柑桔线虫抗性主基因座Tyrl的特异标记开发与遗传作图改进

本文利用先期从BAC文库获得的NBS-LRR类候选抗病基因克隆序列Pt8a和Pt9a,进一步开发与柑桔线虫抗性丰效基因位点Tyrl连锁的分子标记.以Pt8a和Pt9a序列作探针,通过高密度克隆印迹杂交,从BAC文库筛选出200个以上的阳性克隆,以阳性克隆插入序列设计引物,对柑桔抗线虫材料和感线虫材料开展以PCR扩增为基础的集群分离分析,发现一部分克隆序列与柑桔线虫抗性主效基因位点Txrl紧密连锁;再通过染色体步行测序,分别从3个克隆(7A4,4L17和29F20)获得3个完整的NBS-LRR类候选抗病基因序列.从此类序列开发更高特异性的分子标记,并在利用原有分子标记的基础上,对柑桔线虫抗性杂交后代群体(9145 family)构建较高密度的遗传图谱:同时,将新开发的分子标记应用于柑桔衰退病抗性杂交后代群体(9401 family),以初步估算柑桔线虫抗性主效基因位点Tyrl与柑桔衰退病抗性基因Ctv的遗传距离.     

柑桔线虫抗性主基因座Tyr1的特异标记开发与遗传作图改进(英文)

本文利用先期从BAC文库获得的NBS-LRR类候选抗病基因克隆序列Pt8a和Pt9a,进一步开发与柑桔线虫抗性主效基因位点Tyr1连锁的分子标记。以Pt8a和Pt9a序列作探针,通过高密度克隆印迹杂交,从BAC文库筛选出200个以上的阳性克隆,以阳性克隆插入序列设计引物,对柑桔抗线虫材料和感线虫材料开展以PCR扩增为基础的集群分离分析,发现一部分克隆序列与柑桔线虫抗性主效基因位点Tyr1紧密连锁;再通过染色体步行测序,分别从3个克隆（7A4,4L17和29F20）获得3个完整的NBS-LRR类候选抗病基因序列。从此类序列开发更高特异性的分子标记,并在利用原有分子标记的基础上,对柑桔线虫抗性杂交后代群体（9145 family）构建较高密度的遗传图谱;同时,将新开发的分子标记应用于柑桔衰退病抗性杂交后代群体（9401 family）,以初步估算柑桔线虫抗性主效基因位点Tyr]与柑桔衰退病抗性基因Ctv的遗传距离。     

水稻沈农606抗稻瘟病基因遗传分析及SRAP标记筛选

选用抗稻瘟病水稻品种沈农606为抗病亲本,以普感品种丽江新团黑谷为感病亲本配制杂交组合.对两亲本及其F2代单株进行苗期抗病鉴定,结果表明抗病亲本的抗性由核基因控制,受一对显性基因控制.根据F2代单株的抗病鉴定结果,采用BSA法,从110对SRAP引物中筛选出了4对在抗、感池间表现出多态性的引物,表明这些引物可能与沈农606的抗病基因连锁.利用这些标记对F2代112个感病单株进行扩增,根据扩增结果,采用Mapmaker 3.0软件计算遗传距离,结果显示,标记m5e1-500与抗病基因间的遗传距离最近,为2.8 cM.     

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。     

与黄瓜抗黑星病相关基因紧密连锁的SSR标记

本研究以黄瓜抗黑星病母本Q6和感黑星病父本F51及其F2代分离群体为试材,采用BSA法和SSR技术建立了对黑星病的抗病组和感病组,SSR引物CSWCTT02D在抗感组间表现多态性,且呈共显性.经162个F2单株验证,在高抗单株和高感单株中分别仅扩增出246 bp和256 bp的特异片段,而在中间类型个体中同时扩增出了两个特异片段.连锁分析结果表明,该标记与黄瓜黑星病抗病相关基因紧密连锁,距离为3.1 cM.测序结果显示,两个片段的差异在于10个碱基的插入或缺失.     

