Construction Of Chromosomal Map In Brassica Oleracea And Localization Of SI Genes

Fluorescence in situ hybridization (FISH) is a powerful cytogenetic technique that can be used to detect and localize the genes on chromosomes, which is widely used to study the localization of genes and the structure of chromosome, construct the cytogenetic and chromosomal maps. Brassica oleracea is one of the three diploid Brassica species and researched by multinational Brassica Genome Project (MBGP). Therefore, the main content of molecular cytogenetic study is to construct the chromosomal or cytogenetic maps of B.oleracea and directly show the positions of genes and genetically maped markers on chromosomes by FISH. Simultaneously, self-incompatibility is one of the most important means to prevent selfing and thus encourage outcrossing. Besides, the mechanism of signal transduction is controlled by a single locus termed S, which has many different alleles, it is closely related to the other signal transduction elements (MLPK,ARC1, SSP, et al). However, the linkage relationships between these elements and SI genes and their positions on chromosomes are poorly described and further reseached. In this research, self-incompatibility B.oleracea var. acephala was used as the experimental material, The probes of the repeated sequence 5S rDNA, markers (pO147, pR94 and pN120) and SI genes (MLPK and SRK) were located on chromosomes of B.oleracea by FISH,, our aim is to construct chromosomal and cytogenetic maps and determine the localization on chromosome and the linkage relationship of SI genes, it is very important to MBGP and the mechanism of SI.The study results were as follows:1. Target DNA preparations:it was successfully prepared different extension degree in B.oleracea root tips, buds and Young leaves, including metaphase chromosomes from the mitotic divisions of root tips and tapetal cells, pachytene chromosomes from the pollen mother cells and the extended DNA fibers (EDF). A. Preparations of metaphase chromosomes of root tips:the Synchronous processing of B. oleracea root tips with varying temperature greatly improved cell divisions at metaphase; It could obtain the best pretreatment effect with 0.002 mol/L 8-hydroxy quinoline for 1 h; the root tips were treated with digestion for 40-90 min in the mixture of 2%(W/V) Cellulase and 2%(W/V) Pectinase at 37℃, it was obtained the Good dispersion and low background cell division of root tips.B. Preparations of metaphase chromosomes of tapetal cell and pachytene chromosomes: flower buds were taken from plants and stored in 70%ethanol at 4℃for up to 3 months; the anthers were digested in enzyme mixture 2%cellulase and 2%pectinase at 37℃for 60 min.C. Preparations of extended DNA fibers:nuclei with uniform distribution, equal density and little impurities were obtained by the method of homogenization; the good extension, moderate density and parallel DNA fibers were successfully prepared by molecular combing.2. FISH system:it was found to be applied to targets DNA of different extension degree and different types of probes by the research of correlative parameters during FISH, the procedures of FISH were basically identical in metaphase and pachytene chromosomes. However, the slides of EDFs were not pretreated and pre-denatured.A. Pretreatment of targets DNA on slides:in order to improve the signal-to-noise ratio in hybridization, it must be done by incubation the preparation in RNase A (100μg/mL) at 37℃for 60 min; the protein that surrounds the target nucleic acid was digested for 40 min at 37℃in 2×SSC by pepsin (500 ug/ml); the good hybridization results were obtained by the denaturation for 2min in 70%deionized formamide at 70℃.B. The hybridization mixture:50%deionized formamide; 7.5%dextran sulfate; 2x SSC,1.5 ng/μL probes,0.5%SDS, and 0.5/μg/μL herring sperm DNA.C. Posthybridization washes:for repetitive probes, the slides were washed at 42℃for 8 min; for unique probes, it is washed at 37℃for 5 min; if target DNA is DNA fibers, it is washed at 40℃for 5 min..3. The karyotype analysis of metaphase chromosomes:the chromosome numbers were 18, the karyotype formula was K(2n)=18=12m+6Sm, chromosome 1,2,4,5,6, and 8 are metacentric chromosome, chromosome 3,7,9 were sub-metacentric chromosome, chromosome 7 has a statellite in the short arm; The chromosome type of B.oleracea belonged to 2A type, that is a symmetrical karyotype.4. High-resolution 5S rDNA-FISH:B.oleracea var. acephala was used as the experimental material,5S rDNA probe was labeled by PCR-DIG (PCR-Digoxigenin), By FISH,5S rDNA probe was located on the metaphase chromosomes of tapetal cell, pachytene chromosomes and extended DNA fibers (EDF). The aim is to localize 5S rDNA on the chromosomes of Brassica oleracea var. acephala by FISH and estimate the copy number of 5S rDNA, further provide a basis for the location of genes and the construction of cytogenetic map of chromosome 2 by FISH. In the metaphase chromosomes of tapetal cell and pachytene chromosomes, It was observed that three closely adjacent 5S rDNA hybridization signal sites (a, b, c) located near to the centromere in the long arm of the submetacentric chromosome 2, the intensity of signals is b>a>c. In the extended DNA fibers, it was estimated that the physical size of three different stretches of beads-on-string (a, b, c) is of the order of-257 kb,359 kb and 134 kb respectively. It was proved that B.oleracea var. acephala contains three tandem repeat sites, and estimated that the copies number of three 5S rDNA locus are about 510,712 and 266 respectively.5. Construction of a cytogenetic map of B.oleracea chromosome 2. Three markers (pO147, pR94 and pN120) mapped to linkage group 04 and 5S rDNA as FISH probes, which hybridized to the metaphase chromosomes from tapetal cells and pachytene chromosomes from PMC by double FISH. The aim is to construct the cytogenetic map of B. oleracea chromosome 2 and show the positions of genetically mapped markers on chromosome.A. Labeled probes:the length of probes (pO147, pR94 and pN120) is about 8 kb,.5kb,3.2kb, respectivelyB. The relationship between the rate of detected hybridization signals and the probe length: when a 2 kb fragment was used as a probe, the rate of detection is 9.8%; However, the probes are 4.5kb and 8 kb in length, the rate of detection is 16.7%and 20.2% respectively.C. The cytogenetic map of B.oleracea chromosome 2:The probe pO147 was detected on 2S and mapped at cytogenetic position 2S.13; the probes (pR94 and pN120) mapped at cytogenetic position 2S.9 and 2S.15, respectively.6. Localization of MLPK and SSP genes for self-incompatibility:the MLPK probe was hybridized on the short arm of chromosome 2 and mapped at cytogenetic position 2S.17; SRK signal was detected at a single locus on the long arm of chromosome 4 and mapped at cytogenetic position 4L.80. The rate of the hybridization signals of MLPK and SRK probe is 10.2%and 12.2 in metaphase chromosomes, respectively, in the pachytene chromosomes, however, the rate is 26.9%and 33.7%, respectively.7. Construction of chromosomal map:the markers (pR94,pO147,pN120) on linkage group 04, self-incompatibility genes MLPK and 5S rDNA were detected on chromosome 2, SRK is located in the long arm of chromosome 2.