水稻中大麦Mlo和玉米Hm1抗病基因同源序列的分析和定位

大麦抗病基因Mlo和玉米抗病基因Hm1编码的产物不具有绝大多数植物抗病基因产物所含有的保守结构域。这两个抗病基因的作用机理也不符合基因对基因学说。从水稻中分离克隆了Mlo基因的同源序列OsMlo-1和玉米Hm1基因的同源序列DFR－1。利用水稻分子标记遗传连锁图,将OsMlo-1定位于水稻第六染色体的两俱RZ667和RG424之间;Osmlo-1距离这两个分子标记分别为20．6和6．0cM(centi-Morgan)。将DFR－1定位于水稻第一染色体两个分子标记R2635和RG462之间;DFR－1距离这两个分子标记分别为11．3和23．9cM。参照已发表的水稻分子标记连锁图,发现OsMlo-1和DFR－1的染色体位点分别与两个报道的水稻抗稻瘟病数量性状位点（QTL）有较好的对应关系。结果提示,水稻中与大麦Mlo 和玉米Hml同源的基因可能也参于抗病反应的调控。     

水稻NBS-LRR类R基因同源序列

根据多数抗病基因(R)编码蛋白质的核苷酸结合区(nucleotide binding site, NBS)和富含亮氨酸重复(leucine-rich repeat,LRR)保守区域特点,设计PCR特异扩增引物,从水稻中克隆了大小约为520 bpDNA片段23个.通过序列同源比较分析发现, 它们编码的蛋白质氨基酸序列包括有NBS-LRR类基因所具有的kinase-1a,kinase-2a, kinase-3a和保守的domain 2区域,它们属于R基因同源序列(R gene homologous sequence, 简称RS).聚类结果发现它们分为4类.遗传定位结果表明它们分布在1,3,4,7~11染色体上,其中10个RS位于已知R基因所在的染色体区间.用水稻抗白叶枯病基因Xa4的近等基因系和基因累加系对克隆的NBS-LRR同源序列进行RFLP分析,发现序列RS13可能来自Xa4基因家族.     

用RAPD标记对栽培稻F1花粉不育基因座S-b定位

S－b是栽培稻F1花粉不育基因座痊之一,在S－b基因座,台中65的基因型为Sj/Sj,而台中65（简称T65）的近等基因系TISL2的基因型为Si/Si,通过F2群体和BC1F1群体的遗传分析表明,台中65和TISL2的F1花粉不育性由一个基因座控制,S－b基因座的等位基因s^i和S^j相互作用,导致携带S^j基因的花败败育,产生染败花粉,用187个RFLP标记和500个RAPD引物分析 了T65和TISL2间的多态性,结果只有2个RAPD扩增片段H08－1300和Y09－H08－1300转化为RFLP标记,经F2群体的共分离分析表明,H08－1300和Y09－1500在F2群体中呈明显的偏态分离,用MAPMAKER／EXP3．0计算,H08－1300和Y09－1500与S-b基因座间的遗传 1．3cM和6．6cM,克隆和测定了H08－1300两侧的部分DNA序列,经同源性分析发现,右末端580bp的序列中有101bp 的序列与水稻第五染色体的克隆P0033D06（注册号：AC079357）有94％的同源性,左末端中540bp的序列中P0033D06有86％的同源性,由于P0033D06为由位于水稻第五染色体定位18．8cM处的标记R3166锚定,结果说明S－b基因座位于水稻第五染色体上,与R3166的遗传距离为1．3cM,同时还讨论了对于应用基因组测序的结果进行RAPD标记染色体定位的方法。     

野败型水稻细胞质雄性不育恢复基因Rf-4的分子标记定位

为了用分子标记准确定位野败型水稻细胞质雄性不育恢复基因Rf-4,将日本水稻基因组项目（Rice Genome Program,RGP)构建的水稻遗传连锁图谱第10染色体分子遗传图上的分子标记R1877和G2155之间对应区域YAC物理图上的6个YAC克隆进行了亚克隆,获得119个片段,对这些探针进行多态性探查,获得了2个多态分子标记,用珍汕97A和恢复基因近等基因系的杂种F2分离群体中的117完全不育株进行连锁分析表明,从YAC4892获得的亚克隆Y3－8与Rf－4座位的连锁距离为0．9cM,从YAC4630获得的亚克隆Y1－10与Rf－4座位的连锁距离为3．2cM,根据以上结果把Rf－4座位定位于第10染色体的特定位置,为该基因的分子标记辅助选择和定位克隆打下了基础。     

