Studies On Heterosis Utilization Of Interspecific Hybrid Cotton (Gossypium Hirsutum×G. Barbadense) Based On Cytoplasmic Male Sterility System

Gossypium hirsutum (upland cotton) and G. barbadense (sea island cotton) are modern allotetraploid (2n=4x=52) cottons, which together represent the most extensively cultivated species worldwide. While G. hirsutum is the most widely cultivated species—prized for its higher yield and wider environmental adaptation, G. barbadense boasts vastly superior fiber quality and resistance to Verticillium wilt. The useful heterosis in interspecific (G. hirsutum×G. barbadense) F₁ hybrids which combinated productivity, quality and disease resistance has been reported by many researchers. However, to date, few studies on heterosis of interspecific hybrid cotton based on cytoplasmic male sterility (CMS) system which could produce hybrid seeds in an economically feasible way had been conducted. In this dissertation, the development of CMS line with G. barbadense nucleus background and its 'three lines' (sterile line, maintainer line and restorer line) combination were reported. The performance and heterosis of CMS based interspecific hybrid cotton were evaluated. The heterosis mechanism of CMS based interspecific hybrid cotton were also preliminary studied. The main results were as follows:1. Breeding for CMS line in 6. barbadens and the studies on its three lines' combinationA G. barbadense male-sterility line 'Hai A' with G. harknessii cytoplasm and its maintainer 'Hai B' were developed by replacing nuclear genomes from G. hirsutum to G. barbadense in a continuous backcross breeding procedure. Its male cell abortion was observed using paraffin slice via H.E staining technique under light microscope. Results showed that the abortion in 'Hai A', which mainly took place at the stage of microspore mother cell meiosis, did not significantly change at the cytological level, though G. hirsutum nuclear genome of sterile line was replaced by the G. barbadense nuclear genome. 'Hai A' sterility was very stable because of its early abortion and anthers without pollen. Hybrids between 'Hai A' and G. hirsutum restorers were high in pollen viability, boll setting rate, boll weight and lint percentage which contributed to heterosis of high yield. 'Hai A' is of value in the application of cotton interspecific hybrid seed production.The improved G. hirsutum restorer lines and their hybrids which were produced by crossing them with CMS lines were evaluated in a Randomized Complete Block Design with three replications during 2005-2006 at the experimental farm of Zhejiang University, Hangzhou. The experiment showed that some G. hirsutum restorer lines could be improved effectively by polycross, continuous self-pollination and selection under artificial temperature stress in greenhouse in hot summer at Hangzhou. The improved restorer lines were characterized by high pollen fertility, strong restorability, high boll setting percentage and suitable plant shape. Compared with the original restorer 'LuR', the improved restorer lines was significantly improved in the plant shape, but not significantly in yield traits and fiber quality traits except for the fiber strength. For their hybrids, most of them were significantly improved not only in the plant shape than the check hybrid 'KangA×LuR', but also in yield. Moreover, a few crosses had the same or higher yield than 'LMY15' (a commercial intraspecific hybrid). However, there was no significant difference between these hybrids and the hybrid 'KangA×LuR' in fiber quality traits. It indicated that it was feasible to improve restorability of restorer lines through selection under artificial temperature stress and it was difficult to improve their fiber quality.The study of the inheritance of fertility restoring gene(s) from the G. barbadense restorer line through the analysis of different fertility segregation populations showed that the G. barbadense restorer line had one major dominant gene to control the CMS fertility restoration and this fertility restoration gene functioned at the sporophytic level. The transmission rate of male gamete and female gamete might be influenced by sterile cytoplasm background when restorer gene was recessive. It could be deduced that this fertility restoration gene might come from G. harknessii cotton.2. The performance and heterosis of CMS based interspecific hybrid cottonThe performance of interspecific (G. hirsutum×G. barbadense) and intraspecific (G. hirsutum×G. hirsutum) hybrid cotton based on CMS systems along with a standard check ('LMY15', a commercial intraspecific hybrid) was evaluated for yield and fiber quality traits at Zhejiang University, Hangzhou in a Randomized Complete Block Design with three replications during 2005-2006. Data showed that interspecific hybrids were significantly higher than intraspecific hybrids and inbred lines in plant height, number of fruit branches, number of fruit nodes, number of bolls and percentage of aborted seeds. However, boll weight and lint percentage were significantly lower, especially for boll weight, the maximum only was 3.3g for interspecific hybrids, but for intraspecific hybrids the minimum could reach 4.7g. Generally, lint yield of interspecific hybrids was significantly lower than intraspecific hybrids, but a few of interspecific hybrids presented an acceptable lint yield, for example, there was no significant difference between " KangA×HaiR3 " (an interspecific hybrid) and 'LMY15' . For the fiber properties, interspecific hybrids were significantly better, especially in fiber length, strength and elongation, the maximum of that was 36.06mm 37.63 cN/tex and 7.20%, respectively.The photosynthetic