Chromosome Inheritance Mechanism Based On Repetitive Sequences Of Erianthus Arundinaceus And Development Of Molecular Marker

Sugarcane(Saccharum spp.)is the most important sugar crop throughout the world.However,due to inter-species or intervarietal crossing for a long period,the resistant ability to biotic and abiotic stress was declining sharply in sugarcane cultivars.Erianthus arundinaceus has great utilization potential in sugarcane breeding program as one of the closely related genus of Saccharum with many desirable characters.Thus,it is urgent need to broad the sugarcane basic background by distant hybridization for introgressing the relative species into sugarcane.So far,sugarcane breeders had succeed to introgress the E.arundinaceus chromatid into sugarcane.A specific inheritance of chromosome doubling was found in the BC1 progeny between sugarcane and E.arundinaceus,but the mechanism was not clear.In this study,a specific repetitive DNA library of E.arundinaceus was built by suppression subtractive hybridization(SSH).These repetitive DNA clones were identified and selected for chromosome identification by fluorescence in situ hybridization(FISH)from SSH library.Based on this,we constructed the FISH chromosome karyotype.In the light of the chromosome identification of E.arundinaceus in these two generations,we discussed the mechanism in the F1 and BC1 progeny between sugarcane and E.arundinaceus.Furthermore,we analyzed the performances of both yield traits and quality traits in the F1 and BC1 progeny between sugarcane and E.arundinaceus to investigate the impact of this mechanism.At last,we developed the specific molecular markers that covering all the chromosomes to identify E.arundinaceus chromosomes or chromosomal segments.The main results and conclusions were as follows:1.Construction of specific genomic DNA segment library from E.arundinaceusThe subtracted library of specific genomic DNA segments from E.arundinaceus was constructed by suppression subtractive hybridization(SSH)method to remove the homologous sequences and to efficiently enrich the different sequences between sugarcane and E.arundinaceus.The reverse dot blot were performed to screen out the low copy sequences in the different sequences of E.arundinaceus,then the repetitive sequences of E.arundinaceus were obtained.The specific genomic DNA segment library from E.arundinaceus had the sequences for chromosome identification and development of specific molecular marker from E.arundinaceus,which could be used to investigate the chromosome inheritance and trace E.arundinaceus chromosomes or chromosomal segments in sugarcane background.2.E.arundinaceus chromosome identification and establishment of FISH chromosome karyotype in E.arundinaceus(1)FISH was performed by using 5S rDNA and 45S rDNA as probes in chromosome at metaphase,we found that the signal loci of both 5S rDNA and 45S rDNA were six,but 45S rDNA was located in the short arm end of six chromosomes.Among them,three loci were larger,the remaining three loci were smaller,suggesting the copy number of 45S rDNA was different.5S rDNA was located near the long arm of chromosome centromere,all the 6 loci were similar size,suggesting the copy number of 5S rDNA was similar.However,5S rDNA and 45S rDNA could be used to identify just 2 chromosomes,but not the remaining 8 chromosomes.(2)The repetitive sequences from the subtracted library of specific genomic DNA segments were mapping by FISH,we found most repetitive sequences were located at the ends of chromosomes or centromere,and the copy number of various clones were different.These two types of repetitive sequence probes were mixtured as probe combinations and screened by FISH,a good result was obtained from a probe combination.In the probe combination,these four repetitive sequences generated a similar banding pattern among the homologous chromosomes,but a distinctive similar banding pattern among non-homologous chromosome.These four repetitive sequences of the probe combination were successfully developed for simultaneously accurate identification of each of 10 chromosomes.Therefore,a relatively accurate chromosome karyotype was constructed.(3)Based on the system of chromosome identification from this probe combination,the specific mechanism in the F1 and BC1 progeny between sugarcane and E.arundinaceus was investigated.We found that the BC1 progeny between sugarcane and E.arundinaceus inherited distinct chromosomes from their F1 female parents.In the light of the relationship of chromosome inheritance between these two generations,the mechanism was analyzed.We determined the hypothesis Ⅱ(FDR+NSC)was the possible mechanism,but ruled out the hypothesis Ⅰ(NHC+SDR).In addition,wide segregation was occurred in the BC1 progeny between sugarcane and E.arundinaceus in the the performances of both yield traits and quality traits,which may be relevant to more plentiful combination of E.arundinaceus chromosomes in FDR+NSC.3.Development of specific molecular marker from E.arundinaceusTwo repetitive sequences were identified by FISH,Ea086 was located at all the chromosomes but not the ends of chromosomes,Ea009 was located at the ends of all chromosomes but not 45S rDNA.Hence,we designed the specific E.arundinaceus primers based on Ea086,Ea009,and EaITS,and developed the specific molecular markers of E.arundinaceus.The amplification profiles of specific primer were analyzed in E.arundinaceus,Saccharum officinarum,Saccharum robustum,Saccharum spontaneum,Saccharum sinense,Saccharum bareri,and sugarcane cultivars,suggesting these three primers were specific in these three specific molecular markers.To determine the stability result of specific primers,we conducted PCR assay in the F1,BC1,BC2,and BC3 progeny between sugarcane and E.arundinaceus,which were identified by GISH,suggesting the specific primers of both Ea086-128 and Ea009-257 could be used to steadily identify putative progeny.EaITS-278 could be used as a molecular marker to trace the 45 rDNA-bearing E.arundinaceus chromosome.At last,we used the specific primers of both Ea086-128 and Ea009-257 to identify the putative BC4 progeny between sugarcane and E.arundinaceus from four different cross combinations.In brief,these specific molecular markers could be used to rapidly identify E.arundinaceus chromosomes or chromosomal segments in sugarcane background.