Chromosome G-banding in situ hybridization of RFLP marker umc58 linked with the gene hm1 dictatingHelminthosporium carbonum susceptibility1 in maize

The technique of simultaneous G-banding and in situ hybridization has been developed in plants for the first time. Using this technique, RFLP marker umc58 closely linked with the hm1 gene dictatingHelminthosporium carbonum susceptibility1 was localized onto 1L3 (chromosome 1, long arm, the third band from the centromere to the end of the arm), 5L5 and 9L5. The results demonstrated that umc58 was a triplicated sequence. It was deduced that umc58 probably was in a duplicated region that includes a part ofHelminthosporium carbonum susceptibility genes (hm1 and hm2), as the hybridization sites of umc58 in chromosomes 1 and 9 were those at which the genes localize. The techniques of simultaneous G-banding and ISH in plants are discussed.     

Cytogenetic Mapping of Disease Resistance Genes and Analysis of Their Distribution Features on Chromosomes in Maize

0　IntroductionMaizeisamongthemostintensivelystudiedspeciesingeneticsandoneofagronomicallythemostimportantplants.Therearemanydis easemicrobesandpeststoattackmaize,whichre sultsinlowproductionandbadquality .Withthedevelopmentofverydensegeneticmapconstruc tion ,avarietyoftheimportantdiseaseresistancegenesofmaizeincludingHelminthosporiumtur ciumPassresistancegenesHt1,Htn1andHt2 ,HelminthosporiummaydisNisikresistancegenesRhm1andRhm2 ,maizedwarfmosaicvirusresis tancegeneMdm1,wheatstreakmosaicvi…     

Tracing the location of powdery mildew resistance-related gene Stpk-V by FISH with a TAC clone in Triticum aestivum-Haynaldia villosa alien chromosome lines

Bacterial artificial chromosomes(BACs)or yeast artificial chromosomes(YACs)containing large inserts as probes for fluorescence in situ hybridization(FISH)have been used in the physical mapping of specific DNA sequences,especially for single-or low-copy sequences.Our earlier study identified Stpk-V,a powdery mildew resistance-related gene located on the 6VS chromosome arm of the wild grass Haynaldia villosa(tribe Triticeae),and obtained several Triticum aestivum–H.villosa alien chromosome lines carrying the Stpk-V gene.However,the precise physical location of the Stpk-V gene on chromosome 6VS is not known.In this study,we used TAC-FISH with TAC15 as the probe coupled with sequential genomic in situ hybridization(GISH)to determine the physical location of the Stpk-V gene in different T.aestivum–H.villosa 6V alien chromosome lines,including addition,substitution and translocation lines.The result indicated that the fraction length of the Stpk-V locus is 0.575±0.035 on the 6V chromosome short arm and this was confirmed by FISH using TAC15 as the probe for tracing the Stpk-V gene in other genetic stocks.The cytological mapping strategies used in this study will be of benefit for tracing the alien gene location in the course of introducing desirable traits from wild species.     

Physical location of the ricePi-5(t), Glh andRTSV genes by ISH of BAC clones

Mapping of low or single-copy sequences on plant chromosomes has proven difficult because of very low frequency of signal detection. Rice BAC library is being used widely in rice genome research due to its distinctive advantages over other library systems. In this study, two biotin-labeled rice BAC clones closely linked to a rice blast resistance, green leafhopper resistance and tungro spherical virus resistance gene,Pi-5(t), Glh, RTSV, werein situ hybridized to rice chromosomes. They were located on the long arm and short arm of chromosome 4 with FL value of 40% and 100% respectively. The frequency of signal detection reached 46.8% and 59.2%. The signal location were consistent with the selective marker on rice saturated molecular map. The results demonstrated the advantages to locate BAC clones to chromosomes byin situ hybridization and will facilitate the rice low or single-copy gene location by using the BAC library. Supported by the National Natural Science Foundation of China and the Doctorate Vesting Point Foundation of the Education Department of the People's Republic of China Yan Huimin: born in 1964, Lecturer     

Centromere inactivation in a dicentric rice chromosome during sexual reproduction

