| With the rapid development of the flower market,people have begun to pursue exotic flowers and plants with certain ornamental value and application value.Phalaenopsis equestris has a beautiful shape,bright flowers and light fragrance,and is highly sought after in the flower market.Phalaenopsis equestris has extremely high market application potential,and the demand for new varieties of Phalaenopsis equestris is increasing.However,due to the difficulty of molecular breeding,60Co-y ray radiation was used to mutate in Phalaenopsis equestris,in order to obtain excellent new varieties.In this experiment,15Gy and 20Gy doses of 60Co-γ rays were used to irradiate 21 strains of the Phalaenopsis equestris and the Peloric Mutant Phalaenopsis equestris,and their growth status and physiological indicators were observed and recorded.Interval simple repeat sequence markers(IS SR molecular marker technology)were used to analyze the mutation results and genetic distance after mutagenesis.The numbered S-15Gy-8 line and the T-20Gy-10 line showed specific flowering phenotype,and the fluorescence quantitative expression analysis of PeMADS gene was carried out.In addition,6 Peloric Mutant Phalaenopsis equestris plants were treated with radiation at a dose of 40Gy,and their growth rates were observed.It was proved that the appropriate dose of radiation treatment would stimulate the growth and development of Phalaenopsis equestris.Transcriptome analysis to explore the mechanism of radiation effects on the growth and development of Phalaenopsis equestris.The main results are as follows:1.By observing and recording the growth of the new leaves and mature leaves of Phalaenopsis equestris after radiation treatment,the growth rate was calculated according to the length and width of the leaves recorded for the first time and one month later;the wilting within two months after the radiation treatment was recorded.and the number of necrotic lines.The results showed that:(1)Radiation treatment stimulated the growth and development Phalaenopsis equestris,especially in the young leaves;(2)the growth rate of the Phalaenopsis equestris lines treated with radiation was only partially higher than that of the control group,while the radiation treatment The growth rate of the treated Peloric Mutant Phalaenopsis equestris lines was generally higher than that of the control group;(3)The Phalaenopsis equestris appeared the earliest adverse phenotypes such as wilting and leaf necrosis within two months of radiation treatment,and the occurrence probability was higher than that of the Peloric Mutant Phalaenopsis equestris with petals and Peloric Mutant Phalaenopsis equestris showed higher resistance to radiation treatment,and the probability of death,wilting and other adverse phenotypes was lower than that of Phalaenopsis equestris;(4)The Peloric Mutant Phalaenopsis equestris treated with a dose of 40Gy showed an obvious rapid growth and development phenotype within ten days after radiation treatment.According to the transcriptome analysis,the fluorescent quantitative expression results and transcriptome results of genes related to leaf growth and development were screened out.Consistent.2.Physiological indicators of Phalaenopsis equestris trilipae with different radiation doses were analyzed,and the results showed that:(1)The content of malondialdehyde(MDA)in the 15Gy and 20Gy dose treatment groups was lower than that in the control group,and the 20Gy dose was significantly lower than that in the control group.It is lower than CK,indicating that within the 20Gy dose range,the higher the dose,the cell membrane structure of Phalaenopsis equestris is not damaged;(2)Superoxide dismutase(SOD),peroxidase(POD)and catalase(CAT)activity increased with the increase of radiation dose,and there was an obvious positive correlation trend,indicating that radiation treatment would produce peroxides and other harmful substances in the cells of Phalaenopsis equestris and the higher the dose,the more harmful substances;(3)The soluble sugar content is different,the soluble sugar content of 15Gy treatment group is higher than that of control group,and that of 20Gy treatment group is lower than that of control group.3.The ISSR molecular marker technology was used to analyze the genetic diversity and kinship of the irradiated Phalaenopsis equestris materials.The results showed that:(1)8 primers were screened to amplify a total of 89 clear bands,of which 72 The bar showed polymorphism,and the polymorphism ratio was 80.9%.The primers UBC895 and UBC840 had the highest polymorphism ratio,which were 93.3%and 91.6%,respectively.The genetic similarity between Phalaenopsis equestris was 0.54-0.97,and the genetic similarity between Peloric Mutant Phalaenopsis equestris was 0.54-0.91,indicating that there were abundant genetic polymorphisms among radiation mutant varieties;(2)After radiation,Phalaenopsis equestris can be divided into four groups at the genetic distance L=0.65,and the Peloric Mutant Phalaenopsis equestris was divided into seven groups at the genetic distance L=0.72 after radiation;(3)UPGMA cluster analysis showed that,After radiation mutagenesis,the Phalaenopsis equestris has different degrees of mutation,and the species with the greatest degree of mutation forms its own branch on the genetic distance map.4.To observe and record the flowering phenotype of Phalaenopsis equestris with mutant phenotype and analyze the fluorescence quantitative expression of flower development regulatory genes.The results showed that:(1)S-15Gy-8 strains showed a variety of mutant phenotypes.Except for the strange and unclassifiable phenotypes,the rest of the phenotypes were mainly petal phenotype mutations,which were roughly divided into ’sepalization’,’Lip petalization’;(2)The T-20Gy-10 line first appeared phenotypes such as sepal fusion and petal fusion,and then completely mutated to the same phenotype:morphologically,the upper end was a petal,the lower end was a sepal,and the petals and There was no obvious change in the pistil column between sepals;(3)The expression of PeMADS genes in S-15Gy-8 line was consistent with the mutant expression of petals.There were significant differences in all kinds of genes in the three20Gy-10 line.(4)The new varieties Phalaenopsis equestris ’Xunlan’(application number:20211556),’Feilan’(application number:20211557),and Fanlan’(application number:20211558)have been applied.5.The Peloric Mutant Phalaenopsis equestris was treated with a dose of 40Gy to further reveal the molecular mechanism of radiation treatment to stimulate the growth of Phalaenopsis equestris leaves.RNA-seq analysis was performed on untreated WT and 40Gy treated groups.The results showed that:(1)Principal Component Analysis(PCA)results indicated that there were significant differences between the radiation treatment group and the control group,and there were differences between the samples in the treatment groups;(2)Venn showed the number of differentially expressed genes shared between the wild type and the mutants(3)KEGG enrichment analysis showed that radiation mainly affected the hormone signal transduction and carbohydrate phenylpropane synthesis-related pathways of Phalaenopsis equestris;(4)GO enrichment analysis showed that the effect of radiation Mainly related to biological regulation processes such as cell regulation process and intracellular signal transduction;(5)11 genes related to leaf growth and development were screened out,and the results of fluorescence quantitative expression were consistent with the transcriptome expression. |
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