利用分子标记对14份枸橼种质进行多样性分析

目前枸橼C-05 (Citrus medica L.)是已鉴定的抗溃疡病唯一柑橘属种质,但由于枸橼种质存在多样性,枸橼种质间对柑橘溃疡病的抗病性可能存在差异,且其抗病性与枸橼种质间的亲缘关系是否相关尚不清楚。通过分析枸橼C-05与其他枸橼种质的亲缘关系,期待对进一步解析枸橼C-05对柑橘溃疡病的抗性机理提供参考。本研究利用筛选的14对SRAP引物、16对SSR引物和1对cpSSR引物trnH-trnK,对14份枸橼种质和4份柑橘属非枸橼参照种质(沙田柚,冰糖橙,酸橙和椪柑)进行遗传多样性分析。结果表明:(1)枸橼与非枸橼种质遗传相似度很低,可以聚在不同的两大类;枸橼种质可以聚在7个亚类,除木里香橼和印度大果外的12个枸橼种质遗传相似度高,相互之间的相似系数均在0.8以上;与枸橼C-05亲缘关系最近的是美国枸橼,二者遗传相似系数为0.97;与枸橼C-05亲缘关系最远的枸橼种质是木里香橼,二者遗传相似系数为0.76。(2)在SRAP和SSR标记中筛选出65个种质特异性标记,除爪哇柠檬外,所有枸橼种质有1～8个特异性标记,可以把它们全部鉴别出来;爪哇柠檬没有特异标记,但其与丹娜香橼有1个二者共有的特异标记,结合丹娜香橼的特异标记,也可以把爪哇柠檬鉴别出来。(3)结合三种分子标记可以初步确定,木里香橼可能为杂种起源,父本为枸橼,母本为与柠檬(C. limon (L.) Burm. f)血缘相近的酸橙(C. aurantium L.);印度大果也可能为杂种起源,父本为枸橼,母本为大翼橙亚属小花橙(C. micrantha Wester)或近缘种的杂种起源。综上所述,通过分子标记,14份枸橼种质亲缘关系基本明晰,为探明不同枸橼种质与枸橼C-05亲缘关系提供了理论基础。     

西瓜新品种‘抗病948’和‘申抗988’杂交种纯度及其遗传特异性的SSR标记鉴定

本研究以西瓜杂交新品种‘抗病948’和‘申抗988’及其亲本种质为试验材料,采用SSR分子标记检测技术对其种子纯度及遗传特异性进行了鉴定研究。从38对SSR引物中筛选得到2对特异性引物(引物BVWS00209和BVWS00233)可以鉴定出‘抗病948’和‘申抗988’杂交种的纯度,结果表明:‘抗病948’与‘申抗988’的种子纯度分别为97%和98%。SSR标记鉴定结果与‘抗病948’和‘申抗988’的大田植物学形态特征鉴定结果符合率高达99%以上,并通过分析比较各亲本种质的SSR引物的多态性表现,构建出‘抗病948’和‘申抗988’亲本的DNA指纹图谱。本研究可为开展‘抗病948’和‘申抗988’西瓜新品种的杂交种纯度室内快速检测提供技术支撑。     

甘蓝型油菜 ‘西农18’ 种子纯度的SSR鉴定

为了建立有效的SSR分子标记方法鉴定甘蓝型油菜‘西农18’品种纯度,以提高‘西农18’大田制种纯度提供分子辅助工具.以甘蓝型油菜杂交种‘西农18’及其父母本为材料,利用SSR分子标记方法对材料进行差异性分析.结果表明,在200对SSR引物中,发现引物BRAS023在杂交种和父母本之间存在显著差异且条带清晰可辨,差异性以杂交种带有父母本的特异带型存在.利用该引物对150株杂交种进行纯度鉴定,结果为86%,与大田杂交种纯度鉴定结果89%相比,SSR标记与大田种植鉴定结果基本一致.这个结果证明,引物BRAS023可用于‘西农18’的纯度鉴定.     