T0135 is a variant selected from the progeny of a rice line telotrisomic for the short arm of chromosome 11 （2n＋IIS＇）. Fluores- cent in situ hybridization （FISH） results indicated that T0135 contained two telocentric chromosomes, which have two centro- mere-specific molecular markers （5S rDNA） for chromosome 11; thus T0135 is a newly-described rice chromosome variant with two dicentric chromosomes, named 22＋11L-＋11L＇＋I IS.11S-＋I 1S-11S. （22 represents the 22 chromosomes excluding chromo- some 11 in the rice genome, ＂-＂ represents the centromere）. To investigate the genetic stability of the rice dicentric chromosomes during sexual reproduction, we observed the chromosome types in the progeny. Ninety-four percent of the progeny had the same chromosome type as the parental line. This result indicates that the dicentric chromosomes are mostly stable during mitosis and meiosis. Immunofluorescence analysis for centromere specific histone H3 （CENH3） revealed that only one centromere is active and the other centromere is inactivated in the rice dicentric chromosomes.     

Physical location of terminal markers belonging to five linkage groups in maize RFLP map using in situ hybridization

Ten terminal or subterminal RFLP markers belonging to linkage groups 1, 3, 5, 6, and 10 in maize RFLP map were physically locted onto maize mitotic chromosomes with in situ hybridization. All biotinylated probes from 600 to 2 250 bp were detected by DAB staining. The markers belonging to linkage groups 1, 3, 5, 6, and 10 correspondingly located at the chromosomes 1, 3, 5, 6, and 10. All of the tested markers except bnl6.25 and umc44 were duplicated sequences. Each of them was also labeled on another chromosome besides on the chromosome corresponding to its linkage group. The marker bnl3. 04 was triplicated sequences and the signals were detected on three nonhomologous chromosomes. In the tested ten markers, there were only four located at the ends of corresponding chromosomes. Others were located at sites midway along the chromosome arms or near the centromeres. The region covered by two terminal or subterminal markers in each of linkage groups 1, 3, 5, and 6 occupied 80.02%, 38.25%, 82.30% and 51.16% of the region of both short and long arms in chromosomes 1, 3, 5, and 6 respectively. Only two terminal markers of linkage group 10 covered the whole chromosome 10. In some linkage groups, two terminal or subterminal markers covered a short genetic distance but were physically distant, while two covering a longer genetic distance were physically closer. Supported by The National Natural Science Foundation of China and the Doctorate Vesting Point Foundation of the Education Committec of the People's Republic of China Mao Ninghui: born in 1986, used to be an MS student of Wuhan University in 1992–1995 and now is working in Fudan University, Shanghai 200433     

Mapping of two new brown planthopper resistance genes from wild rice

A brown planthopper (BPH) resistance line, B5, derived its resistance genes from the wild riceOryza officinalis Wall exwatt, was hybridized with Taichung Native 1, a cultivar highly susceptible to BPH. A mapping population composed of randomly selected 167 F₂ individuals was used for determining the BPH resistance genes by the restriction fragment length polymorphism analysis (RFLP). Bulked segregant analysis was conducted to identify RFLP makers linked to the BPH resistance genes in B5. The results indicated that the markers linked to BPH resistance are located at two genomic regions on the long arm of chromosome 3 and the short arm of chromosome 4, respectively. The existence of the two loci was further assessed by the quantitative trait locus (QTL) analysis. We located the two loci at a 3.2 cM interval between G1318 and R1925 on chromosome 3 and a 1.2 cM interval between C820 and S11182 on chromosome 4. Comparison with the BPH genes that have been reported indicated that the BPH resistance genes in B5 are novel. These two genes may be useful BPH resistance resource for rice breeding. Furthermore, the mapping of the two genes is useful for cloning the BPH resistance genes.     

Construction of a genetic map and location of quantitative trait loci for dwarf trait in maize by RFLP markers

Using F₂ population derived from the cross of tall inbred 7922 by dwarf inbred 5003, an RFLP linkage map of maize has been constructed, on which 85 markers are distributed among 10 linkage groups and span maize genome about 1827.8 cM with an average distance (24.4 cM) between markers. 106 F_2:3 lines of the population were grown in a 10 × 11 simple rectangular lattice design of one-raw plots with two replications and evaluated for plant height (PH). With interval mapping procedure, 5 QTLs controlling plant height have been identified and their genetic effects and gene action determined. 2 major QTLs with opposite effect have been discovered. One for increasing plant height isph1 which is located at chromosome 2 and accounts for 51.8% of the total phenotypic variation; the other for decreasing plant height isph3 which is located at chromosome 5 and accounts for 38.6% of the total phenotypic variation. The chromosomal location ofph3 might be the same as or close to the position ofbv1, a dwarf mutant of maize.     

