| In Brassica,self-incompatibility(SI) recognition is controlled by the multiallelic gene complex (S-haplotypes) at the S-locus,which controls the SI signal transduction pathway.However,physical location on Brassica oleracea chromosomes and linkage relationships with S-locus for SI genes hasn't been identified up to now.In addition,Brassica oleracea is one of the three basic species in Brassica.Brassica oleracea chromosomes widely participate in somatic synapsis in the produce course of ecotypes and artificial crossing cultivars of Brassica,so recombination rate among chromosomes is very high,which observably influence transfer and genetic variation of SI genes among Brassica.Therefore,chromosomal localization of SI genes has important significance in the study and application for Brassica SI.In this paper,the localization of MLPK and SSP genes for self-incompatibility of Brassica oleracea on prometaphase chromosomes,early pachytene chromosomes and extended DNA fibers(EDFs) was conducted successfully by fluorescence in situ hybridization.We hope that this research will be helpful to further study on Brassica SI mechanism and genetic variation of SI genes.And the technique and results of localization will lay the foundation for constructing the high-density physical map and cytogenetic map.The main study results were as follows:1.Technique system of FISH targets preparation in Brassica oleracea were established through optimizing correlative parameters during the preparation procedure.Three kinds of cytological targets with different extension degree were successfully prepared including prometaphase chromosomes,early pachytene chromosomes and extended DNA fibers,which were applied to fluorescence in situ hybridization.(1) Treating Brassica oleracea roots with varying temperature was adopted to conduct the synchronized induction of cell division at prometaphase.And division index of prometaphase reached upwards 80%.(2) The best pretreatment could be obtained with 0.002mol/L 8-hydroxy quinoline at 20℃for 1h.(3) Treatment with the enzyme mixture of 2%cellulose and 2%pectase at 37℃for 2h or so and low filter with 0.075mol/L potassium chloride at 25℃for 30min performed best at preparing prometaphase chromosomes.Pachytene chromosomes of well spread and clear background were obtained through the treatment with the enzyme mixture of 2%cellulose and 2%pectase at 37℃for 3h or so.(4) Nuclei with symmetrical distributing and equal density were obtained by improved method. And the treatment time with STE should be at range from 5min to 8min.Smooth and parallel extended DNA fibers were successfully prepared using molecular combing.2.FISH system be applied to three kinds of cytological targets with different extension degree was founded through the study of correlative parameters during FISH.The procedures were basically identical in pro-metaphase and early pachytene chromosomes FISH.However,the EDFs slides were not pretreated and pre-denatured;there were slight differences in the probe concentration and the temperature of post-hybridization washing,the rest of FISH procedures were applied to three kinds of cytological targets.The hybridization effect demonstrated the feasibility of this method.(1) Whether slides were pretreated completely or not had directly effect on the rate of detected hybridization signals and the signal-to-noise ratio.(2) The best hybridization results derived from the denaturation at 70℃for 2min,and pro-metaphase chromosomes were easily affected by the denaturation time than early pachytene chromosomes.(3) The best hybridization mixture(20μL) consisted of 50%deionized formamide,7.5%dextran sulfate,2×SSC,1.5ng/μL DNA for each probe,2%SDS,and 0.5μg/μL herring sperm DNA.(4) Stable hybridization signals could be attained using the three-step washing at 37℃for single or low copy probe,with lower background and higher the signal-to-noise ratio.Nonspecific signals would be decreased and the signal-to-noise ratio be also improved if the washing temperature was enhanced to 40℃for EDFs.(5) The signal cascade amplification using antibody conjugate was very important to improving the rate of detected hybridization signals.3.Hybridization signals of MLPK and SSP probes were detected on only a pair of homologous chromosomes.The results indicated that MLPK probe was hybridized onto the short arm of a pair of homologous submetaeentric chromosomes,and SSP probe was hybridized onto the long arm of a pair of homologous subterminal chromosomes with the satellite.Repeated FISH indicated that both MLPK and 5S rDNA probes were hybridized onto the same chromosomes.Doublet of fluorescent signals was not observed on both chromatids of a chromosome.The hybridization signals of MLPK probe were observed in 11.8%examined cells,and those of SSP probe were observed in 9.1% examined cells.A pair of homogeneous hybridization signals was only observed in an examined cell for the FISH of MLPK and SSP probes on early pachytene chromosomes in Brassica oleracea.The detection rate of MLPK probe was about 31.4%,and those of SSP was about 20.4%.4.In this study,the highest spatial resolution of the EDF was about 1.5kb,and a single green spot signal was represented on each single EDF.It was primarily inferred that both MLPK gene and SSP gene might be located at a single-copy locus on one single EDF(chromosome).5.According to karyotype standard of Armstrong et al and chromosomes parameters,it was primarily inferred that MLPK gene might be located on the chromosome 2,the percent distance from centromere to the signal point was about 53.41±3.16;and SSP gene on the chromosome 7,the percent distance from centromere to the signal point was about 78.36±4.26.Hybridization signals from three kinds of cytological targets with different FISH resolution showed that both MLPK and SSP gene might be located at a single-copy locus in Brassica oleracea genome.The results presumably revealed that neither MLPK nor SSP is linked to S-locus.And they locate respectively on the different chromosomes in Brassica oleracea. |
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