利用SRAP技术获得与抗甘薯茎线虫病基因相关的分子标记

用高感甘薯茎线虫病的甘薯品种徐薯18和高抗茎线虫病的徐78-1杂交,得到其F1分离群体。根据连续3年的抗病鉴定结果,从中挑选出8个高抗和8个高感茎线虫病的株系,构建抗病池和感病池。分别以抗感池基因组DNA为模板,用225对SRAP引物组合进行PCR扩增,其中77对引物组合在抗、感池间表现出多态性。通过一组小群体的进一步筛选,有4对引物组合被认为与甘薯茎线虫病抗性基因相关。这4对引物组合分别对2个亲本、抗感池和F1分离群体中的65个抗病株系和79个感病株系基因组DNA进行SRAP分析,有2对引物组合（a5b12和a9b11）各获得1个与甘薯茎线虫病抗性基因紧密连锁的分子标记SP1和SP2,它们与抗甘薯茎线虫病基因的遗传距离分别为4．86cM和4．17cM。获得的分子标记对克隆抗病基因和利用分子标记辅助选择提高育种效率具有重要的意义。     

柠檬合子胚自交实生苗的鉴定及其对柑橘溃疡病的抗性初步评价

枸橼C-05是抗溃疡病的柑橘种质资源,柠檬是枸橼的后代,其自交后代可能继承枸橼的抗性基因。为分析柠檬自交群体对柑橘溃疡病的抗感性规律,本研究建立了433株的‘尤力克’柠檬自交后代群体,并利用SSR分子标记对合子胚实生苗进行了鉴定,观察合子胚实生苗和珠心胚实生苗叶片形态学的差异,对所有自交后代离体注射接种柑橘溃疡病菌,初步评价了实生后代对柑橘溃疡病的抗病性差异。结果表明‘尤力克’柠檬存在多胚性,从300对SSR引物中筛选到8对可用于‘尤力克’柠檬合子胚鉴定的引物,共鉴定出合子胚实生苗57株,确定珠心胚实生苗4株。叶形指数、叶尖指数、翼叶长、翼叶宽和叶柄长均不能作为区分‘尤力克’柠檬合子胚实生苗与珠心胚实生苗的形态学标记,但合子胚实生苗的翼叶长度变异幅度大于珠心胚。57株合子胚实生苗离体接种柑橘溃疡病菌后,5株表现为抗病,52株表现为感病;4株珠心胚实生苗均表现为感病;在372株未鉴定清楚的自交后代中,8株表现为抗病,364株表现为感病。本研究表明利用SSR分子标记还不能完全鉴定出‘尤力克’柠檬合子胚植株,可能需要更多的引物或更灵敏的分子标记才能把全部实生后代鉴定出来。利用合子胚自交后代接种鉴定,更有利于分析‘尤力克’柠檬对柑橘溃疡病的抗感性遗传规律,挖掘抗性基因,为柠檬的抗病育种提供种质资源。     

用改进的SSAP方法克隆抗甘薯茎线虫病相关的RGA

甘薯新品系农大603是从感茎线虫病品种徐薯18的辐照后代中获得的一个抗茎线虫病的突变体。根据已报道的植物线虫抗性基因的核苷酸结合位点(NBS)保守氨基酸序列设计兼并引物，用自行改进的SSAP(modified sequence-specific amplification polymorphisms)方法，对农大603和徐薯18的基因组进行PCR扩增，从农大603中扩增出含有抗性基因NBS保守序列的差异片段2个(片段54和片段72)。在GenBank上序列搜索和比对发现，克隆序列与已报道的植物抗病基因之间同源性较低。根据克隆片段的DNA序列设计引物，以抗、感茎线虫病的甘薯品种的基因组DNA为模板进行PCR扩增，结果显示由片段72设计的引物在抗、感品种上没有扩增出多态性带；由片段54设计的引物在抗病品种和感病品种之间扩增出多态性带，推测片段54是与甘薯抗茎线虫病有关的RGA。     

