U.S. patent application number 14/647242 was filed with the patent office on 2015-11-26 for molecular markers and methods for identifying date palm genotypes.
The applicant listed for this patent is INSTITUT DE RECHERCHE POUR LE DEVELOPPEMENT (IRD), Laboratoire De Gene Tique Moleculaire,Immunologie Et Biotechnologie Labb02De La Faculte Des Sciences. Invention is credited to Frederique ABERLENC-BERTOSSI, Karina CASTILLO-PEREZ, Nathalie CHABRILLANGE, Emira CHERIF, Jean-Christophe PINTAUD, Salwa ZEHDI.
Application Number | 20150337395 14/647242 |
Document ID | / |
Family ID | 47714303 |
Filed Date | 2015-11-26 |
United States Patent
Application |
20150337395 |
Kind Code |
A1 |
ABERLENC-BERTOSSI; Frederique ;
et al. |
November 26, 2015 |
Molecular markers and methods for identifying date palm
genotypes
Abstract
The present invention concerns two sets of mini- and
micro-satellite molecular markers, and the use thereof to study the
genetic diversity and/or identify the genotypes of date palms. The
invention also concerns method for identifying date palm cultivars
using these sets of molecular markers, and kits for implementing
these methods.
Inventors: |
ABERLENC-BERTOSSI; Frederique;
(Castelnau-Le-Lez, FR) ; PINTAUD; Jean-Christophe;
(Grabels, FR) ; CHABRILLANGE; Nathalie; (Grabels,
FR) ; CHERIF; Emira; (Ariana, TN) ;
CASTILLO-PEREZ; Karina; (Castelnau-Le-Lez, FR) ;
ZEHDI; Salwa; (Mnihla, TN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
INSTITUT DE RECHERCHE POUR LE DEVELOPPEMENT (IRD)
Laboratoire De Gene Tique Moleculaire,Immunologie Et Biotechnologie
Labb02De La Faculte Des Sciences |
Marseille 2
El Manar |
|
FR
TN |
|
|
Family ID: |
47714303 |
Appl. No.: |
14/647242 |
Filed: |
November 26, 2013 |
PCT Filed: |
November 26, 2013 |
PCT NO: |
PCT/EP2013/074728 |
371 Date: |
May 26, 2015 |
Current U.S.
Class: |
435/6.11 |
Current CPC
Class: |
C12Q 1/6895 20130101;
C12Q 2600/156 20130101; H01L 27/1225 20130101; H01L 27/1288
20130101; H01L 29/7869 20130101; H01L 29/45 20130101; H01L 29/41733
20130101; H01L 29/66969 20130101 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 26, 2012 |
FR |
1261226 |
Claims
1. A method for identifying the genotype of a date palm comprising
a step of detecting, using a set of minisatellite and
microsatellite molecular markers, an SSR polymorphism in the genome
of the date palm, wherein the step of detecting the SSR
polymorphism comprises, for each molecular marker of the set:
amplifying, using a pair of primers specific for the marker, a
portion of the genomic DNA of the date palm tested comprising the
minisatellite or microsatellite in order to obtain amplicons; and
analyzing the amplicons obtained in order to determine the genotype
of the date palm tested.
2. The method as claimed in claim 1, wherein the set consists of
the following 19 molecular markers: the mPdCIR010 marker, which, in
the 5'.fwdarw.3' direction, has a (ga) motif, is present in the
sequence having the GenBank accession number AJ571673.1, and is
flanked, in 5', by the nucleotide sequence SEQ ID NO: 1 and, in 3',
by the nucleotide sequence SEQ ID NO: 2; the mPdCIR015 marker,
which, in the 5'.fwdarw.3' direction, has a (ga) motif, is present
in the sequence having the GenBank accession number AJ571674.1, and
is flanked, in 5', by the nucleotide sequence SEQ ID NO: 3 and, in
3', by the nucleotide sequence SEQ ID NO: 4; the mPdCIR016 marker,
which, in the 5'.fwdarw.3' direction, has a (ga) motif, is present
in the sequence having the GenBank accession number AJ571675.1, and
is flanked, in 5', by the nucleotide sequence SEQ ID NO: 5 and, in
3', by the nucleotide sequence SEQ ID NO: 6; the mPdCIR025 marker,
which, in the 5'.fwdarw.3' direction, has a (ga) motif, is present
in the sequence having the GenBank accession number AJ571676.1, and
is flanked, in 5', by the nucleotide sequence SEQ ID NO: 7 and, in
3', by the nucleotide sequence SEQ ID NO: 8; the mPdCIR032 marker,
which, in the 5'.fwdarw.3' direction, has a (ga) motif, is present
in the sequence having the GenBank accession number AJ571677.1, and
is flanked, in 5', by the nucleotide sequence SEQ ID NO: 9 and, in
3', by the nucleotide sequence SEQ ID NO: 10; the mPdCIR035 marker,
which, in the 5'.fwdarw.3' direction, has a (ga) motif, is present
in the sequence having the GenBank accession number AJ571678.1, and
is flanked, in 5', by the nucleotide sequence SEQ ID NO: 11 and, in
3', by the nucleotide sequence SEQ ID NO: 12; the mPdCIR057 marker,
which, in the 5'.fwdarw.3' direction, has a (ga) motif, is present
in the sequence having the GenBank accession number AJ571682.1, and
is flanked, in 5', by the nucleotide sequence SEQ ID NO: 13 and, in
3', by the nucleotide sequence SEQ ID NO: 14; the mPdCIR063 marker,
which, in the 5'.fwdarw.3' direction, has a (ga) motif, is present
in the sequence having the GenBank accession number AJ571683.1, and
is flanked, in 5', by the nucleotide sequence SEQ ID NO: 15 and, in
3', by the nucleotide sequence SEQ ID NO: 16; the mPdCIR078 marker,
which, in the 5'.fwdarw.3' direction, has a (ga) motif, is present
in the sequence having the GenBank accession number AJ571685.1, and
is flanked, in 5', by the nucleotide sequence SEQ ID NO: 17 and, in
3', by the nucleotide sequence SEQ ID NO: 18; the mPdCIR085 marker,
which, in the 5'.fwdarw.3' direction, has a (ga) motif, is present
in the sequence having the GenBank accession number AJ571686.1, and
is flanked, in 5', by the nucleotide sequence SEQ ID NO: 19 and, in
3', by the nucleotide sequence SEQ ID NO: 20; the CpfM12 marker,
which, in the 5'.fwdarw.3' direction, has a (ctaactactata) motif,
is present in the sequence having the GenBank accession number
EU043484.1, and is flanked, in 5', by the nucleotide sequence SEQ
ID NO: 21 and, in 3', by the nucleotide sequence SEQ ID NO: 22; the
PdCUC3-ssr1 marker, which, in the 5'.fwdarw.3' direction, has a