烤烟品种‘Oxford207’青枯病抗性的遗传分析与分子标记初选

为了研究烟草青枯病抗性的遗传特性和开发抗性相关分子标记,以抗青枯病的烟草品种‘Oxford207’,感病品种‘红花大金元’为亲本,构建了F1、F2和BC1群体,并采用苗期恒温水培接种法鉴定了群体青枯病的抗性。结果表明,接种后定期调查的F1、F2和BC1F1的死亡率比较接近,均稳定介于抗病亲本和感病亲本之间。F1、F2和BC1F1死亡率曲线下面积比较接近,同样介于抗病与感病亲本之间。表明‘Oxford207’的青枯病抗性不符合典型的显性或隐性基因控制模型,属于加性基因控制。采用简单重复序列(SSR)和分离群体分组分析法初步筛选了抗性相关的分子标记。从800多条SSR引物中,筛选出263个在抗感亲本间有多态性且在F1中呈共显性的引物、3个在抗感池之间有多态性的引物。     

枸橼C-05抗柑橘溃疡病相关PRR基因LYK5的初步鉴定

为筛选出抗溃疡病病菌相关PRR(Pattern Recognition Receptors)基因,本研究以接种了溃疡病菌Xcc的抗病种质枸橼C-05(JY C-05)和感病种质冰糖橙(BTC)、酸橙(SC)及早蜜椪柑(ZM)为研究对象,利用荧光定量PCR分析PRR基因的表达,结果显示PRR基因中的LYK5(LYSIN MOTIF-CONTAINING RECEPTOR-LIKE KINASE5)基因在枸橼C-05中表达上调而在其它感病种质中表达下调,表明LYK5基因可能与枸橼C-05抗溃疡病相关;对枸橼C-05和感病种质LYK5核酸序列及氨基酸序列进行差异分析,发现枸橼C-05 LYK5基因与冰糖橙、来檬(LM)有差异,但是与大香橼(DXY)和爪哇柠檬(ZWNM)LYK5基因相似度为100%,说明基因结构差异不是枸橼C-05的LYK5基因参与抗溃疡病的原因;进一步对其蛋白关键结构域进行比较分析,显示LYK5蛋白质信号肽第16位氨基酸、LysM基序区域(第203号氨基酸,第209号氨基酸)和蛋白激酶结构域(第394位,第449位,第468位,第492位,第647位氨基酸)存在差异;同时,对其三级结构进行比对发现枸橼C-05 LYK5蛋白结构与感病种质冰糖橙、来檬、爪哇柠檬和大香橼并无差异,进一步表明LYK5基因参与枸橼C-05抗溃疡病的过程不是由其蛋白结构差异所致。分析枸橼C-05和其他柑橘种质的LYK5启动子,大多数元件的差异不能区分抗病和感病柑橘种质,只有枸橼C-05缺失激活分生组织特异性调控元件,其他柑橘种质均有该元件,这是不是LYK5在接种Xcc的不同柑橘中表达差异的原因还有待进一步探究。     

基于SSR分子标记的甜瓜杂交种子纯度及真实性鉴定

本研究以广泛种植的甜瓜杂交种‘金蜜6号’和杂交新品种‘南疆蜜1号’及其亲本为试验材料,采用简单重复序列分子标记(SSR)检测技术,选取18对甜瓜多态性丰富的SSR核心引物进行纯度和真实性鉴定。结果表明,‘金蜜六号’和‘南疆蜜1号’在18对SSR核心引物筛选中均获得多对特异性引物,表现出多态性条带,可以用来鉴定种子纯度,‘金蜜六号’种子的纯度为97.87%,特异性引物：SSR12833(A)、CMBR052(C)、ECM147(I)、CMBR002(K)、CM26(L)、MU9175-1(M)、MU5554-1(N)、SSR00398(O)、TJ10(Q)、CMBR154(R),其中,编号C、I、K、N、O引物条带清晰,特异性强;‘南疆蜜1号’的种子纯度为97.56%,特异性引物：SR12833(A)、CMBR097(F)、MU4104-1(G)、NR38(H)、ECM147(I)、CMBR002(K)、MU9175-1(M)、MU5554-1(N)、CMBR154(R),其中,编号F、G、H、I、N、R引物条带清晰,特异性强。SSR分子标记鉴定结果与田间鉴定结果一致。SSR分子标记方法...     

