水稻Pib基因及其连锁RFLP标记在栽培稻、药用野生稻和玉米中的比较物理定位

比较遗传学研究表明,禾本科不同基因组之间存在着广泛的同线性和共线性.对水稻(Oryza sativa L.)这一模式植物与其他禾本科植物的原位杂交定位可以揭示禾本科植物基因组的共同特点和进化规律,为建立禾本科遗传大体系积累资料.实验以图位克隆法分离的水稻Pib 基因(10.3 kb)和与之连锁的RFLP标记为探针, 研究了Pib及与其连锁的RFLP标记在供试种中的同源性和物理位置. Southern杂交结果表明,Pib在玉米(Zea mays L.)基因组中有同源序列.进一步利用单色和双色荧光原位杂交技术确定了Pib在栽培稻(O.sativa ssp. indica cv. Guangluai 4)、玉米和药用野生稻(O. officinalis Wall ex Watt)染色体上的物理位置.定位结果表明,Pib基因和与之连锁的RFLP标记在这3个供试种基因组中具有同线性.     

水稻Xa21基因在水稻和玉米中的比较物理定位

比较基因组分析证明,禾本科不同种基因组间存在广泛的同线性和共线性。对水稻（ＯｒｙｚａｓａｔｉｖａＬ．）这一模式植物与其它禾本科植物基因的原位杂交比较定位可以揭示禾本科植物基因组结构的共同特点和进化规律。利用含Ｘａ２１基因的ｐＢ８２２作探针筛选水稻的细菌人工染色体（ＢＡＣ）文库,建立了一个包含３个ＢＡＣ克隆的重叠群。用生物素标记其中一个ＢＡＣ克隆,对水稻“广陆矮４号”和玉米自交系黄早四进行了染色体荧光原位杂交。同时,用ｐＢ８２２也作了原位杂交检测。在水稻第１１染色体长臂中间检出了杂交信号,信号与着丝粒的百分距离约为２４。在玉米的第１、３和第８染色体长臂观察到杂交信号,表明玉米基因组中具有三个Ｘａ２１的同源序列座位。ＢＡＣＦＩＳＨ的信号检出率达在４０％以上,大大高于质粒探针ｐＢ８２２的检出率（１５％）,而且可在同源染色体和姊妹染色单体上同时检出杂交信号的比例较高,证明了利用ＢＡＣ克隆荧光原位杂交进行比较物理定位的可行性和优越性。在ＢＡＣＦＩＳＨ中必须用相应基因组的ＣｏｔⅠＤＮＡ封阻,以排除重复序列的干扰。     

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。     

用AFLP标记快速构建遗传连锁图谱并定位一个新基因tms5

报导了一个分子标记连锁图的快速构建方法.通过对水稻(Oryza sativa L.)"安农S-1"和"南京11"的F2分离群体的AFLP分析找到了142个AFLP标记,用这142个AFLP标记以及已定位的25个SSR标记和5个RFLP标记构建了水稻12个染色体的分子标记连锁图,该图覆盖水稻基因组的1 537.4 cM,相邻标记间的平均间距为9.0 cM,这是在国内建立的第一张AFLP标记连锁图.在建立连锁图谱的同时把一个新基因tms5 (水稻温敏核不育基因)定位在第2染色体上.     

Bph3在栽培稻和药用野生稻中的BAC-FISH比较物理定位(英)

