The FISH Analysis Of 6 Members Of MADS-box Gene Family And RDNAs Of Hevea Brasiliensis

Hevea brasiliensis,it is an important tropical crop.The latex,which extracted from Hevea brasiliensis,is the main source of natural rubber,and it plays an important role in industrial raw material and strategic material of our country.MADS-box gene family is widely distributed in eukaryotes,it mainly participates in flower morphogenesis and plays an important role in regulating reproductive growth.rDNA(ribosomal DNA)is a highly conserved tandem repeat sequence,which is responsible for transporting and producing ribosomal RNA(r RNA)in cells,and it is directly related to the formation of nucleoli.Due to the characteristics of rDNA,the distribution of rDNA loci in different germplasm may be different.The difference in the distribution of rDNA between or within species can show the difference of chromosomes,reflect the closeness of the genetic relationship between species.The more similar the distribution pattern of rDNA is,the closer the relationship between species will be.In this study,we used fluorescence in situ hybridization(FISH)to locate six members of MADS-box gene family(HbAGL8,HbAG15,HbAGL30,Hb TT16,HbAP1 and Hb SVP1)on the chromosome of‘Reyan7-33-97’,and discussed 5S and 45 S rDNA among different Hevea brasiliensis germplasm chromosome(3 cultivated germplasm ‘Reyan7-33-97’,‘RRIM600’,‘PR107’and 3 wild germplasm ‘XJA04570’,‘XJA04616’ and ‘XJA02089’)by comparative genomic analysis.In addition,we analyzed the karyotypes of 3 wild germplasms,and combined the data of cultivated germplasm reported by the predecessors for karyotype comparison and analysis.This research provides molecular cytogenetic basis for elucidating its origin,phylogeny and evolution,constructing standard karyotype and breeding excellent hybrids of Hevea brasiliensis.The results are as follows:(1)Through bioinformatic methods,we obtained the DNA sequences of 6 members of MADS-box gene family、Hevea brasiliensis 5S r RNA gene and Wheat 25S-18 S rDNA gene on NCBI.By designing primers,screening and probe labeling,we constructed specific probes for 6 genes of MADS-box and specific DNA probes for Hevea brasiliensis5 S rDNA and 45 S rDNA.(2)We located the six gene on different chromosomes of ‘Reyan7-33-97’,among which HbAGL15,HbAG8,HbAG30 and Hb SVP1 were located on the long arms of chromosomes 4,5,7 and 8 with PDCS(percent distance from centromere to the signal site)11.85,39.71,48.94 and 6.70,respectively.Hb TT16 and HbAP1 genes were located on the short arms of chromosomes 1 and 13 with PDCS 22.19 and 18.01,respectively.The results of the 6 gene were all located on different chromosomes,we speculated that these genes may be genetically independent from each other;we also discussed the linkage between the MADS-box gene family and other different functional genes of Hevea brasiliensis;and we matched the chromosome location of MADS-box gene with its corresponding scaffold which is helpful for the assembly of Hevea brasiliensis genome.(3)We analyzed the karyotypes of 3 Hevea brasiliensis germplasms ‘XJA04570’,‘XJA04616’ and ‘XJA02089’,and obtained the data of karyotype asymmetry coefficient,average arm ratio,chromosome type,chromosome composition and karyotype,and we combined the data of cultivated germplasm ‘Reyan7-33-97’,‘RRIM600’ and ‘PR107’reported by the predecessors for karyotype comparison and analysis,we found that the karyotype of wild germplasm were relatively symmetrical,the cultivated germplasm were relatively asymmetric;the evolution of wild germplasm was lower than cultivated germplasm.(4)Our results showed that the number of 5S rDNA loci on chromosomes of 6 Hevea brasiliensis germplasm was 2～3 pairs,the signal loci were mainly distributed near the centromere and near the end of the chromosome,and a small number of loci were distributed on chromosome arms,and some size of the signal loci were different,indicating that distribution pattern was highly polymorphic.45 S rDNA has 2 pairs of loci on chromosomes of 6 Hevea brasiliensis germplasm,mainly distributed in the end of the short arm or secondary constriction and satellite area of the chromosome,but they are located on different chromosomes,and some size of the signal loci were different.(5)According to the location results of rDNA in 6 Hevea brasiliensis germplasms,we analyzed the size,number and distribution pattern of the signal sites,and found that the copy number of rDNA repeat sequence was quite diversity between different Hevea brasiliensis germplasm and between different chromosomes of the same germplasm.It was provided strong evidence to verify the close relationship between cultivated germplasm‘Reyan7-33-97’ and ‘RRIM600’ and ‘PR107’,and speculated that wild germplasm had more distant genetic relationship with each other,and cultivated germplasm had more distant genetic relationship with wild germplasm.