(gt+ga) motif, is present in the sequence having the GenBank
accession number HM622273.1, and is flanked, in 5', by the
nucleotide sequence SEQ ID NO: 23 and, in 3', by the nucleotide
sequence SEQ ID NO: 24; the PdCUC3-ssr2 marker, which, in the
5'.fwdarw.3' direction, has a (ga) motif, is present in the
sequence having the GenBank accession number HM622273.1, and is
flanked, in 5', by the nucleotide sequence SEQ ID NO: 25 and, in
3', by the nucleotide sequence SEQ ID NO: 26; the PdAG1-ssr1
marker, which, in the 5'.fwdarw.3' direction, has a (ga) motif, is
present in the sequence having the GenBank accession number
KC188338.1, and is flanked, in 5', by the nucleotide sequence SEQ
ID NO: 27 and, in 3', by the nucleotide sequence SEQ ID NO: 28; the
PdAP3-ssr marker, which, in the 5'.fwdarw.3' direction, has a (ga)
motif, is present in the sequence having the GenBank accession
number KC188337.1, and is flanked, in 5', by the nucleotide
sequence SEQ ID NO: 29 and, in 3', by the nucleotide sequence SEQ
ID NO: 30; the mPdIRD13 marker, which, in the 5'.fwdarw.3'
direction, has a (cac) motif, is present in the
PDK.sub.--20s1496731g002.sub.--1 scaffold having the GenBank
accession number GL741615.1, and is flanked, in 5', by the
nucleotide sequence SEQ ID NO: 31 and, in 3', by the nucleotide
sequence SEQ ID NO: 32; the mPdIRD31 marker, which, in the
5'.fwdarw.3' direction, has a (cca) motif, is present in the
PDK.sub.--20s1419261g003.sub.--1 scaffold, and is flanked, in 5',
by the nucleotide sequence SEQ ID NO: 33 and, in 3', by the
nucleotide sequence SEQ ID NO: 34; the mPdIRD33 marker, which, in
the 5'.fwdarw.3' direction, has a (cag) motif, is present in the
PDK.sub.--20s1569281g001.sub.--1 scaffold, and is flanked, in 5',
by the nucleotide sequence SEQ ID NO: 35 and, in 3', by the
nucleotide sequence SEQ ID NO: 36; and the mPdIRD40 marker, which,
in the 5'.fwdarw.3' direction, has a (ccagtg) motif, is present in
the PDK.sub.--20s1327401g002.sub.--1 scaffold, and is flanked, in
5', by the nucleotide sequence SEQ ID NO: 37 and, in 3', by the
nucleotide sequence SEQ ID NO: 38.
3. The method as claimed in claim 2, wherein the amplifications are
carried out with each of the pairs of specific primers that follow:
a forward SSR primer of sequence SEQ ID NO: 1 and a reverse SSR
primer of sequence SEQ ID NO: 2; a forward SSR primer of sequence
SEQ ID NO: 3 and a reverse SSR primer of sequence SEQ ID NO: 4; a
forward SSR primer of sequence SEQ ID NO: 5 and a reverse SSR
primer of sequence SEQ ID NO: 6; a forward SSR primer of sequence
SEQ ID NO: 7 and a reverse SSR primer of sequence SEQ ID NO: 8; a
forward SSR primer of sequence SEQ ID NO: 9 and a reverse SSR
primer of sequence SEQ ID NO: 10; a forward SSR primer of sequence
SEQ ID NO: 11 and a reverse SSR primer of sequence SEQ ID NO: 12; a
forward SSR primer of sequence SEQ ID NO: 13 and a reverse SSR
primer of sequence SEQ ID NO: 14; a forward SSR primer of sequence
SEQ ID NO: 15 and a reverse SSR primer of sequence SEQ ID NO: 16; a
forward SSR primer of sequence SEQ ID NO: 17 and a reverse SSR
primer of sequence SEQ ID NO: 18; a forward SSR primer of sequence
SEQ ID NO: 19 and a reverse SSR primer of sequence SEQ ID NO: 20; a
forward SSR primer of sequence SEQ ID NO: 21 and a reverse primer
of sequence SEQ ID NO: 22; a forward SSR primer of sequence SEQ ID
NO: 23 and a reverse SSR primer of sequence SEQ ID NO: 24; a
forward SSR primer of sequence SEQ ID NO: 25 and a reverse SSR
primer of sequence SEQ ID NO: 26; a forward primer of sequence SEQ
ID NO: 27 and a reverse SSR primer of sequence SEQ ID NO: 28; a
forward SSR primer of sequence SEQ ID NO: 29 and a reverse SSR
primer of sequence SEQ ID NO: 30; a forward SSR primer of sequence
SEQ ID NO: 31 and a reverse SSR primer of sequence SEQ ID NO: 32; a
forward SSR primer of sequence SEQ ID NO: 33 and a reverse SSR
primer of sequence SEQ ID NO: 34; a forward SSR primer of sequence
SEQ ID NO: 35 and a reverse SSR primer of sequence SEQ ID NO: 36;
and a forward SSR primer of sequence SEQ ID NO: 37 and a reverse
SSR primer of sequence SEQ ID NO: 38.
4. The method as claimed in claim 1, wherein the set consists of
the 7 molecular markers CpfM12, mPdIRD031, mPdIRD033, mPdIRD040,
PdCUC3-ssr2, PdAP3-ssr-F4 and mPdCIR078 as defined in claim 2.
5. The method as claimed in claim 4, wherein the amplifications are
carried out with each of the pairs of specific primers that follow:
a forward SSR primer of sequence SEQ ID NO: 17 and a reverse SSR
primer of sequence SEQ ID NO: 18; a forward SSR primer of sequence
SEQ ID NO: 21 and a reverse SSR primer of sequence SEQ ID NO: 22; a
forward SSR primer of sequence SEQ ID NO: 25 and a reverse SSR
primer of sequence SEQ ID NO: 26; a forward SSR primer of sequence
SEQ ID NO: 29 and a reverse SSR primer of sequence SEQ ID NO: 30; a
forward SSR primer of sequence SEQ ID NO: 33 and a reverse SSR
primer of sequence SEQ ID NO: 34; a forward SSR primer of sequence
SEQ ID NO: 35 and a reverse SSR primer of sequence SEQ ID NO: 36;
and a forward SSR primer of sequence SEQ ID NO: 37 and a reverse
SSR primer of sequence SEQ ID NO: 38.
6. The method as claimed in claim 4 or claim 5, wherein the
identification of the genotype of the date palm tested comprises
the identification or the certification of the cultivar of the date
palm tested.
7. The method as claimed in claim 1, wherein the step of analyzing
the amplicons obtained in order to determine the genotype of the
date palm tested comprises, for each of the molecular markers:
separating, as a function of their size, the amplicons obtained by
amplification of a portion of the genomic DNA of the date palm
tested, and comparing the sizes of said amplicons with the sizes of
the amplicons obtained by amplification, under the same conditions,
of a portion of the genomic DNA of control date palms.