用同源序列的染色体定位寻找水稻抗病基因DNA片段

根据已知植物抗病基因的序列以及蛋白激酶序列中的高保守区域设计合成了特异性和简并引物,用聚合酶链反应从水稻（ＯｒｙｚａｓａｔｉｖａＬ．）ＤＮＡ中扩增同源片段,获约１００个大小不同的克隆。以这些克隆作探针进行限制性片段长度多态性（ＲＦＬＰ）分析,已将２６个克隆定位在两个水稻分子标记连锁图１２条染色体的３４个位点上。其中１０个克隆与８个已定位的水稻抗病基因在分子标记连锁图上的位置对应或毗邻。用其中部分与抗稻瘟病基因在染色体位置相对应的克隆作探针,分析抗稻瘟病近等基因系,ＲＦＬＰ带型在抗性基因系和感病亲本间表现出多态性,表明这些克隆与抗病基因在染色体位置上有较好的对应关系。     

一种提高水稻FISH检出率的新方法——RFLP混合标记

分别以水稻1号染色体上混合示记的8个紧密连锁的RFLP（平均约1.7kb）和5号染色体的BAC克隆44B4（137kb）,以及12号染色体单个RFLP RG397（约1.5kb）为探针,在水稻染色体上进行了荧光原位杂交（FISH）。结果表明,RFLP混合标记杂交的检出率为27％,大大高于 RFLP的检出率（7％）。其检出率虽然低于BAC克隆44B4（60％）,但它具有程序简单易行的特点,使基因原位定位更加高效,由于水稻中与已知功能基因紧密边销的RFLP标记具有数量丰富、分布密集等优势,揭示了混合标记的RFLP在禾本科植物同线性和共线性分析中的广阔应用前景。此外,混合标记的RFLP带可以用于染色体的准确识别和核型分析。     

水稻F2不育和抽穗期QTL分析

对台中65（粳稻）/Bhadua（籼稻）杂交F2代群体构建了RFLP连锁图谱,含94个分布较为均匀的标记。对F2小穗不育性状进行单点分析和区间分析的结果基本一致：有两个F2小穗不育QTL座位分别位于染色体1的XNpb113～XNpb346之间和染色体8的G187～XNpb397之间,而且该两个QTL均为新检测出的座位;检测出5个抽穗期TQL,其中3个座位在单点分析和区间分析中的结果一致,分别位于染色体1的XNpb113～XNpb346,染色体4的C891～C335,染色体的8的C166～C1121,另外,染色体6的XNpb27为单点分析结果,染色体10的R716～C405为区间分析结果。由于染色体1上的F2不育QTL和抽穗期QTL重叠,该QTL座位是由于遗传效应所至还是由于环境因素（迟抽穗）所至有待构建近等基因系进一步研究。;位于染色体1和10上的抽穗期QTL座位为新检测的座位。对新检测的F2不育和抽穗期QTL座位正在建立相应的近等基因系以精确定位和克隆上述基因。     

转Xa21基因水稻中T-DNA整合的遗传定位

利用转抗白叶枯病基因Xa21的水稻材料,通过TAIL－PCR方法扩增T－DNA整合的侧翼序列。从中筛选属于水稻基因组DNA的T－DNA整合的侧翼序列作为探针,将外源基因整合位点定位到窄叶青／京系17DH群体构建的水稻分子连锁图谱上。共获得属于水稻基因组DNA的T－DNA侧翼序列22个,其中的19个序列在定位群体的两个亲本之间显示RFLP多态性,分别定位在水稻基因组的第3,4,5,7,9,10,11和12染色体上。带有转基因Xa21的T－DNA整合的定位为研究外源基因在不同染色体位点的位置效应和稳定遗传打下基础。     

