Comparison between genetic and physical maps in Zea mays L. of molecular markers linked to resistance against Diatraea spp

In the pachytene stage, chromosomes are maximally extended and can easily be distinguished. Therefore, by applying fluorescence in situ hybridization (FISH) to pachytene chromosomes, it is possible to generate a high-resolution physical map of chromosome 9 in maize. Molecular markers ( umc105a on the short arm of chromosome 9, csu145a on the long arm) were used that flank quantitative trait loci (QTL) for sugarcane borer (SCB) and southwestern corn borer (SWCB) resistance. As reference markers, a centromere-specific probe (CentC) and a knob-specific probe (pZm4-21) were utilized. Two fluorescent dyes with four probes were used to physically position these markers. Signals of repetitive DNA sequences in cosmid probes were suppressed by chromosome in situ suppression (CISS) hybridization. FISH signals were strong and reproducible for all probes. We measured the distances in micrometers for four subchromosomal regions and estimated the corresponding number of base pairs. The physical locations of the markers were compared on mitotic metaphase and pachytene chromosomes to the genetic map of chromosome 9. Genetic analysis positioned the two markers for SCB resistance in a central interval representing approximately 33.7% of the genetic length. However, the physical distance between these probes was determined to encompass about 70% of the physical length of chromosome 9. The two markers were located at distal positions on opposite arms of chromosome 9. Physical maps provide valuable information for gene isolation and understanding recombination.     

玉米（Zea mays L.）过氧化物酶编码基因px和冷调控蛋白编码基因cld的物理定位

过氧化物酶是植物抗病过程中的关键酶,也参与植物抗低温胁迫以及一些正常的植物生长发育生理过程。冷调控蛋白是植物抗低、高温的重要蛋白。近年的研究表明植物抗病、某些正常生长发育过程。冷调控蛋白是植物细胞程序性死亡有关。以玉米自效系黄早四为材料,采用生物素标记,利用原位杂效技术绎玉米中过氧化物酶和冷调控蛋白编码基因ｐｘ与ｃｌｄ进行了原位杂效定位,ＤＡＢ和荧光检测得到了一致的结果。在２号和７号染色体长臂同时     

Physical localization of single-copy sequences on pachytene chromosomes in maize (Zea mays L.) by chromosome in situ suppression hybridization.

A new approach for locating single-copy DNA sequences on pachytene chromosomes of maize (Zea mays L.) was developed. A cosmid clone with homologous sequences to a molecular marker (umc105a) linked to a quantitative trait locus (QTL) for resistance against sugarcane borer (SCB) was physically mapped by fluorescence in situ hybridization (FISH) to the short arm of chromosome 9. The marker umc105a was genetically placed in the centromeric region. To suppress signals generated by maize repetitive DNA, competitive in situ suppression (CISS) hybridization was necessary to obtain specific signals from umc105a. A centromere specific DNA probe (CentC) was used in a double-labeling technique as a reference marker. Fluorescence signals generated by umc105a cosmid and CentC were specific and highly reproducible. Thus the single-copy DNA sequence of umc105a was physically localized on the short arm of chromosome 9 near the telomere. This is the first report of physical localization of single-copy DNA sequence by CISS hybridization to a maize pachytene chromosome.     

Comparison of genetic and physical maps of group 7 chromosomes from Triticum aestivum L.

We present a high density physical map of homoeologous group 7 chromosomes from Triticum aestivum L. using a series of 54 deletion lines, 6 random amplified polymorphic DNA (RAPD) markers and 91 cDNA or genomic DNA clones from wheat, barley and oat. So far, 51 chromosome segments have been distinguished by molecular markers, and 54 homoeoloci have been allocated among chromosomes 7A, 7B and 7D. The linear order of molecular markers along the chromosomes is almost identical in the A- B- and D-genome of wheat. In addition, there is colinearity between the physical and genetic maps of chromosomes 7A, 7B and 7D from T. aestivum, indicating gene synteny among the Triticeae. However, comparison of the physical map of chromosome 7D from T. aestivum with the genetic map from Triticum tauschii some markers have been shown to be physically allocated with distortion in more distal chromosome regions. The integration of genetic and physical maps could assist in estimating the frequency and distribution of recombination in defined regions along the chromosome. Physical distance did not correlate with genetic distance. A dense map facilitates the detection of multiple rearrangements. We present the first evidence for an interstitial inversion either on chromosome arm 7AS or 7DS of Chinese Spring. Molecularly tagged chromosome regions (MTCRs) provide landmarks for long-range mapping of DNA fragments.     

