Molecular Plant Breeding 2015, Vol.6, No.18, 1
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strategies for improving productivity and for their
survival in drought field conditions. To this end, the
development of a set of suitable informative SNP
DNA markers is important, in order to provide a
technique for rapidly screening cultivars with different
drought tolerances.
Aquaporins (AQPs) are known to facilitate the movement
of water and small solutes across cellular membranes.
MIP (major intrinsic protein) like isoforms genes
involved in drought tolerance mechanisms have been
identified, characterized, and assessed for their
comparative transcriptional activity by using whole-
genome sequencing or expressed sequence tag (EST)
libraries in different model species such as
Arabidopsis
(Johanson et al. 2001) and maize
(Chaumont et al. 2001
).
Higher plant aquaporins proteins
represent a large family of the major intrinsic protein
(MIP) superfamily. Aquaporins consist of five
subfamilies that include; 1- plasma membrane intrinsic
proteins (
PIP
), 2- tonoplast intrinsic proteins (
TIP
), 3-
NOD26-like intrinsic proteins (
NIP
), 4- small basic
intrinsic proteins (
SIP
) and 5- the recently identified X
(or unrecognized) intrinsic proteins (
XIP
) (Park et al.
2010).
Aquaporins are involved in the regulation of water
flow and have been shown to be involved in drought
response. Recently, Secchi et al. (2007a,b) deeply
investigated the molecular mechanisms and the
identification transcription factors, including genes
related to stress response and water transport in olive.
They concluded that aquaporins play a major adaptive
role in olive. Indeed, they analyzed the change in the
expression level of genes related to the aquaporin
family in olive subjected to drought treatment. Three
aquaporin (AQPs) genes have been isolated from cv
Leccino tissues exposed to different environmental
conditions. The authors found a strong down
regulation in these genes following drought stress,
probably resulting in reduced membrane water
permeability and preventing the loss of water in
periods of water stress (Bracci et al. 2011).
Moreover, many genes were identified and annotated
based on EST sequencing in some olive cultivars,
which are involved on fatty acid biosynthesis
(Haralampidis et al. 1998; Hatzopoulos et al. 2002),
triacylglycerols (TAGs) biosynthesis (Banilas et al.
2010), antioxidant biosynthesis (Saimaru et al. 2007),
fruit growth and ripening processes (Martinelli and
Tonutti 2012), pollen allergens (Hamman-Khalifa et al.
2008) and drought stress response (Secchi et al.
2007a,b).
In this study, we describe the genetic diversity in the
TIP
and
PIP2
sub-family of the widespread gene
family of aquaporins in 15 commercial Tunisian olive
cultivars.
2 Results and discussion
2.1 SNPs frequency and nucleotide diversity
Aquaporins candidate genes (
TIP
and
PIP2
) were
selected from literature and in silico
analysis attending
to their putative role in drought stress response. The 2
genomic loci were amplified and were properly
sequenced in 15 genotypes representative of cultivated
olive.
germplasm from distant regions in the country.
Different patterns of polymorphisms were obtained.
All discovered sequences for the two used markers
reported in FASTQ are available in the ENA data-
base
under the accessions
number: for
TIP
gene (Chamlali: HG965187, Zarrazi:
HG965188, Zalmati: HG965189, Chétoui: HG965190,
Toffehi: HG965191, Chamlali-Jerba: HG965192 and
Neb-Jamel HG965193) and for
PIP2
gene:
(Chamlali-Jerba: HG965196). Summarizing, the
levels of DNA polymorphism were found in Table 2.
SNP discovery was validated by genotyping a set of
15 cultivars using direct sequencing of PCR products
and comparative analysis of sequences. A total of 6
candidate regions related to drought stress were
selected for SNP identification and nucleotide
diversity analyses. These two loci comprise 1583 bp
of aligned sequence per individual.
TIP
and
PIP2
genes ranged from 519 and 1094 pb respectively. In
total, we found 119 SNP in both coding and
non-coding regions. The two loci used in this study
were polymorphic and the SNP frequency detected in
our set of olive cultivars was 1 SNP/13.30 bp. Thus,
an average of 1 SNP every 9.8 bp for
TIP
gene and 1
SNP every 16.8 pb for
PIP2
for all regions were
detected. The level of SNPs discovered in this study
was higher than previous reports on olive genetic
DNA analysis (5 SNPs in Santos Macedo et al.
(2009); 9 SNPs in hakim et al. (2010)). These results
