Study On The Inheritance And CDNA-AFLP Of A Dwarf Mutant AS98 From Cotton

The development of dwarf plant cultivars in order to increase resistance to lodging is playing an important role in plant breeding. Since dwarf rice was discovered by Parnell in 1922, different types of dwarf mutants have also been found in wheat, barely, maize, cucumber, pumpkin, watermelon, etc. The studying and application of these dwarf mutants positively promoted the dwarf plant breeding in rice and wheat. To date, five different types of dwarf cotton (Harland, Hutchinson, McMichael, He Jian-xin and Chen Xu-sheng) have been found according to previous reports, since Harland (1918) found a dwarf mutant of Gossypium barbadense L. with crinkled leaves. A novel super-dwarf mutant with extremely short stems and normal leaf shape was discovered in a cotton F2 population of interspecific hybridization in 1998. The phenotype is similar to that of normal cotton from the cotyledon stage to the fourth-leaf stage. After the fourth-leaf stage, it shows dwarfism with normal shape leaf. Compared with the other dwarf cottons that had been reported previously, the main difference in the phenotype is that the leaves of AS98 are normal shape, but the leaves of the other dwarf mutants are crinkled. This type dwarf mutant with normal leaves has not been reported in cotton until now. The main purpose of this study was to study the genetics of plant height, the physiologic effects by exogenously application hormones on a cotton dwarf mutant AS98, the molecular mapping of the dwarf character and the expression difference of genes related to the tips between normal and dwarf mutant plants.1,Inheritance of the super-dwarf mutant AS98We compared the morphological character between the normal type plants and dwarf mutant AS98. The results showed that the plant height and internode length of AS98 was shorter significantly than that of the normal line LHF10W99. However, the internode number was reduced only 3% to 5%. Therefore, the decrease of plant height was the result of a reduction in the internode length.In order to study the genetic of the dwarf trait, AS98 was crossed with the normal plant LHF10W99, and the F1, F2, BC1, and BC2 populations were used for the genetic analysis of the dwarf trait. The plants of the F1 showed a semi-dwarf phenotype and the plants height display unimodal distribution. The F2 plants exhibited a wide range of variation for plant height, and the plant height displayed trimodal distribution. The F2 generation was segregated into three phenotypes:normal, semi-dwarf, and super-dwarf. Based on theχ2 test, the three phenotypes segregation ratios of the F2 generation conformed to the ratio of 1:2:1. The BC1 population ((LHF10W99×AS98)×LHF10W99) showed two phenotypes:normal and semi-dwarf with the expected ratio of 1 normal:1 semi-dwarf. Whereas, in the backcross (BC2) with the dwarf parent AS98, a 1:1 segregation ration of super dwarf plants and semi-dwarf plants was observed. These results confirmed that a single incompletely dominance genes controls the super dwarf trait in AS98 and that super dwarf trait is incompletely dominant over the normal plant height trait.2,the response of AS98 to different exogenous hormonesTo assay the response of AS98 to different exogenous hormones, GA3, BR and IAA were applied with different concentrations. The results indicated that AS98 responded significantly to the application GA3. After four times spraying exogenous hormones continuously, the plant height treated with GA3 was higher significantly than that of the treatment with IAA and BR and the control. The plant height and internodes length treated with 0.28μm.L-1 of GA3 reached to 64.8 cm and 2.82 cm respectively, which was similar to that of the LHF10W99 and was significant difference with the control AS98. The plants height and internodes length treated with BR and IAA were 19.7-24.5 cm and 0.95-1.12 cm, which were no significant difference with the control AS98. This showed that the plant height of AS98 can be rescued by the application GA3 continuously.The regression analysis showed that the regression coefficient between plant height and GA3 was 0.46 and was remarkable, but the regression coefficient between plant height and BR or IAA was no significant. From the slope with the internodes, the similar results were obtained. These results demonstrated that the plant height of the AS98 is markedly interrelated with GA3 and can be recovered by applications exogenous GA3 consecutively. Therefore, we infer that the super-dwarf mutant AS98 respond to GA application and is a GA-sensitive dwarf mutant.3,The physiologic effects by application exogenous hormones on AS98 mutantTo study the physiologic effects by application exogenous hormones on AS98 mutant, the contents of endogenous GA, IAA, ZR and ABA were measured, and the activity ofα-amylase was investigated after AS98 were treated with different exogenous hormones. The results indicated that the contents of endogenous GA, IAA, ZR and ABA were increased in the leaves of AS98 after treating with exogenous GA3. The contents of GA and IAA were promoted by the lower concentration of exogenous IAA. After spraying exogenous IAA, the content of endogenous ABA was increased, but the content of ZR was decreased. Exogenous BR could promote GA and ABA content, decrease ZR content and influence on the content of endogenous IAA which had obvious concentration effect. The content of IAA was increased by application of lower concentration exogenous BR and decreased by application high concentration BR. Theα-amylase activity was up-regulated by the lower concentration exogenous GA3. On the contrary, the a-amylase activity was reduced by application exogenous PP333, and the effect could be rescued by application exogenous GA3. Those results showed that AS98 was a GA deficient dwarf mutant. The application of the exogenous GA3 could significantly increase hormones content which promoting plant growth, up-regulate theα-amylase activity. Moreover, the inhibition effect ofα-amylase activity by application exogenous PP333 could be relieved by application exogenous GA3.4,the SSR and AFLP molecular markers link to the dwarf trait of AS98To find the molecular markers link to the dwarf trait of AS98, an F2 population was constructed by crossing mutant AS98 with normal gene type of LHF10W99.38 polymorphic SSR markers were screened between "LHF10W99" and AS98 from the 6000 simple sequence repeat (SSR) primer and 9 Amplified Fragment Length Polymorphism (AFLP) primers with polymorphism were screened from 256 primers. The polymorphism SSR and AFLP markers were then mapped on the above populations by Joinmap4.0 software. When the LOD was 6.0, the linkage map divided 3 linkage groups with an average distance between markers of 26.28 cM (Kosambi) and covered 657 cM. The dwarf trait was mapped on the 1st linkage group and One SSR marker GH537 and one AFLP marker were identified tightly linked to the dwarf trait with the distance of 5.1 cM and 9.5cM, respectively.5,Analysis of differentially expressed genes in the AS98 mutantThe RNAs of the stem tip, stem and root in the squaring stage were extracted from the normal type plants and the dwarf type plants. cDNA-amplified fragment length polymorphism (cDNA-AFLP) was used to identify differentially expressed between the stem tip of the normal and dwarf plants. There were 32 differentially expressed bands between them. The 32 fragments were cloned and sequenced. Blast analysis showed that the HD61 had a high similarity to arabinogalactan proteins from Arabidopsis. So the primer was designed based on the sequence of cotton arabinogalactan proteins (GhAGP) and the RT-PCR method was used to identify differentially expressed between the stem tip, stem and root of the normal and dwarf plants. The results showed that the GhAGP gene expression level in root and stem of the dwarf plants was similarity to that of the normal plants. But its expression level in the stem tip of the normal plants was significantly higher than that of the dwarf plants. The comparison of gene expression profiles between normal type and dwarf mutant provided the evidence that GhAGP was involved in the cell elongation growth in the stem tip.