用染色体畸变品系对果蝇调控基因E（w^a）的定位

在果蝇中,杏黄色眼基因w^a和它的野生型白眼等位基因w的区别在于前者的转录单位中插入了反转录病毒样转座因子copia。半显性突变基因E（w^a)以反式方式降低w^a的活性,对多余翅脉基因px,E（wa)和翅腋具黑点基因sp 的遗传重组分析进一步确定了E（w^a)与sp的重组图距为0．2图距单位,并将E（w^a)定位于第二染色体右臂的2－106．8处,为了对E（w^a)进行细胞学的基因定位,4个Y;2易位品系,1个1;2易位品系和3个2R缺失品系与适当的E（w^a)品系进行杂交,并对其雄性子代进行复眼眼色和翅黑点的检查,结果表明E（w^a)位于第二染色体右臂60B的端粒端。我们用γ射诱变获得了一个E（w^a)突变体和两个sp 的突变体,并检查了它们的唾腺染色体,前者为E（w^a)的回复子,一段来源是有的染色体片段插入在60B5－13的位置,很可能在60B8－9的位置上,这个位置就是E（w^α）的座位,后者在60C1－2的位置上都看到了染色体的断裂点,表明该位置为sp 的基因座位。综合细胞学基因定位和遗传重组基因定位的资料,E(w α）被定位于60B5－13的位置,很可能在60B8－9的位置,它位于sp基因的左侧,二者相距很近。     

野生稻NBS类抗病基因同源序列的分离与序列分析

为了挖掘野生稻中的抗病资源,根据已克隆的植物抗病基因核苷酸结合位点序列中的保守结构域设计3对简并引物,从疣粒、药用、高秆、宽叶和斑点野生稻基因组DNA中分离出13条NBS类抗病基因类似物,其中11条具有连续的ORF,具有NBS类R基因的保守基元P-loop、kinas-2、kinas-3a和GLPL。在NCBI上进行同源性搜索发现,其中12条RGAs的核苷酸序列与水稻已知的NBS类R基因具有66%～94%的同源性,与其他植物已知R基因具有67%～84%的同源性;其对应的氨基酸序列与水稻已知的NBS类R基因具有43%～93%的同源性,与其他植物已知R基因具有37%～79%的同源性。另外1条的核苷酸序列与水稻假定的NBS类R基因具有76%的同源性,其氨基酸序列与水稻假定的NBS类R基因具有74%的同源性。根据序列分析结果设计6对不同基因特异性引物,并利用RT-PCR技术进行表达分析,结果表明,RN1BD5、RN1BD10、RN1GG2和RN1YY6均能表达,说明这些片段可能是功能性抗病基因的部分序列;而RN1KY9和RN1GG5没有表达,可能是假基因。     

RFLP mapping of the genes controlling hybrid breakdown in rice (Oryza sativa L.)

 Complementary recessive genes hwd1 and hwd2 controlling hybrid breakdown (weakness of F₂ and later generations) were mapped in rice using RFLP markers. These genes produce a plant that is shorter and has fewer tillers than normal plants when the two loci have only one or no dominant allele at both loci. A cultivar with two dominant alleles at the hwd1 locus and a cultivar with two dominant alleles at the hwd2 locus were crossed with a double recessive tester line. Linkage analysis was carried out for each gene independently in two F₂ populations derived from these crosses. hwd1 was mapped on the distal region of rice genetic linkage map for chromosome 10, flanked by RFLP markers C701 and R2309 at a distance of 0.9 centiMorgans (cM) and 0.6 cM, respectively. hwd2 was mapped in the central region of rice genetic linkage map for chromosome 7, tightly linked with 4 RFLP markers without detectable recombination. The usefulness of RFLP mapping and map information for the genes controlling reproductive barriers are discussed in the context of breeding using diverse rice germplasm, especially gene introduction by marker-aided selection.     

水稻苗期耐旱性基因位点及其互作的分析

随着全球水资源的日益贫乏和旱灾的日趋严重,水稻耐旱性的研究越来越重要,对籼稻窄叶青8号（ZYQ8）和粳稻京系17（JX17）以及由它们构建的加倍单倍体（DH）群体,参照国际水稻研究所的耐旱鉴定方法,在苗期进行断水,调查期耐旱性,利用该群体的分子连锁图谱进行数量性状座位（QTL）区间作图分析,共检测到2个耐旱的QTL（qDT-5和qDT-12),分别位于第5染色体的GA41-GA257之间的和第12染色体的RG457-Y12817R之间,这两个QTL的加性效应均来自ZYQ8的等位基因,用Epistat软件检测到2个单位点,即GA257和Y12817R,与区间作图分析的结果一致,Epistat还检测到与GA257互作的3个位点（RG541、G318和G192,分别位于第1、4和8染色体上）和与Y12817R互作的1个位点（CT234,位于第3染色体上）。     

