Analysis of Genetic Diversity in the Traditional Chinese Medicine Plant 'Kushen' ( Ait.).

Kushen root, from the woody legume , 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 . Genetic selection is needed to adapt for cultivation and to improve productivity and product quality. Genetic diversity of 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 . 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 sequence for primer design we used a targeted transcript capture and assembly strategy using publicly available RNA sequencing data. Partial gene sequence analysis of revealed two recently duplicated genes, and , in contrast to the single gene found in the QA-producing legume . We demonstrate high efficiency converting SNPs to Kompetitive Allele Specific PCR (KASP) markers developing 27 new KASP markers, 17 from DArTSeq data, 7 for , and 3 for . To complement this genetic diversity analysis a field trial site has been established in South Australia, providing access to diverse 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 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.