抗病品种选育和抗性种质资源筛选上具有实用价值,利用分子标记进行辅助选择和基因聚合的方法选育抗病性强的优势组合应用于生产切实可行。

玉米弯孢菌叶斑病抗性的QTL分析

通过利用AFLP和SSR标记 ,对丹 340×沈 135的F2∶3 群体 (113个家系 )进行玉米弯孢菌叶斑病抗性基因的遗传作图和QTL分析 ,得到如下结论 :(1)玉米弯孢菌叶斑病抗性是由多基因控制的 ;(2 )应用复合区间作图法 ,对 1999年的玉米全株抗病性 ,检测到 4个QTL ,分别位于第 6、6、8和 10染色体上 ,可解释表型变异的 4 9.9% ;对 2 0 0 0年的玉米全株抗病性 ,检测到 6个QTL ,2个位于第 6染色体上 ,3个位于第 7染色体上 ,1个位于第 10染色体上 ,可解释表型变异的 77.6 % ;第 10染色体上的QTL是 2年间共同的QTL ,来自抗病亲本沈 135 ;(3)对每个QTL(定量特征点位分析 ) ,均检测到加性和显性效应 ,但相对大小有不同 ,各QTL以部分显性、显性和超显性为主要遗传方式 ;(4)控制玉米弯孢菌叶斑病抗性的QTL之间存在上位性互作。

甘蔗中一个NBS-LRR类基因的全长克隆与表达分析

采用RACE技术,从甘蔗高抗黑穗病品种NCo376中克隆了一个NBS-LRR类基因的cDNA全长序列,命名为SNLR。生物信息学分析显示,甘蔗SNLR基因的cDNA全长为2985bp(GenBank登录号为EF155654),包括一个2661bp的完整开放读码框以及一个典型的29bppoly-A。同时,还具有NBS-LRR类抗病基因的所有保守结构域,包括4个NBS区域保守结构域和6个潜在LRR结构域;蛋白疏水性分析和二级、三级结构分析表明,该基因编码的蛋白质为弱碱性蛋白,pI为7.76,无明显的疏水结构域,以卷曲结构和螺旋结构为骨架,三级结构未见明显的跨膜信号蛋白区。定量PCR分析表明,甘蔗SNLR基因的表达受到黑穗病菌、水杨酸和过氧化氢的影响,分别表现出"抑-扬"、"全程抑制"和"扬-抑"的表达模式,也具有抗病基因组成型和组织特异性表达的特点。推测甘蔗SNLR基因可能为抗病相关基因。

玉米抗螟性主基因—多基因混合遗传分析

采用混合分布理论,利用F2:3家系分离世代鉴定玉米抗螟性主基因并估计其遗传效应。结果表明:玉米抗螟性存在主基因,主基因遗传率为90.66%,遗传率较大。因此育种时可以选用含有主效基因的亲本,或者通过QTL基因定位的方法找到与这些基因紧密连锁的分子标记,采用分子标记辅助选择方法来提高玉米抗螟性的育种效果。

基于近等基因导入系发掘玉米抗甘蔗花叶病毒主效基因

利用玉米自交系掖478与中自01构建近等基因导入系群体(BC4F2),通过田间人工接种甘蔗花叶病毒鉴定获得抗病植株。采用38个SSR标记分析抗病株基因型,通过连锁不平衡分析,在玉米第3和6染色体上发掘3个主效抗病QTL。第3染色体上的QTL置信区间为26.1cM-phi053-5cM;第6染色体上的QTL置信区间分别为1.2cM-bnlg161和5.3cM-bnlg1538-7cM。建立了基于近等基因导入系发掘玉米抗甘蔗花叶病毒主效QTL技术,获得了一批含有抗病毒QTL的近等基因导入系,为抗病育种提供了信息和材料。

抑制性消减杂交技术及其在玉米抗病基因研究中的应用

近年来,包括玉米在内的植物功能基因组学研究得到了飞速发展,通过功能基因组学研究,可在全基因组水平上对基因的定位、序列和表达产生全新的认识。基因差异表达技术的日趋完善,也为从发育和动态的角度探讨特定的遗传现象和规律提供了极大的方便,抑制性消减杂交技术(SSH)以其假阳性低、敏感性高、速度快、效率高等优点广泛应用于基因转录水平上的差异表达。论述了抑制消减杂交技术的最新研究进展及在玉米抗病基因研究中的应用。

