Abstract
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.
Highlights
Extracts from roots of the woody deciduous legume kushen (Sophora flavescens Ait.) have long been used in traditional Chinese medicine
An initial round of DArTseq using four DNA samples yielded 2212 single nucleotide polymorphism (SNP)-bearing tag pairs, 1392 of which were scored for all four plants
A combination of techniques has provided resources for the future, with over 10,000 SNPs identified and 27 of these validated through the development of high throughput Kompetitive Allele Specific PCR (KASP) markers
Summary
Extracts from roots of the woody deciduous legume kushen (Sophora flavescens Ait.) have long been used in traditional Chinese medicine. The medicinal use of kushen root preparations was first documented in 200 A.D. in the Chinese book Shen Nong Ben Cao Jing and continues today for treatment of a variety of conditions, including cancers, hepatitis, and heart disease (Sun et al, 2012; He et al, 2015). QA alkaloids are produced by a subset of legumes, including narrow-leaf lupin (Lupinus angustifolius), and they are responsible for the bitter taste of some lupin varieties (Ohmiya et al, 1995; Bunsupa et al, 2016; Carvajal-Larenas et al, 2016). Levels of matrine and oxymatrine have been reported to be three to four times higher in kushen root and seed tissue than in leaf and stem tissue (Zhang et al, 2020)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