8. The method as claimed in claim 1, wherein the method is carried
out on genomic DNA extracted from a sample of the date palm tested,
where the sample of the date palm is a protoplast, callus, embryo,
leaf, stipe, root, shoot or cutting sample of the date palm.
9. A set of pairs of primers specific for minisatellite and
microsatellite molecular markers for identifying the genotype of a
date palm, the set consisting of the following pairs of primers: a
forward SSR primer of sequence SEQ ID NO: 1 and a reverse SSR
primer of sequence SEQ ID NO: 2; a forward SSR primer of sequence
SEQ ID NO: 3 and a reverse SSR primer of sequence SEQ ID NO: 4; a
forward SSR primer of sequence SEQ ID NO: 5 and a reverse SSR
primer of sequence SEQ ID NO: 6; a forward SSR primer of sequence
SEQ ID NO: 7 and a reverse SSR primer of sequence SEQ ID NO: 8; a
forward SSR primer of sequence SEQ ID NO: 9 and a reverse SSR
primer of sequence SEQ ID NO: 10; a forward SSR primer of sequence
SEQ ID NO: 11 and a reverse SSR primer of sequence SEQ ID NO: 12; a
forward SSR primer of sequence SEQ ID NO: 13 and a reverse SSR
primer of sequence SEQ ID NO: 14; a forward SSR primer of sequence
SEQ ID NO: 15 and a reverse SSR primer of sequence SEQ ID NO: 16; a
forward SSR primer of sequence SEQ ID NO: 17 and a reverse SSR
primer of sequence SEQ ID NO: 18; a forward SSR primer of sequence
SEQ ID NO: 19 and a reverse SSR primer of sequence SEQ ID NO: 20; a
forward SSR primer of sequence SEQ ID NO: 21 and a reverse primer
of sequence SEQ ID NO: 22; a forward SSR primer of sequence SEQ ID
NO: 23 and a reverse SSR primer of sequence SEQ ID NO: 24; a
forward SSR primer of sequence SEQ ID NO: 25 and a reverse SSR
primer of sequence SEQ ID NO: 26; a forward primer of sequence SEQ
ID NO: 27 and a reverse SSR primer of sequence SEQ ID NO: 28; a
forward SSR primer of sequence SEQ ID NO: 29 and a reverse SSR
primer of sequence SEQ ID NO: 30; a forward SSR primer of sequence
SEQ ID NO: 31 and a reverse SSR primer of sequence SEQ ID NO: 32; a
forward SSR primer of sequence SEQ ID NO: 33 and a reverse SSR
primer of sequence SEQ ID NO: 34; a forward SSR primer of sequence
SEQ ID NO: 35 and a reverse SSR primer of sequence SEQ ID NO: 36;
and a forward SSR primer of sequence SEQ ID NO: 37 and a reverse
SSR primer of sequence SEQ ID NO: 38.
10. A set of pairs of primers specific for minisatellite and
microsatellite molecular markers for identifying or certifying the
cultivar of a date palm tested, the set consisting of the following
pairs of primers: a forward SSR primer of sequence SEQ ID NO: 17
and a reverse SSR primer of sequence SEQ ID NO: 18; a forward SSR
primer of sequence SEQ ID NO: 21 and a reverse SSR primer of
sequence SEQ ID NO: 22; a forward SSR primer of sequence SEQ ID NO:
25 and a reverse SSR primer of sequence SEQ ID NO: 26; a forward
SSR primer of sequence SEQ ID NO: 29 and a reverse SSR primer of
sequence SEQ ID NO: 30; a forward SSR primer of sequence SEQ ID NO:
33 and a reverse SSR primer of sequence SEQ ID NO: 34; a forward
SSR primer of sequence SEQ ID NO: 35 and a reverse SSR primer of
sequence SEQ ID NO: 36; and a forward SSR primer of sequence SEQ ID
NO: 37 and a reverse SSR primer of sequence SEQ ID NO: 38.
11. The set of pairs of primers as claimed in claim 9 or claim 10,
wherein one of the primers of each of the pairs of primers
comprises a detectable marker.
12. (canceled)
13. (canceled)
14. A kit for identifying the genotype of date palms, comprising a
set of pairs of primers specific for minisatellite and
microsatellite molecular markers and instructions for identifying
the genotype using a method as claimed in claim 2, wherein the set
consists of the following pairs of primers: a forward SSR primer of
sequence SEQ ID NO: 1 and a reverse SSR primer of sequence SEQ ID
NO: 2; a forward SSR primer of sequence SEQ ID NO: 3 and a reverse
SSR primer of sequence SEQ ID NO: 4; a forward SSR primer of
sequence SEQ ID NO: 5 and a reverse SSR primer of sequence SEQ ID
NO: 6; a forward SSR primer of sequence SEQ ID NO: 7 and a reverse
SSR primer of sequence SEQ ID NO: 8; a forward SSR primer of
sequence SEQ ID NO: 9 and a reverse SSR primer of sequence SEQ ID
NO: 10; a forward SSR primer of sequence SEQ ID NO: 11 and a
reverse SSR primer of sequence SEQ ID NO: 12; a forward SSR primer
of sequence SEQ ID NO: 13 and a reverse SSR primer of sequence SEQ
ID NO: 14; a forward SSR primer of sequence SEQ ID NO: 15 and a
reverse SSR primer of sequence SEQ ID NO: 16; a forward SSR primer
of sequence SEQ ID NO: 17 and a reverse SSR primer of sequence SEQ
ID NO: 18; a forward SSR primer of sequence SEQ ID NO: 19 and a
reverse SSR primer of sequence SEQ ID NO: 20; a forward SSR primer
of sequence SEQ ID NO: 21 and a reverse primer of sequence SEQ ID
NO: 22; a forward SSR primer of sequence SEQ ID NO: 23 and a
reverse SSR primer of sequence SEQ ID NO: 24; a forward SSR primer
of sequence SEQ ID NO: 25 and a reverse SSR primer of sequence SEQ
ID NO: 26; a forward primer of sequence SEQ ID NO: 27 and a reverse
SSR primer of sequence SEQ ID NO: 28; a forward SSR primer of
sequence SEQ ID NO: 29 and a reverse SSR primer of sequence SEQ ID
NO: 30; a forward SSR primer of sequence SEQ ID NO: 31 and a
reverse SSR primer of sequence SEQ ID NO: 32; a forward SSR primer
of sequence SEQ ID NO: 33 and a reverse SSR primer of sequence SEQ
ID NO: 34; a forward SSR primer of sequence SEQ ID NO: 35 and a
reverse SSR primer of sequence SEQ ID NO: 36; and a forward SSR
primer of sequence SEQ ID NO: 37 and a reverse SSR primer of
sequence SEQ ID NO: 38.