Fine mapping of the <Emphasis Type="Italic">Ht2 (Helminthosporium turcicum</Emphasis> resistance 2) gene in maize

Fine mapping of Helminthosporium turcicum resistance gene Ht2 is extremely valuable for map-based cloning of the Ht2 gene,gaining a better knowledge of the distribution of resistance genes in maize genome and marker-assisted selection in maize breeding.An F2 mapping population was developed from a cross between a resistant inbred line 77Ht2 and a susceptible inbred line Huobai.With the aid of RFLP marker analyses,the Ht2 gene was mapped between the RFLP markers UMC89 and BNL2.369on chromosome 8,with a genetic distance of 0.9cM to BNL2.369.There was a linkage between SSR markers UMC1202,BNLG1152,UMC1149 and the Ht2 gene by SSR assay,Among the SSR markers,the genetic distance between UMC1149 and the Ht2 gene was 7.2cM,By bulked segregant analysis 7 RAPD-amplified products which were probably linked to the Ht2 gene were selected after screening 450 RAPD primers and converted the single-copy ones into SCAR markers.Linkage analysis showed that the genetic distance between the SCAR marker SD-06633 and the Ht2 gene was 0.4cM.From these results,a part of linkage map around the Ht2 gene was constructed.     

Breeding of T. aestivum-Ag. intermedium translocation lines with T. timopheevii cytoplasm and characterization by GISH

A male-sterileT. aestivum-Ag. intermedium partial amphiploid with cytoplasm ofT. timopheevii as a female parent was crossed to common wheat. The hybrid was backcrossed to the male parent several times continually and setf-crossed at last. Two stable lines with common wheat phenotype, H96269-2 and H96278, have been obtained. The chromosome numbers of the two lines are all2n = 42 in somatic cetls. By inoculation test, the two lines show a high levet of resistance to yetlow rust. Through genomicin situ hybridization (GISH) withAg. intermedium total genomic DNA as a probe, it is demonstrated that the two stable lines are all small segmental translocation lines, and the translocated chromosome segments fromAg. intermedium are located on the short arm terminals of wheat chromosomes. Genetics analysis suggests that the yetlow rust resistance gene(s) are probably located on the translocated chromosome segments ofAg. intermedium.     

Studies inin situ PCR detected by the BrdU antibody technique

Using the BrdU antibody technique followed by an immuno-chemical staining (BAT), the amplification of DNA fragments specific to human Y chromosome on cell specimen slides was efficiently detected. Whether direct BrdU incorporation into PCR products orin situ hybridization with PCR products on slides, the amplified target DNA fragments of specimen were visualized by BAT under the microscope. The availability of BAT and differences in the sensitivity and efficiency between BAT and dig-11-dUTP labeling in cellin situ PCR were discussed. Zhang Xiyuan: born in Oct. 1935, Professor, Current research interest in Cellular and Molecular Biology Supported by the National Natural Science Foundation of China     

Ubi1 intron-mediated enhancement of the expression of Bt cry1Ah gene in transgenic maize （Zea mays L.）

The cry1Ah gene was one of novel insecticidal genes cloned from Bacillus thuringiensis isolate BT8. Two plant expression vectors containing cry1Ah gene were constructed. The first intron of maize ubiqutinl gene was inserted between the maize Ubiquitin promoter and cry1Ah gene in one of the plant expressing vectors （pUUOAH）. The two vectors were introduced into maize immature embryonic calli by microprojectile bombardment, and the reproductively plants were acquired. PCR and Southern blot analysis showed that foreign genes had been integrated into maize genome and inherited to the next generation stably. The ELISA assay to T1 and T2 generation plants showed that the expression of CrylAh protein in the construct containing the ubil intron （pUUOAH） was 20% higher than that of the intronless construct （pUOAH）. Bioassay results showed that the transgenic maize harboring cry1Ah gene had high resistance to the Asian corn borers and the insecticidal activity of the transgenic maize containing the ubil intron was higher than that of the intronless construct. These results indicated that the maize ubil intron can enhance the expression of the Bt cry1Ah gene in transgenic maize efficiently     

