Studies On The Selection Of Hami Melon Hermaphroditic Mutant Induced By Space Flight And Its Mutagenesis Mechanism

Xinjiang thick-skinned melon, commonly known as hami Melon, is very well-known both in and outside China for its unique flavor of being crisp, fragrant, sweet and so on. As an intermediate or high-grade fruits, it is a pillar industry in Xinjiang agriculture and a foreign exchange earning product. However, due to variety degeneration and serious plant diseases, the commercial quality of hami melon is reduced, thus affecting hami melon's production and consumption in recent years. Therefore the cultivation of new varieties of high quality hami melon becomes an urgent yet practical issue for us. In melon germplasm resources, the vast majority of thick-skinned melon cultivars belong to andromonoecious type, only the partial thin-skinned varieties of melon and wild melon being hermaphrodite type. In order to obtain new varieties of excellent melon and new breeding material with good comprehensive traits, sexual type selection of the melon is one of its important directions.Space mutation breeding, a new breeding method, causes strong vacillation and the mutagenesis of the biological hereditary feature through the spatial environment comprehensive physical factors and the condition, thus obtaining the specific germplasm resources which are difficult to get with the convention method in ground, and breeding new varieties. Through space mutation of hami melon seed, we have obtained a hermaphrodite mutant after selective breeding. This research, taking the original hami melon variety, the mutant and mutant's offsprings as materials, studied the heredity rule of hermaphrodite type, from the perspective of morphological, cytological and molecular aspects to know more about mutation mechanism and speed up material utilization. Meanwhile we have cloned two members of gene of the hami melon 1- amino cyclopropane -1- carboxylic acid synzyme (ACS), and analyzed these two genes in this mutant and the original variety changes, aimed at understanding these two genes and differentiation of flower-type of relationship to explore the ACC synthase function in the melon. The results are following:1. Flower sexual type of hami melon and their offsprings was investigated by space mutation. A stable hermaphrodite inbred line was obtained by screening a hermaphrodite mutant in the SP2 generation, and carrying on three generations of continual inbred lines of it. 2. The mutant SP3-SP5 generation of the flowered organ was observed. Compared with the hermaphrodite flower ovary of the original variety, H8 strain's ovary of SP3 generation is minimum, only one-third of the original species in size, looking like a staminate flower; H2-3 strain's ovary of SP4 generation is seemingly spindle; H2-2-3 strain's ovary of SP5 generation is a cone.Compared with stigma of the original variety, hermaphrodite mutants appear all kinds of"forked type", the abnormal stigmas.3. The hermaphrodite flower's pollens of the original variety, H2-2-3 strain of SP5 generation and H8 strain of SP3 were scanned and observed by electron microscope. The results demonstrated flower's pollen grain of original variety and mutant H2-2-3 strain of SP5 generation is oblate triangle, with four symmetrical surface of radial symmetric shape, and three germination holes, having circular aperture, thickened surrounding, and bulged middle, and a clear reticular pattern wall. But H8 strain's pollen grain of SP3 generation is distorted and shrunken. This is possibly one of the reasons regarding the fruit set difficulty of H8 strain of SP3 generation.4. Bulked segregation analysis (BSA) was employed to identify randomly amplified polymorphic DNA (RAPD) and Simple Sequence repeats (SSR) markers linked to the hermaphrodite type in hami melon. Only one RAPD marker S1254750 was found to be linked to the hermaphrodite type, the genetic distance being 4.5 cM; Two SSR markers CMTC12350 and CMTC158200 were found to be linked to the hermaphrodite type in hami melon, the genetic distance being 6.3 cM and 8.8 cM respectively. Two SSR markers were cloned into a pMD18-T vector and the sequence analysis was conducted.5. The main factors affecting the SSR-PCR amplification system were included in a multi-gradient optimization experiment, and an SSR reaction system suitable for hami melon was established. The 25μl reaction system contained 1.5mmol/L Mg2+, 1U Taq polymerase, 200μmol/L dNTP and 0.50μmol/L primer. The comparison between touch-down PCR and ordinary PCR showed that touch down PCR amplification is a reliable step to find the best method of annealing temperature.6. In this study, using RT-PCR and RACE technology, a full-length gene cDNA sequence was obtained from ACS3 hami melon, and analysis revealed that the total length of the gene is 1895bp, open reading frame 1446bp, encoding 482 amino acids, and has been submitted to GenBank(accession number for FJ383171). The homology was analyzed through Clustal X software, showing that ACS3 has more than 70% similarity with and Cucurbita maxima, mung beans, especially the similarity is up to 98% with cucumber females master genes ACS1G. The phylogenetic trees constructed by software MEGA 4 showed that ACS3 gene sequences were closest to the ACS of cucumber, closer to the ACS gene of plum, white lupine, etc., which is in conformity with the evolution relationship classification imbedded in the Origin of Species Evolution. The amino acid sequence analysis of ACS3 gene showed that its secondary structure is mainly Alpha helix and random coil, with a relatively large number of random coil, indicating instable biochemistry character; the similarity is up to 79% with Apple ACS protein tertiary structure. Functional amino acid sequence analysis of ACS3 revealed that the N-and C- terminal of ACS3 has a high hydrophilicity, non-transmembrane domain, and non-secreted protein.7. The ACS3 obtained in this experiment from the original male, female parent, original variety and hermaphrodite mutant demonstrates high gene sequence similarity, identical exon sequences, certain differences in intron region. Mutant TB-ACS3 lacks an A base at 265bp site in the first intron region, a T base at 1783bp site in the 3'UTR, but there is an extra 3'UTR at 1817bp site. Such non-coding region of the mutation may directly or indirectly affect the ACS3 gene in melon flower sexual type expression, and may be also one of the factors causing transformation of flower sexual type.8. The ACS3 obtained in this experiment from the original male, female parent, original variety and hermaphrodite mutant demonstrates identical CDS sequence, being T bases at 170bp site, consistent with studies of the ACS7 gene sequence with the melon andromonoecious line's(genotype aaGG) by Adnane Boualem et al; but the gene ORF sequence of ACS7 has two differences with the melon monoecious line's(genotype A_GG) , that is, at 170bp and the 1730bp site the ACS7 (A_GG) is C with monoecious line, while those of the original male, female parent, original variety and hermaphrodite mutant are T. This further proves that the mutation of ACS7 gene at base 170 is the direct cause of the transformation of the melon flower sexual type from A_ to aa.