Tagging and combining bacterial blight resistance genes in rice using RAPD and RFLP markers

Four genes of rice,Oryza sativa L., conditioning resistance to the bacterial blight pathogenXanthomonas oryzae pv.oryzae (X. o. pv.oryzae), were tagged by restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) markers. No recombinants were observed betweenxa-5 and RFLP marker lociRZ390, RG556 orRG207 on chromosome 5.Xa-3 andXa-4 were linked to RFLP locusXNpb181 at the top of chromosome 11, at distances of 2.3 cM and 1.7 cM, respectively. The nearest marker toXa-10, also located on chromosome 11, was the RAPD locusO07 ₂₀₀₀ at a distance of 5.3 cM. From this study, the conventional map [19, 28] and two RFLP linkage maps of chromosome 11 [14, 26] were partially integrated. Using the RFLP and RAPD markers linked to the resistance genes, we selected rice lines homozygous for pairs of resistance genes,Xa-4 +xa-5 andXa-4 +Xa-10. Lines carryingXa-4 +xa-5 andXa-4 +Xa-10 were evaluated for reaction to eight strains of the bacterial blight pathogen, representing eight pathotypes and three genetic lineages. As expected, the lines carrying pairs of genes were resistant to more of the isolates than their single-gene parental lines. Lines carryingXa-4 +xa-5 were more resistant to isolates of race 4 than were either of the parental lines (quantitative complementation). No such effects were seen forXa-4 +Xa-10. Thus, combinations of resistance genes provide broader spectra of resistance through both ordinary gene action expected and quantitative complementation.     

QTL analysis and mapping of pi21, a recessive gene for field resistance to rice blast in Japanese upland rice

Field resistance is defined as the resistance that allows effective control of a parasite under natural field conditions and is durable when exposed to new races of that parasite. To identify the genes for field resistance to rice blast, quantitative trait loci (QTLs) conferring field resistance to rice blast in Japanese upland rice were detected and mapped using RFLP and SSR markers. QTL analysis was carried out in F₄ progeny lines from the cross between Nipponbare (moderately susceptible, lowland) and Owarihatamochi (resistant, upland). Two QTLs were detected on chromosome 4 and one QTL was detected on each of chromosomes 9 and 12. The phenotypic variation explained by each QTL ranged from 7.9 to 45.7% and the four QTLs explained 66.3% of the total phenotypic variation. Backcrossed progeny lines were developed to transfer the QTL with largest effect using the susceptible cultivar Aichiasahi as a recurrent parent. Among 82 F₃ lines derived from the backcross, resistance segregated in the expected ratio of resistant 1 : heterozygous 2 : susceptible 1. The average score for blast resistance measured in the field was 4.2 ± 0.67, 7.5 ± 0.51and 8.2 ± 0.66, for resistant, heterozygous and susceptible groups, respectively. The resistance gene, designated pi21, was mapped on chromosome 4 as a single recessive gene between RFLP marker loci G271 and G317 at a distance of 5.0 cM and 8.5 cM, respectively. The relationship to previously reported major genes and QTLs conferring resistance to blasts, and the significance of marker-assisted selection to improve field resistance, are discussed. Received: 8 June 2000 / Accepted: 24 November 2000     

药用野生稻转育后代一个抗白叶枯病新基因的定位

从药用野生稻渗入后代选育的水稻株系B5表现为高抗褐飞虱、白背飞虱和白叶枯病。对B5与籼稻品种明恢63杂交组合的187个重组自交系(RILs)进行了抗白叶枯病接种鉴定,采用分离集团分析法(Bulked Segregant Analysis,BSA),在第1染色体上筛选到与水稻抗白叶枯病基因相连锁RFLP分子标记。利用RILs抗病性表现型鉴定资料和构建的分子标记连锁图谱,将抗白叶枯病基因定位在第1染色体短臂的C904和R596之间,这两个分子标记间遗传距离为1．3cM。该基因对RILs群体抗病性变异的贡献率为52．96％,是一效应值较大的主效基因。这一抗白叶枯病基因不同于已报道的抗白叶枯病基因的位点,因此将其命名为Xa29(t)。     

