Molecular Plant Breeding 2015, Vol.6, No.18, 1
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are in contrast comparable to the frequency obtained
by Besnard and El Bakkali (2014) who found on two
olive subspecies one substitution per 100 bp. On the
other hand, other agronomically important crops like
sunflower (1 SNP/69 bp) (Fusari et al. 2008) and
cultivated and wild grapevine (1 SNP/63) (Riahi et al.
2013) presented a higher SNP frequency than the olive
cultivars surveyed in this work.
Nevertheless, the
discrepancy between nucleotide genetic studies could
be caused by differences in gene sampling. Moreover,
the number of SNPs varied also between coding and
non-coding regions: 53 SNPs were found in 392 bp of
coding regions whereas 66 SNPs were detected in
1191 bp of non-coding sequences: hence, the SNP
frequency was 1 SNP/7.39 bp in coding regions and
1SNP/18.04 bp in non-coding regions. These results
suggest that coding regions are more conserved (less
SNP frequency) than non-coding regions, most
probably due to purifying selection (Fusari et al. 2008).
Genetic variation at the nucleotide level was estimated
from nucleotide diversity (π = 0.0024 and π = 0.0017)
and from the number of segregating sites as indicated
by Watterson (1975) (θ
W
= 0.0020 and θ
W
= 0.0013)
for
TIP
and
PIP2
genes respectively. The recorded
level of nucleotide diversity in this study is
comparable to that reported by Audigeos et al. (2010)
in Neotropical trees belonging to
Eperua falcata
and
Virola sebifera
species which the genetic diversity
ranged between 2.20 and 2.72 and higher than in
Eperua grandiflora
(π=1.21) in aquaporins PIP gene
family studies.
The average level of nucleotide diversity was lower
than those observed in two wild olive subspecies
(suspp.
Europaea
and
cuspidata
), (π=0.0057; π=0.0031,
respectively) (Besnard and El Bakkali 2014). Indeed,
Sabetta et al. (2013) showed higher nucleotide
diversity at nuclear genes as ω-3 fatty acid desaturases
gene (
Fad7
) in Italian olive cultivars. Nevertheless,
the discrepancy between olive studies on nucleotide
diversity could be explained by the genetic divergence
of the materials analyzed as
olea
species have a wide
genetic diversity and could be caused by the difference of
genomic sequenced regions since genomic analysis,
generally depends on the nature of the study and their
fittingness to sample different parts of the genome.
Indeed, in this study we used generated primers from
highly conserved regions of candidate genes involved
in drought response from model species. The transfer
ability of information for olive could be a source of
variation due the scarce of olive genome sequences,
the big size of olive genome (1,800 Mb), is a diploid
species (2n = 2x = 46) and predominantly allogamous
(Rugini et al. 2011). This difference is not surprising
as studied cultivars were selected for covering varying
geographic and environmental ranges and as observed
by Secchi et al. (2007a), there is a relationship between the
expression patterns of AQPs and physiological
responses in olive during drought and up and
down-regulation of this protein allows plants to
respond to environmental changes and to maintain
their water status which intrinsic plant factors depend
from development of plant architecture and rootstock
(Secchi et al. 2007b).
The average nucleotide polymorphism and nucleotide
diversity of non-coding regions (π = 0.0020) was
slightly lower, although non-significant, than diversity
estimates in coding regions (π = 0.0021). Average
synonymous-sites (π = 0.0026) was lower than
non-synonymous (π = 0.0028) for the two genes
suggesting that the diversity of these regions is
governed by purifying selection and an excess of
synonymous mutations was occurred in this candidate
gene.
A trend for a higher observed homozygosity (65%)
and expected heterozygosity (24.3%) in
TIP
gene was
observed than in
PIP2
gene (H
o
= 55%; H
e
= 18.7%
respectively). As indicated by Riahi et al. (2013), the
SNP molecular marker system show usually lower
expected heterozygosity due to its bi-allelic nature.
The genetic variability was higher in
TIP
gene than in
PIP2
locus for the 15 Tunisian cultivars determined
by SNP molecular markers. The high levels of
diversity at
TIP
gene than
PIP2
gene reveal more
details on the processes shaping olive diversity and
provide information on the link between genetic
diversity and ecological conditions. However, the non
uniformity between the length regions sequences
(coding, non-coding) for the two studies genes could
influence different molecular processes in plant and
statistical tests seem to be mostly restricted at the
exon-intron boundary (Audigeos et al. (2010).
2.2 Neutrality tests
Neutrality tests were applied to identify departures
from the standard neutral patterns of evolution in the