15. A kit for identifying or certifying the cultivar of date palms,
comprising a set of pairs of primers specific for minisatellite and
microsatellite molecular markers and instructions for identifying
or certifying the cultivar using a method as claimed in claim 4,
wherein the set consists of the following pairs of primer: a
forward SSR primer of sequence SEQ ID NO: 17 and a reverse SSR
primer of sequence SEQ ID NO: 18; a forward SSR primer of sequence
SEQ ID NO: 21 and a reverse SSR primer of sequence SEQ ID NO: 22; a
forward SSR primer of sequence SEQ ID NO: 25 and a reverse SSR
primer of sequence SEQ ID NO: 26; a forward SSR primer of sequence
SEQ ID NO: 29 and a reverse SSR primer of sequence SEQ ID NO: 30; a
forward SSR primer of sequence SEQ ID NO: 33 and a reverse SSR
primer of sequence SEQ ID NO: 34; a forward SSR primer of sequence
SEQ ID NO: 35 and a reverse SSR primer of sequence SEQ ID NO: 36;
and a forward SSR primer of sequence SEQ ID NO: 37 and a reverse
SSR primer of sequence SEQ ID NO: 38.
Description
RELATED PATENT APPLICATIONS
[0001] The present application is filed pursuant to 35 U.S.C
.sctn.371 as a U.S. National Phase application of international
application No. PCT/EP2013/074728, which was filed on Nov. 26,
2013, claiming the benefit of French patent application number FR
12 61226 filed on Nov. 26, 2012. The International application was
published as PCT Publication No. WO 2014/080034 on May 30, 2014.
The entire contents of each of the aforementioned applications is
incorporated herein by reference in their entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been filed electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on May 26, 2015, is named 47855.sub.--00.sub.--2002_SL.txt and is
8,780 bytes in size.
FIELD OF THE INVENTION
[0003] The present invention concerns two sets of molecular markers
specific for the genetic variety of date palms and the use thereof
for identifying date palm genotypes and cultivars.
CONTEXT OF THE INVENTION
[0004] The date palm (Phoenix dactylifera L.) is a monocotyledonous
plant of the family Arecaceae which is widely cultivated for its
fruits, which are dates. In arid regions, the date palm plays not
only a major economic role, through the production of dates which
make up the basis of the human and animal diet, but also an
ecological role since it gives the oasis its structure. The date
palm has been cultivated by humans for thousands of years; the
first documents and drawings confirm the cultivation thereof going
back to 6000 years BC. Suitable for cultivation in arid and
semi-arid regions, it has gradually been introduced on the five
continents.
[0005] The cultivation of the date palm has evolved from a
traditional, rich and diversified system to an industrial system
centered on a single-variety oligoculture. This constitutes a
potential risk of genetic erosion of the inheritance. The loss of
genetic diversity is worsened by the biotic stresses (diseases and
ravaging pests) and abiotic stresses (drought and soil salinity) to
which palm groves are subjected. Consequently, it becomes
essential, for many producing countries, to characterize the
diversity of the genetic resources, in order to organize the
preservation and safeguarding thereof.
[0006] The characterization of the diversity of date palm cultivars
can be obtained using morphological markers or molecular markers of
RAPD (Random Amplified Polymorphic DNA), RFLP (Restriction Fragment
Length Polymorphism), AFLP (Amplified Fragment Length Polymorphism)
and SSR (Simple Sequence Repeat) type. During the last decade, the
development and use of molecular markers made it possible to
improve the study of genetic resources in plant organisms by
reducing the uncertainty due to the variation in morphological and
agronomic characters under the effect of environmental factors.
[0007] Microsatellite markers or SSRs are particularly
advantageous: they are abundant in the genome; they have high
levels of polymorphism, which provides great reliability in the
characterization of individuals; and they are codominant and
transmitted according to Mendelian law.
[0008] In the date palm, 16 dinucleotide microsatellite markers,
(GA).sub.n, have been generated from a DNA library (Billotte et
al., Molecular Ecology Notes, 2004, 4: 256-258). These markers have
been validated with respect to their accuracy in distinguishing the
various cultivars of Tunisia (Zehdi et al., Hereditas, 2004, 141:
278-287) and of Sudan (Elshibli et al., Genetica, 2008, 134:
251-260). However, the majority of the date palm diversity studies
have been carried out using various sets of markers. Furthermore,
the microsatellite markers have generally been validated on
samplings carried out at the local geographic level. It follows
that the results obtained by the various teams are not
internationally compatible. In addition, some dinucleotide markers
are difficult to analyze because of the size of the alleles
generated, sometimes varying by a single nucleotide. Since 2011,
the partial sequencing of the date palm genome, which was recently
published (Al-Dous et al., Nature Biotechnology, 2011, 29:
521-527), has offered the possibility of gaining access to a large
number of markers with various repeating motifs.
[0009] It is therefore important to develop new strategies,
validated on samplings representing the worldwide diversity of the
species, which make it possible to standardize the identification
procedures with the objective of characterizing the agrodiversity
of the date palm. Such strategies would open up new horizons for
the reintroduction of biodiversity in palm groves and
implementation of programs for genetic improvement. There is also a
need for tools enabling the certification of vegetatively
propagated plants sought by farmers in palm groves and vitroplant
production laboratories.
SUMMARY OF THE INVENTION
[0010] Generally, the present invention relates to sets of
optimized molecular markers, composed of a limited number of
markers selected for their precision and their capacity to generate
a high polymorphism. These sets of markers have been validated on a
collection of genotypes representing the worldwide diversity of the
date palm. More specifically, the present invention relates to a
first set of 19 mini- and microsatellite markers making it possible
to analyze 100% of genotypes. This set is particularly useful for
research on the analysis of genetic diversity, the identification
of genotypes, the analysis of hybrids and gene flows within
Phoenix. The present invention also relates to a second set of 7
mini- and microsatellite markers making it possible to identify 99%
of genotypes. This second set is particularly useful for the
certification of cultivars. The inventors have also designed a
reference cultivar library covering the worldwide diversity of the
date palm and a database which manages all the information
generated.