High-resolution mapping of loci conferring resistance to sugarcane mosaic virus in maize using RFLP, SSR, and AFLP markers

Sugarcane mosaic virus (SCMV) is one of the most important virus diseases of maize in Europe. Genetic analysis on backcross five (BC5) progeny derived from the cross FAP1360A (resistant) × F7 (susceptible) confirmed that at least two dominant genes, Scm1 and Scm2, are required for resistance to SCMV in the progeny of this cross. With the aid of RFLP and SSR marker analyses, Scm1 was mapped in the region of 8.7 cM – between the nucleolus organizer region (nor) and RFLP marker bnl6.29 on the short arm of chromosome 6, while Scm2 was mapped to an interval of 26.8 cM flanked by the RFLP markers umc92 and umc102 near the centromere region of chromosome 3. Both chromosome regions were further enriched for AFLP markers by successful application of a bulked segregant analysis to this oligogenic trait. A total of 23 linked AFLP markers were identified, clustered in chromosome regions adjacent to either Scm1 or Scm2. Seven AFLP markers linked to Scm1 resided within the nor-bnl6.29 interval, and one of them, E3M8-1, showed no recombination with Scm1. Three AFLP markers linked to Scm2 are located between umc92 and umc102. Received: 13 October 1998 / Accepted: 18 January 1999     

程序性细胞死亡抑制基因Dad-1在水稻和玉米中的FISH定位(英文)

Dad-1是一种在动物和植物中都非常保守的细胞程序性死亡 (PCD) 抑制基因。作者利用 FISH (荧光原位杂交)首次把单拷贝水稻Dad-1基因物理定位在水稻第2号染色体短臂的端部(Fig.2 A,B&C)。我们还分析了它在玉米基因组中的同源序列。Southern 杂交结果显示在玉米基因组中确实存在水稻Dad-1 的同源序列(Fig.1)。FISH进一步展示了三个杂交信号分别在玉米4、5号染色体长臂和9号染色体短臂上(Fig.2 D,E&F),其信号距着丝粒的百分距离(FL值)分别为 91、98和96。其杂交位点的位置与水稻Dad-1所处的相对位置是相似的,它们都处于染色体臂的端部。这表明在一定的程度上,Dad-1基因不仅在序列同源性上而且在所处的染色体位置上具有保守性。 水稻Dad-1基因在水稻中的杂交信号检出率 (38%) 高于玉米中的。这表明与玉米相比,水稻Dad-1 基因的编码序列更容易与水稻染色体杂交;它与玉米中的相应序列可能只是部分同源。     

Order and genomic distances among members of the carcinoembryonic antigen (CEA) gene family determined by fluorescence in situ hybridization.

Fluorescence in situ hybridization was used to establish the order of, and to estimate genomic distances among, members of the carcinoembryonic antigen (CEA) and pregnancy-specific glycoprotein (PSG) subgroups on chromosome 19. Fluorescence in situ hybridization to metaphase chromosomes localized the PSG subgroup telomeric to the CEA subgroup. Cosmid clones containing sequences for individual genes in the CEA and PSG subgroups were also hybridized to human sperm pronuclear and somatic interphase nuclear chromatin targets. The mapping results lead to the gene order cen-CGM7-CEA-NCA-CGM1-BGP-CGM9-CGM8-PSG-te l. The genomic distances between selected pairs of gene family members were estimated from the physical distances between hybridization sites measured in pronuclei. The CEA-PSG gene family region is estimated to span 1.1 to 1.2 Mb.     

