Journal: Ecology and Evolution

Article Title: 3 RAD ‐Guided SNP Discovery for Species Identification and Conservation of the Medicinal Southern African Tree Genus Greyia Hook. & Harv.

doi: 10.1002/ece3.73412

Figure Lengend Snippet: Two‐phase strategy for the development of an SNP‐based species identification method for the genus Greyia . The ascertainment panel was made up of 2–3 trees of each species G. radlkoferi , G. sutherlandii , and G. flanaganii sampled from geographically separated “core populations.” The SNP assay design started with extracting 200 SNP subsets from the full SNP dataset based on F ST ranges (0.3–0.5, 05–0.7, 0.8–1). In this study, the subset in the 0.3–0.5 range was selected based on PCA and co‐ancestory analyses. An optional step was including informative SNPs from standard barcoding genes in the final SNP assay design. Proof of concept evaluation (testing) of the 12–24 SNP panel is carried out on the Flex Six Genotyping integrated fluidic circuit (IFC) run on the Biomark HD instrument. Screening of large numbers of unknown samples on the Biomark HD instrument was performed on the 96.96 Dynamic Array IFC to obtain technical replicates of assays/samples in a single run. Final species identifications made with the allele scoring matrix were confirmed with other genetic clustering methods (e.g., PCoA, Admixture, and DAPC). This framework of SNP discovery and SNP assay design can be adapted to develop a 12–24 SNP panel for species identification/genotype differentiation in other plants/organisms.

Article Snippet: The 23 SNP Type assays on the Biomark HD microfluidics system were successfully designed and optimized for standardized genetic species identification of Greyia trees (Table ).

Techniques:

Journal: Ecology and Evolution

Article Title: 3 RAD ‐Guided SNP Discovery for Species Identification and Conservation of the Medicinal Southern African Tree Genus Greyia Hook. & Harv.

doi: 10.1002/ece3.73412

Figure Lengend Snippet: Greyia 23 SNP Type TM assays differentiate the ascertainment panel trees into three species groups. Principal coordinates analysis (PCoA) with the 23 SNP data from the Biomark HD microfluidics system is shown for the Greyia ascertainment panel trees with three additional trees from each of the core geographic sites in Limpopo, KwaZulu‐Natal, and Eastern Cape representing G. radlkoferi (GRA), G. sutherlandii (GSU) and G. flanaganii (GFL), respectively (n=17). Sample IDs refer to the following Greyia trees with ascertainment panel members in bold: G. sutherlandii — P090_G60 , P241_G64A, P242_G64 , P243_G56, P244_G57, P245_G65; G. radlkoferi —P001_G58, P002_G59, P003_G65A, P004_G66, P005_G67, P023_G61; G. flanaganii —P157_G52, P158_G55, P159_G62A, P160_G62, P161_G63 . Red dots = GSU, green dots = GRA, blue dots = GFL, purple dots = additional trees. PC1 explains 54.66% of the variability, while PC2 explains 34.44% of the variability.

Article Snippet: The 23 SNP Type assays on the Biomark HD microfluidics system were successfully designed and optimized for standardized genetic species identification of Greyia trees (Table ).

Techniques:

Journal: Ecology and Evolution

Article Title: 3 RAD ‐Guided SNP Discovery for Species Identification and Conservation of the Medicinal Southern African Tree Genus Greyia Hook. & Harv.

doi: 10.1002/ece3.73412

Figure Lengend Snippet: Wild‐growing Greyia trees form three genetic clusters with 23 SNP Type assay. (A) Principle Components Analysis with the 23 SNP data show that the 73 wild‐growing trees form three Greyia species clusters: Cluster I—“Radlkoferi,” Cluster II—“Sutherlandii,” Cluster III—“Flanaganii.” The G. radlkoferi , G. sutherlandii , G. flanaganii species‐representative ascertainment panel members are indicated by triangles: GRA (green); GSU (red); GFL (blue). The other wild‐growing Greyia trees are indicated by gray circles. PC1 and PC2 describe 37.95% and 21.45% of the variation, respectively. (B) Admixture analysis of the same SNP dataset as (A) shows that the 73 wild‐growing Greyia trees form the same three clusters of inferred ancestory ( K = 3) visualized with an ADMIXTURE bar plot. (C) UPGMA dendrogram calculated from the matrix of shared alleles of the same SNP dataset as (A) groups the 73 wild‐growing trees into three Greyia species clades indicated by the color coding: GRA (green); GSU (red); GFL (blue). Greyia tree IDs correspond to the trees listed in the Appendix A: Table . The scale bar illustrates the amount of genetic distance expressed as the proportion of differing alleles.

