| Bacterial wilt(BW),caused by Ralstonia solanacearum,is the most important soilborne bacterial disease of potato which commonly occurs in tropical and subtropical regions.However,there are no effective means to control the disease that usually results in a serious yield loss.Cultivated potato(Solanum tuberosum L.)is lack of bacterial wilt resistance,worse is the specific cross incompatibility that impedes the use of wild and closely related species,development of the new resistant germplasms is,hence,the most priority.We previously regenerated the somatic hybrids of potato + eggplant,part of those have BW resistance derived from the eggplant fusion parent.Aiming at to explore the genetic basis of BW resistance for efficient use of these novel germplasm,the present research conducted the genomic in situ hybridization(GISH)and,in combination of the speciesspecific marker selection,clarified the genome composition of the somatic hybrids;associated it to the BW resistance of the hybrids and identified the resistance loci introduced from eggplant;carried out the transcriptomic and expression analyses between the resistant and susceptible somatic hybrids and pointed out the candidate gene,as well as the markers for the resistance selection;and investigated systematically the traits of the hybrid in both cytologic and botanic levels and suggested the strategy of using the somatic hybrids in potato breeding for BW resistance.The main results of this study are as follows:1.BW resistance test selected the resistant somatic hybrids that may harbor the resistance introgressed from the eggplant fusion parent.Plantlets cultured in vitro of 90 somatic hybrids,obtained from the protoplast fusion of BW-susceptible potato AC142(dihaploid of S.tuberosum)and BW-resistant eggplant 508.3(S.melongena),and their fusion parents were inoculated with R.solanacearum isolate HZAU007(race 2,biochemical II B/I).The results showed that 6 somatic hybrids reached medium resistance and above,24 were medium susceptible,and the other 60 were susceptible.This result indicates that the bacterial wilt resistance derived from eggplant was successfully transferred to part of the somatic hybrids,providing a material base for the present research and for the germplasm development.2.The ploidy level test suggested that complicated variations in ploidy level may be the genetic basis for trait diversity of the somatic hybrids.By using of the flow cytometry and chromosome counting it was determined that 45 of the 90 somatic hybrids are tetraploids,4 are heptaploids,2 are octaploids,and the rest are aneuploids.The complexity in ploidy may reflect the genetic diversity of the somatic hybrids for the trait segregation.3.GISH with species-specific marker analyses indicated the genetic loci associated with BW resistance of the hybrids.In this study,the eggplant-specific SSR retention of somatic hybrids was analyzed using 44 eggplant-specific SSRs selected from 236 eggplant SSRs published,and the chromosomal composition of the somatic hybrids was analyzed by GISH.The results showed that all hybrids contain full set of the potato genome while the eggplant genome retained could be detected as intact by GISH or partial by only the SSRs.There are 12 somatic hybrids integrated intact eggplant genome whereas 78 retain different numbers of eggplant DNA fragments.In addition,loss of potato chromosome and rearrangement of potato and eggplant chromosomes were also observed.The results suggest there may be unknown factors influencing the genome combination in a somatic fusion between distant parents.Seven eggplant-specific SSR loci were identified as their closer link with BW resistance than the others,that could a base for the resistance gene mining.4.Transcriptome analysis selected and preliminarily confirmed the candidate gene that contributes to BW resistance of the somatic hybrids.The somatic hybrids,containing only small fragments of eggplant DNA but diverse in BW resistance,were separately pulled as the resistant and susceptible pools and subjected to the transcriptome analysis.The results demonstrated 15 eggplant genes that were differentially expressed between resistant and susceptible pools.Alignment of these genes and the 7 SSR loci identified above suggested two genes,Sm PGH1 and Sm GSTZ5,that may be potentially associated with BW resistance.Further expression in the resistant and susceptible somatic hybrids confirmed that Sm PGH1 may be a critical contributor to BW resistance of the somatic hybrids since it could be induced by the pathogen in only the resistant hybrids.5.Provided with plant phenotype foundation and molecular selection markers for employing the BW resistant somatic hybrids in potato breeding programs.The plant phenotyping was conducted and showed that most somatic hybrids have potato morphology and formed tubers.Some BW resistant hybrids could flower with active pollens which laid a foundation for their application in the resistance improvement.Meanwhile,a marker assistant selection strategy has been approached according to the Sm PGH1 sequence and the linked markers. |
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