与苹果Co基因紧密连锁的RAPD标记的筛选及其SCAR标记转换

以短枝富士(Spur Fuji)X舞姿(Telamon)的105株F1群体为试材,利用RAPD技术,结合集群分类分析法(BSA)进行了苹果柱型基因(Co)分子标记的研究。通过对300条随机引物的筛选,获得一个与Co基因紧密连锁的RAPD标记S1142682,连锁距离为2．86cM。对该标记片段进行序列测定,然后根据序列特点设计了4条特异引物(其中正向引物与反向引物各两条)。PCR结果显示,这4条引物的4种组合都可以扩增出柱型性状的特征带。选其中之一进行群体上的分析,结果表明该SCAR标记特征带与柱型性状的共分离行为与原RAPD标记表现一致。可见,此组合的引物可以作为该SCAR标记的特异引物。通过对S1142682标记片段序列分析发现,在 45～ 251区域含有一个可编码68个氨基酸残基的ORF。     

小麦抗白粉病基因Pm23的RAPD标记

小麦抗白粉病基因Pm23对世界上很多麦区流行的白粉病表现高抗或免疫.本研究以Pm23和Chancellor为抗感亲本,用集群分离分析法对抗性基因Pm23进行了RAPD分析,从320个十碱基随机引物中筛选到一个与Pm23紧密连锁的相引相标记OPE051100. 对F2分离群体进行RAPD分析表明,该标记与Pm23基因之间的连锁距离为10.65±3.25 cM.该标记可以有效用于小麦育种分子标记辅助选择中.     

利用RAPD标记构建美洲黑杨×欧美分子标记连锁图谱

本文利用RAPD标记和美洲黑杨(Populus deltoides)×欧美杨(P.euramericana)的F1群体,构建了美洲黑杨×欧美杨的分子标记连锁图谱。实验过程中对1040个寡核苷酸随机引物进行了重复筛选,共选出127个引物用于作图群体(包括双亲共92个无性系)的随机扩增,这127个引物产生229个多态基因座,其中符合“拟测交”1∶1分离的有214个。利用多点连锁分析,形成19个连锁群及6个三连体和14个连锁对。由19个连锁群构成的图谱含标记129个,总图距为1914.2cM,覆盖杨树基因组约73.62％。标记间的平均间距为14.84cM。本研究获得了中等密度的美洲黑杨×欧美杨的一个连锁框架。 Abstract:A molecular linkage map was constructed for the parents of a P.deltoides × P.euramericana F1 family based on random amplified polymorphic DNA(RAPD)markers.A set of 1040 random oligonucleotide primers were screened and 127 primers were selected to generate RAPD markers within a sample of 90 F1 progenies.A total of 229 segregating loci were identified.Among the 229 loci,15 loci were found distorted from the normal 1∶1 ratio.Using multiple analysis,the 214 markers formed 19 main Linkage groups (including 129 markers)and b triples and 14 pairs.The resulting Linkage map of Populus deltoides × P.euramericana (including 129 markers)spanned 1914.2cM(73.62％ coverage of genome length)with an average distance of 14.84cM between markers.     

利用RAPD标记构建美洲黑杨×欧美分子标记连锁图谱

本文利用RAPD标记和美洲黑杨(Populus deltoides)×欧美杨(P.euramericana)的F1群体,构建了美洲黑杨×欧美杨的分子标记连锁图谱。实验过程中对1040个寡核苷酸随机引物进行了重复筛选,共选出127个引物用于作图群体(包括双亲共92个无性系)的随机扩增,这127个引物产生229个多态基因座,其中符合“拟测交”1∶1分离的有214个。利用多点连锁分析,形成19个连锁群及6个三连体和14个连锁对。由19个连锁群构成的图谱含标记129个,总图距为1914.2cM,覆盖杨树基因组约73.62％。标记间的平均间距为14.84cM。本研究获得了中等密度的美洲黑杨×欧美杨的一个连锁框架。 Abstract:A molecular linkage map was constructed for the parents of a P.deltoides × P.euramericana F1 family based on random amplified polymorphic DNA(RAPD)markers.A set of 1040 random oligonucleotide primers were screened and 127 primers were selected to generate RAPD markers within a sample of 90 F1 progenies.A total of 229 segregating loci were identified.Among the 229 loci,15 loci were found distorted from the normal 1∶1 ratio.Using multiple analysis,the 214 markers formed 19 main Linkage groups (including 129 markers)and b triples and 14 pairs.The resulting Linkage map of Populus deltoides × P.euramericana (including 129 markers)spanned 1914.2cM(73.62％ coverage of genome length)with an average distance of 14.84cM between markers.     

