水稻花药培养植株后代的DNA变异

对籼稻圭６３０和粳稻０２４２８及其Ｆ１通过花药培养获得的８１个ＤＨ系进行了ＲＦＬＰ分析,有２８个探针揭示了ＤＮＡ变异。８１个ＤＨ系不同程度地发生了变异,并具有以下特点：（１）ＤＮＡ变异类型包括限制性片段长度的变化、９ＮＡ片段的丢失以及ＤＮＡ序列的扩增;（２）变异发生在籼稻圭６３０供体片段中的频率高于粳稻０２４２８,表现出基因型差异;（３）染色体组中第３、８、９和１０染色体较少发生变异,在其它染色体上均存在易变异位点;（４）在染色体的一些区段,相邻的探针均揭示了ＤＮＡ变异,表明在染色体上存在ＤＮＡ易变异区域;（５）变异位点和变异类型具有特异性,在同一位点不同的ＤＨ系中发生相同的变异。     

两种泥鳅中PdSox8和PdSox9基因的染色体定位

应用染色体原位杂交技术,以地高辛标记的大鳞副泥鳅ＰｄＳｏｘ８和ＰｄＳｏｘ９基因片段为探针,研究了二者在泥鳅和大鳞副泥鳅染色体组中的定位。结果表明,在大鳞副泥鳅中ＰｄＳｏｘ８、ＰｄＳｏｘ９分别于端部着丝粒染色体第４和第２号即１４和１２染色体上,信号位点距着丝粒的相对距离分别为４０．２％和６７．５％,在泥鳅中,则分别位于ｔ９、ｔ６上,信息位点距着丝粒的相对蹁分别为５８．３％和３０．８％。     

Ds因子在烟草染色体插入位点旁邻顺序的克隆

用Ｎｅｓｔｅｄ－ＰＣＲ方法从含Ｄｓ因子的转基因烟草ＤＮＡ中克隆了Ｄｓ因子在烟草染色体插入位点的９个旁邻ＤＮＡ片段,以这些片段作探针,和野生型烟草的ＤＮＡ进行Ｓｏｕｔｈｅｒｎ杂交,以检测这些片段在烟草染色体上的低拷贝ＤＮＡ。另外,对这些ＤＮＡ片段进行核苷酸序列测定,并将它们的顺序与Ｇｅｎｂａｎｋ数据中已有的核苷酸序列相比较,其中长度为１２８核苷酸的片段１和荷兰芹的４ＣＬ－２基因的一个区段有５７．８％     

Ds因子在烟草染色体插入位点旁邻顺序的克隆

用ＮｅｓｔｅｄＰＣＲ 方法从含Ｄｓ 因子的转基因烟草ＤＮＡ 中克隆了Ｄｓ 因子在烟草染色体插入位点的９ 个旁邻ＤＮＡ 片段,以这些片段作探针,和野生型烟草的ＤＮＡ 进行Ｓｏｕｔｈｅｒｎ 杂交,以检测这些片段在烟草基因组中的拷贝数,结果表明它们都属于烟草染色体上的低拷贝ＤＮＡ。另外,对这些ＤＮＡ 片段进行核苷酸序列测定,并将它们的顺序与Ｇｅｎｂａｎｋ 数据库中已有的核苷酸序列相比较,其中长度为１２８ 核苷酸的片段１ 和荷兰芹的４ＣＬ２ 基因的一个区段有５７ ．８ ％ 同源性,而另一长度为１６９ 核苷酸的片段３ 和百合中的反转座子的一个区段有６０ ．９ ％ 的同源性。这都表明Ｄｓ 因子在异源植物烟草中插入染色体单拷贝基因的机率很高,这对转座子标签法克隆基因是非常有利的。     