水稻抗白叶枯病基因Xa4位点跨叠BAC克隆群的构建

水稻白叶枯病抗性基因Xa4已被定位于第11染色体长臂末端的分子标记VG181和L1044之间,并与抗性基因同源序列片段RS13共分离。利用这3个标记筛选IRBB56的BAC文库,共得到128个阳性BAC克隆,其中RS13获得18个阳性克隆,这18个克隆中有4个和6个我隆分别同时为G181和L1044的阳性克隆,选其中的12克隆进行分析,构建了一个从G181到L1044区间的BAC跨叠克隆,全长420kb,并且56M22、106P13和104B153个BAC克隆可覆盖整个跨叠克隆群。这一研究结果为进一步分离Xa4基因打下基础。     

Identification of a YAC clone carrying the Xa-1 allele, a bacterial blight resistance gene in rice

Map-based cloning methods have been applied for isolation of Xa-1, one of the bacterial blight resistance genes in rice.Xa-1 was previously mapped on chromosome 4 using molecular markers. For positional cloning of Xa-1, a high-resolution genetic map was made for theXa-1 region using an F₂ population of 402 plants and additional molecular markers. Three restriction fragment length polymorphism (RFLP) markers, XNpb235, XNpb264 and C600 were found to be linked tightly to Xa-1, with no recombinants, and U08 ₇₅₀ was mapped 1.5 cM from Xa-1. The screening of a yeast artificial chromosome (YAC) library using theseXa-1-linked RFLP markers resulted in the identification of ten contiguous YAC clones. Among these, one YAC clone, designated Y5212, with an insert of 340 kb, hybridized with all three tightly linked markers. This YAC was confirmed to possess the Xa-1 allele by mapping the Xa-1 gene between both end clones of this YAC (Y5212R and Y5212L).     

NBS-LRR resistance gene homologues in rice

Twenty three DNA fragments with a size of about 520 bp have been cloned from rice genome by PCR amplification using primers designed according to the conserved region of most plant resistance (R) genes which have Nucleotide Binding Site (NBS) and Leucine-Rich Repeat (LRR) domains. Homologous comparison showed that these fragments contained typical motifs of the NBS-LRR resistance gene class, kinase 1a, kinase 2a, kinase 3a and domain 2. Thus they were named R gene homologous sequences (RS). These RS were divided into 4 groups by clustering analysis and mapped onto chromosomes 1, 3, 4, 7, 8, 9, 10 and 11, respectively, by genetic mapping. Ten RS were located in the chromosomal intervals where known R genes had been mapped. Further RFLP analysis of an RS, RS13, near the bacterial blight resistance gene Xa4 locus on chromosome 11 among near isogenic lines and pyramiding lines of Xa4 showed that RS13 was possibly amplified from the gene family of Xa4.     

A genome-wide analysis of wide compatibility in rice and the precise location of the S5 locus in the molecular map

 The discovery of wide-compatibility varieties (WCVs) that are able to produce normal fertility hybrids when crossed both to indica and japonica rice has enabled the fertility barrier between indica and japonica subspecies to be broken and provided the possibility of developing inter-subspecific hybrids in rice breeding programs. However, a considerable variation in the fertility level of hybrids from the same WCV crossed to different varieties has often been observed. One hypothesis for this variable fertility is that additional genes are involved in hybrid fertility besides the wide-compatibility gene (WCG). To assess such a possibility, we performed a genome-wide analysis by assaying a large population from a three-way cross ‘02428’/‘Nanjing 11’//‘Balilla’ using a total of 171 RFLP probes detecting 191 polymorphic loci distributed throughout the entire rice linkage map. Our analysis recovered 3 loci conferring significant effects on hybrid fertility. The major locus on chromosome 6 coincided in chromosomal location with the previously identified S ₅ locus, and the 2 minor loci that mapped to chromosomes 2 and 12, respectively, were apparently distinct from all previously reported hybrid sterility genes. Interaction between the indica and japonica alleles at each of the loci caused a reduction in hybrid fertility. The joint effect of the 2 minor loci could lead to partial sterility even in the presence of the WCG. The location of the S ₅ locus on the molecular marker linkage map was determined to be approximately 1.0 cM from the RFLP locus R2349. This tight linkage will be useful for marker-aided transfer of the WCG in hybrid rice breeding and for map-based cloning. Received: 5 February 1997 / Accepted: 4 April 1997