植物抗病性信号传导调控基因的克隆与表达检测方法的研究

建立了从不同植物中克隆抗病性信号传导调控基因及检测其表达的方法 ,并优化了相关条件。用RT PCR和PCR方法从拟南芥、烟草、番茄、中华鳖中克隆了 7个信号传导通路中 18个调控基因的cDNA和DNA序列 ,它们分别是 :抗病基因Pto介导的蛋白激酶级联中的Pti4、Pti5和Pti6 ,细胞编程死亡通路中的Hin1和hsr2 0 3,水杨酸介导的系统性获得抗病性通路中的NPR1,乙烯信号通路中的ETR1、ERS1、CTR1、EIN2和ERF1,茉莉酸信号通路中的COI1,核黄素信号通路中的RFBP和LR ,脱落酸信号通路中的ABF3、ABF4和ABH1,以及调控不同信号通路的通调基因NDR1。进一步研究了这些基因表达的定量测定方法 :用细菌激发子harpinEa处理植物 ,提取RNA作为cDNA第1链合成的模板 ,以在真核生物中广泛同源和高度保守的细胞伸长翻译因子基因EF1α为内参 ,用半定量RT PCR法测定基因mRNA的积累 ,可以比较准确地检测不同基因的相对表达水平。

转水稻NibT基因玉米植株的获得及抗病性研究

以玉米（Zea maysL.）自交系金黄96B为受体材料,供体为质粒pWM101并携带有水稻矮缩病病毒复制酶基因Nib的提前终止突变体基因NibT,采用超声波处理花粉介导植物基因转化方法将NibT-基因导入受体,经PCR检测和Southern杂交分析证实获得转基因植株,进而对T1－T3代转基因植株（株系）进行分子分析、田间抗病鉴定和农艺性状调查.逐代分子检测分析结果证明,目的基因可稳定遗传.抗病鉴定结果证明转基因植株（株系）各代抗病水平基本一致,抗病性比对照提高3级.农艺性状调查分析表明,与对照比较,转基因植株株高增加7－18cm、穗位高增高0－13 cm、穗长增加0.7－2.1 cm、穗粒数多8－35粒、百粒重增加1.1－2.6 g,转基因株系与阴性对照间、各代转基因株系相互间都差异显著（P〈0.05）;调查还发现转基因植株的株高和穗位高随着世代的增加,与对照间的差异逐代减少.研究也说明,超声波处理花粉介导植物基因转化方法是一种简捷、快速和有效的植物转化工具.

白粉菌诱导的华东葡萄环化cDNA文库中抗病基因筛选

【目的】从环化的中国华东葡萄白河35-1经白粉菌接种诱导后的叶片所构建的cDNA文库中,筛选抗病相关基因序列。【方法】根据植物抗病基因在氨基酸水平上的保守结构域NBS的P-loop区和LRR设计特异引物,采用三维文库筛选法,对环化的葡萄白粉菌接种诱导后的叶片cDNA文库进行PCR筛选。【结果】共建组池24个,每个组池中含有单菌落768个,包含18432个克隆。从中筛选出具有抗病基因保守序列的阳性克隆585个,其中具有NBS保守序列引物筛选出330个,LRR保守序列引物筛选出255个。对获得的阳性克隆中的插入片段进行了测序,经生物信息学分析去除重复序列后,获得422条具有抗病基因保守结构域的基因序列,其中具有NBS保守序列的250条,具有LRR保守序列的172条,共有NBS和LRR保守序列的25条。【结论】经翻译分析,其中8条编码产物包含NBS的P-loop结构域(GW787739—787742、GW787746、GW787747、GW787749、GW787758),5条编码产物包含LRR结构(GW787743、GW787744、GW787750、GW787751、GW787753),1条华东葡萄特有基因(GW787754)。从中选出具有不同抗病结构域的10条基因进行病源侵染过程中的基因实时定量表达分析,发现其中5条抗病相关基因的表达可被白粉菌接种所诱导,可能与白粉病侵染过程有关。

玉米真菌性病害抗性遗传、基因定位与克隆研究进展

玉米真菌性病害在玉米病害中占有重要的地位,给农业生产带来巨大的损失。概述了近年来国内外玉米真菌性病害抗性遗传、基因定位与克隆的研究进展,并提出了发展对策。