Allele frequency profiling of candidate adaptive genes for landscape genomics in perennial ryegrass

Dynamic changes in the genomic composition of populations can provide insight
in the genetic response to short-term selection pressure, or the mechanisms
underlying adaptation to a changing environment. To characterize such genetic
responses, we have implemented a high-multiplex amplicon sequencing method
to quantify allele frequencies in populations, based on the use of pooled leaf
samples to represent populations. First, we used probe capture enrichment to resequence a panel of 550 genes in a diverse set of 736 perennial ryegrass
(Lolium perenne) genotypes. From this catalogue of genetic diversity we
identified highly divergent regions in 26 candidate genes expected to drive
adaptation to differing environmental conditions, and developed primers in
flanking conserved domains to amplify 85 DNA fragments of around 500 bp. Next, we split these primers over two multiplex sets and used unique sample-
indexes for the parallel sequencing of thousands of pool-samples in a single
HiSeq2500 lane. We validated the method by comparing allele frequencies determined in pool-samples versus those determined using the individual plant
s. We used the same strategy to develop a multiplex set targeting six regio
ns in the chloroplast genome. This set allows determining phylogeographic re
lations in perennial ryegrass. Finally, we used these three multiplex sets to es
timate allele frequencies of 26 candidate adaptive genes and six chloroplast regions in 576 natural populations covering the European distribution range of
L. perenne. These data are used to determine phylogeographic relationships and
genetic signatures of adaptation of the species across Europe as part of the Eur
opean network research project GrassLandscape (http://www.faccejpi.com/FACCE-Joint-activities/ERA-NET-Plus-on-Climate-Smart-Agriculture/Grasslandscape).