玉米两个RFLP标记的原位单杂交与共杂交定位的比较

ＲＦＬＰ标记ｂｎ１８．２３和 ｕｍｃ１１１位于玉米遗传日第 ４连锁群近端,彼此密切连锁但次序尚未确定。用生物素标记对它们进行了原位单杂交和共杂交的比较定位。在植物中,这类原位共杂交的研究为首次报道。在单一探针的原位杂交中 ｕｍｃ１１１被定位在第 １、 ４和９染色体长上,与着丝粒的百分距离分别是７．３６±２．６５、６３．６７±１．０７、４７．８７±２．９０。ｂｎ１８．２３被定位在第４和８染色体长臂上,与着丝粒的百分距离分别是８７．４２±２．４５和２７．６０±１．７５,清楚地表明了这两个标记在第４染色体上的次序。ｂｎ１８．２３和ｕｍｃ１１１分别与编码过氧化氢酶的ｃａｔ３基因和编码丝氨酸／苏氨酸蛋白激酶的ｃｄｅ２Ａ基因紧密连锁。根据供试ＲＦＬＰ标记检出位点推断了基因ｃｄｃ２Ａ和ｃａｔ３的物理位置。原位共杂交在第 ４染色体长臂上同时显示出了ｕｍｃ１１１和ｂｎ１８．２３两个标记的杂交信号,它们的位置分别与单一探针原位杂交的位置基本吻合。这为低拷贝或单一拷贝等小片段ＤＮＡ物理定位的可靠性以及它们共杂交的可行性提供了令人信服的证据。     

玉米优异早熟种质单330开花相关性状的QTL分析

玉米开花相关性状与玉米的成熟期和产量有密切的联系。通过对玉米CN165×单330(早熟种质)群体的130个F2:3家系开花相关性状在3个环境下进行分子鉴定和数量性状位点(QTL)分析,结果表明,在3个环境中检测到控制抽雄天数的10个QTL,分别位于第2、3、4、5、7、8染色体上,在第8染色体上同一区域在3种环境下都检测到了QTL;检测到控制散粉天数的10个QTL,分别位于第1、2、3、5、7、8染色体上,在第8染色体上同一区域在2种环境下都检测到了QTL;检测到控制吐丝天数的4个QTL,分别位于第4、5、8染色体上,在第8染色体不同环境下都检测到了2个QTL;仅仅在一个环境中检测到控制ASI的2个QTL,分别位于第6、9染色体上。这些QTL的基因效应以部分显性和超显性为主。研究表明,第8染色体上ph i060-um c2401区域(8.03~8.04)是一个研究开花相关性状的重要基因组区段,涉及到的标记可以作为分子标记辅助选择的重要候选标记。     

海岛棉原位杂交及核型比较

采用Ａ染色体组（Ａ genome)棉种亚洲基因组ＤＮＡ（gDNA)为探针,对海岛棉体细胞染色体进行荧光原位杂交（ＦＩＳＨ）,结果发现５２条染色体中有杂交信号与否的刚好各一半,从而直观地证实了海岛棉异源双二倍体起源的理论,但是,染色体的长度Ａ亚组的并非全部大于Ｄ亚组的。海岛棉基于ＦＩＳＨ图像的核型公式为：２n=4x=52=38m 14sm(sat)。３对随体染色体序号分别是Ａ亚组第１１、Ｄ亚组第２２和２５,均属于近中部着丝点（sm)类型,随体均在各自杂色体的短臂上,而且与所有染色体无关晨同一亚组起源。Ａ亚组第５、６和９对染色体长臂发生长了片段的易位,易位的片段较大,占所在染色体和蔗的百分率依次为１９．２１％、１７．６９％和１２．８８％,在Ｄ亚组１３对染色体中,最少５对的着丝点区域多或少地显示出与亚洲棉gDNA探针杂交的红色荧光信号,意味着有Ａ亚组染色体的交换。     

水稻分蘖角度的ＱＴＬs分析

分蘖角是水稻株型构成的重要性状之一,在育种上人有极其重要的意义,利用分蘖角度差异显著的１对籼粳亲本,将其杂交Ｆ１代花培加倍,构建了１个ＤＨ群体,考察了１１５个ＤＨ株系的分蘖角度,并使用该群体构建的分子图谱进行数量性状座痊（ＱＴＬs)分析。分别在第９和１２个染色体上检测３个ＱＴＬs(qTA-9a、qTA－９b和qTA－１２）,贡献率分别为２２．７％、１１．９％和２０．９％,其加性效应均为负,表明由分蘖角度较大的窄占青８号的基因控制,并讨论了这种由主产和微效基因控制的分蘖性状在育种学上的应用。     

人类染色体8q24.1带特异性微小卫星DNA的筛选

本研究运用人类高分辨染色体显微切割、ＰＣＲ技术获得的８ｑ２４．１带特异性探针池,构建了该区带的ｐＵＣ１９文库,从中筛选出４８个含ＣＡ重复顺序的微小卫星ＤＮＡ的克隆,已完成１２个克隆的序列分析,发现了一个世界上至今未曾报道过的、在正常人群中已检出１１个等位片段的、杂合度在中国汉族人群和美国盎格鲁撤克逊族人群中分别达０．８４和０．８３的高度多态的微小卫星ＤＮＡ（编号：Ｄ８Ｓ７Ｆ）,经ＰＣＲ检测人鼠杂种细胞系列,证实其来源于人８号染色体。     

