美国玉米自交系对4种病原茎腐病的抗性鉴定及遗传多样性分析

对外引玉米种质进行抗病鉴定是对其充分利用并选育抗病品种的基础和前提。本研究于2017-2018年连续2年采用人工接种技术评价了149个美国自交系对分别由禾谷镰孢(Fusarium graminearum)、拟轮枝镰孢(Fusarium verticillioides)、层出镰孢(Fusarium proliferatum)和芒孢腐霉(Pythium aristosporum)4种病原菌引起的茎腐病的抗性表现,并选择30个抗性较好的与6个骨干自交系进行遗传多样性分析。结果表明,149个美国自交系中,对禾谷镰孢、拟轮枝镰孢、层出镰孢和芒孢腐霉表现中抗及以上抗性的自交系分别有62个、44个、41个和54个,其中对4种茎腐病全部表现高抗的自交系有23个,全部表现中抗及以上抗性的自交系有32个,对3种镰孢菌茎腐病全部表现高抗的自交系有27个;所有供试材料中,88个自交系对4种茎腐病的抗性表现一致,61个自交系对4种茎腐病的抗性存在差异;共筛选出20条多态性引物,每个引物可检测出5～11个等位基因,平均等位变异数为7.7个,聚类分析将36个材料划分为6个类群,其中2FACC、J8608、黄早四分别被单独聚为一类,剩余自交系被划分为3个类群,分别包含自交系个数为26、5和2,第Ⅰ类群又分为3个亚群。本研究结果明确了外引美国自交系对不同病原菌引起的玉米茎腐病的抗性差异,为抗病育种中抗源的选择提供参考。     

不同种植密度下玉米茎秆纤维性状和抗倒性相关分析

以290份遗传多样性丰富的自交系为材料,研究不同种植密度下茎秆抗推力和纤维品质性状与茎秆抗倒性之间的关系;对不同杂种优势群的抗推力和纤维素含量进行多重比较,并筛选茎秆抗推力和纤维品质性状优良的自交系。结果表明:不同种植密度和不同自交系间的抗推力的显著性差异均达到显著水平。抗推力与纤维素含量在高密度和低密度条件下均呈极显著正相关关系。多重比较结果显示,高密度条件下的纤维素含量在不同类群间没有显著性差异,高密度和低密度条件下的抗推力和低密度条件下的纤维素含量存在类群间的显著性差异。不同杂种优势群中,抗推力和纤维素含量在两个密度下均表现稳定优良的自交系,瑞德群分别包括4个和3个,兰卡斯特分别包括1个和5个,P群分别包括2个和2个,旅大红骨分别包括2个和1个,塘四平头分别包括2个和1个。     

基于染色体片段导入系发掘抗玉米丝黑穗病主效QTL

选用抗玉米丝黑穗病自交系Mo17和SH15为供体,与受体感病自交系黄早四和昌7-2构建回交群体(BC3F1\BC4F2),通过田间人工接种玉米丝黑穗病原菌鉴定抗病性表现,评价群体抗病性。研究结果显示黄早四×(黄早四×Mo17)BC4F2群体发病率明显高于BC3F1群体;两个BC4F2黄早四×(黄早四×Mo17)和昌7-2×(昌7-2×SH15)群体的发病率差异较大。采用SSR标记分析抗病株的供体染色体导入片段,发现随着回交次数的增多,导入片段数量减少,但不同回交群体中供体导入片段数目明显不同。通过连锁不平衡分析,在染色体2.09和3.04区段发掘和验证2个抗玉米丝黑穗病主效QTL,连锁标记分别为umc2077和phio53或bnlg1965。本文研究结果为抗丝黑穗病基因精细定位和分子聚合育种提供了信息和材料。     

