Comprehensive Evaluation Of Germplasm Resources And Variety Breeding Of Isatis Indigotica Fort.

Isatis indigotica Fort.is a biennial herb of the cruciferous family Isatis genus.Its roots and leaves are used as medicine.The roots are radix isatidis and the leaves are folium isatidis.The wild germplasm resources of I.indigotica are scarce,and the types of cultivated populations are rich and diverse.They are mainly produced in Heilongjiang Province,Gansu Province,Hebei Province,Ningxia Hui Autonomous Region,Henan Province,Xinjiang Uygur Autonomous Region and Anhui Province,and so on.There are few good varieties popularized and applied in production,and most of the seeds are kept and grown by themselves,and the agronomic traits,yield,medicinal quality and quality of germplasms in different regions were quite different.In order to ensure the quality of medicinal materials from the source and improve the clinical efficacy,we collected germplasm resources from the main producing areas and distribution areas of I.indigotica across the country.The 27 germplasms were comprehensively evaluated by grey correlation analysis and DTOPSIS method,and high-quality germplasms were screened.2008-11 which is a new line of I indigotica was bred by systematic breeding,and studied from the aspects of agronomic traits,genetic indicators,photosynthetic characteristics,disease resistance,content of pharmacodynamic substances and accumulation rules.At the same time,combined transcriptome and metabolome exploration To analyze the molecular basis of the accumulation of antiviral active components lignans in roots of 2008-11.The main findings are as follows:Combining 18 Agronomic and Traditional Chinese Medicine Indicators,Grey relational grade analysis and DTOPSIS method were used to evaluate 27 I.Indigotica germplasm resources,The results showed that the order of weight of each trait were calculated as follows:Guanosine content（0.0643）>Uridine content（0.0608）>Length of taproot（0.0601）>Height of lateral root（0.0589）>Length of leaf（0.0587）>Width of plant（0.0576）>Number of blades（0.0573）>Diameter of root（0.0559）>Height of plant（0.0552）>（R,S）-goitrin content（0.0550）>Leaf yield（0.0547）>Adenosine content（0.0540）>Number of lateral roots（0.0539）>Root yield（0.0533）>Weight of root（0.0526）>Width of leaf（0.0521）>weight of Leaf（0.0490）>Weight of lateral root（0.0490）.The results of grey relational grade analysis and DTOPSIS are basically consistent,which is more comprehensive and practical than those of yield or medicinal ingredients.Hebei2018-1 and 2008-11 were selected from 27 germplasm resources,which had better comprehensive performance and could be used as breeding materials or fine lines production,the evaluation method of Grey relational analysis and DTOPSIS can be used to evaluate the germplasm resources of I.indigotica and other Chinese medicinal materials.2.The new line 2008-11 was bred by systematic breeding.The main features are as follows:（1）The leaves of 2008-11 are medium in size,the leaf length is 31.0cm,the leaf width is 6.0cm,the number of leaves is 14.8,the fresh weight of the leaf per plant is 50.76g,the leaf color is light green,the leaf shape is obovate;the root is straight,the main root is cylindrical,The number of lateral roots is small,and the lateral roots are placed at a lower position on the main root,the main root length is 27.1cm,the root diameter is 9.88mm,the fresh root weight per plant is 24.53g,the number of lateral roots is 2.5,the lateral root height is 5.8cm,and the lateral root weight is 4.70g.（2）The yield of 2008-11 was as follows:In the two-year comparison test of 2017 and 2018,the average leaf yield was798.6kg·666.7m^-2,an increase of 42.04%compared with the control;the average root yield was 438.6kg·666.7m^-2.Compared with the control,the yield increased by 23.52%;In the two-year regional tests in 2019 and 2020,the average root yield for the two years was 219.0kg·666.7m^-2,an increase of 49.88%compared with the control;In the 2021 production trial,the average root yield was 34.3%higher than the control.（3）The net photosynthetic rate,stomatal conductance and transpiration rate of 2008-11 were higher than the control,but there was no statistical difference;the intercellular CO₂ concentration and chlorophyll content of 2008-11 had extremely significant differences compared with the control,which increased by 82.0%and 35.9%,respectively.（4）the diseased plant rate of root rot was21.12%,the disease index was 12.15,and it was a disease-resistant variety.（5）The contents of uridine and guanosine in the roots of 2008-11 were significantly lower than those of the control;the content of（R,S）-goitrin was 0.1512%,which was significantly higher than that of the control.3.The Illumina platform was used to sequence the transcriptomes of 15 samples of Radix isatidis in five developmental stages.After quality control,a total of 104.56Gb Clean Data was obtained.The Clean Data of each sample was over 5.87Gb,and the percentage of Q30 bases was over 94.73%.The alignment efficiency of Isatis indigotica reference genome is between86.27%and 94.73%;the differentially expressed genes of each group are functionally annotated in 8 functional databases（including:COG,GO,KEGG,KOG,NR,Pfam,Swiss-Prot and egg NOG）),a total of 6033 DEGs were obtained.A total of 31 genes were screened in the phenylpropane biosynthesis pathway to be related to the biosynthesis of the medicinal substance lignin.The AB4500 Q TRAP UPLC/MS/MS system was used to analyze the metabolites of 15samples,and a total of 719 metabolites were detected.Different metabolites changed greatly in different periods.A total of 289 differential metabolites were screened in each group,and there were no common differential metabolites in 5 periods.Thirty metabolites were screened in radix isatidis,including 14 metabolites related to lignan biosynthesis,13 alkaloid metabolites and 3nucleotides.Based on the phenylpropane synthesis pathway related to the synthesis of lignans,combined with transcriptome and metabolome analysis,a total of 4 differential metabolites were screened.In the C vs D group,8 genes were related to the synthesis of isoeugenol.In the D vs E group,33 genes were enriched and related to the synthesis regulation of L-phenylalanine,isoeugenol,sinapic acid and spermidine,among which L-phenylalanine（31 genes were related）,isoeugenol（26 genes related）and sinapic acid（24 genes related）are the precursors or intermediates of lignan synthesis.The dynamic changes of these genes and metabolites revealed possible pathways of lignin biosynthesis.