[0011] Consequently, in a first aspect, the present invention
relates to a first set of mini- and microsatellite markers specific
for the date palm, characterized in that the set consists of the
following 19 microsatellite markers: [0012] the mPdCIR010 marker,
which, in the 5'.fwdarw.3' direction, has a (ga) motif, is present
in the sequence having the GenBank accession number AJ571673.1, and
is flanked, in 5', by the nucleotide sequence SEQ ID NO: 1 and, in
3', by the nucleotide sequence SEQ ID NO: 2; [0013] the mPdCIR015
marker, which, in the 5'.fwdarw.3' direction, has a (ga) motif, is
present in the sequence having the GenBank accession number
AJ571674.1, and is flanked, in 5', by the nucleotide sequence SEQ
ID NO: 3 and, in 3', by the nucleotide sequence SEQ ID NO: 4;
[0014] the mPdCIR016 marker, which, in the 5'.fwdarw.3' direction,
has a (ga) motif, is present in the sequence having the GenBank
accession number AJ571675.1, and is flanked, in 5', by the
nucleotide sequence SEQ ID NO: 5 and, in 3', by the nucleotide
sequence SEQ ID NO: 6; [0015] the mPdCIR025 marker, which, in the
5'.fwdarw.3' direction, has a (ga) motif, is present in the
sequence having the GenBank accession number AJ571676.1, and is
flanked, in 5', by the nucleotide sequence SEQ ID NO: 7 and, in 3',
by the nucleotide sequence SEQ ID NO: 8; [0016] the mPdCIR032
marker, which, in the 5'.fwdarw.3' direction, has a (ga) motif, is
present in the sequence having the GenBank accession number
AJ571677.1, and is flanked, in 5', by the nucleotide sequence SEQ
ID NO: 9 and, in 3', by the nucleotide sequence SEQ ID NO: 10;
[0017] the mPdCIR035 marker, which, in the 5'.fwdarw.3' direction,
has a (ga) motif, is present in the sequence having the GenBank
accession number AJ571678.1, and is flanked, in 5', by the
nucleotide sequence SEQ ID NO: 11 and, in 3', by the nucleotide
sequence SEQ ID NO: 12; [0018] the mPdCIR057 marker, which, in the
5'.fwdarw.3' direction, has a (ga) motif, is present in the
sequence having the GenBank accession number AJ571682.1, and is
flanked, in 5', by the nucleotide sequence SEQ ID NO: 13 and, in
3', by the nucleotide sequence SEQ ID NO: 14; [0019] the mPdCIR063
marker, which, in the 5'.fwdarw.3' direction, has a (ga) motif, is
present in the sequence having the GenBank accession number
AJ571683.1, and is flanked, in 5', by the nucleotide sequence SEQ
ID NO: 15 and, in 3', by the nucleotide sequence SEQ ID NO: 16;
[0020] the mPdCIR078 marker, which, in the 5'.fwdarw.3' direction,
has a (ga) motif, is present in the sequence having the GenBank
accession number AJ571685.1, and is flanked, in 5', by the
nucleotide sequence SEQ ID NO: 17 and, in 3', by the nucleotide
sequence SEQ ID NO: 18; [0021] the mPdCIR085 marker, which, in the
5'.fwdarw.3' direction, has a (ga) motif, is present in the
sequence having the GenBank accession number AJ571686.1, and is
flanked, in 5', by the nucleotide sequence SEQ ID NO: 19 and, in
3', by the nucleotide sequence SEQ ID NO: 20; [0022] the CpfM12
marker, which, in the 5'.fwdarw.3' direction, has a (ctaactactata)
motif, is present in the sequence having the GenBank accession
number EU043484.1, and is flanked, in 5', by the nucleotide
sequence SEQ ID NO: 21 and, in 3', by the nucleotide sequence SEQ
ID NO: 22; [0023] the PdCUC3-ssr1 marker, which, in the
5'.fwdarw.3' direction, has a (gt+ga) motif, is present in the
sequence having the GenBank accession number HM622273.1, and is
flanked, in 5', by the nucleotide sequence SEQ ID NO: 23 and, in
3', by the nucleotide sequence SEQ ID NO: 24; [0024] the
PdCUC3-ssr2 marker, which, in the 5'.fwdarw.3' direction, has a
(ga) motif, is present in the sequence having the GenBank accession
number HM622273.1, and is flanked, in 5', by the nucleotide
sequence SEQ ID NO: 25 and, in 3', by the nucleotide sequence SEQ
ID NO: 26; [0025] the PdAG1-ssr1 marker, which, in the 5'.fwdarw.3'
direction, has a (ga) motif, is present in the sequence having the
GenBank accession number KC188338.1, and is flanked, in 5', by the
nucleotide sequence SEQ ID NO: 27 and, in 3', by the nucleotide
sequence SEQ ID NO: 28; [0026] the PdAP3-ssr marker, which, in the
5'.fwdarw.3' direction, has a (ga) motif, is present in the
sequence having the GenBank accession number KC188337.1, and is
flanked, in 5', by the nucleotide sequence SEQ ID NO: 29 and, in
3', by the nucleotide sequence SEQ ID NO: 30; [0027] the mPdIRD13
marker, which, in the 5'.fwdarw.3' direction, has a (cac) motif, is
present in the PDK.sub.--20 s1496731g002.sub.--1 scaffold (GenBank
accession number GL741615.1), and is flanked, in 5', by the
nucleotide sequence SEQ ID NO: 31 and, in 3', by the nucleotide
sequence SEQ ID NO: 32; [0028] the mPdIRD31 marker, which, in the
5'.fwdarw.3' direction, has a (cca) motif, is present in the
PDK.sub.--20 s1419261g003.sub.--1 scaffold (described in
http://qatar-weill.cornell.edu/research/datepalmGenome/download.html),
and is flanked, in 5', by the nucleotide sequence SEQ ID NO: 33
and, in 3', by the nucleotide sequence SEQ ID NO: 34; [0029] the
mPdIRD33 marker, which, in the 5'.fwdarw.3' direction, has a (cag)
motif, is present in the PDK.sub.--20 s1569281g001.sub.--1 scaffold
(described in
http://qatar-weill.cornell.edu/research/datepalmGenome/download.html),
and is flanked, in 5', by the nucleotide sequence SEQ ID NO: 35
and, in 3', by the nucleotide sequence SEQ ID NO: 36; and [0030]
the mPdIRD40 marker, which, in the 5' .fwdarw.3' direction, has a
(ccagtg) motif, is present in the PDK.sub.--20 s1327401g002.sub.--1
scaffold (described in
http://qatar-weill.cornell.edu/research/datepalmGenome/download.html),
and is flanked, in 5', by the nucleotide sequence SEQ ID NO: 37
and, in 3', by the nucleotide sequence SEQ ID NO: 38.
[0031] The present invention also relates to a second set of mini-
and microsatellite markers specific for the date palm,
characterized in that the set consists of the following 7
microsatellite markers: CpfM12, mPdIRD031, mPdIRD033, mPdIRD040,
PdCUC3-ssr2, PdAP3-ssr and mPdCIR078 as defined above.
[0032] In another aspect, the present invention relates to a method
for identifying the genotype of a date palm by detecting, using the
set of 19 mini- and microsatellite markers, an SSR polymorphism in
the genome of the date palm, characterized in that the detecting of
the SSR polymorphism comprises, for each molecular marker of the
set: [0033] amplification of a portion of the genomic DNA of the
date palm tested which comprises the mini- or microsatellite, using
a pair of primers specific for the marker in order to obtain
amplicons; and [0034] analysis of the amplicons obtained in order
to determine the genotype of the date palm tested.
[0035] In some embodiments, the analysis of the amplicons comprises
the analysis of the amplicon sizes.