用栽培稻 (OryzasativaL .)遗传图第四连锁群中与抗褐稻虱基因Bph3紧密连锁的RFLP标记RZ6 9及筛选出来的BAC克隆 38J9作探针 ,对药用野生稻 (O .officinalisWellexWatt)和栽培稻荧光原位杂交 ,供试标记RZ6 9及38J9均被定位于药用野生稻和栽培稻第 4染色体的短臂上 ,药用野生稻杂交信号的百分距分别为 2 2 .12± 3.4 4和2 0 .0 0± 5 .4 0 ,而栽培稻均为 0。在栽培稻中 ,信号检出率相应地为 6 .2 9%和 5 6 .10 % ,在药用野生稻中则为 6 .14 %和 5 0 .0 0 %。BAC克隆和RFLP标记探针杂交信号的百分距十分接近 ,说明在栽培稻和野生稻中RFLP标记RZ6 9都在同一BAC克隆的大插入片段中。由此推知 ,药用野生稻与抗性基因Bph3的同源顺序就在第 4染色体信号出现的相应位置。在未封阻的情况下 ,药用野生稻的BAC杂交在多条染色体上具有信号 ,这表明它和栽培稻的Cot_1DNA重复顺序也在一定程度上具有同源性。药用野生稻第 4染色体是根据栽培稻与药用野生稻的比较遗传图选用与Gm_6连锁的RG2 14通过FISH确定的。讨论了栽培稻BAC克隆对药用野生稻比较原位杂交物理作图的可行性问题。     

Bph3在栽培稻和药用野生稻中的BAC-FISH比较物理定位

用栽培稻(Oryza sativa L.)遗传图第四连锁群中与抗褐稻虱基因Bph3紧密连锁的RFLP标记RZ69及筛选出来的BAC克隆38J9作探针,对药用野生稻(O.officinalis Well ex Watt)和栽培稻荧光原位杂交,供试标记RZ69及38J9均被定位于药用野生稻和栽培稻第4染色体的短臂上,药用野生稻杂交信号的百分距分别为22.12±3.44和20.00±5.40,而栽培稻均为0.在栽培稻中,信号检出率相应地为6.29%和56.10%,在药用野生稻中则为6.14%和50.00%.BAC克隆和RFLP标记探针杂交信号的百分距十分接近,说明在栽培稻和野生稻中RFLP标记RZ69都在同一BAC克隆的大插入片段中.由此推知,药用野生稻与抗性基因Bph3的同源顺序就在第4染色体信号出现的相应位置.在未封阻的情况下,药用野生稻的BAC杂交在多条染色体上具有信号,这表明它和栽培稻的Cot-1 DNA重复顺序也在一定程度上具有同源性.药用野生稻第4染色体是根据栽培稻与药用野生稻的比较遗传图选用与Gm-6连锁的RG214通过FISH确定的.讨论了栽培稻BAC克隆对药用野生稻比较原位杂交物理作图的可行性问题.     

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号染色体上混合标记的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RFLPRG397（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%,大大高于单个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连锁的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还可以用于染色体的准确识别和核型分析。 Abstract：Using mix-labeled 8 RFLPs (average length is about 1.7 kb) as a probe,the autho rs carried out fluorescence in situ hybridization in rice, and detected the sing le RFLP RZ397 (1.5 kb)and the BAC clone 44B4 (137 kb) simultaneously. The detec tion rate of mix-labeled RFLP was 27%, much higher than single-labeled RFLP (7%) , though lower than BAC clone (60%). Mix-labeled RFLP is an easy and effective m ethod to locate genes for its simplicity and sufficiency of RFLPs linked with th e functional genes. In addition, Mix-labeled RFLP groups can be used as effectiv e markers in karotype analysis of rice and the analysis of colinearity or synten y among cereals.     

利用分子标记和水稻籼粳交双单倍体群体进行遗传作图研究

利用“圭 630”(籼稻 ,Oryza sativa subsp.indica) /“0 2 4 2 8”(粳型广亲和品种 ,O.sativa subsp.japonica)的 F1代花药培养双单倍体植株 ( DH)群体和 RFLP标记构建了一张水稻分子连锁图谱。图上有 2 33个标记 ,覆盖基因组约 2 0 70 c M( centimorgan) ,定位了 2 5个 RFLP新标记、2个染色体端粒和水稻落粒性基因 sh- 2。亲本间的 RFLP主要来源于碱基取代 ,少数来源于 DNA结构的变化。RFLP标记在图谱上的排序与其它图谱基本相同 ,但标记在染色体间和染色体内分配不均 ,这可能与染色体间的遗传稳定性和染色体内的区段变异性 ,以及各区段交换重组活性不同有关。     