Ipi-t,Ipi-3(t)在水稻中的物理定位及水稻第12染色体的识别

选用与水稻12染色体上Ipi-t,Ipi-3(t)和Pi-4(t)及着丝粒连锁的RFLP标记RZ670对水稻进行了荧光原位杂交。清楚显示了第12染色体着丝粒所在的位置,为水稻染色体的准确识别提供了一种新的方法。     

Analysis of F1 hybrid and BC1 monosomic alien addition line plants from Brassica oleracea × Sinapis alba by GISH

Sterile and semi-fertile F₁ plants were obtained by intergeneric sexual hybridization between paternal Brassica oleracea var. alboglabra (genome CC, 2n=18) and maternal Sinapis alba (genome SS, 2n=24), BC₁ plants were obtained by backcrossing between paternal B. oleracea and maternal semi-fertile F₁ plants. Genomic in situ hybridization (GISH) combined with dual-colour fluorescence in situ hybridization (dcFISH) showed that sterile F₁ plants contained 21 chromosomes consisting of one B. oleracea chromosome set and one S. alba chromosome set, belonging to expected hybrids, and semi-fertile F₁ plants contained 30 chromosomes consisting of two B. oleracea chromosome sets and one S. alba chromosome set. It is obvious that the semi-fertile F₁ plants belong to unexpected hybrids. 1―3 trivalents were detected at meiotic metaphase I of semi-fertile F₁ pollen mother cells (PMCs). Different separation ratios of S chromosomes were detected at anaphase I. A monosomic alien addition line (MAAL) was identified by GISH-dcFISH from BC₁ plants; it contained 19 chromosomes consisting of 18 C chromosomes and 1 S chromosome. At meiotic metaphase I, 9 divalents from B. oleracea and one univalent from S. alba could be detected. Sometimes, one putative C-S trivalent could also be detected. The achievement of B. oleracea-S. alba monosomic alien addition lines lays a foundation for gene introgression, location and cloning.     

Isolation and polymorphic frequency detection of a novel STR on human chromosome 14q24.3

A single copy fragment (FD14-ca 1) cantaining 22 CA repetitive units was isolated with (CA)₁₅ oligonucleotide probe from a human chromosome 14q24.3 probe pool generated by microdissection, and proved to be a new short tandem repeat (STR) sequence through querying in GenBank of NCBI. The STR has 11 alleles in Chinese. and its polymorphic information content (PIC) is 0.85. Mendelian segregation was shown in 2 Chinese pedigrees with two generations; This STR has been accurately relocalized on chromosome 14q24.3 by fluorescencein situ hybridization (FISH). The accession numbers of this STR in GDB and Gnknk database are D14S1435 and G31413 respectively. This STR would be able to be regarded as a novel genetic marker which can increase the genetic map accuracy in this chromosome region and improve the gene diagnosis on some genetic diseases located in chromosome 14q24.3 band.     

Repetitive DNA sequences from the X chromosome of the spiny eel (Mastacembelus aculeatus)

To investigate the characters of repetitive DNA sequence in the sex chromosomes of the spiny eel (Mastacembelus aculeatus), the X chromosomal library was screened and a family of repetitive sequence, consisting of Ma 1-Ma 6, was isolated. The fluorescence in situ hybridization (FISH) result confirmed that Ma 1 - Ma 5 dispersed over sex chromosomes and all autosomes, whereas, Ma 6 is sex chromosome-specific and distributed only on the C-band positive regions of X chromosome, and Ma 6 maybe the main components of the heterochromatic regions of X chromosome. This study provides additional information about the evolution of sex chromosomes in lower vertebrates such as fish.      