高粱抗蚜基因的RAPD分析

应用RAPD技术BSA法,对高梁抗蚜基因进行分析,筛选了500个随机引物,共扩增出1614条谱带,得到了10个具有稳定多态性标记的引物,分别为OPA－01、OPP－09、OPP－14、OPH－19、OPN－08、OPN－07、OPN－20、OPY－14、OPS－20、OPJ－06。     

利用RAPD标记构建美洲黑杨×欧美杨分子标记连锁图谱

本文利用RAPD标记和美洲黑杨(Populusdeltoides)×欧美杨(P.euramericana)的F1 群体 ,构建了美洲黑杨×欧美杨的分子标记连锁图谱。实验过程中对1040个寡核苷酸随机引物进行了重复筛选 ,共选出127个引物用于作图群体(包括双亲共92个无性系)的随机扩增 ,这127个引物产生229个多态基因座 ,其中符合“拟测交”1∶1分离的有214个。利用多点连锁分析 ,形成19个连锁群及6个三连体和14个连锁对。由19个连锁群构成的图谱含标记129个 ,总图距为1914 2cM ,覆盖杨树基因组约73 62 %。标记间的平均间距为14 84cM。本研究获得了中等密度的美洲黑杨×欧美杨的一个连锁框架。     

小麦耐盐基因的标记和标记的克隆

普通小麦（Triticum aesticum L）耐盐农家品种茶淀红与盐敏感品种农大85021杂交,对杂交后代141株F2分离个体进行耐盐性鉴定。通过遗传分析和卡方检验证实小麦耐盐农家品种茶淀红含有一个主效耐盐基因,杂交后代符合1:2:1的分离规律。依据混合群体分组分析法（Bulked Segregate Analysis,BSA）建立耐盐基因池和盐敏感基因池,通过RAPD实验,从520个引物中筛选出了一个在两池间具有多态性的引物OPZ09,用双亲、F1、F2单株DNA进行RAPD实验证明了该引物扩增出的特异性片段OPZ09-590是一个与茶淀红耐盐基因连锁的RAPD标记,用JOINMAP Version1.4计算基因与标记间的重组值为5.674%,连锁距离为6.557cM。从琼脂糖凝胶回收OPZ09-590与载体pUCm-T连接,并转入受体菌JM109,对克隆片段测序表明其实际大小为591bp,故此小麦茶淀红耐盐基因的RAPD标记为OPZ09-591。     

油菜单显性核不育及其不育基因的RAPD标记

以陕西省杂交油菜研究中心选育的单显性核不育油菜分离群体为材料,对单显性核不育油菜的育性特征、花器形态进行了多代跟踪调查;对其遗传规律进行了探讨,确定该不育性状受一对显性核基因控制;利用集群分离法(BSA)对该油菜单显性核不育基因进行了RAPD分析。在所选用的300个随机引物中,获得引物S243(5’CTATGCCGAC 3’)在可育集团与不育集团问扩增出多态性产物S-2431500。通过对该分离群体及其姐妹系分离群体进行单株验证,均获得相同的扩增结果,表明S-2431500与单显性核不育基因相连锁。     