玉米种质资源的抗倒伏性评价及鉴定指标筛选

倒伏是影响玉米高产稳产的主要问题之一。本研究以181份玉米自交系为试验材料,对22个与抗倒伏性紧密相关的性状进行测定,采用相关性分析、主成分分析、聚类分析、逐步判别分析和岭回归分析等方法,综合评价玉米自交系的抗倒伏性。相关性分析的结果表明,22个性状间存在不同程度的相关性。主成分分析的结果表明,前7个主成分代表了181份玉米自交系的22个性状74.460%的信息,其贡献率分别为25.700%、12.369%、9.782%、8.159%、7.782%、5.490%和5.177%。聚类分析的结果表明,当遗传距离为3.5时,181份玉米自交系被聚为4大类,第Ⅰ类群有35份自交系、第Ⅱ类群有47份自交系、第Ⅲ类群有49份自交系、第Ⅳ类群有50份自交系。逐步判别分析的结果表明,175份玉米自交系被正确判别,判对概率为96.69%;6份玉米自交系被误判,误判率为3.31%,这说明聚类分析的结果是准确可靠的。进一步利用岭回归方法筛选出半纤维素含量、穿刺强度、第3节间长度、第3节间直径、穗位高和维管束总面积6个性状,建立了可靠的玉米自交系抗倒伏性评价回归模型。在181份玉米自交系中,以AHU24、e220、7026B等35份自交系的抗倒伏能力最强。本研究结果为玉米抗倒伏种质资源的遗传改良和组配抗倒伏杂交种提供借鉴。     

抗甘薯黑斑病优异种质资源的筛选与评价

通过对1107份甘薯种质资源抗黑斑病鉴定,筛选出高抗品种8份,占鉴定品种总数的0.7%;抗病品种128份,占11.6%.并进一步对这些抗性资源的兼抗病性、抗逆性、产量及品质进行分析研究,从中筛选出了兼抗两种或两种以上病害的优异种质资源若干份,以期为育种和生产提供依据.     

240份玉米自交系纹枯病抗性鉴定与评价

在人工接种条件下,连续3年对240份玉米自交系纹枯病抗性进行鉴定和评价,分析了玉米纹枯病抗性与主要农艺性状的相关性。结果表明,玉米纹枯病抗性资源较为缺乏,240份自交系中无免疫或高抗的材料,有中抗自交系4份、感病自交系18份、高感自交系218份。旅大红骨、Reid、PA和塘四平头类群自交系中未发现玉米纹枯病抗源,PB类群和Lancaster类群自交系纹枯病抗性相对较好,今后应主要从这两类种质中寻找玉米纹枯病抗源。玉米纹枯病病情指数与株高、穗位高、穗位高/株高、穗下节间数和穗下平均节间长均呈极显著负相关,这些表型可以作为非接种条件下筛选抗玉米纹枯病种质的参考指标。     

辣椒31个优良自交系的亲本类群分析

以包含我国重要尖椒品种的亲本材料在内的31份优良自交系为材料, 利用SRAP标记和基因型值分析技术开展了辣椒自交系间遗传差异的分析与类群划分研究。结果表明: 在30个引物组合中, 27个引物组合可以 在自交系间扩增出多态性条带, 共扩增出310个多态性条带, 平均每个引物组合产生11.5个多态性条带, 显示出SRAP技术具有较强的分析效率; 基于SRAP标记和Yule相似系数对这些自交系进行的聚类分析中, 可以基本区分辣椒的2个变种(C. annuum var. grossum和C. annuum var. longum), 而且可以反映出自交系间的亲缘及系谱关系; 在相似系数为0.67处, 可将这31个自交系分为4个类群; 基于基因型值和标准Euclidean距离对这些自交系进行的聚类分析可成功地将辣椒的两个变种完全区分; 在遗传距离约4.5处, 可将这31个自交系分为4个类群; 自交系间基于SRAP标记与基因型值的遗传距离存在一定的相关性。     

