| The medicinal A.paniculata is the aerial part of A.paniculata(Burm.f.)Nees,a plant of the Acanthaceae family.Clinically,it is usually applied to prevent and treat infections of the upper respiratory,gastrointestinal and hepatobiliary diseases.A.paniculata is mainly artificially cultivated in China,and the planting area is relatively wide.However,the variety of A.paniculata is single,severely degraded,and the effective ingredients vary widely and unstable due to single source of the A.paniculata seeds from Guangxi province,which will prevent the breeding process of A.paniculata.In this paper,the space mutagenic seeds of A.paniculata were selected to study the effects of space mutation on the agronomic traits and active component in the SP3 and SP4 generations of A.paniculata.Meanwhile,the specific expressions of WRKY family genes in the leaves of A.paniculata variant strains were studied,and whole genome re-sequencing analysis was carried out in 5 strains of A.paniculata with obvious phenotypic variations,which provided a theoretical basis for the breeding of high-quality and high-yield A.paniculata.The main results are as follows:1.There is still a big variation between the agronomic traits and active ingredients in the SP3 and SP4 generations of A.paniculata.Study shows that the phenotypes and active ingredients of in the SP3 and SP4 generations of A.paniculata are genetically unstable,and there are significant differences among the parameters.Moreover,there is an obvious correlation between the agronomic traits in each generation of A.paniculata and the effective ingredients.For example,the leaf length of A.paniculata in SP3 generation is extremely significantly correlated with leaf width and number of leaves,and is significantly correlated with plant height,fresh weight and dry weight;The leaf length and leaf width of SP4 A.paniculata were extremely significantly correlated,plant height and neoandrographolide were extremely significantly negatively correlated,and fresh weight and dry weight were extremely significantly correlated.According to the main active components of the agronomic traits of A.paniculata,the following lines were selected as the focus of subsequent breeding by correlation analysis: mutagenesis lines of “Long March 7”:H4,H29,H31,H36,H59,H88,H99,H119 and H120;The mutant lines of “Shenzhou11” were D45,D85,D106,D127 and D133.2.Five specific mutagenic strains of A.paniculata were selected in the SP4 generation.Through phenotypic analysis,we obtained 5 strains with the following specific charateristics: three-lobed whorled(H47);The number of leaves is the most(H47-2);main branches(H4-3,H29-2 and H3-2)in the SP4 generation,among which andrographolide content in leaves or stems of the strain H29-2 was significantly higher than that of the control group;the fresh weight and dry weight of the H47-2strain were significantly higher than the control group.Based on the variation of the morphological characteristics of A.paniculata,we took H4-3,H29-2,H47,H47-2 and H29-2 as the focus of SP5 breeding.3.The WRKY gene expressions of the 5 specific mutagenic strains of A.paniculata in the SP4 generation were obviously different.In this study,59 ApWRKY genes were identified,which were unevenly distributed on 23 chromosomes of A.paniculata;and through phylogenetic analysis,the WRKY domains of A.paniculata were divided into three groups,and the ApWRKY transcription factor has a certain species specificity during the evolution process.The specific expression of ApWRKYs in the leaves of five strains was studied by real-time fluorescent quantitative PCR(qRT-PCR).The results show that mutagenesis has obvious differences in the expression of most ApWRKY genes.For example,the expression of ApWRKY11 gene of strain H29-2 in leaves is 25 times higher than that of the CK.4.The contents and morphology of the four main active ingredients in the leaves of the 5 mutant strains of A.paniculata selected from the SP4 generation changed to varying degrees,indicating that mutagenesis can affect the synthesis and morphological variation of the active ingredients of A.paniculata.The whole genome resequencing analysis was carried out on 5 mutant lines.The results showed that a total of 228,694 SNPs,1,913,310 Indels and 15,875 SVs were detected in the five samples,and a total of 67,383 gene variants were found when compared with the reference genome.The major variants were found to be concentrated in the secondary metabolic pathway by KEGG pathway annotation comparison. |
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