Plant materials and sequencing
A total of 631 A. corniculatum individuals from 18 sites in the
Indo-West Pacific region were collected (Figure 1a, Table 1). The
sampled individuals were at least 5 m apart, and one leaf was collected
from each individual. DNA of each individual was extracted using the
cetyltrimethylammonium bromide (CTAB) method (Doyle & Doyle, 1987).
Referencing the transcriptome of an individual from Hainan, we newly
designed 93 pairs of primers anchored at the nuclear genome of A.
corniculatum , with each pair of primers anchoring at exons but spanning
at least one intron. Six genes (A189, A245, A383, A414, A440, and C058)
were amplified in 10 to 37 individuals from the 18 populations (423 in
total) (Figure 1, Table 1). The amplicons were sequenced using the
Sanger method on an ABI 3730 platform at Huada Genomic Institute (BGI)
(Shenzhen). The PCR procedure for amplification was as follows: 4 min at
94°C; 30 cycles of 15 s at 94°C, 15 s of annealing at 53°C, and
extension at 72°C for 2 min; and a final 10 min extension at 72°C. The
reactions were held at 10 ℃ before the PCR products were subjected to
electrophoresis on 1% agarose gels.
To corroborate the pattern uncovered by the six Sanger-sequenced genes,
we sequenced all 93 genes against 11 populations (491 individuals in
total) on an Illumina GA platform at Huada Genomic Institute (BGI)
(Shenzhen). For each population, we pooled equal amounts of DNA from all
the individuals of the population.
For each of the 93 genes, we amplified the pooled DNA using the same
procedure described above, and the purified PCR products of all 93
nuclear loci from one population were further pooled in equal quantities
to reach a total of 10 μg for
sequencing.