properties and its related physiological characters of different types cotton (interspecific hybrid, intraspecific hybrid, G. barbadense and G. hirsutum) were detected. The results showed that there was no significant difference between interspecific hybrid and other G. hirsutum. When the viable pollens percentage was over 80%, there was no close relationship between starch, soluble sugars, soluble protein contents of anthers and viable pollens percentage in different types cotton. It might be helpful to improve the photosynthetic capability of interspecific hybrid by breeding and selecting the G. barbadense parent lines with higher photosynthetic capability.Two types of topcross hybrids, G. hirsutum sterile line/G, barbadense maintainer line//G. hirsutum restorer lines and G. hirsutum sterile line//G. hirsutum restorer line/G. barbadense restorer line, were produced and were evaluated in a Randomized Complete Block Design with three replications during 2005-2006 at Hangzhou. The results showed that topcross hybrids all had excessive vegetative growth and late maturity. The lint yield and seed cotton yield of topcross hybrids were lower, because of their significantly lower in boll weight and lint percentage than intraspecific hybrid. The boll weight and lint percentage of topcross hybrids were not enhanced, even though the G. hirsutum genome proportion in topcross hybrids were increased compared with that in interspecific hybrids. It might be difficult to enhance yield through increasing G. hirsutum genome proportion in topcross hybrids.3. The studies on heterosis mechanism of CMS based interspecific hybrid cottonGenetic distances (GD) among the parents were calculated from 56 random amplified polymorphic DNAs (RAPD) and 66 simple sequence repeat (SSR) markers data, and their correlation with hybrid performance and heterosis were analyzed. The parents could be discriminated into G. hirsutum and G. barbadense clusters by cluster analysis based on both RAPD and SSR markers data. The correlation (r = 0.503, p<0.05) was calculated between GD_rapd (GD based on RAPD markers) and GD_ssr (GD based on SSR markers). Correlation of GD with hybrid performance and heterosis differed considerably between intra- and interspecific hybrids. The correlation between GD and hybrid performance was non-significant for most of traits within the hybrids of G. hirsutum species. However, It is significantly and positively correlated for fiber length, fiber strength and elongation in interspecific hybrids. The relationship between GD and heterosis was observed positively significant for boll weight within hybrids of G. hirsutum and significant and negative correlation for fiber length and elongation. However, It is significantly and positively correlated for fiber length in interspecific hybrids. In conclusion, the power of prediction hybrid performance using molecular markers in cotton is low. But, the relationship between SSR marker heterozygosity and hybrid performance can be used to predict fiber length during interspecific hybrids cotton breeding. The functional genes related to cotton fiber expansion and fiber secondary cell wall synthesis were selected to analyses their expression level in different type (interspecific hybrid, G. barbadense and G. hirsutum) cotton fibers at the key stage of fiber development by real-time quantitative RT-PCR (RT-qPCR) technology. Eight genes that include two cell wall extensibility related genes endo-xyloglucan transferase gene (EXT) and endo-l,4-beta-glucanase gene (E14G); five turgor related genes delta-TIP homolog gene (MIP), sucrose synthase gene (SuSy), H+-pyrophosphatase gene (VPP), phosphoenolpyruvate carboxylase2 gene (PEPC2) and Plasma membrane H+-ATPase gene (PMA); one cytoskeleton related gene ACTIN1 (ACT1) were selected to evaluate their expression level in different type cotton fibers at 10 DPA. And two cell wall synthesis related genes endo-1,3-beta-glucase gene (E13G) and cellulose synthase 1 gene (celA1) were also studied at 24 DPA. The correlation between genes expression level and related fiber quality in different type cotton was calculated. EXT and E14G showed the highest level of expression in interspecific hybrid, medium in G. barbadense and the lowest in G. hirsutum. The expression level was positively associated with the fiber length in different type cotton. It refered that the cell wall extensibility related genes played an important role in fiber length formation. SuSy expressed higher level in interspecific hybrid and G. barbadense than in G. hirsutum, and its expression trends was as same as sucrose synthase activity in different type cotton. For the MIP, the highest level were found in interspecific hybrid cotton, and there were similar level in G. barbadense and G. hirsutum. In contrast, VPP, PEPC2 and PMA showed the highest level of expression in interspecific hybrid cotton, medium in G. barbadense and the lowest in G. hirsutum. E13G and celA1 had higher expressin level in interspecific hybrid and G. barbadense than in G. hirsutum. It showed that the expression level of cell wall synthesis related genes had an important role to fiber strength formation. There were significant correlation between the expressin level of genes related to the primary fiber expansion (10 DPA) and fiber length. It means that the expression level of genes related to cotton fiber expansion played an important role to fiber elongation. The correlation between the expressin level of cell wall synthesis related genes (E13G and celA ) in the secondary cell wall synthesis 24 DPA was highly significant. It revealed that the relationship between the expressin level of cell wall synthesis related genes and fiber strength was close.