Development of polymorphic EST-SSR markers and characterization of the autotetraploid in sainfoin (Onobrychis viciifolia)
- Published
- Accepted
- Subject Areas
- Agricultural Science, Molecular Biology, Plant Science
- Keywords
- Onobrychis viciifolia, Autotetraploidy, Polymorphism, Genetic diversity, EST-SSR
- Copyright
- © 2018 Shen et al.
- Licence
- This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ Preprints) and either DOI or URL of the article must be cited.
- Cite this article
- 2018. Development of polymorphic EST-SSR markers and characterization of the autotetraploid in sainfoin (Onobrychis viciifolia) PeerJ Preprints 6:e27232v1 https://doi.org/10.7287/peerj.preprints.27232v1
Abstract
Background: Sainfoin (Onobrychis viciifolia) is a highly nutritious, tannin-containing, and tetraploid forage legume. Due to the lack of detailed transcriptomic and genomic information on this species, genetic and breeding projects for sainfoin improvement have been significantly hindered.
Methods: In this study, a total of 24,630,711 clean reads were generated from 14 different sainfoin tissues using Illumina paired-end sequencing technology and deposited in the NCBI SRA database (SRX3763386). From these clean reads, 77,764 unigene sequences were obtained and 6,752 EST-SSRs were identified using denovo assembly. A total of 2,469 primer pairs were designed, and 200 primer pairs were randomly selected to analyze the polymorphism in five sainfoin wild accessions.
Results: Further analysis of 40 sainfoin individuals from the five wild populations using 61 EST-SSR loci showed that the number of alleles per locus ranged from 4 to 15, and the expected heterozygosity varied from 0.55 to 0.91. Additionally, by counting the EST-SSR band number and sequencing the three or four bands in one sainfoin individual, sainfoin was confirmed to be autotetraploid. This finding provides a high level of information about this plant.
Discussion: Through this study, 61 EST-SSR markers were successfully developed and shown to be useful for genetic studies and investigations of population genetic structures and variabilities among different sainfoin accessions.
Author Comment
This is a preprint submission to peerj preprints
Supplemental Information
Figure 1: Tissues used in this study. Samples were collected as described in the Materials and Methods section.
(A) Callus cells. (B) An emerging tidbit. (C) A young inflorescence. (D) An inflorescence. (E) Mature inflorescence. (F) Developing seed pods. (G) Mature seed pods. (H) Roots. (I) A germinated seed. (J) Young stems. (K) Stems. (L) Mature stems. (M) A young compound leaf. (N) A mature compound leaf. Bar = 1 cm.
Figure 2: STRUCTURE analysis of the genetic structures of five vetch sainfoin
Six different colors represent six different clusters. Pink, cluster 1; Green, cluster 2; Cyan, cluster 3; Blue, cluster 4; Yellow, cluster 5; and Red, cluster 6. Genetic structure of eight individuals in each of the five sainfoin sample populations is inferred by STRUCTURE using the EST-SSR markers dataset.
Figure 3: Phylogenic relationships among the 40 wild sainfoin individuals
The phylogeny tree was constructed using a neighbor-joining dendrogram in the Darwin software. The starting dataset was represented by the 61 best EST-SSRs. I-V, group number representing five different sampling locations. 1-8, sample number representing eight individual samples in the same group.
Figure 4: Comparative analysis of the DNA fragment peak spectrum for two selected EST-SSR loci among the same unit of Onobrychis viciifolia
Table1 Summary of the de novo assembled sainfoin EST-SSRs
Table 2: Functional annotation of sainfiontranscriptome
Supplemental Figure S1: Length distribution of all unigenes
Supplemental Figure S2: COG analysis of the unigene sequences
Supplemental Figure S3: Summary of GO analysis of the unigene sequences
The y-axis on the right indicates the number of genes in a category. The y-axis on the left indicates the number of genes in a specific category.
Supplemental Figure S4: Statistics of the EST-SSR length distribution
Supplemental Figure S5: Gene ontology (GO) term enrichment status for SSR repeat-containing unigenes in sainfoin
Supplemental Figure S6: Variations of EST-SSR markers by PCR using the PCR primer set Vo61 and Vo157 and the 40 wild sainfoin individuals collected from the five different geographic locations
The letter M denotes a molecular marker of 300 and 200bp size (top to bottom).The red triangle indicates the individual plant used to cut the polyacrylamide gel, Vo61selected No. 2 individual from site 2, Vo157 selected No.1 individual from site 2.
