We present here a study of the genetic diversity and
population structure in a collection of Trifolium
pratense using GBS to produce the molecular marker data. GBS is a high
throughput and cost effective procedure that can be used to identify and select
target plants for breeding programs (Kim, et al., 2016).
GBS was performed on a collection of 640 individuals from 75 collections. However,
10 genotypes did not pass the sequencing quality scores and were therefore
omitted from the analysis. ApeKI was used to reduce the complexity of the
genome and a high number of tags were identified in conjunction with the red
clover genome assembly (De Vega, et al., 2015).
ApkeI was selected as the restriction enzyme as it is partially methylation
sensitive and rarely cuts retrotransposons. Therefore, ApeKI digestion products
are fragments preferentially from low-copy genomic regions (Elshire, et al., 2011, Sonah, et al., 2013),
and are more likely to be genic in origin.
1.8 million tags were produced in TASSEL5v2. Within these
tags 8,118 high quality SNP were identified across the 640 genotypes. For SNP
to pass into the final cohort there had to be a minimum of 10 reads per variant
tag. This ensured that heterozygous calling was stable and reliable across the
genome. Red clover is a heterozygous plant and this was reflected in the number
of heterozygous biallelic SNP identified. The 8,118 SNP were predominantly
transition SNP as opposed to transversion SNP (4999 v 3119, Table 3), this
phenomenon has also been reported amongst others in chickpea (Kujur, et al., 2015).
This bias towards transitional SNP is advantageous during natural selection as
these SNP are more likely to conserve protein structure than are transvertion
SNP (Wakeley, 1996).
Phylogenetic relationships and genetic diversity
This paper describes the genetic diversity and population
structure of red clover; and indicates a strong relationship between geography
and accession. The germplasm was a collection of material from ecotype
populations from Europe and Asia and five breeding lines originating from
Europe. There were some anomalies in the genetic structure analysis, and there
is some indecision as to how many groupings best explains this collection of
red clover from the IBERS gene bank. According to k-means clustering, the
change in slope identified either a two or a four groupings structure, this also
was reflected in the PCA. However, STRUCTURE analysis identified the two groupings
structure, but a more likely population of nine groupings. In all cases, the
two grouping structure separated Asia from Europe; the four group structure
differentiated Asia, UK, Iberia and the rest of Europe, with no discernible
genetic variation between the cultivars and their region of origin. The nine
groups structure again separated Asia from Europe, however the European
accessions were less well defined with the likely scenario being a division of
Iberia and UK from the rest of Europe.
Landraces and natural ecotype populations, and to some
extent cultivars, are heterogeneous populations derived from cross-pollination
of many individuals (Bowley, et al., 1984, Taylor and Quesenberry, 1996).
Therefore high levels of within population variability are expected (Kolliker, et al., 2003). This has been shown in
studies of alfalfa (Mengoni, et al., 2000), and many other legume
species (Smykal, et al., 2015). AMOVA analysis revealed high
levels of within population variation with a range of 8-15% of the total
variation, and low levels between accessions (