Development of T. aestivum L.eH. californicum Alien Chromosome Lines and Assignment of Homoeologous Groups of Hordeum californicum Chromosomes

Hordeum californicum(2n=2x=14, HH) is resistant to several wheat diseases and tolerant to lower nitrogen. In this study, a molecular karyotype of H. californicum chromosomes in the Triticum aestivum L. cv. Chinese Spring(CS)eH. californicum amphidiploid(2n=6x=56, AABBDDHH) was established. By genomic in situ hybridization(GISH) and multicolor fluorescent in situ hybridization(FISH) using repetitive DNA clones(pTa71, pTa794 and pSc119.2) as probes, the H. californicum chromosomes could be differentiated from each other and from the wheat chromosomes unequivocally. Based on molecular karyotype and marker analyses, 12 wheatealien chromosome lines, including four disomic addition lines(DAH1, DAH3, DAH5 and DAH6), five telosomic addition lines(MtH7L,MtH1 S, MtH1 L, DtH6 S and DtH6L), one multiple addition line involving H. californicum chromosome H2, one disomic substitution line(DSH4) and one translocation line(TH7S/1BL), were identified from the progenies derived from the crosses of CSeH. californicum amphidiploid with common wheat varieties. A total of 482 EST(expressed sequence tag) or SSR(simple sequence repeat) markers specific for individual H. californicum chromosomes were identified, and 47, 50, 45, 49, 21, 51 and 40 markers were assigned to chromosomes H1, H2, H3, H4, H5, H6 and H7, respectively. According to the chromosome allocation of these markers, chromosomes H2,H3, H4, H5, and H7 of H. californicum have relationship with wheat homoeologous groups 5, 2, 6, 3, and 1, and hence could be designated as 5Hc, 2Hc, 6Hc, 3Hcand 1Hc, respectively. The chromosomes H1 and H6 were designated as 7Hcand 4Hc, respectively, by referring to SSR markers located on rye chromosomes.     

Identification of Zea diploperennis Chromosome Fragments Introgressed to Maize via Genomic in Situ Hybridization

Zea diploperennis Doebley (DP) chromosome fragments introgressed to maize ( Zea mays L. ) were identified by genomie in situ hybridization in the stable alloplasmic pure line 540 and its hybrid Fl, Yidan 6 was obtained by crossing with maize inbred line. Diaminobenzidine tetrahydrochloride (DAB) and fluorescence staining systems were utilized for detection of the hybridization signals respectively, and both of them gave almost the same results. The hybridization signals of DP elm)matin were showed on the long arms of two members for each of chromosomes 1, 2, 5, and 8 in pure line 540 and on those of only one member for each of chromosomes 1, 2, and 8 in Yidan 6. Not only located DP chromatin on the same chromosome arms but also their percentage distances from the centromeres to the hybridization sites were close to each other for chromosomes I and 2 between pure line 540 and Yidan 6. The percentage distance of the signal on the long ann of chromosome 8 was notably shorter in Yidan 6 than in 540. The differences of the signal distribution between pure line 540 and Yidan 6 were discussed.     

Physical Localization of Genes for CaM and Ca2+- ATPase in Rice by in situ Hybridization

CaM and Ca2 + -ATPase genes are important components of signal transduction chains which affect the regulation of' gene expression and development in plants. These two genes are tunctionally closely related. The rice ( Oryza sativa L. )cDNA probes C419 and SSU304 for these two genes, which are small single-copy ones and 0.8 and 0.3 kb in size respectively, were first physically mapped on rice chromosomes by biotin-labeled in situ hybridization. Both probes were detected on chromosome 5. The detection rate was 6.18 %, and the average chromosome ann ratios and standard deviations of detected chromosomes for probes C419 and SSU304 were 1.79 ± 0.06 and 1.91 ± 0.08 respectively. Probe C419 for CaM gene was located at the end of the long ann, and probe SSU304 for Ca2 + -ATPase gene -- on the short arm near the centromere. As it was reported before, they were closely linked in the high density genetic map. This demonstrated that there was a large discrepancy between the results of genetic and physical mapping of genes, and it indicated that the region between the functionally related genes could be the cold spot. What relationship there is between the region and gene expression is to be shudied further. The nonradioactive in situ hybridization technique about the physical mapping of low/single copy, short DNA fragments is also discussed.     