水稻叶绿素含量动态QTL分析

为剖析水稻不同生育时期叶绿素含量的变化动态及遗传机制,以‘Sasanishiki’（粳稻）、‘Habataki’（籼稻）及其杂交衍生的85个回交重组自交系（BILs）群体为材料,对控制水稻叶片叶绿素含量的数量性状基因位点（QTL）变化动态进行了分析。共检测到39个QTL,包括26个非条件QTL和13个条件QTL,分布在除第7和第11号染色体以外的10条染色体上,平均每个时期检测到3．25个非条件QTL。其中生育前期和生育中后期检测到的QTL位点较少,仅为1—3个;在生育中期（盛期）检测到的QTL位点相对较多,一般为4～5个。在生育期的中期和后期均能在第1和2号染色体上检测到控制叶绿素含量的QTL,并且这些QTL位点在1和2号染色体上呈现聚集现象。本研究同时发现了一些新的QTL位点,这些QTL将有助于我们更全面地了解叶绿素在不同发育时期的遗传基础。     

Segmental Duplications Are Common in Rice Genome

Segmental duplications on rice (Oryza sativa L.) chromosomes 8, 9, 11, and 12 were studied by examining the distributions of sequences resolved by 13 probes detecting multiple copies of DNA sequences. Four of the hybridization bands detected by a repetitive sequence probe, rTRS, were mapped to the ends of all the four chromosomes. Two or three of the bands detected by each of the other 12 probes were also mapped to different chromosomes. The bands detected by the same probe usually occurred in similar locations of different chromosomes. Loci detected by different DNA probes were often similarly arranged on different chromosomes. Chromosomes 8 and 9 showed colinearity of marker loci arrangement indicating a possible common origin. A segment on chromosome 9 was also very similar to the previously reported duplicated fragments on the ends of chromosomes 11 and 12 which were also detected in this study, indicating a likely common origin. Moreover, the various degrees of distributional similarity of the segments suggest a complex relationship among the chromosomes in the evolution of the rice genome. These results support the proposition that chromosome duplication and diversification may be a mechanism for the origin and evolution of the chromosomes in the rice genome.     

Integrated cytogenetic map of mitotic metaphase chromosome 9 of maize: resolution,sensitivity, and banding paint development

To study the correlation of the sequence positions on the physical DNA finger print contig (FPC) map and cytogenetic maps of pachytene and somatic maize chromosomes, sequences located along the chromosome 9 FPC map approximately every 10 Mb were selected to place on maize chromosomes using fluorescent in situ hybridization (FISH). The probes were produced as pooled polymerase chain reaction products based on sequences of genetic markers or repeat-free portions of mapped bacterial artificial chromosome (BAC) clones. Fifteen probes were visualized on chromosome 9. The cytological positions of most sequences correspond on the pachytene, somatic, and FPC maps except some probes at the pericentromeric regions. Because of unequal condensation of mitotic metaphase chromosomes, being lower at pericentromeric regions and higher in the arms, probe positions are displaced to the distal ends of both arms. The axial resolution of FISH on somatic chromosome 9 varied from 3.3 to 8.2 Mb, which is 12-30 times lower than on pachytene chromosomes. The probe collection can be used as chromosomal landmarks or as a "banding paint" for the physical mapping of sequences including transgenes and BAC clones and for studying chromosomal rearrangements.     