The expression of vasa gene during zebrafish ( Danio rerio ) oogenesis

vasa gene expression pattern during oogenesis of zebrafish was examined usingin situ hybridization and fluorescent quantitative RT-PCR. During zebrafish oogensis,vasa mRNA is expressed strongly and uniformly distributed in the cytoplasm in stage II oocytes, followed by a distribution among vacuome in stage III. Later in stage IV and V,vasa mRNA is enriched at the cortex and finally localized at the cortex. The fluorescent quantitative RT-PCR shows that the quantity ofvasa mRNA decreases from stage II to stage III, but remains relatively invariable from stage III to stage V. The observed differences invasa mRNA expression in the different stages of zebrafish oogenesis suggest thatvasa gene plays an important role during oogenesis. Foundation item: Supported by the National Natural Science Foundation of China (30370744, 30150005) Biography: XIANG Fang (1979-), male, Master candidate, research direction: molecular development of animals.     

Generating of rice OsCENH3-GFP transgenic plants and their genetic applications

In order to investigate rice functional centromeres, OsCENH3-GFP chimeric gene was constructed and transformed into the indica rice variety, Zhongxian 3037, mediated by Agrobacturium. The integration of the exogenous genes in the transgenic plants was confirmed by PCR and Southern blotting. The transgenic plants grow normally during their whole life time, just like Zhongxian 3037. No significant defects were detected in either mitosis or meiosis of the transgenic plants. The overlapping of GFP signals and anti-CENH3 foci in both mitotic and meiotic cells from T0 and T1 generation plants indicated that GFP had been successfully fused with CENH3, so the GFP signals can well represent the CENH3 locations on each chromosome. To evaluate the applicability of the transgenic plants to other genetic studies, fluorescence in situ hybridization （FISH） using rice centromeric tandem repetitive sequence CentO as the probe was conducted on the zygotene chromosomes of pollen mother cells （PMCs）. It has been revealed that the GFP signals are overlapping with CentO FISH signals, showing that CentO is one of the key elements constituting rice functional centromeres. Immunofluorescent staining using anti-o-tublin antibody and anti-PAIR2 antibody on the chromosomes during mitosis and meiosis stages of the transgenic plants further reveals that OsCENH3-GFP transgenic plants can be widely used for studying rice molecular biology, especially for tagging functional centromeres in both living cells and tissues.     

Isolation and identification of the three rice monotelosomics

In order to obtain rice monotelosomic, the progeny of 24 telotrisomics, derived from an indica rice variety, Zhongxian 3037, were screened. The variants that differed morphologically from the diploids and the original primary trisomics as well as the telotrisomics were collected for cytological identification. The variants with 24 chromosomes were selected according to the prometaphase chromosomes. From these variants, three monotelosomies with one chromosome arm deletion in each were verified by fluorescence in situ hybridization （FISH） using a rice centromeric BAC clone of 17p22 as a marker probe. The three monotelosomics were derived from telotrisomic 1S, 4L and 11L, respectively. Further identification was conducted on the prometaphase or pachytene chromosomes of the three variants, which were probed with the same centromeric BAC clone together with the corresponding chromosome arm specific makers, a0059H02 （on the short arm of chromosome 1）, a0034E24 （on the long arm of chromosome 4）, and a0071H11 （on the long arm of chromosome 11）. The results indicated that the telocentric chromosomes in the three monotelosom. ics were derived from their respective corresponding telotrisomics. According to the telocentric chromosomes of the variants, they were monotelosomic 1S （one long arm of chromosome 1 was lost）, monotelosomic 4L （one short arm of chromosome 4 was lost） and monotelosomic 11L （one short arm of chromosome 11 was lost）, respectively.     

Rapid diagnosis with FISH for chromosomal abnormality of fetal pyelectasia

Fluorescence in situ Hybridization （FISH） was used to investigate whether the chromosome of the fetus prenatally diagnosed as pyelectasis was normal or not. Amniotic fluid was taken from the pregnant woman whose fetus was detected with pyelectasia by prenatal examination. The chromosome of the amniotic fluid cell without culture was examined with FISH. The result shows that compared with the traditional amniotic fluid cell culture, FISH has the advantages of more rapid, higher sensitivity and specificity, and was 10-12 days earlier to complete the diagnosing than the traditional method. The fetuses detected chromosomal abnormality in each groups were induced during the middle and late trimester, while those fetuses with normal chromosome continued pregnancy, the rate of spontaneous disappearance of pyelectasia decreased as the severity of pyelectasia increased. FISH can satisfy the urgent need in the clinical prenatal diagnosis due to its rapidity to determine whether fetus with pyelectasia was accompanied with chromosomal.