与小麦白粉病抗性基因Pm2紧密连锁RAPD标记的筛选研究

以２５６个随机引物对含小麦抗白粉病基因Ｐｍ２近等基因系进行ＲＡＰＤ分析,发现１７个随机引物的扩增产物在抗、感ＮＩＬｓ材料间表现多态性,且其中５个引物经４次以上重复,均获相同结果,其多态性标记分别为ＯＰＭ０８(１６００)、ＯＰＩ０４(１７００)、ＯＰＨ１９(１１００、ＯＰＥ０９(９００)及ＯＰＭ１６(８５０)。当以这５个随机引物对１４个已知含Ｐｍ２基因的抗病材料及９个不含Ｐｍ２基因的感病材料进行检测时,只有标记ＯＰＩ０４(１７００)在１２个抗病材料中出现（另两个抗病材料中未检测到）,而在９个感病材料中均未出现。进一步用 ＯＰI(０４)对１０２株（Ｃｈａｎｃｅｌｌｏｒｘ Ｕｋａ／８＊Ｃｃ）Ｆ２分离群体进行分析,估算出标记ＯＰＩ０４(１７００)与Ｐｍ２基因间的遗传距离为１２．２±３．３ｃＭ。     

RAPD and SCAR Markers Linked to a Gene Conferring Resistance to Angular Leaf Spot in Common Bean

Angular leaf spot, caused by the fungus Phaeoisariopsis griseola , is one of the most important bean diseases in Brazil. The objectives of this study were to determine the inheritance of angular leaf spot resistance and to identify random amplified polymorphic DNA (RAPD) and sequence-characterized amplified region (SCAR) markers linked to the resistance gene present in cv. Cornell 49-242, in a cross between this cultivar and susceptible cv. Rudá. The parents, F₁, F₂ and backcross-derived plants were inoculated with P. griseola pathotype 31-17 under environmentally controlled greenhouse conditions. The results indicate that one single dominant gene controls the resistance in Cornell 49–242. Two RAPD markers, OPN 02_890c and OPE 04_650c, were found to be linked in the coupling phase, at 3.2 and 12.5 c m of the resistance gene, respectively. To increase the reproducibility of the detection of marker OPN02_890c it was converted into a SCAR marker. It is proposed that the designation of Phg-2 be used for the angular leaf spot resistant gene present in Cornell 49-242.     

Screening of RAPD Markers Linked to the Photoperiod-Sensitivity Gene in Rice Chromosome 6 Using Bulked Segregant Analysis

Bulked segregant analysis was used to determine randomly amplifiedpolymorphic DNA (RAPD) markers in a specific interval in themiddle of chromosome 6 of rice for tagging the photoperiod sensitivitygene.Two pools of F₂ individuals (japonica cv. Nipponbare and indicacv. Kasalath) were constructed according to the genotypes ofthree restriction fragment length polymorphism (RFLP) markerslocated at both ends and the middle of the targeted interval.Then another pair of pools were constructed based on the "graphicalgenotype," which was made with our high density linkage map.RAPD analysis was performed using these DNA pools as templates,and polymorphic fragments were detected and mapped. Using 80primers, either singlyor pairwise, we tested 2,404 primer pairsand established 14 markers tightly linked to the photoperiodsensitivitygene. The obtained RAPD markers were converted intosequence-tagged sites bycloning and sequencing of the polymorphicfragments and they can be used directlyfor construction of physicalmaps. This bulked segregant method can be applied for any speciesand any region of interest in which detailed linkage maps orphysical maps are needed.     

玉米弯孢叶斑病菌的形态学分类与RAPD分析

对16个供试弯孢分离株根据其分生孢子的形态进行分类,其中12个为新月弯孢(Curvularia luna-ta),其余4个是画眉草弯孢(C.eragrostidis)。对这些分离株基因组DNA进行RAPD分析发现,一些分属于两种不同弯孢菌的分离株间比同种弯孢的分离株间具有更近的遗传距离,即具有更近的亲缘关系。因此,根据弯孢菌的形态分类和利用RAPD标记的分类结果存在不一致现象。     

Molecular Genetics of Rust Resistance in Poplars (Melampsora Larici-Populina Kleb/Populus Sp.) by Bulked Segregant Analysis in a 2 X 2 Factorial Mating Design

With random amplified polymorphic DNA (RAPD) markers, we have tagged a genomic region in Populus sp. involved in qualitative resistance to Melampsora larici-populina. Our approach was based on three steps: use of RAPD markers that can be quickly and efficiently researched; application of ``bulked segregant analysis' technique on individuals of one interspecific family P. trichocarpa X P. deltoides to search for RAPD markers linked to resistance; and validation of these markers in two other families linked with the first one in a 2 X 2 factorial mating design. Of five detected markers, only one marker M03/04_480 was polymorphic in the three segregating families, involving 89 individuals and four different parents. We have estimated the recombination value of 1 cM with 1 cM sampling error.     

