Analysis of Genetic Diversity in the Traditional Chinese Medicine Plant ‘Kushen’ (Sophora flavescens Ait.)

Kushen root, from the woody legume Sophora flavescens, is a traditional Chinese medicine that is a key ingredient in several promising cancer treatments. This activity is attributed in part to two quinolizidine alkaloids (QAs), oxymatrine and matrine, that have a variety of therapeutic activities in vitro. Genetic selection is needed to adapt S. flavescens for cultivation and to improve productivity and product quality. Genetic diversity of S. flavescens was investigated using genotyping-by-sequencing (GBS) on 85 plants grown from seeds collected from 9 provinces of China. DArTSeq provided over 10,000 single nucleotide polymorphism (SNP) markers, 1636 of which were used in phylogenetic analysis to reveal clear regional differences for S. flavescens. One accession from each region was selected for PCR-sequencing to identify gene-specific SNPs in the first two QA pathway genes, lysine decarboxylase (LDC) and copper amine oxidase (CAO). To obtain SfCAO sequence for primer design we used a targeted transcript capture and assembly strategy using publicly available RNA sequencing data. Partial gene sequence analysis of SfCAO revealed two recently duplicated genes, SfCAO1 and SfCAO2, in contrast to the single gene found in the QA-producing legume Lupinus angustifolius. We demonstrate high efficiency converting SNPs to Kompetitive Allele Specific PCR (KASP) markers developing 27 new KASP markers, 17 from DArTSeq data, 7 for SfLDC, and 3 for SfCAO1. To complement this genetic diversity analysis a field trial site has been established in South Australia, providing access to diverse S. flavescens material for morphological, transcriptomic, and QA metabolite analysis. Analysis of dissected flower buds revealed that anthesis occurs before buds fully open suggesting a potential for S. flavescens to be an inbreeding species, however this is not supported by the relatively high level of heterozygosity observed. Two plants from the field trial site were analysed by quantitative real-time PCR and levels of matrine and oxymatrine were assessed in a variety of tissues. We are now in a strong position to select diverse plants for crosses to accelerate the process of genetic selection needed to adapt kushen to cultivation and improve productivity and product quality.

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Assessing the equivalence between traditional medicinal varieties and nontraditional medicinal varieties of Citri reticulata Semen

10.21203/rs.3.rs-27851/v1 ◽

2020 ◽

Author(s):

Bin Xian ◽

Li Wang ◽

Rui Wang ◽

Chaoxiang Ren ◽

Jiang Chen ◽

...

Keyword(s):

Genetic Diversity ◽

Molecular Markers ◽

Chinese Medicine ◽

Traditional Chinese Medicine ◽

Genetic Relationship ◽

Citrus Reticulata ◽

Significant Difference ◽

Stereo Microscope ◽

New Varieties ◽

Shape And Size

Abstract Background Juhe, which is derived from seeds of citrus (Citri reticulata Semen), is wildly used as a traditional Chinese medicine. Its original plants have numinous variety, and new varieties are constantly emerging, especially in recent years. The decline in the market share of traditional varieties each year, has resulted in a shortage of Juhe resources, and nontraditional Juhe varieties have been used clinically without research. Thus Juhe resources vary significantly. Furthermore, most of new varieties are hybrids, with significant differences in genes, active ingredients. Therefore, a further research is needed on Nontraditional medicinal varieties. Methods In this study, the genetic diversity, shape and size, and flavonoids content of 35 different batches of Juhe samples, from different source, were analyzed base on SRAP molecular markers, a stereo microscope, and HPLC. Then correlation analysis was performed using these data. Results The genetic backgrounds of traditional medicinal varieties (TMV) and nontraditional medicinal varieties (N-TMV) differed significantly, and TMV were closely related to some Citrus ‘Ponkan’ samples. Seeds of TMV and N-TMV differ based on shape, size and number. In addition, flavonoid compounds and level in the seeds of TMV and N-TMV also showed a significant difference. Five batches of C. reticulata ‘Ponkan’ samples collected from Zigong, Meishan, Neijiang, Nanchong and Jiangyou were closely related to the traditional medicinal variety Citrus reticulata ‘Dahongpao’,based on genetic relationship, shape and sizes, as well as flavonoids content. Conclusins: A total of 5 C. reticulata ‘Ponkan’ samples, including Zigong No. 7, Meishan No. 21, Neijiang No. 27, Nanchong No. 32, and Jiangyou No. 34, were closely related to the traditional medicinal variety C. reticulata ‘Dahongpao’ in genetic relationship, seeds’ characteristics and sizes, and flavonoids content. These varieties could serve as a new medicinal variety.

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