[0036] In some embodiments, the method is characterized in that the
amplifications are carried out with each of the pairs of specific
primers that follow: [0037] a forward SSR primer of sequence SEQ ID
NO: 1 and a reverse SSR primer of sequence SEQ ID NO: 2; [0038] a
forward SSR primer of sequence SEQ ID NO: 3 and a reverse SSR
primer of sequence SEQ ID NO: 4; [0039] a forward SSR primer of
sequence SEQ ID NO: 5 and a reverse SSR primer of sequence SEQ ID
NO: 6; [0040] a forward SSR primer of sequence SEQ ID NO: 7 and a
reverse SSR primer of sequence SEQ ID NO: 8; [0041] a forward SSR
primer of sequence SEQ ID NO: 9 and a reverse SSR primer of
sequence SEQ ID NO: 10; [0042] a forward SSR primer of sequence SEQ
ID NO: 11 and a reverse SSR primer of sequence SEQ ID NO: 12;
[0043] a forward SSR primer of sequence SEQ ID NO: 13 and a reverse
SSR primer of sequence SEQ ID NO: 14; [0044] a forward SSR primer
of sequence SEQ ID NO: 15 and a reverse SSR primer of sequence SEQ
ID NO: 16; [0045] a forward SSR primer of sequence SEQ ID NO: 17
and a reverse SSR primer of sequence SEQ ID NO: 18; [0046] a
forward SSR primer of sequence SEQ ID NO: 19 and a reverse SSR
primer of sequence SEQ ID NO: 20; [0047] a forward SSR primer of
sequence SEQ ID NO: 21 and a reverse primer of sequence SEQ ID NO:
22; [0048] a forward SSR primer of sequence SEQ ID NO: 23 and a
reverse SSR primer of sequence SEQ ID NO: 24; [0049] a forward SSR
primer of sequence SEQ ID NO: 25 and a reverse SSR primer of
sequence SEQ ID NO: 26; [0050] a forward primer of sequence SEQ ID
NO: 27 and a reverse SSR primer of sequence SEQ ID NO: 28; [0051] a
forward SSR primer of sequence SEQ ID NO: 29 and a reverse SSR
primer of sequence SEQ ID NO: 30; [0052] a forward SSR primer of
sequence SEQ ID NO: 31 and a reverse SSR primer of sequence SEQ ID
NO: 32; [0053] a forward SSR primer of sequence SEQ ID NO: 33 and a
reverse SSR primer of sequence SEQ ID NO: 34; [0054] a forward SSR
primer of sequence SEQ ID NO: 35 and a reverse SSR primer of
sequence SEQ ID NO: 36; and [0055] a forward SSR primer of sequence
SEQ ID NO: 37 and a reverse SSR primer of sequence SEQ ID NO:
38.
[0056] In another aspect, the present invention relates to a method
for identifying or certifying a date palm cultivar by detecting,
using the set of 7 mini- or microsatellite markers, an SSR
polymorphism in the genome of the date palm, characterized in that
the detecting of the SSR polymorphism comprises, for each molecular
marker of the set: [0057] amplification of a portion of the genomic
DNA of the date palm tested which comprises the mini- or
microsatellite, using a pair of primers specific for the marker in
order to obtain amplicons; and [0058] analysis of the amplicons
obtained in order to determine the genotype of the date palm
tested.
[0059] In some embodiments, the analysis of the amplicons comprises
the analysis of the amplicon sizes.
[0060] In some embodiments, the method is characterized in that the
amplifications are carried out with each of the pairs of specific
primers that follow: [0061] a forward SSR primer of sequence SEQ ID
NO: 17 and a reverse SSR primer of sequence SEQ ID NO: 18; [0062] a
forward SSR primer of sequence SEQ ID NO: 21 and a reverse SSR
primer of sequence SEQ ID NO: 22; [0063] a forward SSR primer of
sequence SEQ ID NO: 25 and a reverse SSR primer of sequence SEQ ID
NO: 26; [0064] a forward SSR primer of sequence SEQ ID NO: 29 and a
reverse SSR primer of sequence SEQ ID NO: 30; [0065] a forward SSR
primer of sequence SEQ ID NO: 33 and a reverse SSR primer of
sequence SEQ ID NO: 34; [0066] a forward SSR primer of sequence SEQ
ID NO: 35 and a reverse SSR primer of sequence SEQ ID NO: 36; and
[0067] a forward SSR primer of sequence SEQ ID NO: 37 and a reverse
SSR primer of sequence SEQ ID NO: 38.
[0068] In some embodiments of the methods according to the
invention, the step of analyzing the amplicons obtained in order to
determine the genotype or the cultivar of the date palm tested
comprises, for each of the markers of the set used: [0069]
separation, as a function of their size, of the amplicons obtained
by amplification of a portion of the genomic DNA of the date palm
tested, and [0070] comparison of the sizes of said amplicons with
the sizes of the amplicons obtained by amplification, under the
same conditions, of a portion of the genomic DNA of control date
palms.
[0071] In some embodiments, the methods according to the invention
are carried out on genomic DNA extracted from a sample of the date
palm tested, where the sample of the date palm is a protoplast,
callus, embryo, leaf, stipe, root, shoot or cutting sample of the
date palm.
[0072] In another aspect, the present invention relates to a first
set of pairs of primers specific for a microsatellite marker for
identifying the genotype of a date palm, the set consisting of the
following pairs of primers: [0073] a forward SSR primer of sequence
SEQ ID NO: 1 and a reverse SSR primer of sequence SEQ ID NO: 2;
[0074] a forward SSR primer of sequence SEQ ID NO: 3 and a reverse
SSR primer of sequence SEQ ID NO: 4; [0075] a forward SSR primer of
sequence SEQ ID NO: 5 and a reverse SSR primer of sequence SEQ ID
NO: 6; [0076] a forward SSR primer of sequence SEQ ID NO: 7 and a
reverse SSR primer of sequence SEQ ID NO: 8; [0077] a forward SSR
primer of sequence SEQ ID NO: 9 and a reverse SSR primer of
sequence SEQ ID NO: 10; [0078] a forward SSR primer of sequence SEQ
ID NO: 11 and a reverse SSR primer of sequence SEQ ID NO: 12;
[0079] a forward SSR primer of sequence SEQ ID NO: 13 and a reverse
SSR primer of sequence SEQ ID NO: 14; [0080] a forward SSR primer
of sequence SEQ ID NO: 15 and a reverse SSR primer of sequence SEQ
ID NO: 16; [0081] a forward SSR primer of sequence SEQ ID NO: 17
and a reverse SSR primer of sequence SEQ ID NO: 18; [0082] a
forward SSR primer of sequence SEQ ID NO: 19 and a reverse SSR
primer of sequence SEQ ID NO: 20; [0083] a forward SSR primer of
sequence SEQ ID NO: 21 and a reverse primer of sequence SEQ ID NO:
22; [0084] a forward SSR primer of sequence SEQ ID NO: 23 and a
reverse SSR primer of sequence SEQ ID NO: 24; [0085] a forward SSR
primer of sequence SEQ ID NO: 25 and a reverse SSR primer of
sequence SEQ ID NO: 26; [0086] a forward primer of sequence SEQ ID
NO: 27 and a reverse SSR primer of sequence SEQ ID NO: 28; [0087] a
forward SSR primer of sequence SEQ ID NO: and a reverse SSR primer
of sequence SEQ ID NO: 30; [0088] a forward SSR primer of sequence
SEQ ID NO: and a reverse SSR primer of sequence SEQ ID NO: 32;
[0089] a forward SSR primer of sequence SEQ ID NO: 33 and a reverse
SSR primer of sequence SEQ ID NO: 34; [0090] a forward SSR primer
of sequence SEQ ID NO: 35 and a reverse SSR primer of sequence SEQ
ID NO: 36; and [0091] a forward SSR primer of sequence SEQ ID NO:
37 and a reverse SSR primer of sequence SEQ ID NO: 38.