小麦抗白粉病基因Pm6的RAPD标记

从提莫菲维小麦转移到普通小麦中的小麦白粉病抗性基因Ｐｍ６是小麦白粉病（Ｅｒｙｓｉｐｈｅ ｈｒａｍｉｎｉｓ ｆ ｓｐ．ｔｒｉｔｉｃｉ）的有效抗性基因。用７００个随机引物对Ｐｍ６近等基因系进行ＲＡＰＤ分析,发现引物ＯＰＶ２０可在抗病近等基因系中产生大小为２ｋｂ的稳定的多态片段。用该引物检测１０个其他携Ｐｍ６的渐渗系材料,均可稳定扩增出该２ｋｂ的多态片段。理一步用ＯＰＶ２０对Ｐｍ６Ｆ２（ＩＧＶ１－４６３     

Identification of the New Pathogen (Stemphylium lycopersici) Causing Leaf Spot on Pepino (Solanum muricatum)

Pepino (Solanum muricatum var. pepino) plants were found affected by an extensive leaf spot caused by plant pathogenic fungi during a survey in the Cameron highlands, Pahang state, Malaysia. Symptomatic leaf samples were collected from infected pepino plants and cultivated on PDA medium, and the pathogen was isolated and purified; then, consequently, all isolates were identified as Stemphylium lycopersici on the basis of their cultural and morphological characteristics and combined sequences of the internal transcribed spacer (ITS) and glyceraldehyde‐3‐phosphate dehydrogenase (gpd) regions. A pathogenicity assay on detached leaves further confirmed that S. lycopersici causes leaf spot disease. To the best of our knowledge, this is the first report of S. lycopersici causing leaf spot on pepino in Malaysia and worldwide.     

Combining Next Generation Sequencing with Bulked Segregant Analysis to Fine Map a Stem Moisture Locus in Sorghum (Sorghum bicolor L. Moench)

Sorghum is one of the most promising bioenergy crops. Stem juice yield, together with stem sugar concentration, determines sugar yield in sweet sorghum. Bulked segregant analysis (BSA) is a gene mapping technique for identifying genomic regions containing genetic loci affecting a trait of interest that when combined with deep sequencing could effectively accelerate the gene mapping process. In this study, a dry stem sorghum landrace was characterized and the stem water controlling locus, qSW6, was fine mapped using QTL analysis and the combined BSA and deep sequencing technologies. Results showed that: (i) In sorghum variety Jiliang 2, stem water content was around 80% before flowering stage. It dropped to 75% during grain filling with little difference between different internodes. In landrace G21, stem water content keeps dropping after the flag leaf stage. The drop from 71% at flowering time progressed to 60% at grain filling time. Large differences exist between different internodes with the lowest (51%) at the 7^th and 8^th internodes at dough stage. (ii) A quantitative trait locus (QTL) controlling stem water content mapped on chromosome 6 between SSR markers Ch6-2 and gpsb069 explained about 34.7-56.9% of the phenotypic variation for the 5^th to 10^th internodes, respectively. (iii) BSA and deep sequencing analysis narrowed the associated region to 339 kb containing 38 putative genes. The results could help reveal molecular mechanisms underlying juice yield of sorghum and thus to improve total sugar yield.     

