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...
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Frontiers Media S.A.
2021-08-01
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| Series: | Frontiers in Plant Science |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fpls.2021.704201/full |
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| author | Carolyn J. Schultz Shashi N. Goonetilleke Jianping Liang Jelle Lahnstein Kara A. Levin Tina Bianco-Miotto Rachel A. Burton Diane E. Mather Kenneth J. Chalmers |
| author_facet | Carolyn J. Schultz Shashi N. Goonetilleke Jianping Liang Jelle Lahnstein Kara A. Levin Tina Bianco-Miotto Rachel A. Burton Diane E. Mather Kenneth J. Chalmers |
| author_sort | Carolyn J. Schultz |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-19T21:01:48Z |
| format | Article |
| id | doaj.art-63fb84124169427bb69db95174ad2579 |
| institution | Directory Open Access Journal |
| issn | 1664-462X |
| language | English |
| last_indexed | 2024-12-19T21:01:48Z |
| publishDate | 2021-08-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Plant Science |
| spelling | doaj.art-63fb84124169427bb69db95174ad25792022-12-21T20:05:48ZengFrontiers Media S.A.Frontiers in Plant Science1664-462X2021-08-011210.3389/fpls.2021.704201704201Analysis of Genetic Diversity in the Traditional Chinese Medicine Plant ‘Kushen’ (Sophora flavescens Ait.)Carolyn J. Schultz0Shashi N. Goonetilleke1Jianping Liang2Jelle Lahnstein3Kara A. Levin4Tina Bianco-Miotto5Rachel A. Burton6Diane E. Mather7Kenneth J. Chalmers8School of Agriculture, Food and Wine, Waite Research Institute, University of Adelaide, Adelaide, SA, AustraliaSchool of Agriculture, Food and Wine, Waite Research Institute, University of Adelaide, Adelaide, SA, AustraliaDepartment of Chinese Medicine, College of Life Sciences, Shanxi Agricultural University, Shanxi, ChinaSchool of Agriculture, Food and Wine, Waite Research Institute, University of Adelaide, Adelaide, SA, AustraliaSchool of Agriculture, Food and Wine, Waite Research Institute, University of Adelaide, Adelaide, SA, AustraliaSchool of Agriculture, Food and Wine, Waite Research Institute, University of Adelaide, Adelaide, SA, AustraliaSchool of Agriculture, Food and Wine, Waite Research Institute, University of Adelaide, Adelaide, SA, AustraliaSchool of Agriculture, Food and Wine, Waite Research Institute, University of Adelaide, Adelaide, SA, AustraliaSchool of Agriculture, Food and Wine, Waite Research Institute, University of Adelaide, Adelaide, SA, AustraliaKushen 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.https://www.frontiersin.org/articles/10.3389/fpls.2021.704201/fullquinolizidine alkaloidstraditional Chinese medicineDArT sequencinglysine decarboxylasecopper amine oxidasesingle nucleotide polymorphisms |
| spellingShingle | Carolyn J. Schultz Shashi N. Goonetilleke Jianping Liang Jelle Lahnstein Kara A. Levin Tina Bianco-Miotto Rachel A. Burton Diane E. Mather Kenneth J. Chalmers Analysis of Genetic Diversity in the Traditional Chinese Medicine Plant ‘Kushen’ (Sophora flavescens Ait.) Frontiers in Plant Science quinolizidine alkaloids traditional Chinese medicine DArT sequencing lysine decarboxylase copper amine oxidase single nucleotide polymorphisms |
| title | Analysis of Genetic Diversity in the Traditional Chinese Medicine Plant ‘Kushen’ (Sophora flavescens Ait.) |
| title_full | Analysis of Genetic Diversity in the Traditional Chinese Medicine Plant ‘Kushen’ (Sophora flavescens Ait.) |
| title_fullStr | Analysis of Genetic Diversity in the Traditional Chinese Medicine Plant ‘Kushen’ (Sophora flavescens Ait.) |
| title_full_unstemmed | Analysis of Genetic Diversity in the Traditional Chinese Medicine Plant ‘Kushen’ (Sophora flavescens Ait.) |
| title_short | Analysis of Genetic Diversity in the Traditional Chinese Medicine Plant ‘Kushen’ (Sophora flavescens Ait.) |
| title_sort | analysis of genetic diversity in the traditional chinese medicine plant kushen sophora flavescens ait |
| topic | quinolizidine alkaloids traditional Chinese medicine DArT sequencing lysine decarboxylase copper amine oxidase single nucleotide polymorphisms |
| url | https://www.frontiersin.org/articles/10.3389/fpls.2021.704201/full |
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