[0092] The present invention also relates to a second set of pairs
of primers specific for a microsatellite marker for identifying or
certifying the cultivar of a date palm, the set consisting of the
following pairs of primers: [0093] a forward SSR primer of sequence
SEQ ID NO: 17 and a reverse SSR primer of sequence SEQ ID NO: 18;
[0094] a forward SSR primer of sequence SEQ ID NO: 21 and a reverse
SSR primer of sequence SEQ ID NO: 22; [0095] a forward SSR primer
of sequence SEQ ID NO: 25 and a reverse SSR primer of sequence SEQ
ID NO: 26; [0096] a forward SSR primer of sequence SEQ ID NO: 29
and a reverse SSR primer of sequence SEQ ID NO: 30; [0097] a
forward SSR primer of sequence SEQ ID NO: 33 and a reverse SSR
primer of sequence SEQ ID NO: 34; [0098] a forward SSR primer of
sequence SEQ ID NO: 35 and a reverse SSR primer of sequence SEQ ID
NO: 36; and a forward SSR primer of sequence SEQ ID NO: 37 and a
reverse SSR primer of sequence SEQ ID NO: 38.
[0099] In some embodiments, a set of pairs of primers according to
the invention is characterized in that one of the primers of each
of the pairs of primers comprises a detectable marker.
[0100] One aspect of the invention relates to the use of the first
set of pairs of primers for identifying the genotype of a date
palm.
[0101] Another aspect of the invention relates to the use of the
second set of pairs of primers for identifying or certifying the
cultivar of a date palm.
[0102] Finally, the invention provides a first kit for identifying
the genotype of date palms, comprising the first set of 19 pairs of
primers according to the invention and instructions for carrying
out a method for identifying the genotype according to the
invention.
[0103] The invention also provides a second kit for identifying or
certifying the cultivar of date palms, comprising the second set of
7 pairs of primers according to the invention and instructions for
carrying out a method for identifying the cultivar according to the
invention.
[0104] A more detailed description of some preferred embodiments of
the invention is given below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0105] FIG. 1 depicts an identification key to identify 99% of the
genotypes.
DETAILED DESCRIPTION OF THE INVENTION
[0106] As mentioned above, the present invention relates to two
sets of molecular markers for studying the genetic biodiversity of
the date palm and identifying cultivars.
I--Sets of Markers for Genotyping the Date Palm
[0107] The inventors have assembled a novel optimized collection of
mini- and microsatellites defined by the exploitation of
microsatellite markers described in the literature or identified
from the available genomic resources.
[0108] The term "minisatellite" is intended to mean herein a DNA
sequence formed by a continuous repetition of motifs composed of 10
to 100 nucleotides and most commonly flanked by conserved regions.
The term "microsatellite" is intended to mean herein a DNA sequence
formed by a continuous repetition of motifs composed of 1 to 10
nucleotides and most commonly flanked by conserved regions.
Generally, the length of these (minisatellite or microsatellite)
SSR sequences, i.e. the number of repetitions of the motif, is
variable from one species to another, from one individual to
another and from one allele to another in the same individual. All
of the information relating to the lengths of sequences of the
mini- and microsatellite markers is indicative of the genotype of
the date palm.
[0109] The first set of markers according to the invention
comprises 19 SSR markers: CpfM12, mPdIRD031, mPdIRD033, mPdIRD040,
PdCUC3-ssr2, PdAP3-ssr, mPdCIR078, mPdCIR015, mPdCIR016, mPdCIR032,
mPdCIR035, mPdCIR057, mPdCIR085, PdAG1-ssr, mPdCIR010, mPdCIR025,
mPdCIR063, PdCUC3-ssr1 and mPdIRD013.
[0110] The second set of markers according to the invention
comprises 7 SSR markers: CpfM12, mPdIRD031, mPdIRD033, mPdIRD040,
PdCUC3-ssr2, PdAP3-ssr and mPdCIR078.
[0111] The mPdCIR078, mPdCIR015, mPdCIR016, mPdCIR032, mPdCIR035,
mPdCIR057, mPdCIR085, mPdCIR010, mPdCIR025 and mPdCIR063 markers
are (GA)n dinucleotide loci which have been described by Billotte
et al. (Molecular Ecology Notes, 2004, 4: 256-258).
[0112] The mPdIRD031, mPdIRD033, mPdIRD040 and mPdIRD013 markers
are microsatellite tandem repeats of 3 or 6 base pairs that the
present inventors have identified in silico in coding sequences of
the sequence of the date palm genome (Al-Dous et al., Nature
Biotechnology, 2011, 29:
521-527--http://qatar-weill.cornell.edu/research/datepalmGenome/download.-
html). These 4 loci were selected among 47 for their amplification
capacity and the polymorphism that they generate.
[0113] The PdCUC3-ssr1 and PdCUC3-ssr2 markers are dinucleotide
sequences identified in CUC genes (Adam et al., Molecular Biology
and Evolution, 2010, 28: 1439-1454).
[0114] The PdAG1-ssr1 and PdAP3-ssr1 markers are dinucleotide
sequences identified by the present inventors in the AG1 gene and
the AP3 gene (not published) respectively.
[0115] The CpfM12 marker is a minisatellite marker of the
chloroplast genome (Henderson et al., Conservation Genetics, 2006,
7: 213-223).
[0116] The invention relates to two sets of mini- and
microsatellite markers specific for the genetic diversity of the
date palm, where the markers are identified either by their motif
and the sequence in which they are contained, or by their motif and
the conserved nucleotide sequences which flank them in 5' and in
3'. The terms "nucleotide sequence", "sequence" and "nucleic
sequence" are used herein without implied distinction. These terms
are intended to denote a precise succession of nucleotides which
makes it possible to define a region of a nucleic acid and which
can correspond both to a double-stranded DNA or a single-stranded
DNA and to products of transcription of these DNA molecules.
