Early Identification And Genetic Basis Of Resistance To Bacterial Wilt In Tobacco

Tobacco(Nicotiana tabacum L.)is an important economic crop as well as an important model plant.Tobacco bacterial wilt(TWB)is one of the most devastating diseases in tobacco production.TWB is a soil-borne bacterial disease caused by Ralstonia solanacearum.TWB is epidemic mainly in the southern China's major tobacco production area,but also shows a trend to spread northward.Genetic improvement with disease-resistant genes is an effective way to control tobacco bacterial wilt.In this thesis,identification method at early stage and genetic basis of TWB resistance were studied,aiming to lay a technical and theoretical foundation for TWB resistance breeding.The main results are as follows:(1)A major QTL qBWR17a for TWB resistance was previously mapped on Chromosome 17 by our lab.Six SSR markers located in the vicinity of this QTL within 4 cM were selected to genotype 23 tobacco cultivars(lines).At the same time,these varieties(lines)were also tested for TWB resistance in the field.Two SSR markers that could generate clear PCR bands in all the cultivars(lines)were screened out.Among them,marker PT51333 showed the band-type of the resistant control Yanyan 97 in all the resistant cultivars(lines),the band-type of the susceptible control CB-1 in all the susceptible cultivars(lines),and a third band-type in the mid-resistant cultivars(lines).According to correlation analysis by T-test,TWB resistance shows significant difference between cultivars(lines)with and without resistant band-type at PT51333.The results verifies the reliability of the major QTL qBWR17a in germplasm resources.(2)Thirteen cultivars(lines)were used for the experiment of TVWB test at seedling stage.The 13 cultivars(lines)were chosen from those having been tested for TWB resistance in the field.The results indicated that by irrigating seedlings of sand culture with 106 cfu/ml bacteria suspension at the five-leaf stage,the TWB resistance of different cultivars(lines)could be distinguished within 25 days.The results were highly correlated with those obtained from the field test,suggesting that the method of early identification of TWB resistance is reliable.(3)The roots of seedlings of resistant variety D101 and susceptible variety CBH were soaked into the suspension of Ralstonia solanacearum and the corresponding autoclaved suspension(as control),respectively,with three replicates.Total RNA was extracted from the roots 3 h after the inoculation and the RNA samples were sent for transcriptome sequencing.A total of 73 and 519 differentially expressed genes(DEGs)were detected in D101 and CBH,respectively,of which 55 and 304 were up-regulated,with 27 overlapped;18 and 215 were down-regulated,with 4 overlapped.The number of DEGs in CBH was significantly higher than that in D101.This is consistent with the fact that CBH showed apparent symptoms after its roots were soaked for 3h.GO and KEGG analyses showed that the enriched GO terms and metabolic pathways were quite different between the two varieties.It is noteworthy that the D101 up-regulated genes were enriched in the metabolism of aromatic and other resistant substances,suggesting that disease-resistant varieties might resist bacterial infection by producing resistant substances.