九个中华类红肉猕猴桃品种的亲缘关系及其溃疡病抗性分析

为明确中华类红肉猕猴桃品种的亲缘关系并从中筛选溃疡病抗性种质,本研究使用SCo T分子标记对九个红肉猕猴桃品种的亲缘关系进行鉴定,同时使用离体枝条鉴定的方法对各品种的溃疡病抗性进行评价。结果表明:除红阳系品种‘红阳’、‘红昇’、‘红实1号’、‘红实2号’、‘东红’、‘脐红’等明显聚为一类外,野外实生选育品种‘湘吉红’也与红阳系有较近的亲缘关系,甚至比东红更优先聚类,说明其可能具有‘红阳’的遗传背景。而‘楚红’和‘桂红’与红阳系品种具有明显较远的遗传距离,甚至远于对照品种‘Hort16A’和‘Hayward’,说明其与红阳系有着明显不同的遗传背景。红阳系除‘红实2号’表现为感病外,其余均表现为高感。‘楚红’和‘桂红’被证明对溃疡病的抗性较强,甚至强于耐病对照品种‘Hayward’。研究明确了九个中华类红肉猕猴桃品种的亲缘关系及其对溃疡病的抗性,筛出了两个对溃疡病具有显著抗性的红肉猕猴桃资源,为进一步培育具有溃疡病抗性的红肉猕猴桃新品种提供了重要的物质基础。     

基于SSR标记的牡丹杂交子代真实性鉴定

SSR分子标记技术在杂交后代真实性的鉴定中具有重要意义。本试验以湖南本土牡丹‘宝庆红’为亲本,通过常规人工杂交育种得到5个不同杂交组合共69株杂交子代实生苗为材料,利用SSR分子标记技术对其进行早期真伪性鉴定。结果发现,4对SSR引物从69株杂交子代中筛选出5个单株具有父母本特异性互补条带,鉴定为真杂种。研究表明,SSR分子标记技术在对牡丹杂交子代真实性鉴定中具有准确、高效等特点,在后续牡丹种质资源创新以及新品种的选育过程中具有应用价值。     

冬瓜杂种种子纯度鉴定的SSR分析

种子纯度是种子质量的核心指标,目前冬瓜、节瓜杂种优势已被广泛地应用于生产。探索出一种快速、准确的冬瓜、节瓜品种纯度鉴定方法是确保种子质量的技术保证,也是品种纯度检测实现商业化的前提。本研究以‘绿优’冬瓜和‘梅花8号’节瓜及其各自亲本为试验材料,利用SSR分子标记技术对亲本及杂种之间的多态性进行分析。结果显示,从200对SSR引物中筛选出2对多态性引物scaffold4775和scaffold5674,用于‘绿优’冬瓜和‘梅花8号’节瓜杂种种子纯度的鉴定。其扩增片段电泳条带清晰可辨,杂交种中呈现双亲的互补带型,SSR标记表现出稳定的显性和共显性,能将‘绿优’冬瓜和‘梅花8号’节瓜与其父母本区分开来。分子检测平均纯度均为98%,田间纯度鉴定分别为98%和99%,结果基本一致,表明引物scaffold4775和scaffold5674可用于‘绿优’冬瓜和‘梅花8号’节瓜的纯度鉴定。SSR分子标记呈现出经济快速、准确等特点,在冬瓜、节瓜品种纯度鉴定上其应用前景十分广泛。     

利用SSR分子标记鉴定玉米杂交种‘吉祥1号’纯度

纯度是种子质量的重要指标。‘吉祥1号’玉米杂交种是在甘肃省选育并大面积栽培的主推玉米品种。为鉴定该品种的纯度,本研究开展了‘吉祥1号’纯度鉴定SSR标记方法研究。通过比较快速提取法和改良CTAB法2种DNA提取方法在SSR分子标记纯度鉴定中的应用,结果表明:2种方法提取的DNA均适用于SSR分子标记纯度检测,但快速提取法具有简单、快速的特点,更适用于分子标记纯度鉴定。从20对SSR引物中筛选出2个标记bnlg2291k4和bnlg2305k4,用于‘吉祥1号’品种的纯度鉴定,2个标记均能明显的区分亲本和杂交种。在‘吉祥1号’杂交种中人为混入双亲,采用本研究确定的方法可准确鉴定出混入的自交系。     

Thorn as a possible genetic marker to distinguish zygotic from nucellar seedlings in citrus

Summary The presence or absence of thorns was examined as a genetic marker to differentiate between zygotic and nucellar seedlings following hybridization of some citrus species or varieties. Shamouti orange and Eureka lemon are thornless, and when used as female parents, their seedlings with thorns are likely zygotes. Thornless seedlings from thorned varieties are also likely zygotes.Contribution from The Volcani Institute of Agricultural Research, Bet Dagan, Israel. 1971 Series, No 1946-E.     