大白菜的适宜AFLP-Pst I/Mse I引物组合

基于PCR的DNA指纹鉴定技术AFLP广泛应用于生物多样性、遗传图谱构建、基因定位、分子系统发育和品种鉴定等方面.为了筛选出大白菜适宜的AFLP引物组合,研究以抗根肿病大白菜双单倍体CR Shinki、感病自交系94SK及其后代F2构成的纯合抗病和纯合感病DNA混合池为材料,利用256对Pst I/Mse I引物组合进行了AFLP分析.结果表明:平均每个组合检测到55条带,共扩增13 079条带,平均每1 000条带检测到1.30个根肿病抗性基因连锁标记;多态性带数为3 167条,双亲间多态性频率约为0.24;扩增带数与多态性带数呈线性正相关,引物组合中的不同GC含量与扩增带数和多态性带数存在显著性差异,并表现出线性负相关.筛选出的适宜53对引物组合共扩增出4 381条带,占总扩增片断的33.50%,平均每对扩增出83条,其中9个组合扩增出与抗根肿病基因连锁标记.     

甜瓜抗霜霉病基因SSR分子标记

为了研究甜瓜抗霜霉病资源T115抗性遗传规律,找到与抗病基因连锁的分子标记,并对抗病基因进行定位。以抗病资源T115和感病哈密瓜农家品种SP红心脆、F1、BCr、BCs、F2为材料,苗期接种甜瓜霜霉病进行抗性鉴定,基于ICu GI已构建遗传连锁图谱,应用集团分离法和1 090对甜瓜SSR引物进行连锁遗传分析。结果表明,T115对霜霉病的抗性属显性单基因控制,引物DM0073扩增出的特异性片段与抗病基因表现连锁关系,该片段大小为120 bp,与抗病基因遗传连锁距离为3.6 c M,并将抗病基因定位在LG1上。DM0073可作为甜瓜抗霜霉病分子育种的分子标记。     

花生黄曲霉抗性与类受体蛋白激酶的相关性分析

LRR类受体激酶RH4基因在花生黄曲霉敏感品种发育种子的种皮中上调表达.基于这类受体激酶多由于序列的变异导致抗性的变异,笔者将数据库中花生抗黄曲霉和敏感品种相似基因编码的蛋白进行序列比较分析,发现抗性品种与敏感品种的类似蛋白序列有很大差别.用荧光定量PCR方法对抗性品种KB153与敏感品种JH1012发育中不同时期的果...     

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。     

Regeneration and characterization of somatic hybrid plants of Citrus sinensis (sweet orange) and Citrus micrantha, a progenitor species of lime

Most commercial citrus fruit species and cultivars are inter- and intra-specific hybrids. Conventional hybridization in citrus is largely handicapped by apomixis and long juvenility. As an alternative, somatic hybridization via protoplast fusion has been employed to create novel citrus germplasm. Witches' broom disease of lime (WBDL) emerged in Oman during the 1970s, which has been spreading to the neighboring countries. The disease is extremely destructive. A possible solution to the WBDL problem is to develop resistant hybrids. Resistance is available among the citrus relatives of lime i.e., sweet orange. Somatic hybrids combining sweet orange with lime have been produced but the fruit characteristics are different from lime. Herein, we report the development of somatic hybrids between a progenitor of lime (Citrus micrantha) and sweet orange (Citrus sinensis) in efforts to recreate a lime-like fruit using sweet orange as a donor of disease resistance gene(s). Successful somatic hybridization was verified by ploidy analyses using flow cytometry and RAPD analyses. This is the first report of using a progenitor species in somatic hybridization experiments in efforts to resynthesize an improved lime.     