吉林省部分玉米种质资源抗玉米弯孢菌叶斑病鉴定研究

玉米弯孢菌叶斑病主要是由弯孢菌（Curvularia lunata）引起的侵染性病害。近年来,该病害在我国玉米主产区发生面积逐年扩大,已成为玉米重要病害之一。本文研究了吉林省主要玉米杂交种和自交系抗病性差异,结果表明：玉米杂交种间以及自交系间抗病性差异明显;吉63、黄早4、自330、丹9046、丹9041、铁7922、掖478、沈5003、丹340和E28等一批应用多年的种质资源均为感病类型（MS-HS）;而新选育的各类群一些种质和具有热带、亚热带血缘的78599等一些外来种质资源均为抗病类型（R～HR）。杂交种的抗病性与双亲的抗病性有密切关系;玉米杂交种苗期较抗病,抽丝期极为感病,这表明杂交种抗性随植株生长而递减。     

白菜紫色性状RAPD连锁标记的筛选与染色体定位研究

以紫菜薹自交系95T2-5、大白菜自交系94S17-1及其F1、F2、BC1植株为材料,进行RAPD分析,从640个随机引物中筛选出2个引物S79和S123,分别能扩增出与紫色性状连锁的条带S79-934和S123-750。连锁分析发现,标记S79-934和S123-750与紫色基因间的遗传距离分别为13.73cM和18.65cM,并且位于紫色基因的两边。回收S79-934特异带,克隆转化并测序,比较分析表明其与大白菜1号染色体上已知克隆KBrH077A05的全序列(113253bp)有99%的相似性,初步推断控制紫色性状的主基因位于大白菜1号染色体上。     

120份欧美玉米自交系的遗传多样性分析

为了拓宽黄淮海区玉米自交系的遗传基础,加快欧美优异种质的融入与利用,本研究利用SSR分子标记对120份来自美国和塞尔维亚及2份中国的玉米自交系进行遗传多样性和聚类分析。结果表明:29个多态性SSR标记共检测到115个等位位点,平均3.97个,位点多态性信息指数(PIC)平均为0.50,较好地揭示了自交系间的遗传多样性;观测杂合度(Ho)仅为0.03,表明参试自交系遗传稳定、纯合度高;美国SS、美国NSS、塞尔维亚和中国骨干自交系4个群之间相比,美国NSS群的等位位点数(3.55)、Shannon信息指数(0.93)最高,而塞尔维亚群的有效等位位点数(2.37)最高,表明美国NSS和塞尔维亚自交系群比其他两个群遗传多样性高;4个自交系群间的遗传距离介于0.1403~0.4695之间,美国NSS群与美国SS群、塞尔维亚群之间较小(0.1419,0.1403),与中国骨干自交系群之间最大(0.4695),4个群的遗传一致度介于0.6253~0.8691之间,美国NSS群与美国SS、塞尔维亚两个群之间的遗传一致度较高,表明美国与塞尔维亚自交系之间基因交流频繁,亲缘关系较近;聚类分析将122份玉米自交系分为9大主要类群,美国SS种质、NSS种质自交系被明显的区分开,并且SS种质被分为2个主要类群(Ⅰ和Ⅸ),NSS种质被分为6个主要类群(Ⅱ-Ⅶ),来自塞尔维亚的材料分散在美国NSS种质类群。本研究结果为来自欧美的自交系在玉米育种中合理利用提供可靠依据。     

大白菜—结球甘蓝1号二体异附加系分子标记鉴定及其自交后代的性状分析

利用与抗霜霉病、抗芜菁花叶病毒基因连锁的SCAR标记和能够区分大白菜和结球甘蓝FLCs基因的特异引物对大白菜—结球甘蓝1号二体异附加系(AC1d)及其亲本进行分析,结果表明:AC1d及其双亲中均含有与抗霜霉病基因连锁的标记;AC1d和亲本结球甘蓝中含有与抗芜菁花叶病毒基因连锁的标记,亲本大白菜中无该标记;AC1d除具有4个大白菜Br FLCs基因外,同时还添加了结球甘蓝Bo FLC3基因。AC1d自交后代的株高、株展、球高、维生素C含量、可溶性蛋白质及其7种硫苷组分含量超出了亲本大白菜和结球甘蓝;叶形指数、可溶性糖含量和对小菜蛾的抗性等超出了亲本大白菜。为利用AC1d后代选育携有目标性状的易位系提供了依据。     

