EST-SSR Development,Genetic Mapping Construction In Sesame And Cloning Of Genes Related With Fatty Acid Metabolism In Cotton

Sesame is an important oil crop worldwide. Compared with other crops, the molecular biology research in sesame was behindhand, and no molecular marker map and transgenic variety for sesame have yet been published; Cotton is also an important cash crop with fat and protein rich in seed. Although molecular biology research in cotton was fast, the rsearch has mainly focused on improving the fiber yield and quality. Correspondingly, the research on improving the cotton seed oil and the cotton cold resistance by genetic engineering of fatty acid matabolism have not been regarded until now. Therefore, the present study mainly did the research on molecular genetic mapping construction in sesame and cloning of genes related with fatty acid metabolism in cotton. This study will lay the foundation not only for the sesame marker-assisted selection (MAS), quantitative trait loci (QTL) analysis and gene localization, but also for the transgenic breeding and comparative genomics research between sesame and cotton.The results are as follows:1. Development of sesame molecular markersOwing to the late genome research and the little information of same genome, to construct the sesame genetic map we analyzed and developed molecular markers based on the sesame transcription and genome information. On the one hand, EST-SSR characterization, markers development and utilization using publicly available sesame EST data were performed. A total of 1,785 non-redundant EST sets were assembled among the 3,328 identified sesame ESTs. The total length of the non-redundant EST sequences was 774.266 kb, on average one EST-SSR each 3.09 kb. The characteristics and distribution of the EST-SSR markers were showed as follows. Among the SSR (≥18bp), the dinucleotide repeat motif was the most abundant among them, with a frequency of 50.0%, pentaucleotide repeat motif was the least, with a frequency of 2.2%. Dinucleotide AG/TC was the most abundant (occurring 58 times), with frequency of 37.42%. According to these EST sequences containing SSR,120 primer pairs were designed. On the other hand, according to the characterization of the SAMPL markers, we developed a new and efficient marker:RSAMPL (Random Selective Amplification of Microsatellite Polymorphic Loci), which is a modified AFLP technique and SSR primers are used as one of the AFLP primers in the second selective amplification. Due to the SSR primer can be used randomly, whatever it belong to any species and any position, the primer combinations of RSAMPL marker are almost infinite and can spread over all genome sequence and could be used as a new molecular marker in other species research.2. Construction of sesame genetic linkage mapThe first genetic linkage map based on an intraspecific cross between two inbred lines of sesame (COI1134 and RXBS) has been constructed with 96 F2 population using EST-SSR, AFLP and RSAMPL markers. A total of 220 markers were mapped in 30 linkage groups covering a genetic length of 936.72cM, with the average interval was 4.93cM. The number of markers varied from 2 to 33 per group, with average 7.33 markers in each group. The length of the linkage groups ranged from 6.44 to 74.52cM, with average 31.22cM each group. The estimated genome length of sesame was 1,232.53cM, with the genome coverage was 76%. This is the first reported linkage map in the genus Sesamum, and in the Pedaliaceae family as well. Although the genetic maps presented here are preliminary, they provide a starting point for the eventual construction of high-density maps, and the mapping of economically important genes and QTLs in the sesame.3. Cloning of genes related with fatty acid metabolism in cottonSeven genes which play an important role in the process of fatty acid metabolism had been cloned and identified by homologic sequences cloning, in silico cloning and RACE. (1) Beta-ketoacyl-ACP synthase II (KASII). Sequence analyses showed that the size of cDNA was 2,085bp, and it included an open reading frame of 1,73 1bp that encoded a polypeptide of 576 amino acids. BlastP analysis indicated GhKASⅡhad 93%,91% and 88% homologies with beta-ketoacyl-ACP synthase II of Glycine max, Brassica napus and Helianthus annuus respectively. (2) Beta-ketoacyl-ACP synthase III (KASIII). Sequence analyses showed that the size of cDNA was 1,751bp, and it included an open reading frame of 1,206bp that encoded a polypeptide of 401 amino acids. BlastP analysis indicated GhKASIII had 89%,88% and 85% homologies with beta-ketoacyl-ACP synthase III of Ricinus communis, Perilla frutescens and Arabidopsis thaliana respectively. (3) Omega-3 fatty acid desturase (ω-3FAD). Sequence analyses showed that the size of cDNA was 1,905bp, and it included an open reading frame of 1,341bp that encoded a polypeptide of 446 amino acids. BlastP analysis indicated Ghω-3FAD had 84%,82% and 78% homologies with omega-3 fatty acid desaturase of Nicotiana tabacum, Helianthus annuus and Arabidopsis thaliana respectively. (4) Glycerol-3-phosphate acyltransferase (GPAT). Sequence analyses showed that the size of cDNA was 1,845bp, and it included an open reading frame of 1,503 bp that encoded a polypeptide of 500 amino acids. BlastP analysis indicated GhGPAT had 91% and 78% homologies with glycerol-3-phosphate acyltransferase of Arabidopsis thaliana and Oryza sativa (japonica cultivar-group) respectively. (5) 4-coumarate:CoA ligase (4CL). Sequence analyses showed that the size of cDNA was 1,909bp, and it included an open reading frame of 1,725bp that encoded a polypeptide of 574 amino acids. BlastP analysis indicated Gh4CL had 88%,87% and 82% homologies with 4-coumarate:CoA ligase of Camellia sinensis, Glycine max and Arabidopsis thaliana respectively. (6) Acyl-CoA oxidase (ACX). Sequence analyses showed that the size of cDNA was 2,268bp, and it included an open reading frame of 1,995bp that encoded a polypeptide of 664 amino acids. BlastP analysis indicated GhACX had 92%,92% and 91% homologies with acyl-CoA oxidase of Lycopersicon Esculentum, Vitis vinifera and Glycine max respectively. (7) Fatty acid beta-oxidation multifunctional protein (MFP). Sequence analyses showed that the size of cDNA was 2,455bp, and it included an open reading frame of 2,166bp that encoded a polypeptide of 721 amino acids. BlastP analysis indicated GhMFP had 89%,86% and 83% homologies with fatty acid beta-oxidation multifunctional protein of Vitis vinifera, Arabidopsis thaliana and Oryza sativa (japonica cultivar-group) respectively. Among the seven genes described above, genes no.1-4 may take part in fatty acid anabolism, they adjust the biosynthesis of store lipid and membrane lipid, and play roles in the rate of fatty acid synthesize and cold resistance of cotton; genes no.5-7 may take part in fatty acid catabolism, they adjust the decomposition of fatty acid and its derivative, and play roles in seed germination, seeding building and defending against exoteric stimulation. In this paper, the further study shoud be needed to confirm the function of the seven genes.