Determination of self-incompatible genotypes in sweet cherry (Prunus avium L.) accessions and cultivars of the German Fruit Gene Bank and from private collections

Sweet cherries are self-incompatible because of a gametophytic self-incompatibility system. S alleles in the style and pollen determine the crossing relationships. Knowledge of the S allele constitution of cultivars is very important for cherry growers and breeders, and recently, molecular methods have been developed to distinguish the S alleles in sweet cherries. The S allele genotypes of 149 sweet cherry cultivars and clones, including 126 not previously genotyped, were determined by using PCR analysis. Thirteen different S alleles in 40 combinations were distinguished and nine new incompatibility groups were documented. Two new S alleles were identified in five local sweet cherry processing cultivars from southwestern Germany using the second intron primers. The sequence of these alleles was determined and compared to all known sequences available in the NCBI database. The sequences obtained showed high similarities to the alleles S ₁₉ and S ₂₂, previously described only in wild cherries, Prunus avium L.     

碱处理对甜高粱秸秆渣组成结构及酶解糖化效果的影响

以甜高粱品种M81为试验材料,以清洗烘干后未经处理的甜高粱渣为对照,在常温、高温高压、微波条件下Ca(OH)₂和常温条件下NaOH处理甜高粱秸秆渣,调查处理后甜高粱秸秆渣木质纤维素组成结构及纤维素酶酶解糖化情况。结果表明,采用的4种处理都能有效地改变甜高粱渣木质纤维素组成结构,其中氢氧化钠常温长时间处理对于木质素与半纤维素的溶降效果最好,3种石灰处理对半纤维素的溶解也均有一定作用。扫描电镜观察石灰高温高压与氢氧化钠常温两种处理对于木质纤维素结构的改变不同,前者木质纤维素表层木质素结构被侵蚀严重,呈破碎状附着在纤维素表面,内部纤维结构仍紧密排列,后者木质纤维素束状结构溶胀降解,表层木质素成分被大量去除,被包裹的纤维素组分显露,纤维素网断裂且纤维素表面出现许多小孔。经这4种方式处理后的甜高粱渣,木质纤维素中纤维素与半纤维素经纤维素酶酶解糖化,葡萄糖和木糖产物浓度都有所提高,分别达到对照的1.5、2.1、1.9、4.2倍和3.1、5.0、4.9、2.4倍;木质纤维素纤维组分的直接转化率与相对转化率的含义与计算方法不同,两种计算方式对于甜高粱木质纤维素生物质原料预处理方式的选择和效果的综合评价具有指导意义。     

甘薯基因组NBS-LRR类抗病家族基因挖掘与分析

NBS-LRR类基因家族是植物抗病R基因(Resistance gene)数量最多的一类,具有NBS (Nucleotide-binding site)和LRR (Leucine-leucine-repeat)结构域。甘薯(Ipomoea batatas)栽培种基因组已完成测序,但尚未注释,本研究对甘薯基因组序列进行外显子预测,得到甘薯染色体组全基因组蛋白序列,在此基础上进一步对NBS-LRR家族基因鉴定和分析表明,甘薯基因组中含有379个NBS-LRR家族基因,占全基因组基因总数的0.212%,其中N型亚家族120个,NL型103个, CNL型133个, TNL型22个, PN型1个。所有染色体上均有NBS-LRR家族基因分布,但数量明显不同,其中有60.9%的NBS-LRR基因序列呈簇状分布。NBS-LRR基因序列有15个保守结构域,在N端较为保守。研究结果为甘薯进一步开展NBS-LRR家族基因的功能研究和抗性育种提供了参考。     

植物生长调节剂对3个柑橘品种形态建成的影响

摘 要：本研究以墨西哥来檬、甜橙和香橼为材料,采用茎段培养方法,研究了植物生长调节剂不同配比对3个柑橘品种腋芽萌发、增殖和生根的影响,结果表明,以MT为基本培养基,3个柑橘品种在附加BAP 2.0 mg/L和IBA 0.1 mg/L的培养基中腋芽萌发效果最好,离体植株在附加BAP 1.0 mg/L和IBA 0.2 mg/L的培养基中增殖系数最大。此外,以1/2MT为基本培养基,附加NAA 2.0 mg/L 和IBA 0.25 mg/L时,适宜于墨西哥来檬和甜橙的生根,而单独使用NAA 3.0 mg/L则易于香橼的生根。离体小植株的移栽成活率均在90%以上。     