叶片结构与柑橘溃疡病抗性的初步研究

摘要: 对5个柑橘品种（稻叶温州蜜柑[Citrus unshiu (L.) var. Inaba]、春见[(C. reticulata × C. sinensis) var. Okitsu No. 44]、卡拉卡拉红肉脐橙[C. sinensis (O.) var. Cara Cara]、玫瑰橙[C. Sinensis var. Meiguicheng]和HB柚[C. grandis (L.) var. HB]） 的夏梢老熟叶片气孔及其显微结构进行解剖构造的观察和测定。结果表明：不同品种间叶片的气孔密度差异显著,并且与品种抗病性成正相关,其相关系数为0.888;可以把叶片气孔的密度作为抗柑橘溃疡病的形态结构的初步鉴定指标。不同品种间叶片的显微结构特征差异不显著,与品种抗病性相关性不显著,品种间变化规律性不强。     

A quick methodology to identify sexual seedlings in citrus breeding programs using SSR markers

In citrus breeding and genetics, it is very important to distinguish between zygotic and nucellar seedlings in order to eliminate unwanted genotypes. Usually, isozyme marker shave been employed to determine the genetic origin of young plants. In this work we propose the use of SSR markers as an alternative methodology and compare them with isozymes in this kind of screenings. Two different populations were analysed: one derives from an interspecific cross and the other from selfing. We conclude that, in most cases, microsatellites are more efficient than isozymic markers to identify the sexual origin of citrus seedlings, given their higher level of polymorphism and the scarce number of polymorphic isozymes in some populations. We describe a quick and efficient methodology for SSR analysis, including a fast DNA extraction in microcentrifuge tubes, and visualization through silver staining, which eliminates the need for a labelling step. This revised version was published online in July 2006 with corrections to the Cover Date.     

Identification of citrus hybrids through the combination of leaf apex morphology and SSR markers

Citrus breeding programs normally face several biological obstacles such as apomixis and polyembryony that result in a cumbersome identification of hybrid seedlings. The main purpose of this work is to describe the combined use of visual selection based on the leaf apex morphology and SSR analysis to differentiate hybrid from nucellar seedlings derived from the cross between the ‘Murcott’ tangor [Citrus reticulata Blanco × Citrus sinensis (L.) Osb.] and ‘Pêra’ sweet orange [Citrus sinensis(L.) Osb.]. A new morphological variable named leaf apex morphometric index is also described as the quantitative basis of the visual selection. The efficiency of visual selection of hybrids was tested under two growth conditions, seedlings germinated in seedbeds and in plastic tubes. Putative hybrid seedlings were also confirmed through the analysis of simple sequence repeats (SSR). The visual selection of hybrid seedlings resulted in an increase of 87.2% (p < 0.01) and 202.2% (p <0.001) in the number of correctly identified hybrids when compared to the method of random picking of seedlings in seedbeds and plastic tubes,respectively. The results indicate that the combination of visual selection and SSR analysis for the identification of hybrids derived from the cross of polyembryonic citrus cultivars will improve the accuracy of the selection,save time, and reduce the costs involved in the use of molecular markers alone in citrus breeding programs. This revised version was published online in August 2006 with corrections to the Cover Date.     

大豆灰斑病1号生理小种抗性基因的SSR标记

针对中国大豆灰斑病1号生理小种,以抗所有生理小种的品系东农40566为母本,以感所有生理小种的品种东农410为父本配制杂交组合,杂交得到F2代后连续自交3代得到F5代群体。该群体经人工接种灰斑病1号生理小种后,利用BSA法对500个SSR标记进行筛选,其中3个标记Satt565、SOYGPATR和Satt396在抗、感池间表现出稳定的多态性,并且在F2代个体中表现出抗性与多态性协同分离的趋势。3个标记与抗性基因的连锁顺序为Satt565—SOYGPATR—Hrcs1—Satt396,它们与抗性基因的连锁距离分别为12.7cM、6.5cM、14.7cM。推测抗大豆灰斑病1号生理小种的基因可能位于C1连锁群上。     

Screening of Tomato Somaclones for Resistance to Bacterial Canker (Clavibacter michiganensis subsp. michiganensis)

