Research Article |
Corresponding author: Joeri Sergej Strijk ( jsstrijk@hotmail.com ) Academic editor: Hugo de Boer
© 2019 Damien Daniel Hinsinger, Joeri Sergej Strijk.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Hinsinger DD, Strijk JS (2019) Plastome of Quercus xanthoclada and comparison of genomic diversity amongst selected Quercus species using genome skimming. PhytoKeys 132: 75-89. https://doi.org/10.3897/phytokeys.132.36365
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The genus Quercus L. contains several of the most economically important species for timber production in the Northern Hemisphere. It was one of the first genera described, but genetic diversity at a global scale within and amongst oak species remains unclear, despite numerous regional or species-specific assessments. To evaluate global plastid diversity in oaks, we sequenced the complete chloroplast of Quercus xanthoclada and compared its sequence with those available from other main taxonomic groups in Quercus. We quantify genomic divergence amongst oaks and performed a sliding window analysis to detect the most variable regions amongst members of the various clades, as well as divergent regions occurring in specific pairs of species. We identified private and shared SNPs amongst oaks species and sections and stress the need for a large global assessment of genetic diversity in this economically and ecologically important genus.
Phylogenomics, Quercus, Plastid genome, Genomic Diversity, Diversification, Sections, Taxonomy, Fagaceae, SNPs, Genomic Resources
The genus Quercus in Fagaceae, a large and locally dominant family of trees in temperate forests of the Northern Hemisphere and in major regions of the tropics and subtropics, is one of the first plant genera described in history (
Chloroplast DNA loci have been widely used in plant studies, both for evolutionary studies and for identification purposes, due to their natural abundance in plant cells (≈3–5% of the cell DNA content), when compared to nuclear DNA. In angiosperms, the chloroplast genome is a circular molecule (76–217 kilobases), with a conserved structure of two inverted repeats (IR) separated by small (SSC) and large (LSC) single-copy regions (
Genomic DNA was extracted from 0.1 g of silica gel-dehydrated leaves using a protocol modified from
Following the recommended best practices for complete organellar sequencing (
We used the retrieved oak chloroplasts to estimate the genetic divergence amongst chloroplasts using MEGA6 (
62,060 trimmed reads were mapped on the Q. rubra chloroplast sequence, for a total linear length of 162,328 bp. The main sequencing depth was 47.7 (min: 2; max: 90; S.D.: 12.5), covering 100% of the reference sequence (161,304 bp). The mean mapping confidence was 35.3, with 94.5% of the bases with mapping quality > Q20, and 89.0% with quality > Q30. The frequencies of each nucleotide were 31.1% (A), 18.7% (C), 18.0% (G) and 32.2% (T), with 439 positions unresolved (N). The properties of the Quercus xanthoclada plastid are shown in Table
Characteristics of the complete chloroplasts used in this study, showing the length, the GC content of each regions and the number of coding, tRNA and rRNA loci. LSC: Large Single Copy region; SSC: Small Single Copy region; IR: Inverted Repeats. Data from
Species | length | LSC | SSC | IR | GC total | GC LSC | GC SSC | GC IR | coding | tRNA | rRNA |
---|---|---|---|---|---|---|---|---|---|---|---|
Q. xanthoclada | 160,988 | 90,353 | 18,955 | 25,840 | 36.9 | 34.8 | 31.1 | 42.8 | 90 | 31 | 8 |
Q. spinosa | 160,825 | 90,371 | 18,732 | 25,861 | 36.87 | 34.7 | 31.2 | 42.6 | 87 | 29 | 8 |
Q. aliena | 160,921 | 90,258 | 18,980 | 25,841 | 36.9 | 34.8 | 31.3 | 42.7 | 89 | 39 | 8 |
Q. aquifolioides | 160,415 | 89,856 | 18,935 | 25,812 | 37.0 | 36.6 | 31.2 | 42.8 | 78 | 29 | 8 |
Q. rubra | 161,304 | 90,542 | 19,025 | 25,869 | 36.8 | 34.6 | 30.9 | 42.7 | 89 | 41 | 8 |
The maximum likelihood (ML) tree of ten Quercus chloroplasts available in GenBank shows that Q. xanthoclada is closely related to Q. spinosa and to the clade formed by Lithocarpus, Castanea and Castanopsis (Fig.