RTSV和Glh在药用野生稻中的物理定位

药用野生稻具有多种抗病虫性,是水稻品种改良重要的种质资源之一.本研究采用与栽培稻遗传图第4连锁群中与RTSV和Glh紧密连锁的RFLP标记RZ262及其筛选出来的BAC克隆作探针,对药用野生稻进行荧光原位杂交,供试探针均被定位于药用野生稻第4染色体短臂的中部,百分距分别为74.86±3.72和73.98±4.44,信号检出率为7.6%和45.1%.BAC克隆和RFLP标记探针杂交位置几乎一致,说明在栽培稻和野生稻中RFLP标记RZ262都存在同一BAC克隆的大插入片段中,药用野生稻与抗性基因RTSV和Glh的同源顺序就在第4染色体信号出现的相应位置,从而为作物育种开发和利用野生资源的抗性基因提供了理论依据.     

玉米mir1基因在玉米和薏苡中的比较物理定位

玉米基因mir1编码一种抗秋季黏虫的半胱氨酸蛋白酶。利用RFLP作图mir1基因被定位在玉米第 6号染色体短臂上 ,但它在第 6号染色体短臂上的物理位置还不知道。实验以mir1和 4 5SrDNA为探针 ,通过双色荧光原位杂交技术确定了mir1基因在玉米细胞分裂中期和粗线期第 6号染色体上的物理位置。Southern杂交结果表明 ,在薏苡基因组中存在mir1基因的同源序列 ,进一步利用荧光原位杂交的方法确定mir1基因的同源序列定位于薏苡第 7号染色体长臂的近末端 ,其信号与着丝粒的百分距离为 73 33± 0 15。     

A rice blast-resistance genetic resource from wild rice in Yunnan, China.

Compared to Pi-ta(-) alleles, Pi-ta(+) alleles can cause blast resistance response. In this work, Pi-ta gene in multiple rice materials, including local rice cultivars, different types of O. rufipogon and O. longistaminata was detected by molecular cloning and sequence analysis. Results indicated that Pi-ta(+) alleles were rare alleles, because in all the tested materials, only the 'Erect' type of O. rufipogon (ETOR) from Jinghong county in Yunnan province contains a Pi-ta(+) allele. Another rice blast resistance gene, Pib, confers resistance to the Japanese strain of M. grisea, was also confirmed to be functional in this type of O. rufipogon. The results of pathogen inoculation test show that ETOR is more strongly resistant to the tested blast pathogen races than other types of O. rufipogon. The resistance of ETOR may at least partially depend upon the functioning of Pi-ta and Pib gene. As O. rufipogon has the same type of genome with the cultivated rice (O. sativa), Pi-ta(+) and Pib gene in Erect type of O. rufipogon can be used to improve the tolerance of cultivated rice to blast, either by traditional hybridization or by genetic engineering.     

Fluorescence in situ hybridization mapping of Avena sativa L. cv. SunII and its monosomic lines using cloned repetitive DNA sequences.

Fluorescent in situ hybridization (FISH) employing multiple probes was used with mitotic or meiotic chromosome spreads of Avena sativa L. cv. SunII and its monosomic lines to produce physical chromosome maps. The probes used were Avena strigosa pAs120a (which hybridizes exclusively to A-genome chromosomes), Avena murphyi pAm1 (which hybridizes exclusively to C-genome chromosomes), A. strigosa pAs121 (which hybridizes exclusively to A- and D-genome chromosomes), and the wheat rDNA probes pTa71 and pTa794. Simultaneous and sequential FISH employing two-by-two combinations of these probes allowed the unequivocal identification and genome assignation of all chromosomes. Ten pairs were found carrying intergenomic translocations: (i) between the A and C genomes (chromosome pair 5A); (ii) between the C and D genomes (pairs 1C, 2C, 4C, 10C, and 16C); and (iii) between the D and C genomes (pairs 9D, 11D, 13D, and 14D). The existence of a reciprocal intergenomic translocation (10C-14D) is also proposed. Comparing these results with those of other hexaploids, three intergenomic translocations (10C, 9D, and 14D) were found to be unique to A. sativa cv. SunII, supporting the view that 'SunII' is genetically distinct from other hexaploid Avena species and from cultivars of the A. sativa species. FISH mapping using meiotic and mitotic metaphases facilitated the genomic and chromosomal identification of the aneuploid chromosome in each monosomic line. Of the 18 analyzed, only 11 distinct monosomic lines were actually found, corresponding to 5 lines of the A genome, 2 lines of the C genome, and 4 lines of the D genome. The presence or absence of the 10C-14D interchange was also monitored in these lines.     