Genetic and physical mapping of telomeres and macrosatellites of rice

Telomeres and telomere-associated satellites of rice were genetically and physically analyzed by pulsed-field gel electrophoresis (PFGE) using Arabidopsis telomeric DNA and rice satellite sequences as probes. We demonstrate that Arabidopsis telomeric sequences hybridize to rice telomeres under the conditions of high stringency. Using the Arabidopsis probe, multiple, discrete telomeric fragments could be identified on pulsed-field gel blots of rice DNAs digested with rare-cutting restriction enzymes. Most of the telomeric bands larger than 300 kb are physically linked with satellite bands as revealed by PFGE. Some of the telomeric and satellite bands segregate in a Mendelian fashion and are highly reproducible. Three such telomeric bands have been mapped to the distal ends of RFLP linkage groups: Telsm-1 on chromosome 8, Telsa-1 on chromosome 9 and Telsm-3 on chromosome 11. One segregating satellite band was mapped to an internal region of chromosome 10. Telomeric fragments were shown not only to be genetically linked to but also physically linked (based on PFGE) to the terminal RFLP markers. The physical distance from telomeric sequences to a distal RFLP marker, r45s gene, on chromosome 9, is 200 kb while the distance from telomeric sequences to RG98, a terminal RFLP marker on chromosome 11, is 260 kb. Physical maps of the telomere regions of chromosome 9 and chromosome 11 are presented.     

Extensive sequence homologies between Y and other human chromosomes

Twenty-six human Y-chromosome-derived DNA sequences, free of repetitive material, were used to probe male and female genomic blots. We present data from a detailed analysis and chromosomal location of the bands detected by such probes, which demonstrate extensive DNA sequence homology between the mammalian sex chromosomes and autosomes. Under stringent conditions, nine Y-derived probes reacted exclusively with the Y chromosome, 12 probes detected homologous sequences present on both the Y and the X, four probes detected homologies between Y and autosome(s) without any X counterpart and, finally, one probe hybridized to homologous sequences on Y, X and autosome(s). These data are consistent with the hypothesis of a common evolutionary origin for the mammalian sex chromosomes and reveal structural similarities between Y-located and autosomal non-repetitive sequences.     

Karyotype and identification of all homoeologous chromosomes of allopolyploid Brassica napus and its diploid progenitors

Investigating recombination of homoeologous chromosomes in allopolyploid species is central to understanding plant breeding and evolution. However, examining chromosome pairing in the allotetraploid Brassica napus has been hampered by the lack of chromosome-specific molecular probes. In this study, we establish the identification of all homoeologous chromosomes of allopolyploid B. napus by using robust molecular cytogenetic karyotypes developed for the progenitor species Brassica rapa (A genome) and Brassica oleracea (C genome). The identification of every chromosome among these three Brassica species utilized genetically mapped bacterial artificial chromosomes (BACs) from B. rapa as probes for fluorescent in situ hybridization (FISH). With this BAC-FISH data, a second karyotype was developed using two BACs that contained repetitive DNA sequences and the ubiquitous ribosomal and pericentromere repeats. Using this diagnostic probe mix and a BAC that contained a C-genome repeat in two successive hybridizations allowed for routine identification of the corresponding homoeologous chromosomes between the A and C genomes of B. napus. When applied to the B. napus cultivar Stellar, we detected one chromosomal rearrangement relative to the parental karyotypes. This robust novel chromosomal painting technique will have biological applications for the understanding of chromosome pairing, homoeologous recombination, and genome evolution in the genus Brassica and will facilitate new applied breeding technologies that rely upon identification of chromosomes.     

Chromosomal mapping of four different integration sites of Moloney murine leukemia virus including the locus for alpha 1(I) collagen in mouse

Infection of mouse embryos with Moloney murine leukemia virus (M-MuLV) has yielded several mouse substrains with stable germ line integration of retroviral DNA at distinct chromosomal loci (Mov loci; Jaenisch et al., 1981). There is evidence that flanking DNA sequences can have an effect on virus expression and, conversely, inserted viral DNA may affect the expression of adjacent host genes. As part of our studies on the interaction of inserted M-MuLV with the mouse genome, we have chromosomally mapped four different Mov loci by hybridizing single-copy mouse sequences, flanking the proviral DNA, to interspecies somatic cell hybrids. Furthermore, these sequences were assigned regionally by in situ hybridization to mouse metaphase chromosomes. In Mov-13 mice, M-MuLV had inserted into the alpha 1(I) collagen gene leading to early embryonic death in homozygotes. We have assigned this locus to the distal region of chromosome 11. Thus, the alpha 1(I) collagen gene is part of an evolutionarily conserved linkage group with the homologous genes on human chromosome 17. Three other proviral integration sites were mapped to chromosome 1, bands BC (Mov-7), chromosome 11, bands BC (Mov-9), and chromosome 3, bands FG (Mov-10). The Mov-10-specific probe detects an EcoRI-specific restriction fragment length polymorphism, which can make this probe a useful genetic marker.     