Results of the Tajima relative rate test. X2 test statistic value indicated, p-value in parenthesis. p-values < 0.01 in bold. For significant rate heterogeneity comparisons, the species with slower evolutionary rate is indicated: Qsp: Q. spinosa, Qal: Q. aliena, Qaq: Q. aquifolioides, Qru: Q. rubra. For clarity, p-values lower than 0.005 are not indicated.
Species | Q. xanthoclada | Q. spinosa | Q. aliena | Q. aquifolioides | Q. rubra |
---|---|---|---|---|---|
Q. xanthoclada | / | 17.37 – Qsp | 30.35 – Qal | 10.37 – Qaq | 32.64 – Qru |
Q. spinosa | / | 4.17 (0.04) – Qal | 0.02 (0.89) | 3.47 (0.06) | |
Q. aliena | / | 4.99 (0.03)-Qal | 0.06 (0.81) | ||
Q. aquifolioides | / | 3.30 (0.07) | |||
Q. rubra | / |
All five oak species exhibited high overall similarities (99.4–99.6%) (Table
Sliding window analysis of the whole plastomes of five oak species. (window length: 500 bp, step size: 250 bp). X-axis: position of the mid-point of the window, Y-axis: number of SNPs (solid line) and indels (dashed line) positions of each window in bp. Coding regions and directions of transcription are indicated by arrows, inverted repeats by grey areas. Putative barcode loci are highlighted for SNPs and indels. For readability, only a few major genes are indicated.
Estimates of p-distance amongst oak species. The number of base differences per site is shown. All positions containing gaps and missing data were eliminated. There were a total of 158480 positions in the final dataset.
Species | Q. xanthoclada | Q. spinosa | Q. aliena | Q. aquifolioides | Q. rubra |
---|---|---|---|---|---|
Q. xanthoclada | / | 0.005 | 0.006 | 0.006 | 0.006 |
Q. spinosa | / | 0.005 | 0.005 | 0.005 | |
Q. aliena | / | 0.005 | 0.004 | ||
Q. aquifolioides | / | 0.005 | |||
Q. rubra | / |
However, this overall pattern varied when considering each species pair (Fig.
The genome-skimming approach is now widely used to reconstruct chloroplasts in angiosperms (
In addition to the complete chloroplast sequence, the genome skimming can also be use to retrieve nuclear regions found in high copy number in the genome, such as the nuclear ribosomal cistrons (NRC) (
In our study, the plastome of Q. rubra was the largest in size in oaks (161,304 bp), but in other studies, it was described as the second smallest in oaks (
Quercus is widespread throughout the whole of the northern hemisphere, but in our study, three of the four available species came from Asia. To fully capture the chloroplast sequence diversity on a global scale, future inclusion of and genomic comparison with American and European Quercus is needed. Although species in this study are members of different recognised sections and subgenera, it is likely that they represent only a fraction of the total diversity within each of these groups and inclusion of additional members will reveal more about the extent and distribution of plastome diversity on various taxonomic and global scales.
Interestingly, one of the regions showing a relatively high level of variation amongst oak species is the rpl32-trnL spacer. As the observed variations are located in different portions of the region, it is likely that these SNPs and indels represent section specific diagnostic regions. Indeed, rpl32-trnL has been hypothesised as a DNA-barcode locus in several groups (
Most of the SNPs were either specific to one species or shared by four species and only a few shared by only two species (Fig.
Although these results, in combination with those obtained in Tajima’s relative rate test, seem to suggest a relative distinction of the Cyclobalanopsis species and their previous separation as a separate subgenus in the genus Quercus, this is not corroborated by any other data in our study. Neither the complete chloroplast phylogeny, nor previous studies based on nuclear and chloroplast loci (
Our work will allow for the development of new loci to be used in comparative phylogeography of the different section and subgenera (i.e. primers that are easily transferable amongst the different sections that can occur in sympatry), as well as open up new perspectives for conservation, management and the use of DNA fingerprinting to aid tracking of wood products from subtropical and tropical Asian oak species.
We would like to acknowledge R. Saunders, L. Wong and students from Hong Kong University, G. Fischer from Kadoorie Farm and Botanical Garden (HK) and K. Pang from the Agriculture, Fisheries and Conservation Department (AFCD) for their generous assistance in our ongoing collecting and sampling efforts of the Hong Kong flora.