Red-Light-Induced Resistance in Broad Bean (Vicia faba L.) to Leaf Spot Disease Caused by Alternaria tenuissima

The effect of different light wavelengths on the development of lesions induced by Alternaria tenuissima in broad bean leaves was investigated. Lesion development was completely suppressed in red‐light‐irradiated broad bean leaflets, irrespective of isolate or spore concentration. Pre‐treatment of leaflets with red light for 24 h before inoculation also suppressed lesion development. Alternaria tenuissima failed to produce infection hyphae in red‐light‐irradiated broad bean leaflets. These results indicate that disease suppression in broad bean leaflets is due to light‐induced resistance. Spore germination fluid (SGF) of A. tenuissima allowed non‐pathogenic Alternaria alternata to infect wounded and unwounded broad bean leaflets kept in the dark, results suggesting that SGF induced susceptibility. Red light suppressed susceptibility induced by A. tenuissima SGF; thus, lesion formation and development were suppressed when leaflets inoculated with the spores of A. alternata suspended in A. tenuissima SGF were kept under red light. From these results, we conclude that red light induced resistance in broad bean to leaf spot disease caused by A. tenuissima, and that SGF induced susceptibility of broad bean leaflets to a non‐pathogenic isolate of A. alternata.     

First Report of Alternaria Leaf Spot on Gerbera (Gerbera jamesonii H. Bolux ex J. D. Hook) in Bulgaria

Alternaria leaf spots of gerbera (Gerbera jamesonii H. Bolus ex J. D. Hook) were observed on plants from different greenhouses on commercial plants in Bulgaria. The symptoms of the disease on the leaves were characterized by the development of brown, small, scattered dots, which gradually enlarged and coalesced to form large, oval, circular or irregular, brown to black lesions with concentric rings. Affected plants showed lower vitality, suppressed development and fewer, smaller, distorted in shape flowers. Alternaria isolates, obtained from infected leaf tissues were grown in pure culture and the morphological characteristics of the colony and sporulation apparatus were determined. DNA, extracted from the fungal isolates was subjected to polymerase chain reaction (PCR) with primers ITS1/ITS4, amplifying the internal transcribed spacer (ITS) regions. The resulting products were sequenced and compared for homology with other species in the GeneBank. The isolates showed 94% homology of the ITS region with either Alternaria alternata, A. arborescens, A. tenuissima, A. longipes, A. lini or A. smyrnii. None of the studied isolates was amplified with the A. alternata specific primers AAF2/AAR3, indicating that they are pathogenic varieties of it or belong to another species. Pathogenicity tests on 10 gerbera cultivars revealed that all of them were susceptible to Alternaria leaf spot. Additional tests on nine other crops (Solanum lycopersicum, Calendula officinalis, Capsicum annuum, Celosia argantea, Pelargonium spp., Petunia hybrida, Nicotiana tabacum, Cucurbita moscata and Raphanus sativus var. radicina) and on tomato or pepper fruits, potato tubers and carrot roots also indicated that all tested plant species were potential hosts of the disease. This is the first report of highly virulent isolates of Alternaria spp. in Bulgaria that cause leaf spots on gerbera in greenhouses.     

Identification and Fine Mapping of rhm1 Locus for Resistance to Southern Corn Leaf Blight in Maize

rhm1 is a major recessive disease resistance locus for Southern corn leaf blight (SCLB).To further narrow down its genetic position,F 2 population and BC 1 F 1 population derived from the cross between resistant (H95 rhm) and susceptible parents (H95) of maize (Zea mays) were constructed.Using newly developed markers,rhm1 was initially delimited within an interval of 2.5 Mb,and then finally mapped to a 8.56 kb interval between InDel marker IDP961-503 and simple sequence repeat (SSR) marker A194149-1.Three polymorphic markers IDP961-504,IDP B2-3 and A194149-2 were shown to be co-segregated with the rhm1 locus.Sequence analysis of the 8.56 kb DNA fragment revealed that it contained only one putative gene with a predicted amino acid sequence identical to lysine histidine transporter 1 (LHT1).Comparative sequence analysis indicated that the LHT1 in H95 rhm harbors a 354 bp insertion in its third exon as compared with that of susceptible alleles in B73,H95 and Mo17.The 354 bp insertion resulted in a truncation of the predicted protein of candidate resistance allele (LHT1-H95 rhm).Our results strongly suggest LHT1 as the candidate gene for rhm1 against SCLB.The tightly linked molecular markers developed in this study can be directly used for molecular breeding of resistance to Southern corn leaf blight in maize.