cDNA sequence, interspecies comparison, and gene mapping analysis of argininosuccinate lyase

A cDNA clone of the argininosuccinate lyase gene (ASL) was isolated from an adult human liver library by probing with synthetic oligonucleotide probes. This clone and a yeast genomic DNA fragment containing the ASL gene were sequenced using the M13-dideoxynucleotide method. Comparison of the yeast and human clones at the nucleotide and putative amino acid sequence levels indicated identities of 50 and 54%, respectively. The most conserved region of the yeast gene was used to detect human clones in the liver cDNA library to test phylogenetic screening capabilities of conserved genes. ASL was mapped to human chromosome 7pter----q22 using human-mouse somatic cell hybrid DNA and further mapped by in situ hybridization to chromosome 7cen----q11.2 on human metaphase chromosomes. The probe also detected a sequence on chromosome 22. Somatic cell hybrid DNA digested with PvuII revealed a mouse polymorphism between Balb/c and C3H mice in the ASL gene.     

Identification of Chromosomes from Multiple Rice Genomes Using a Universal Molecular Cytogenetic Marker System

To develop reliable techniques for chromosome identification is critical for cytogenetic research, especially for genomes with a large number and smaller-sized chromosomes. An efficient approach using bacterial artificial chromosome （BAC） clones as molecular cytological markers has been developed for many organisms. Herein, we present a set of chromosomal arm-specific molecular cytological markers derived from the gene-enriched regions of the sequenced rice genome. All these markers are able to generate very strong signals on the pachytene chromosomes of Oryza sativa L. （AA genome） when used as fluorescence in situ hybridization （FISH） probes. We further probed those markers to the pachytene chromosomes of O. punctata （BB genome） and O. officinalis （CC genome） and also got very strong signals on the relevant pachytene chromosomes. The signal position of each marker on the related chromosomes from the three different rice genomes was pretty much stable, which enabled us to identify different chromosomes among various rice genomes. We also constructed the karyotype for both O. punctata and O. officinalis with the BB and CC genomes, respectively, by analysis of 10 pachytene cells anchored by these chromosomal arm-specific markers.     

Two human genes encoding zinc finger proteins,ZNF12 (KOX 3) and ZNF 26 (KOX 20), map to chromosomes 7p22-p21 and 12q24.33, respectively

Summary Two members of the human zinc finger Krüppel family, ZNF 12 (KOX 3) and ZNF 26 (KOX 20), have been localized by somatic cell hybrid analysis and in situ chromosomal hybridization. The presence of individual human zinc finger genes in mouse-human hybrid DNAs was correlated with the presence of specific human chromosomes or regions of chromosomes in the corresponding cell hybrids. Analysis of such mouse-human hybrid DNAs allowed the assignment of the ZNF 12 (KOX 3) gene to chromosome region 7p. The ZNF 26 (KOX 20) gene segregated with chromosome region 12q13-qter. The zinc finger genes ZNF 12 (KOX 3) and ZNF 26 (KOX 20) were localized by in situ chromosomal hybridization to human chromosome regions 7p22-21 and 12q24.33, respectively. These genes and the previously mapped ZNF 24 (KOX 17) and ZNF 29 (KOX 26) genes, are found near fragile sites.     

Identification of a homeologous chromosome pair by in situ DNA hybridization to ribosomal RNA loci in meiotic chromosomes of cotton (Gossypium hirsutum).

