| Sugarcane is one of the world's most important crops,producing about 70%of the world's total sugar.S.spontaneum contributed to genetic background of stress resistance for modern sugarcane cultivar.The S.spontaneum chromosome number is large(2n = 40-128),and their chromosomes are small with similar morphology.Therefore,it is very challenging to discern between the different chromosomes based on traditional cytogenetic methods,this hinders the development of the research work in cytology.In this study,based on the high-collinear between Saccharum and sorghum,we adopted the method of comparative genomics to screen the low copy sequence of S.spontaneum,and to develop the chromosome-specific cytological markers.The results are as follows.:(1)Uesd comparative genomics,we searched the low copy BAC clones sequences based on the sorghum genome sequences as a reference.Total 164 single low copy sequences with single BLAST hit in sorghum genome corresponding to the different arms of sorghum NO.1-NO.10 chromosomes were selected as candidate probes for BAC-FISH.Furthermore,a two-step PCR method was used to screen the 2D dimension pools of the BAC library.Totally,and 84 PCR positive clones were achieved.(2)The DNA of 84 positive clones were labeled as probes for BAC-FISH analysis,and 42 probes presented specific signals on S.spontaneum(AP85-441,2n = 4x= 32)chromosomes with the number of signals varied from 1 to 4.Of the 42 BAC probes,2 probes located on Sb2,Sb5,Sb9 and Sb10,4 probes located on Sb4 and Sb8,5 probes located on Sb1,6 probes located on Sb8,7 probes located on Sb3,and 8 probes located on Sb6.This specific probes provide a basis for the S.spontaneum chromosome identification and karyotype analysis.(3)Ten BAC probes corresponding to the nine sorghum chromosomes were identified to be chromosome specific cytological markers and were used to identify the chromosomes:BAC-52(Sb1),BAC-58(Sb1),BAC-65(Sb2),BAC-80(Sb3),BAC-97(Sb4),BAC-117(Sb6),BAC-125(Sb7),BAC-134(Sb8),BAC-141(Sb9)and BAC-155(Sb10).In previous our study,a genetic map based on the F1 population of S.spontaneum revealed that Sb05 was divided into two segments,and the two segments were merged with Sb06 and Sb07 respectively.Besides,that Sb08 was also divided into two segments,and the two segments were merged with Sb02 and Sb09 respectively.Based on the BAC-FISH results and the genetic map,the eight S.spontaneum chromosomes could be idenfitied by eight chromosomal specific BAC probes:SsChrl(BAC-52,BAC-58),SsChr2(BAC-65,BAC-134),SsChr3(BAC-80),SsChr4(BAC-97),SsChr5(BAC-117),SsChr6(BAC-125),SsChr7(BAC-141)and SsChr8(BAC-155).(4)After divergence of sorghum and S.spontaneum,S.spontaneum has serveral times chromosomal recombination events,and the basic chromosome was reduced from x = 10 to x = 8.BAC-13 was located in the recombination interval of sorghum chromosome 7,and was used to test the genomic divergence of Saccharum speices.BAC-13 was labeled probe and hybridized with different polyploid S.spontaneum,AP85-441(2n = 4x = 32),SES208(2n = 8x = 64),AP83-108(2n=8x ? 64)and Fujian 89-1-16(2n = 12x = 96),obtained chromosome-specific signals,2,4,4 and 4.However,there were more than 10 signals on the pentaploid S.spontaneum(Nepal-Tibet,2n = 5x= 40)and other species,S.robustum(2n = 8x = 80)and S.officinarum(2n = 8x = 80).The difference in the number of hybridization signals between different ploidy S.spontaneum,indicate that repeated sequences were eliminated to maintain the stability of the S.spontaneum genome structure during the polyploid progress.In addition,the results from different Saccharum species is similar,such as Nepal-Tibet,S.robustum,S.officinarum,illustrated that BAC-13 occurred different evolution after separation of the three Saccharum species. |
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