| As an important economic crop, cotton plays an important role in the economy of our country. Verticillium wilt is one of the most destructive diseases in cotton. In recent years, many cotton fields in China have been severely damaged by this disease, being recognized as a serious threat to cotton production. It has been confirmed that breeding for highly tolerant or resistant varieties to Verticillium wilt is the most effective and economical method to control this disease. Despite long-term efforts to use plant resistant varieties and cultivated management techniques to control Verticillium wilt in cotton, losses have remained relatively constant. Major challenges have included the lack of effective resistant material, inadequate knowledge of genetic mechanism, and the need for more knowledge regarding pathogen distribution and race differentiation. Therefore, further studies about the inheritance and the molecular markers linking to Verticillium wilt resistance, dynamic process of Verticillium dahliae infection, tissue distribution of pathogen in cotton and resistance mechanism of each material are needed urgently.In this study, we used major gene-polygene mixed inheritance models and joint analysis methods for six generations to analyze the resistance genetic law of upland cotton cultivar in different development stages and illustrated location of major resistance gene and its distribution characteristic by composite interval mapping. Furthermore, we transformated green fluorescent protein (GFP) gene to Verticillium dahliae through construction of the fungus expression vector for providing detection marker to dynamic process of Verticillium dahliae infection. The results are following:1,Genetic analysis on resistance gene for upland cottonUsing resistant cultivar 5026,60182 and a susceptible cultivar Junmianl, we obtained Pi, P2, Fi, Bi, B2 and F2 generations from the cross 5026×Junmianl and 60182xJunmianl. We reported phenotypic distributions of infected leaf percentage in these six generations inoculated with BP2, VD8, T9, or their isoconcentration mixture. Genetic segregation analysis for infected leaf percentage trait were conducted by using major gene-polygene mixed inheritance models and joint analysis method of P1, P2, F1, B1, B2 and F2 generations. We found that resistance of upland cotton cultivar 5026 to nondefoliating strains of V. dahliae was controlled by two major genes plus polygenes, however, one major gene plus polygenes when inoculated with defoliating strains of V. dahliae, or a mixture of defoliating and nondefoliating strains of V. dahliae. The results suggest that resistance of upland cotton cultivar 60182 to pathogens BP2, VD8, T9 and the isoconcentration mixture was controlled primarily by two major genes. The inheritance of this trait could be explained with two major genes plus polygenes models when inoculated with BP2, VD8, T9 and the isoconcentration mixture, respectively. The heritability of the major gene was dominant, no matter what type of pathogen.2,Construction of upland cotton intraspecific molecular linkage mapUsing a resistant cultivar 60182 and a susceptible cultivar Junmianl, we obtained 229 F2 individuals as mapping population. One hundred and ninety primer pairs out of a total of 6,771 SSR primers (2.81%) amplified polymorphisms and produced one hundred and ninety-one loci between the two parents. Of these,139 loci were assigned to 31 linkage groups with a total map distance of 1,165 cM and an average distance of about 8.38 cM between the two markers, covering approximately 25.88% of the total recombinational length (4500cM) of the cotton genome. According to the information from our backbone linkage maps,22 of 31 linkage groups were tentatively assigned to 19 chromosomes, while 9 groups remained unattached. This linkage map provided a good foundation of further screening resistance gene to Verticillium wilt3,QTLs mapping for resistance to Verticillium wilt in upland cottonLeaves disease grades were measured in F2:3 family lines in mid-June and late July as seedling resistance, and lesion area in the vascular bundle in mid-December as maturity resistance, for screening QTL of Verticillium wilt resistance. Based on the genetic linkage map, QTLs resistant to Verticillium wilt were screened using multiple interval mapping method. The result showed that QTLs were clustered on chromosomes D9 and D7 according to the data of three different developmental stages inoculated with BP2, VD8, T9 and the isoconcentration mixture.Inoculating with BP2, we have detected eight QTLs on D7 and D9 chromosome. Four of these were on D7, one from June data accounting for 24.1% of phenotypic variance, two from July data accounting for 5.8% and 13.6% of phenotypic variance and one from maturity stage accounting for 32.0% of phenotypic variance. Other four QTLs were on D9, one common QTLs in June and July accounting for 24.5% and 19.8% of phenotypic variance, one in July accounting for 7.8% of phenotypic variance and one at maturity stage accounting for 25.3% of phenotypic variance.Using VD8, we have detected fourteen QTLs on D7 and D9 chromosome. Five of these were on D7, one common QTLs in June and July accounting for 30.8% and 12.1% of phenotypic variance, another one in July accounting for 7.5% of phenotypic variance, and two at maturity stage accounting for 31.5% and 27.7% of phenotypic variance. Other nine QTLs were on D9, a common QTLs in June and July accounting for 11.9% and 32.1% of phenotypic variance, a common QTLs in June,July and maturity stage accounting for 16.5%,16.8% and 32.1% of phenotypic variance, another one in June accounting for 11.8% of phenotypic variance, one in July accounting for 15.7% of phenotypic variance and two QTLs at maturity stage accounting for 13.1% and 18.6% of phenotypic variance.For T9, we have detected nine QTLs on D7 and D9 chromosome. Four of these were on D7, one common QTLs in June and July accounting for 19.1% and 10.2% of phenotypic variance, another one in July accounting for 16.5% of phenotypic variance, and one at maturity stage accounting for 24.0% of phenotypic variance. Other five QTLs were on D9, one common QTLs in June and July accounting for 13.0% and 18.8% of phenotypic variance, another one in June accounting for 20.2% of phenotypic variance, two in July accounting for 19.3% and 11.8% of phenotypic variance.For mixed pathogen, we have detected ten QTLs on D7 and D9 chromosome. Three of these were on D7, one from June data accounting for 24.6% of phenotypic variance, one from July data accounting for 33.4% of phenotypic variance and one from maturity stage accounting for 23.4% of phenotypic variance. Other seven QTLs were on D9, two common QTLs in June and maturity stage accounting for 23.1% and 12.7% of phenotypic variance in June and 9.0% and 33.2% of phenotypic variance in July, two in July accounting for 9.7% and 20.6% of phenotypic variance, one at maturity stage accounting for 8.9% of phenotypic variance.As a result, some QTL in same marker interval were detected to be common resistance QTL to four types of V. dahliae. Three QTL were thought as broad-spectrum no matter what type of pathogen BP2,VD8,T9 or their isoconcentration mixture. It is suggested that these three QTL possess broad-spectrum resistance to V. dahliae. One was presumably specific resistance QTL to isolate BP2, one was specific resistance QTL to isolate VD8, one was speculated as specific resistance QTL to defoliating isolates and two for mixed pathogen.In summary, the tagging of two QTLs clusters on chromosomes D9 and D7, either in different developmental stages or when inoculated with different isolate treatments, implied the existence of broad-spectrum and stable resistance to V. dahliae pathogens with different isolates in 60182. However, QTLs for different pathogen were located in different interval.4,Transformation of green fluorescent protein (GFP) gene to Verticillium dahliaeConstruction of GFP fungus expression vector was completed. In following research, we may inoculate cotton with Verticillium dahliae containing expressed GFP to clarify dynamic process of Verticillium dahliae infection, tissue distribution of pathogen in cotton and resistance mechanism of each material. |
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