稻属不同染色体组着丝粒BAC克隆的分离和鉴定

着丝粒在真核生物有丝分裂和减数分裂染色体正常的分离和传递中起着重要的作用。通过构建5个稻属二倍体野生种的基因组BAC文库, 采用菌落杂交和FISH技术, 筛选和鉴定了各染色体组着丝粒克隆, 并且分析了这些克隆在不同基因组间的共杂交情况, 结果表明: (1) C染色体组的野生种O. officinalis 和F染色体组的野生种O. brachyantha具有各自着丝粒特异的卫星DNA序列, 并且O. brachyantha着丝粒还具有特异的逆转座子序列; (2) A、B和E染色体组的野生稻O. glaberrima、O. punctata和O. australiensis着丝粒区域都含有与栽培稻着丝粒重复序列CentO和CRR同源的序列; (3) C染色体组野生稻O. officinalis的2条体细胞染色体着丝粒具有CentO的同源序列, 同时也发现其所有着丝粒区域都包含栽培稻CRR的同源序列。这些结果对克隆稻属不同染色体组的着丝粒序列、研究不同染色体组间着丝粒的进化关系和稻属不同着丝粒DNA序列与功能之间的关系均具有重要意义。     

Simple sequence repeat analyses of interspecific hybrids and MAALs of <Emphasis Type="Italic">Oryza </Emphasis><Emphasis Type="Italic">officinalis</Emphasis> and <Emphasis Type="Italic">Oryza sativa</Emphasis>

Wild rice is a valuable resource for the genetic improvement of cultivated rice (Oryza sativa L., AA genome). Molecular markers are important tools for monitoring gene introgression from wild rice into cultivated rice. In this study, Simple sequence repeat (SSR) markers were used to analyze interspecific hybrids of O. sativa-O. officinalis (CC genome), the backcrossing progenies and the parent plants. Results showed that most of the SSR primers (335 out of 396, 84.6%) developed in cultivated rice successfully amplified products from DNA samples of wild rice O. officinalis. The polymorphism ratio of SSR bands between O. sativa and O. officinalis was as high as 93.9%, indicating differences between the two species with respect to SSRs. When the SSR markers were applied in the interspecific hybrids, only a portion of SSR primers amplified O. officinalis-specific bands in the F(1) hybrid (52.5%), BC(1) (52.5%), and MAALs (37.0%); a number of the bands disappeared. Of the 124 SSR loci that detected officinalis-specific bands in MAAL plants, 96 (77.4%) showed synteny between the A and C-genomes, and 20 (16.1%) showed duplication in the C-genome. Sequencing analysis revealed that indels, substitution and duplication contribute to the diversity of SSR loci between the genomes of O. sativa and O. officinalis.     

云南元江普通野生稻中Pi-ta和Pib同源基因的克隆和分析

用高保真PCR技术从云南元江普通野生稻中克隆了抗稻瘟病Pi-ta同源基因的编码区及Pib基因的部分同源序列。Pi-ta同源基因的编码区序列与报道的栽培稻有99.7%的同源性。根据前人的结果,从元江普通野生稻的Pi-ta基因推导的氨基酸序列中918位点为丝氨酸,属于Pi-ta~-等位基因,不能对含有AVRPita基因的稻瘟病菌产生抗性。与Pi-ta基因相比,元江普通野生稻中的Pib同源基因第一外显子与栽培稻的相应序列间存在较大差异,其中有一段87 bp的DNA序列缺失,而且不能按正常的Pib基因序列的阅读框进行翻译。因此认为,元江普通野生稻不具有基于Pi-ta和Pib基因的抗稻瘟病遗传基础。     