Development of co-dominant amplified polymorphic sequence markers for resistance of sunflower to downy mildew race 730

The inheritance of the reaction of sunflower to downy mildew was investigated using resistant and susceptible near isogenic lines (NILs) and their F₃ families. Resistance to race 730 was evaluated using the whole seedling inoculation technique. Seventy-three F₃ families were inoculated, among which 54 families were resistant and 19 susceptible, fitting a 3 : 1 segregation ratio. F₃ families were also studied using several PCR markers. Ten markers at the Pl6 locus, specific for the resistant line, also segregated in F₃ families with a 3 : 1 ratio. The same segregation ratio occurred for microsatellite haplotypes that resembled the resistant parent, and were amplified with ORS 166 and ORS 1043. The only common fragment that was observed between resistant and susceptible parental lines was one of the TIR-NBS-LRR resistance gene analogue markers, having a restriction site. Two co-dominant cleaved amplified polymorphic sequence (CAPS) markers were obtained. The mapping data indicate that several dominant markers and two CAPS markers, developed here, completely co-segregate with the Pl6 gene conferring resistance to race 730. CAPS markers will facilitate efficient marker-assisted selection for sunflower resistance to downy mildew race 730.     

Chloroplast DNA variation in cultivated and wild Prunus avium L: a comparative study

Chloroplast DNA variation in 96 Prunus avium L. cultivars was assessed and compared with the results of a previous study of cpDNA diversity in 23 wild populations of the species. The polymerase chain reaction‐restriction fragment length polymorphism method was used in these studies. Approximately 9% of the chloroplast genome was analyzed, using five universal primer pairs and three restriction enzymes. Ten polymorphic fragments were common to both the wild and sweet cherry; eight polymorphic fragments were found only in the wild cherry. In the cultivars, all mutations were small (5‐30 bp) indels. In the wild populations, a point mutation was also detected in addition to indels. The mutational combinations revealed three haplotypes in the cultivars, which are the main haplotypes in the wild cherry populations. Chloroplast DNA diversity in wild cherry is higher (16 haplotypes) than in sweet cherry cultivars (three haplotypes). The probable wild origin of the sweet cherry cultivars in the maternal line, on the basis of haplotypic similarity, was discussed.     

The integration and insertion site of GbVe1 gene in the genome of transgenic cotton (Gossypium hirsutum)

[Objective] The aim of this study was to obtain the flanking sequences of T-DNA in the transgenic cotton containing a GbVe1 over-expression cassette. [Method] The T-DNA insertion copy number in the transgenic GbVe1 cotton was analyzed by southern blot. Flanking sequences of the transgenic lines with putative single T-DNA insertion copy were obtained using high-efficiency Thermal asymmetric interlaced polymerase chain reaction (hiTAIL-PCR). The T-DNA insertion sites were further confirmed by PCR with specific primers. [Result] RB-flanking sequences (119-1 018 bp) and LB-flanking sequences (243-516 bp) were obtained from three transgenic lines with low copy number of T-DNA insertion. The AT content was more than 63% in these flanking sequences. A same single insertion site in the intron of Gohir.D01G157600.1 was found in the two transgenic lines 7/100826-152 and 12/100826-393, while two separated insertion sites, one also in the intron of Gohir.-D01G157600.1 and the other in the intergenic region of A12 chromosome, were found in the transgenic line 1/w-ch14. A deletion of 21 bp was found in the insertion site in the intron of Gohir.D01G157600.1. The T-DNA insertion in the intron of Gohir.D01G157600.1 was further confirmed by the specific PCR. [Conclusion] The flanking sequences of T-DNA in the transgenic GbVe1 cotton were obtained and the specific transformation event in the intron of Gohir.D01G157600.1 was further confirmed by PCR.