与大白菜霜霉病抗性主效QTL连锁的分子标记开发

霜霉病是危害大白菜的三大病害之一,该病的发生会严重影响大白菜的产量及品质,因而研究与霜霉病抗性QTL紧密连锁的分子标记对大白菜抗病新品种培育具有重要意义。该研究在前期工作的基础上,选用高感霜霉病株系91-112、高抗霜霉病株系T12-19以及由二者为双亲构建的DH群体为实验材料,针对大白菜霜霉病抗性主效QTL——BrDW所在的标记区间,利用已有的大白菜基因组信息发展与抗性QTL紧密连锁的分子标记,通过Blast和IMap分析,将与BrDW连锁的RAPD标记K14-1030定位于大白菜KBrB058M10上(位于Contig214上),根据KBrB058M10附近的BAC及BAC-end序列设计引物,结合限制性内切酶酶切及HRM分析方法,筛选得到5个与BrDW连锁的分子标记,包括1个Indel标记Brb062-Indel230,3个CAPS标记Brb094-DraⅠ787、Brb094-AatⅡ666和Brb043-BglⅡ715,1个SNP标记Brh019-SNP137;同时,通过筛选与目标区域具有同源性的Unigene序列得到了1个与BrDW紧密连锁的SSR标记bru1209。标记Brb062-Indel230、Brb094-DraⅠ787、Brb094-AatⅡ666、Brb043-BglⅡ715、Brh019-SNP137和bru1209与RAPD标记K14-1030之间的遗传距离分别为4.3 cM、1.7 cM、5.9 cM、5.9 cM、4.6 cM和0.8 cM,在对DH群体中的抗性株系选择上准确率分别为69.7%、70.9%、72.4%、72.4%、58.3%和74.2%,可应用于分子标记辅助选择,为霜霉病抗性分子育种奠定了良好基础。     

Screening and evaluation of resistance to downy mildew (Peronospora parasitica) and clubroot (Plasmodiophora brassicae) in genetic resources of Brassica oleracea

52 entries including landraces, old cultivars and wild accessions of B. oleracea and closely related Brassica species were screened for resistance against downy mildew and clubroot. Several accessions resistant to downy mildew and a few to clubroot were found. Genetic inheritance of the resistance in downy mildew was investigated by screening F1 and BC1F1 offspring from three resistant landrace accessions crossed with both a resistant and a susceptible father. The seedling resistance against downy mildew was found to be inherited recessively. This is a bit surprising as earlier papers mostly report of inheritance controlled by a single dominant gene. Previous screenings of B. oleracea resistance against downy mildew at the cotyledon stage have been done with P. parasitica isolated from B. oleracea as the original host plant. The recessive nature of the cotyledon resistance found in this screening might be due to the fact that the P. parasitica isolate was collected from B. napus fields. The clubroot seedling resistance was found to be controlled by recessive inheritance after screening the F1 offspring, this in agreement with earlier results/reports.     

不结球白菜抗根肿病渐渗系的分子辅助选育

该研究基于与大白菜抗根肿病连锁的分子标记,设计特异引物,获得简便实用的SCAR标记,并用于分子标记辅助选择,创制不结球白菜抗根肿病新材料。结果发现,在设计的8对特异引物中,有1对特异引物在抗、感亲本间表现出多态性。F2群体验证发现,该标记与已有SSR标记及根肿病抗性共分离,能够用于抗根肿病鉴定,定名为CRb-R-25。通过亚种间杂交并回交,利用标记CRb-R-25辅助选择将大白菜根肿病抗性转入不结球白菜中,获得抗根肿病不结球白菜渐渗系材料TQ14-1-15。     