Genetic Mapping of Rice Using RFLP Markers and a Double Haploid Population of a Cross Between indica and japonica Varieties

A genetic linkage map of rice was constructed using a double haploid (DH) population from "Gui 630” (Oryza sativa subsp, indica)/"02428" (O. sativa subsp, japonica, wide compatibility variety) and RFLP markers. It consists of 233 loci and covers rice genomes about 2070 cM (centimorgan), and compares well with the other published rice maps. 25 RFLP markers, 2 telomeres and sh-2 (shattering ability) gene were first located on the molecular map of rice. RFLPs between "Gui 630' and "02428' mainly came from base substitution and a few DNA construction variance, not distributed evenly among chromosomes and on chromosome. This was probably resulted from the difference genetic stability among chromosomes and regions, in exchanging recombination ability in different segments of chromosome.     

Phylogenetic relationships in the genus Oryza based on mitochondrial RFLPs.

Restriction fragment length polymorphism (RFLP) of mitochondrial DNA in the genus Oryza was surveyed using 20 accessions including 11 species and a single endonuclease, EcoRI. RFLPs were visualized by Southern hybridization with eight rice mitochondrial DNA probes labeled non-radioactively with digoxigenin-dUTP. A total of 66 bands were obtained from all of the accessions. The total number of fragments per plant was higher in diploid A-genome species (an average of 35.3) than that in diploid B- and C-genome species and allotetraploid BC- and CD-genome species (an average of 28.2). The extent of the polymorphism in the RFLP patterns was various depending on the probes used. A diverse polymorphism was observed with most of the probes used, i.e. the cob, cox I, atp6, rrn18, rrn26 and atp9 regions, whereas, no polymorphic band was observed with a probe for the coxII region. The genus Oryza was separated into two large clusters. One cluster was comprised of A-genome species and the other cluster was comprised of B-, BC-, C-, and CD- genome species. Within A-genome species, the genetic variation was relatively high. Even in O. sativa species, the RFLP patterns of japonica and indica subspecies were clearly different from each other when three probes were used. However, there was no polymorphism between O. glaberrima and O. barthii. Within the genomes of B, BC, C, and CD, RFLP patterns were similar to each other and they showed a closer affinity except for O. minuta (BBCC). Within the BC genome species, the patterns of O. punctata and O. minuta were largely different from each other and separated into two different subclusters. Thus, the mitochondrial genomes of the two BC species (O. punctata and O. minuta) apparently evolved independently. Among CD genome species (O. latifolia and O. alta), the patterns of one accession, O. alta W0017 were largely different from those of the other accessions of CD genome species.     

Long-range and targeted ectopic recombination between the two homeologous chromosomes 11 and 12 in Oryza species

Whole genome duplication (WGD) and subsequent evolution of gene pairs have been shown to have shaped the present day genomes of most, if not all, plants and to have played an essential role in the evolution of many eukaryotic genomes. Analysis of the rice (Oryza sativa ssp. japonica) genome sequence suggested an ancestral WGD ～50-70 Ma common to all cereals and a segmental duplication between chromosomes 11 and 12 as recently as 5 Ma. More recent studies based on coding sequences have demonstrated that gene conversion is responsible for the high sequence conservation which suggested such a recent duplication. We previously showed that gene conversion has been a recurrent process throughout the Oryza genus and in closely related species and that orthologous duplicated regions are also highly conserved in other cereal genomes. We have extended these studies to compare megabase regions of genomic (coding and noncoding) sequences between two cultivated (O. sativa, Oryza glaberrima) and one wild (Oryza brachyantha) rice species using a novel approach of topological incongruency. The high levels of intraspecies conservation of both gene and nongene sequences, particularly in O. brachyantha, indicate long-range conversion events less than 4 Ma in all three species. These observations demonstrate megabase-scale conversion initiated within a highly rearranged region located at ～2.1 Mb from the chromosome termini and emphasize the importance of gene conversion in cereal genome evolution.