In situ DNA hybridization with 18S-28S and 5S ribosomal DNA probes was used to map 18S-28S nucleolar organizers and tandem 5S repeats to meiotic chromosomes of cotton (Gossypium hirsutum L.). Mapping was performed by correlating hybridization sites to particular positions in translocation quadrivalents. Arm assignment required translocation quadrivalents with at least one interstitial chiasma and sufficient distance between the hybridization site and the centromere. We had previously localized a major 18S-28S site to the short arm of chromosome 9; here we mapped two additional major 18S-28S sites to the short arm of chromosome 16 and the left arm of chromosome 23. We also identified and mapped a minor 18S-28S site to the short arm of chromosome 7. Two 5S sites of unequal size were identified, the larger one near the centromere of chromosome 9 and the smaller one near the centromere of chromosome 23. Synteny of 5S and 18S-28S sites indicated homeology of chromosomes 9 and 23, while positions of the other two 18S-28S sites supplement genetic evidence that chromosomes 7 and 16 are homeologous.     

Characterization of Aegilops uniaristata chromosomes by comparative DNA marker analysis and repetitive DNA sequence in situ hybridization

RFLP analyses were performed on wheat-Aegilops uniaristata Vis. addition and translocation lines to confirm the identity of added N-genome chromosomes. Complete 1N, 3N, 4N, 5N and 7N chromosome additions were identified, while the complete long arm and only part of the short arm was identified for chromosome 2N. There were no wheat-like 4/5 and 4/7 translocations in the Ae. uniaristata chromosomes. Chromosome 3N carried an asymmetric pericentric inversion, and the translocation line was a product of centric fusion between the long arms of chromosomes 3B and 3N. Chromosome-specific RAPD and microsatellite markers were also identified for all the added Ae. uniaristata chromosomes available in this set of addition lines. A new genomic in situ hybridization protocol combining pre-annealing of probe and blocking DNA and prehybridization with blocking DNA was developed to differentiate the very closely related genomes of Ae. uniaristata and wheat. Hybridization sites for the repetitive DNA sequences pAs1, pSc119.2 and pTa71 were identified on the N-genome chromosomes of Ae. uniaristata using the fluorescent in situ hybridization technique. Results showed deviation from the previously published ideogram of this species. A new ideogram, which shows the hybridization sites for the above sequences, was produced in which the chromosomes are arranged according to their homoeologous group. Received: 23 April 1999 / Accepted: 6 August 1999     

Two subsets of human alphoid repetitive DNA show distinct preferential localization in the pericentric regions of chromosomes 13, 18, and 21

We have isolated and characterized two human middle repetitive alphoid DNA fragments, L1.26 and L1.84, which localize to two different sets of chromosomes. In situ hybridization revealed both repeats to have major and minor binding sites on the pericentric regions of several chromosomes. Probe L1.26 maps predominantly to chromosomes 13 and 21. Probe L1.84 locates to chromosome 18. Minor hybridization sites for both probes include chromosomes 2, 8, 9, and 20; in addition, L1.26 revealed minor sites on chromosomes 18 and 22. The binding to these sites strongly depends on hybridization conditions. In Southern blot hybridizations to total human DNA, both L1.26 and L1.84 give the same ladder pattern, with a step size of 170 bp, indicating their presence as tandem repeats, but with different band intensities for each probe. The chromosome-specific nature of particular multimers was confirmed by Southern blot analyses of a human-rodent hybrid cell panel. We conclude that L1.26 and L1.84, with their related sequences, constitute subfamilies of alphoid DNA that are specific for subsets of chromosomes and, in some cases, possibly even for single chromosomes.     

Homologies in human and Macasa fuscata chromosomes revealed by in situ suppression hybridization with human chromosome specific DNA libraries

We established chromosomal homologies between all chromosomes of the human karyotype and that of an old world monkey (Macaca fuscata) by chromosomal in situ suppression (CISS) hybridization with human chromosome specific DNA libraries. Except for the human chromosome 2 library and limited cross-hybridization of X and Y chromosome libraries all human DNA libraries hybridized to single GTG-banded macaque chromosomes. Only three macaque chromosomes (2, 7, 13) were each hybridized by two separate human libraries (7 and 21, 14 and 15, 20 and 22 respectively). Thus, an unequivocally high degree of synteny between human and macaque chromosomes has been maintained for more than 20 million years. As previously suggested, both Papionini (macaques, baboons, mandrills and cercocebus monkeys, all of which have nearly identical karyotypes) and humans are chromosomally conservative. The results suggest, that CISS hybridization can be expected to become an indispensable tool in comparative chromosome and gene mapping and will help clarify chromosomal phylogenies with speed and accuracy.by E.R. Schmidt     