松花菜种质资源评价及根肿病、黑斑病兼抗材料筛选

松花菜是目前我国花椰菜消费的主要类型。根肿病和黑斑病是直接影响松花菜产量及品质的重要病害。选育商品性好、高抗多抗品种是松花菜的主要育种目标之一。本研究首次建立了松花菜种质资源花球性状评价体系,从花球紧实度、球形、球面光滑度、球面颜色、球面蕾粒大小、二级侧枝长度、花梗颜色、球面长毛情况8个方面对66份松花菜自交系进行评价,并对不同性状赋值以便统计、比较。利用上述体系共筛选出11份花球性状优良的材料。采用苗期人工接种技术分别对松花菜根肿病和黑斑病抗性进行鉴定评价,结果显示无免疫根肿病材料,有2份高抗材料,9份抗病,16份中抗;无免疫和高抗黑斑病材料,有4份抗病材料,10份中抗。另外,兼抗两种病害的材料较少,其中GY-40对两种病害均表现抗病,GY-21对根肿病表现抗病,对黑斑病表现中抗,GY-39对两种病害均表现中抗。值得一提的是,GY-40性状优良且同时对根肿病和黑斑病具有较高抗性,是选育商品性好、抗病性强松花菜品种的理想亲本。本研究结果为评价松花菜种质资源花球相关性状提供了参考,对于规范资源的收集、整理和保存具有重要意义,同时为开展抗根肿病和黑斑病松花菜品种选育提供了优异材料。     

Variation in abundance of predicted resistance genes in the Brassica oleracea pangenome

Brassica oleracea is an important agricultural species encompassing many vegetable crops including cabbage, cauliflower, broccoli and kale; however, it can be susceptible to a variety of fungal diseases such as clubroot, blackleg, leaf spot and downy mildew. Resistance to these diseases is meditated by specific disease resistance genes analogs (RGAs) which are differently distributed across B. oleracea lines. The sequenced reference cultivar does not contain all B. oleracea genes due to gene presence/absence variation between individuals, which makes it necessary to search for RGA candidates in the B. oleracea pangenome. Here we present a comparative analysis of RGA candidates in the pangenome of B. oleracea. We show that the presence of RGA candidates differs between lines and suggests that in B. oleracea, SNPs and presence/absence variation drive RGA diversity using separate mechanisms. We identified 59 RGA candidates linked to Sclerotinia, clubroot, and Fusarium wilt resistance QTL, and these findings have implications for crop breeding in B. oleracea, which may also be applicable in other crops species.     

Identification of the BrRHP1 locus that confers resistance to downy mildew in Chinese cabbage (Brassica rapa ssp. pekinensis) and development of linked molecular markers

Inheritance of resistance to downy mildew (Hyaloperonospora parasitica) in Chinese cabbage (Brassica rapa ssp. pekinensis) was studied using inbred parental lines RS1 and SS1 that display strong resistance and severe susceptibility, respectively. F(1), F(2), and BC(1)F(1) populations were evaluated for their responses to downy mildew infection. Resistance to downy mildew was conditioned by a single dominant locus designated BrRHP1. A random amplified polymorphic DNA (RAPD) marker linked to BrRHP1 was identified using bulked segregant analysis and two molecular markers designated BrPERK15A and BrPERK15B were developed. BrPERK15B was polymorphic between the parental lines used to construct the reference linkage map of B. rapa, allowing the mapping of the BrRHP1 locus to the A1 linkage group. Using bacterial artificial chromosome clone sequences anchored to the A1 linkage group, six simple polymerase chain reaction (PCR) markers were developed for use in marker-assisted breeding of downy mildew resistance in Chinese cabbage. Four simple PCR markers flanking the BrRHP1 locus were shown to be collinear with the long-arm region of Arabidopsis chromosome 3. The two closely linked flanking markers delimit the BrRHP1 locus within a 2.2-Mb interval of this Arabidopsis syntenic region.     