Article Snippet: The 23 SNP Type assays on the Biomark HD microfluidics system were successfully designed and optimized for standardized genetic species identification of Greyia trees (Table ).

Techniques:

Journal: Ecology and Evolution

Article Title: 3 RAD ‐Guided SNP Discovery for Species Identification and Conservation of the Medicinal Southern African Tree Genus Greyia Hook. & Harv.

doi: 10.1002/ece3.73412

Figure Lengend Snippet: Discriminant Analysis of Principle Components (DAPC) shows that wild‐growing Greyia trees form three genetic clusters. (A) DAPC plot generated with the 23 SNP Type TM assay data of the 73 wild‐growing trees illustrates the three Greyia species clusters: Cluster I—“Radlkoferi” (GRA, green circles), Cluster II—“Sutherlandii” (GSU, red diamonds), Cluster III—“Flanaganii” (GFL, blue triangles). (B) Density plot of the DAPC illustrating the three species clusters in the same color coding as A.

Article Snippet: The 23 SNP Type assays on the Biomark HD microfluidics system were successfully designed and optimized for standardized genetic species identification of Greyia trees (Table ).

Techniques: Generated

Journal: Ecology and Evolution

Article Title: 3 RAD ‐Guided SNP Discovery for Species Identification and Conservation of the Medicinal Southern African Tree Genus Greyia Hook. & Harv.

doi: 10.1002/ece3.73412

Figure Lengend Snippet: Biogeographical distribution of wild‐growing Greyia plants and identification of cultivated Greyia plants with 23 SNP Type assay. (A) Map of South Africa with locations of 73 wild‐growing Greyia trees, color coded by the species assignment from the 23 SNP Type assay. Trees identified as G. radlkoferi (green) are located in Limpopo and Mpumalanga provinces; G. sutherlandii (red) trees in southern Mpumalanga, KwaZulu‐Natal, and north‐east Eastern Cape provinces, and G. flanaganii (blue) in the Eastern Cape province. (B) Principle Components Analysis with the 23 SNP data show that the 33 cultivated trees cluster in one of the three Greyia species clusters: Cluster I—“Radlkoferi,” Cluster II—“Sutherlandii,” Cluster III—“Flanaganii.” The G. radlkoferi , G. sutherlandii , G. flanaganii wild‐growing trees are indicated by triangles: GRA (green); GSU (red); GFL (blue). The cultivated Greyia trees are indicated by gray circles. PC1 and PC2 describe 42.58% and 18.22% of the variation, respectively. The species assignments, identities and location of the cultivated trees are given in Appendix A: Table .

Article Snippet: The 23 SNP Type assays on the Biomark HD microfluidics system were successfully designed and optimized for standardized genetic species identification of Greyia trees (Table ).

Techniques:

Journal: Ecology and Evolution

Article Title: 3 RAD ‐Guided SNP Discovery for Species Identification and Conservation of the Medicinal Southern African Tree Genus Greyia Hook. & Harv.

doi: 10.1002/ece3.73412

Figure Lengend Snippet: Admixture analysis indicates that cultivated and wild‐growing Greyia trees form three genetic clusters with the 23 SNP Type TM assay. (A) ADMIXTURE error rates from 20‐fold cross‐validation tests to determine the optimal number of clusters (ancestral populations (K)) based on the 23 SNP data of 33 cultivated and 73 wild‐growing Greyia trees. The purple box illustrates that the lowest error rates were obtained for K = 3. Each box indicates the interquartile range, while the horizontal line within the box shows the median. Whisker bars show the maximum and minimum values, with outliers shown with open circles. (B) STRUCTURE plot illustrating that K = 3 was the optimal number of inferred ancestral populations for the SNP dataset of cultivated and wild‐growing Greyia trees. The ascertainment panel trees are labelled GRA, GSU, and GFL. The wild‐growing and cultivated trees are labelled as WIL and CUL, respectively.

Article Snippet: The 23 SNP Type assays on the Biomark HD microfluidics system were successfully designed and optimized for standardized genetic species identification of Greyia trees (Table ).

Techniques: Biomarker Discovery, Whisker Assay