To facilitate testing large numbers of tomato soma-clones for resistance to bacterial canker, a fast screening procedure was developed. Two inoculation methods were investigated, i.e. excision with an infected scalpel of I) one cotyledon of 2-week-old seedlings, and II) the first true leaf of 3-week-old seedlings, followed by applying a drop of inoculum on the wound. Inoculation by method II discriminated well between populations of partially resistant and susceptible tomato genotypes. A criterion for the selection of single, putatively resistant plants, based on rating scores of the severity of wilting symptoms, was proposed and tested with plants of a Lycopersicon peruvianum accession with a relatively high level of resistance to bacterial canker. Progenies of 279 somaclones, derived from tissue explants of the susceptible tomato cultivar ‘Moneymaker’, were evaluated for resistance. The presence of somaclonal variation for morphological characters in these populations was previously shown. However, no differences were detected between the somaclone population and controls in either mean susceptibility or distribution of plants over disease severity categories. Moreover, plants with major increases in resistance were not detected. These results suggest that the potential of somaclonal variation as a source of resistance to bacterial canker may be limited.     

基于SSR分子标记技术的葡萄种间杂交F1代鉴定

SSR分子标记技术以其多态性高、重复性好和检测方便快捷等优点,被广泛应用于植物F1代真假杂种的鉴定。为了长期有效的开展葡萄杂交育种工作,本研究以葡萄杂交组合‘北冰红’伊‘贵人香’F1代159个单株及‘双红’伊‘贵人香’F1代121个单株为试验材料,利用SSR分子标记技术结合田间形态学性状对葡萄杂交后代的真伪进行鉴定。在12对SSR引物中筛选出同时在亲本间具有特异性位点的引物,对F1代进行扩增检测,结果表明:‘北冰红’伊‘贵人香’及‘双红’伊‘贵人香’2个杂交组合中分别有63和94个单株具有双亲特异性条带,结合田间形态学鉴定,认为其为真杂种,故2个杂交群体的杂种率分别为40.38%和77.69%,该结果可为今后开展葡萄遗传连锁图铺构建及数量性状的QTL定位提供依据。     

柑橘多胚性砧木枳橙同源四倍体的发掘与SSR鉴定

为发掘枳橙同源四倍体资源,从而为柑橘砧木遗传改良提供新种质,本研究通过初步的形态学观察,从具多胚性的柑橘重要砧木资源枳橙实生苗群体750个单株中初步筛选出具典型多倍体特征的植株24株,其中流式细胞仪鉴定为枳橙四倍体22株,群体四倍体自然发生率达2.93%。下表皮气孔分析表明枳橙四倍体气孔密度显著低于二倍体对照。对获得的四倍体植株进行了SSR分子鉴定,19对引物扩增结果均显示枳橙四倍体与其二倍体对照的带型完全一致,表明获得的四倍体枳橙均为同源四倍体。本研究表明将形态学初选与流式细胞仪快速鉴定及SSR分析相结合是从多胚性柑橘资源实生群体获得同源四倍体的简便有效途径,本研究发掘的同源四倍体枳橙是柑橘砧木遗传改良相关研究的良好资源。     

Analysis of genetic variability in various tissue culture-derived lemon plant populations using RAPD and flow cytometry

Five populations of lemon plants [Citrus limon (L.) Burm] obtained from undeveloped ovules through different tissue culture procedures were examined for the presence of somaclonal and irradiation-induced genetic variation. Tested groups were: (1) nucellar seedlings; (2) organogenic, regenerated via adventitious buds from nucellar seedling internodes; (3) embryogenic population, regenerated from non-irradiated nucellar callus via somatic embryogenesis; (4) embryogenic population, regenerated from irradiated nucellar callus via somatic embryogenesis; and (5) protoplast-derived, regenerated via somatic embryogenesis. Genomic DNA samples from 360 plants (72 from each group) were screened for polymorphism among RAPD fingerprints amplified by 10 decamer primers. Among all tested plants, genetic variation was detected only within the group of plants recovered from irradiated embryogenic calli. Out of 72 plants from that group, three had RAPD fingerprints different from the rest of the population, and fourth plant was found to be cytochimeric, consisting of diploid and tetraploid cells as revealed by flow cytometry. In all other populations of regenerated plants, we did not come across any plants with changed ploidy level.