Mapping of the transferrin gene in laboratory rats, mice and man by direct in situ hybridization

Mapping of the gene coding for transferrin was carried out in metaphase chromosomes from bone marrow of laboratory mice and rats as well as from PHA-stimulated human lymphocytes using direct in situ hybridization technique. Plasmid pRTf-17 carrying the insert of rat transferrin cDNA was nick-translated with [125I]dCTP and used as a specific hybridization probe. The total number of silver grains and their distribution along differentially stained chromosomes were determined in 464 metaphase plates (114, 263 and 87 from rat, mouse and man, respectively). The data obtained enable us to assign transferrin gene to chromosome 3 in human and chromosome 9 in mouse. For the first time, the rat transferrin gene was localized on chromosome 7. The most probable sites of transferrin gene localization are 7q31-34, 9F1-3 and 3q21 in rat, mouse and human chromosomes, respectively.     

Development and application of EST-based markers specific for chromosome arms of rye (Secale cereale L.)

To develop a set of molecular markers specific for the chromosome arms of rye, a total of 1,098 and 93 primer pairs derived from the expressed sequence tag (EST) sequences distributed on all 21 wheat chromosomes and 7 rye chromosomes, respectively, were initially screened on common wheat 'Chinese Spring' and rye cultivar 'Imperial'. Four hundred and fourteen EST-based markers were specific for the rye genome. Seven disomic chromosome addition lines, 10 telosomic addition lines and 1 translocation line of 'Chinese Spring-Imperial' were confirmed by genomic in situ hybridization and fluorescencein situ hybridization, and used to screen the rye-specific markers. Thirty-one of the 414 markers produced stable specific amplicons in 'Imperial', as well as individual addition lines and were assigned to 13 chromosome arms of rye except for 6RS. Six rye cultivars, wheat cultivar 'Xiaoyan 6' and accessions of 4 wheat relatives were then used to test the specificity of the 31 EST-based markers. To confirm the specificity, 4 wheat-rye derivatives of 'Xiaoyan 6 × German White', with chromosomes 1RS, 2R and 4R, were amplified by some of the EST-based markers. The results indicated that they can effectively be used to detect corresponding rye chromosomes or chromosome arms introgressed into a wheat background, and hence to accelerate the utilization of rye genes in wheat breeding.     

QTL mapping of resistance to Fusarium ear rot using a RIL population in maize

Fusarium ear rot is a prevalent disease in maize, reducing grain yields and quality. Resistance breeding is an efficient way to minimize losses caused by the disease. In this study, 187 lines from a RIL population along with the resistant (87-1) and susceptible (Zong 3) parents were planted in Zhengzhou and Beijing with three replications in years 2004 and 2006. Each line was artificially inoculated using the nail-punch method. Significant genotypic variation in response to Fusarium ear rot was detected in both years. Based on a genetic map containing 246 polymorphic SSR markers with average genetic distances of 9.1 cM, the ear-rot resistance QTL were firstly analyzed by composite interval mapping (CIM). Three QTL were detected in both Zhengzhou and Beijing in 2004; and three and four QTL, respectively, were identified in 2006. The resistant parent contributed all resistance QTL. By using composite interval mapping and a mixed model (MCIM), significant epistatic effects on Fusarium ear rot as well as interactions between mapped loci and environments were observed across environments. Two QTL on chromosome 3 (3.04 bin) were consistently identified across all environments by the two methods. The major resistant QTL with the largest effect was flanked by markers umc1025 and umc1742 on chromosome 3 (3.04 bin), explaining 13–22% of the phenotypic variation. The SSR markers closely flanking the major resistance QTL will facilitate marker-assisted selection (MAS) of resistance to Fusarium ear rot in maize breeding programs.