| Onion(Allium cepa L. 2n=2x=16) is a biennial vegetable crop that belongs to Allium, Liliaceae family. Onion is one of the most important vegetable crops grown in the world. Although the history of planting onions is shorter than other vegetables in China, it has developed rapidly and is widely cultivated. Onion is a representative cross-pollinated vegetable with obvious heterosis. Onion inbreds are often produced through inbreeding using self and sib pollinations, requiring 6 to 10 years of time and resource investments. The use of haploidization methods in breeding hybrid cultivars is of particular interest in long cycle species. Production of double haploid(DH) lines is a rapid method of obtaining completely homozygous inbred lines for onion. Doubled haploids can be exploited not only to produce new cultivars, but also to construct genetic maps, locate genes of important agronomic characters and develop markers for trait selection.Intermediate-day onion inbred lines, open-pollinated and hybrid cultivars from Japan and SAAS were tested for gynogenic induction. SLAF-seq approach was employed to develop SNPs for onion using male sterile double haploid DH-1, fertile doubled haploid DH-17 and their 5 F1 plants. The main results were as follows:Unopened flower buds of three intermediate-day hybrid cultivars of onion from Japanese seed company were cultured in vitro using B5 medium which supplied with 100 g·L-1 sucrose and 2,4-D and 6-BA. The suitable media for gynogenic embryo induction of onion were B5 medium with 1.5 mg·L-1 2,4-D + 1.5 mg·L-1 6-BA and B5 medium with 2.0 mg·L-1 2,4-D + 2.0 mg·L-1 6-BA. The highest induction rate was 4.00%.The optimization of induction culture for the unopened flower buds in onion was studied. The results showed that the size of the flower buds had a great influence on the gynogenesis of the onion. The embryo rate of flower buds with the diameter of 2.1-3.0 mm was significantly lower than that of other diameter and flower buds with diameter 3.1-4.0 mm have the highest induction rate. After pretreatment 1 day at 4℃, embryo rate of ‘EARTH’ was significantly higher than that of non-pretreated, but begins to drop significantly after 3 day pretreatment at 4℃.The genotype of the onion donor plant determined the efficiency of gynogenic embryo production. The hybrid cultivar ‘EARTH’ has the highest embryo rate in the 9 onion genotypes of tested, followed by ‘TABAO’ and ‘ATON’ which embryo rate was 4.33% and 4.00% respectively. Six and 8 embryos were induced from two open-pollinated cultivar ’Tianzheng hongyu’ and ’Tianzheng huangjin’. Thirteen and 2 embryos were induced from inbred lines ‘217’ and ‘503’, which induction rate was significantly higher than that of the open-pollinated cultivars. In general, the embryo rate of hybrid cultivars was higher than inbred lines, and the induction rate of inbred lines was higher than that of the open-pollinated cultivars.The gynogenic embryos inoculated to the medium 1/2B5+0.5 mg·L-1NAA containing 30 g·L-1 sucrose showed the highest response with 86.67% plant regeneration. The DNA relative content of regenerated plants was determinated by flow cytometry. Separation peak of normal diploid sample was located at 140 relative fluorescence intensity. It is concluded that the separation peak at 70 is haploid material. Chromosome counts of root-tip also confirmed ploidy of regenerated plants. Diploid plants which identified by flow cytometry have chromosome number 2n=2x=16 and the haploid have chromosomes n=x=8. Identification results was exactly as the same as by flow cytometry.Effects of different colchicine concentration and treatment time on the chromosome doubling for onion haploid were studied. The results show that at the same treatment time, as the concentration of colchicine was increased the regenerated plants survival rate were reduced, but the diploid rate increases. Under the same concentration of colchicine, with the prolongation of treatment time, plant survival rate was reduced, but diploids rate was increased. After 48 h of treatment with 200 mg L-1colchicine, the chromosome doubling effect was the best for the two tested cultivars. The survival rates were 61.11% and 50.00% respectively. Eight and 7 double haploid plants were induced from 18 haploid plants, and doubling rates were 44.44% and 38.89% respectively.In order to identify regenerated plants originated from gynogenesis, DNF-566, RNS-357 and AcSKP1 markers were used. Eleven plants of ‘ATON’ and 15 plants of ‘EARTH’ were homozygous at Ms locus. Of the 16 regenerated plants from ‘TABAO’, 13 plants were homozygous and 3 plants were heterozygous at the Ms locus.The regenerated plants were transplanted into the net house of experimental base. Most of regenerated plants survived showed normal morphology. Onion haploid plants can grow normally, but the plant was short and thin. Flowering phase of haploid was as the same as the diploid plants, but the flower stalk was small and short. The whole inflorescence was small which have fewer flower buds than the diploidy plants. The plants from the haploid produced very little pollen in the anther. The traits of double haploid plants were in line with the diploid and blossom normally. It was found that the doubled-haploid plants with Ms Ms genotype were male fertile and the doubled-haploid plants with msms genotype were male sterile. After flowering, the doubled-haploid plants with normal fertility were bagged with parchment bag and put flies into them. Douled-haploid seeds were obtained by self-pollination. The sterile double haploid plants crossed with fertile doubled-haploid plants and F1 seeds were harvested.Molecular markers by SLAF-seq based on Reduced-Representation Genomic Deep Sequencing in onion with two double haploids and their F1 plants were developed. The scheme of the experiment was designed based on bioinformatics technology. Taking onion transcriptome as the reference, specific size of DNAs were chosen to construct the SLAF-seq library. After high-throughput sequencing, a great amount of sequences were used to obtain the polymorphism SLAF tags by software alignment, then found the distribution of specific SNP sites. The results coming from the alignment between the sequences of Arabidopsis and its reference genome indicated that the construction of SLAF library fitted well to the standard, with its paired-end mapped reads reaching 80.60%. With the help of sequencing alignment as well as other bioinformatics technologies, a total of 125.98 M reads were obtained, the total number of the reads of each sample varied from 29736 to 30825419. The average value of Q30 was 89.87% and all the sample of Q30 were beyond 80%. The average GC content of each sample was 35.77% which indicated the average level of GC content low enough to process the sequencing.After sequence alignment and cluste analysis, 294911 high quality SLAFs were detected, of which 14776 were polymorphic, giving a polymorphism rate of only 5%. After genotyping analysis and filtering out the SLAFs lacking parent information, 6384 were classified into eight segregation patterns. In a total of 5354 polymorphic markers fell into aa×bb segregation pattern. 36109 SNP sites were obtained based on the analysis of polymorphic SLAFs.Genetic relationship identification of the samples showed that the proportion of the number of abnormal labels were far less than 0.5%. It can be determined that the sample F1-1, F1-2, F1-3, F1-4, F1-5 were offspring of DH-17 and DH-1. |
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