Towards map-based cloning of the barley stem rust resistance genes Rpg1 and rpg4 using rice as an intergenomic cloning vehicle

The barley stem rust resistance genes Rpg1 and rpg4 were mapped in barley on chromosomes 1P and 7M, respectively and the syntenous rice chromosomes identified as 6P and 3P by mapping common probes in barley and rice. Rice yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) and cosmid clones were used to isolate probes mapping to the barley Rpg1 region. The rice BAC isolated with the pM13 probe was a particularly excellent source of probes. A high-resolution map of the Rpg1 region was established with 1400 gametes yielding a map density of 3.6 markers per 0.1 cM. A detailed physical map was established for the rice BAC fragment containing the Rpg1-flanking markers pM13 and B24. This fragment covers a barley genetic distance of 0.6 cM and a rice DNA physical distance of ca. 70 kb. The distribution of barley cross-overs in relation to the rice DNA physical distances was extremely uneven. The barley genetic distance between the pM13 marker and Rpg1 was 0.1 cM per ca. 55 kb, while on the proximal side it was 0.5 cm per ca. 15 kb. Three probes from the distal end of the pM13 BAC mapped 3.0 cm proximal of Rpg1 and out of synteny with rice. These experiments confirm the validity of using large insert rice clones as probe sources to saturate small barley (and other large genome cereals) genome regions with markers. They also establish a note of caution that even in regions of high microsynteny, there may be small DNA fragments that have transposed and are no longer in syntenous positions.     

Quantification of total genomic DNA and selected repetitive sequences reveals concurrent changes in different DNA families in indica and japonica rice

This paper describes a fluorescence in situ hybridization (FISH) analysis of three different repetitive sequence families, which were mapped to mitotic metaphase chromosomes and extended DNA fibers (EDFs) of the two subspecies of rice (Oryza sativa), indica and japonica (2n=2x=24). The repeat families studied were (1) the tandem repeat sequence A (TrsA), a functionally non-significant repeat; (2) the [TTTAGGG]_n telomere sequence, a non-transcribed, tandemly repeated but functionally significant repeat; and (3) the 5S ribosomal RNA (5S rDNA). FISH of the TrsA repeat to metaphase chromosomes of indica and japonica cultivars revealed clear signals at the distal ends of twelve and four chromosomes, respectively. As shown in a previous report, the 17S ribosomal RNA genes (17S rDNA) are located at the nucleolus organizers (NORs) on chromosomes 9 and 10 of the indica cultivar. However, the japonica rice lacked the rDNA signals on chromosome 10. The size of the 5S rDNA repeat block, which was mapped on the chromosome 11 of both cultivars, was 1.22 times larger in the indica than in the japonica genome. The telomeric repeat arrays at the distal ends of all chromosome arms were on average three times longer in the indica genome than in the japonica genome. Flow cytometric measurements revealed that the nuclear DNA content of indica rice is 9.7% higher than that of japonica rice. Our data suggest that different repetitive sequence families contribute significantly to the variation in genome size between indica and japonica rice, though to different extents. The increase or decrease in the copy number of several repetitive sequences examined here may indicate the existence of a directed change in genome size in rice. Possible reasons for this phenomenon of concurrent evolution of various repeat families are discussed. Received: 9 August 1999 / Accepted: 29 December 1999     

Evolutionary implications of the human aldolase-A, -B, -C, and -pseudogene chromosome locations.

The aldolase genes represent an ancient gene family with tissue-specific isozymic forms expressed only in vertebrates. The chromosomal locations of the aldolase genes provide insight into their tissue-specific and developmentally regulated expression and evolution. DNA probes for the human aldolase-A and -C genes and for an aldolase pseudogene were used to quantify and map the aldolase loci in the haploid human genome. Genomic hybridization of restriction fragments determined that all the aldolase genes exist in single copy in the haploid human genome. Spot-blot analysis of sorted chromosomes mapped human aldolase A to chromosome 16, aldolase C to chromosome 17, the pseudogene to chromosome 10; it previously had mapped the aldolase-B gene to chromosome 9. All loci are unlinked and located on to two pairs of morphologically similar chromosomes, a situation consistent with tetraploidization during isozymic and vertebrate evolution. Sequence comparisons of expressed and flanking regions support this conclusion. These locations on similar chromosome pairs correctly predicted that the aldolase pseudogene arose when sequences from the aldolase-A gene were inserted into the homologous aldolase location on chromosome 10.     

Isolation and characterization of five rice telomere-associated sequences

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