Haplotyping and mapping a large cluster of downy mildew resistance gene candidates in sunflower using multilocus intron fragment length polymorphisms

Downy mildew (Plasmopara halstedii (Farl.) Berlese et de Toni) is a serious foliar pathogen of cultivated sunflower (Helianthus annuus L.). Genetic resistance is conditioned by several linked downy mildew resistance gene specificities in the HaRGC1 cluster of TIR-NBS-LRR resistance gene candidates (RGCs) on linkage group 8. The complexity and diversity of the HaRGC1 cluster was assessed by multilocus intron fragment length polymorphism (IFLP) genotyping using a single pair of primers flanking a hypervariable intron located between the TIR and NBS domains. Two to 23 bands were amplified per germplasm accession. The size of the included intron ranged from 89 to 858 nucleotides. Forty-eight unique markers were distinguished among 24 elite inbred lines, six partially isogenic inbred lines, nine open-pollinated populations, four Native American land races, and 20 wild H. annuus populations. Nine haplotypes (based on 24 RGCs) were identified among elite inbred lines and were correlated with known downy mildew resistance specificities. Sixteen out of 39 RGCs identified in wild H. annuus populations were not observed in elite germplasm. Five partially isogenic downy mildew resistant lines developed from wild H. annuus and H. praecox donors carried eight RGCs not found in other elite inbred lines. Twenty-four HaRGC1 loci were mapped to a 2-4 cM segment of linkage group 8. The multilocus IFLP marker and duplicated, hypervariable microsatellite markers tightly linked to the HaRGC1 cluster are powerful tools for distinguishing downy mildew resistance gene specificities and identifying and introgressing new downy mildew resistance gene specificities from wild sunflowers.     

Wall-associated kinase BrWAK1 confers resistance to downy mildew in Brassica rapa

The plant cell wall is the first line of defence against physical damage and pathogen attack. Wall-associated kinase (WAK) has the ability to perceive the changes in the cell wall matrix and transform signals into the cytoplasm, being involved in plant development and the defence response. Downy mildew, caused by Hyaloperonospora brassicae, can result in a massive loss in Chinese cabbage (Brassica rapa L. ssp. pekinensis) production. Herein, we identified a candidate resistant WAK gene, BrWAK1, in a major resistant quantitative trait locus, using a double haploid population derived from resistant inbred line T12–19 and the susceptible line 91–112. The expression of BrWAK1 could be induced by salicylic acid and pathogen inoculation. Expression of BrWAK1 in 91–112 could significantly enhance resistance to the pathogen, while truncating BrWAK1 in T12–19 increased disease susceptibility. Variation in the extracellular galacturonan binding (GUB) domain of BrWAK1 was found to mainly confer resistance to downy mildew in T12–19. Moreover, BrWAK1 was proved to interact with BrBAK1 (brassinosteroid insensitive 1 associated kinase), resulting in the activation of the downstream mitogen-activated protein kinase (MAPK) cascade to trigger the defence response. BrWAK1 is the first identified and thoroughly characterized WAK gene conferring disease resistance in Chinese cabbage, and the plant biomass is not significantly influenced by BrWAK1, which will greatly accelerate Chinese cabbage breeding for downy mildew resistance.     

Molecular characterization of the CRa gene conferring clubroot resistance in Brassica rapa

Clubroot disease is one of the major diseases affecting Brassicaceae crops, and a number of these crops grown commercially, such as Chinese cabbage (Brassica rapa L. ssp. pekinensis), are known to be highly susceptible to clubroot disease. To provide protection from this disease, plant breeders have introduced genes for resistance to clubroot from the European turnip into susceptible lines. The CRa gene confers specific resistance to the clubroot pathogen Plasmodiophora brassicae isolate M85. Fine mapping of the CRa locus using synteny to the Arabidopsis thaliana genome and partial genome sequences of B. rapa revealed a candidate gene encoding a TIR-NBS-LRR protein. Several structural differences in this candidate gene were found between susceptible and resistant lines, and CRa expression was observed only in the resistant line. Four mutant lines lacking clubroot resistance were obtained by the UV irradiation of pollen from a resistant line, and all of these mutant lines carried independent mutations in the candidate TIR-NBS-LRR gene. This genetic and molecular evidence strongly suggests that the identified gene is CRa. This is the first report on the molecular characterization of a clubroot Resistance gene in Brassicaceae and of the disease resistance gene in B. rapa.