Genome-Wide Association Study Of The Heritable Variation Of The Potassium Content In Tobacco

In China, tobacco(Nicotiana.tabacum) is an important cash crop and the potassium content in tobacco leaf has an important impact on the economic value of tobacco. Compared with the high grade tobacco leaf in the overseas market, the potassium content of tobacco leaf in China is lower, which restricts severely increase of economic value of tobacco leaf and makes the farmers in remote mountainous areas get fortune slowly. In view of the problem of low-potassium content in tobacco leaf, the effect of traditional cultivation and fertilization is not ideal. Thus, these measures, like exploiting related potassium-absorbing and potassium-transporting genes, developing functional markers and using molecular marker-assisted breeding, can increase potassium content in tobacco leaf.This study selected 96 flue-cured tobacco materials on the basis of core tobacco germplasm resources to construct the test population, as population Ⅰ. In addition, builded a verification population which included 130 tobacco materials, as group Ⅱ. Used 454 SSR markers that evenly distributed in the genome to analyse the genetic diversity and population structure of population Ⅰ. Then, based on the results of association analysis, selected candidate genes and SSR markers that related to potassium absorption and potassium transportation. And constructed a molecular markers screening system which was use to screen high-potassium tobacco varieties. The main results as follows:(1) Analysis about the potassium content in tobacco leaf under different environments showed that the potassium content in tobacco leaf in Xichang was significantly higher than that in Zhucheng. In contrast, in terms of the coefficient of variation of the content potassium in tobacco leaf, Xichang was obviously less than Zhucheng. It indirectly indicated that Xichang was more potential than Zhucheng in tobacco quality. What’s more, in Xichang and Zhucheng, there is no difference for the potassium content in tobacco leaf between domestic varieties and foreign varieties.(2)The experiments used 454 SSR markers that uniformly distributed in 24 linkage groups to scan the whole genome in 96 flue-cured tobacco materials. Then filtered the rare allelic variation at a proportion of 5%. After that, there were 417 polymorphic markers and 1038 allelic variations. Each marker had 2.5 allelic variations on average and the amplitude of variation was from 2 to 6. PIC value was 0.299 on average, whose amplitude of variation was from 0.044 to 0.698.(3) Cluster analysis based on Nei’s genetic distance showed that 96 tobacco materials could be divided into three categories. Besides, structural analysis by Structure software also showed that three subpopulation existed in 96 tobacco materials. This further illustrated that there were three genetic backgrounds in the population.(4) By using association analysis, there were 16 markers excavated, whose-log P value≥2.00. The value of PT61367 was highest, and reached 4.46. What’s more, PT51364, PT53362, PT55319 had a good reproducibility, and associated phenotypes in two environments. In addition, according to the 16 markers positioning results in the tobacco genome database, 4 candidate genes related to potassium absorption and transport in tobacco were found, and they were Ntab0898590, Ntab0163850, Ntab0171930, Ntab0562640. The function of Ntab0898590 and Ntab0163850 is similar to potassium channel genes, and the function of Ntab0171930 and Ntab0562640 is similar to potassium transporter genes.(5)We analysed high-potassium favorable allels from 11 markers whose-log P values≥2.50. By analyzing significance testing of difference of the potassium content and regularity of distribution of the potassium content in different environments for the alleles of each marker, ultimately, we selected five SSR markers to construct a marker-assisted screening system of high-potassium tobacco varieties. The 5 SSR marker are PT61367, PT51364, PT30132, PT55319, PT53362, whose favorable alleles are respectively 101, 102, 103, 101 & 103, 101. In addition, in population Ⅰ, we found that the more favorable alleles the test materials had, the higher the potassium content is in tobacco leaf. After verifying with the populaton Ⅱ, we drew the same result. It indicated the five markers can apply to molecular marker-assisted selecting of high-potassium tobacco germplasm.