[0117] The information provided below for defining each SSR marker
of a set of markers according to the invention concerns the
5'.fwdarw.3' strand of the region of the genome in which the SSR is
located. As those skilled in the art will recognize, given that an
SSR sequence is a double-stranded DNA sequence, the invention also
encompasses the complementary motif and the complementary locus
(which are on the 3'.fwdarw.5' strand).
[0118] The mPdCIR010 marker is characterized in that, in the
5'.fwdarw.3' direction, it has a (ga) motif, it is present in the
sequence having the GenBank accession number AJ571673.1 (Phoenix
dactylifera microsatellite DNA, clone CIR010), and it is flanked,
in 5', by the nucleotide sequence SEQ ID NO: 1 and, in 3', by the
nucleotide sequence SEQ ID NO: 2.
[0119] The mPdCIR015 marker is characterized in that, in the
5'.fwdarw.3' direction, it has a (ga) motif, it is present in the
sequence having the GenBank accession number AJ571674.1 (Phoenix
dactylifera microsatellite DNA, clone CIR015), and it is flanked,
in 5', by the nucleotide sequence SEQ ID NO: 3 and, in 3', by the
nucleotide sequence SEQ ID NO: 4.
[0120] The mPdCIR016 marker is characterized in that, in the
5'.fwdarw.3' direction, it has a (ga) motif, it is present in the
sequence having the GenBank accession number AJ571675.1 (Phoenix
dactylifera microsatellite DNA, clone CIR016), and it is flanked,
in 5', by the nucleotide sequence SEQ ID NO: 5 and, in 3', by the
nucleotide sequence SEQ ID NO: 6.
[0121] The mPdCIR025 marker is characterized in that, in the
5'.fwdarw.3' direction, it has a (ga) motif, it is present in the
sequence having the GenBank accession number AJ571676.1 (Phoenix
dactylifera microsatellite DNA, clone CIR025), and it is flanked,
in 5', by the nucleotide sequence SEQ ID NO: 7 and, in 3', by the
nucleotide sequence SEQ ID NO: 8.
[0122] The mPdCIR032 marker is characterized in that, in the
5'.fwdarw.3' direction, it has a (ga) motif, it is present in the
sequence having the GenBank accession number AJ571677.1 (Phoenix
dactylifera microsatellite DNA, clone CIR032), and it is flanked,
in 5', by the nucleotide sequence SEQ ID NO: 9 and, in 3', by the
nucleotide sequence SEQ ID NO: 10.
[0123] The mPdCIR035 marker is characterized in that, in the
5'.fwdarw.3' direction, it has a (ga) motif, it is present in the
sequence having the GenBank accession number AJ571678.1 (Phoenix
dactylifera microsatellite DNA, clone CIR035), and it is flanked,
in 5', by the nucleotide sequence SEQ ID NO: 11 and, in 3', by the
nucleotide sequence SEQ ID NO: 12.
[0124] The mPdCIR057 marker is characterized in that, in the
5'.fwdarw.3' direction, it has a (ga) motif, it is present in the
sequence having the GenBank accession number AJ571682.1 (Phoenix
dactylifera microsatellite DNA, clone CIR057), and it is flanked,
in 5', by the nucleotide sequence SEQ ID NO: 13 and, in 3', by the
nucleotide sequence SEQ ID NO: 14.
[0125] The mPdCIR063 marker is characterized in that, in the
5'.fwdarw.3' direction, it has a (ga) motif, it is present in the
sequence having the GenBank accession number AJ571683.1 (Phoenix
dactylifera microsatellite DNA, clone CIR063), and it is flanked,
in 5', by the nucleotide sequence SEQ ID NO: 15 and, in 3', by the
nucleotide sequence SEQ ID NO: 16.
[0126] The mPdCIR078 marker is characterized in that, in the
5'.fwdarw.3' direction, it has a (ga) motif, it is present in the
sequence having the GenBank accession number AJ571685.1 (Phoenix
dactylifera microsatellite DNA, clone CIR078), and it is flanked,
in 5', by the nucleotide sequence SEQ ID NO: 17 and, in 3', by the
nucleotide sequence SEQ ID NO: 18.
[0127] The mPdCIR085 marker is characterized in that, in the
5'.fwdarw.3' direction, it has a (ga) motif, it is present in the
sequence having the GenBank accession number AJ571686.1 (Phoenix
dactylifera microsatellite DNA, clone CIR085), and it is flanked,
in 5', by the nucleotide sequence SEQ ID NO: 19 and, in 3', by the
nucleotide sequence SEQ ID NO: 20.
[0128] The CpfM12 marker is characterized in that, in the
5'.fwdarw.3' direction, it has a (ctaactactata) motif, it is
present in the sequence having the GenBank accession number
EU043484.1, and it is flanked, in 5', by the nucleotide sequence
SEQ ID NO: 21 and, in 3', by the nucleotide sequence SEQ ID NO:
22.
[0129] The PdCUC3-ssr1 marker is characterized in that, in the
5'.fwdarw.3' direction, it has a (gt+ga) motif, it is present in
the sequence having the GenBank accession number HM622273.1
(Phoenix dactylifera cup-shaped cotyledon 3-related protein (CUC3)
gene, partial cds), and it is flanked, in 5', by the nucleotide
sequence SEQ ID NO: 23 and, in 3', by the nucleotide sequence SEQ
ID NO: 24.
[0130] The PdCUC3-ssr2 marker is characterized in that, in the
5'.fwdarw.3' direction, it has a (ga) motif, it is present in the
sequence having the GenBank accession number HM622273.1 (Phoenix
dactylifera cup-shaped cotyledon 3-related protein (CUC3) gene,
partial cds), and it is flanked, in 5', by the nucleotide sequence
SEQ ID NO: 25 and, in 3', by the nucleotide sequence SEQ ID NO:
26.
[0131] The PdAG1-ssr1 marker is characterized in that, in the
5'.fwdarw.3' direction, it has a (ga) motif, it is present in the
sequence having the GenBank accession number KC188338.1, and it is
flanked, in 5', by the nucleotide sequence SEQ ID NO: 27 and, in
3', by the nucleotide sequence SEQ ID NO: 28.
[0132] The PdAP3-ssr marker is characterized in that, in the
5'.fwdarw.3' direction, it has a (ga) motif, it is present in the
sequence having the GenBank accession number KC188337.1, and it is
flanked, in 5', by the nucleotide sequence SEQ ID NO: 29 and, in
3', by the nucleotide sequence SEQ ID NO: 30.
[0133] The mPdIRD13 marker is characterized in that, in the
5'.fwdarw.3' direction, it has a (cac) motif, it is present in the
PDK.sub.--20 s1496731g002.sub.--1 scaffold (GenBank accession
number GL741615.1), and it is flanked, in 5', by the nucleotide
sequence SEQ ID NO: 31 and, in 3', by the nucleotide sequence SEQ
ID NO: 32.
[0134] The mPdIRD31 marker is characterized in that, in the
5'.fwdarw.3' direction, it has a (cca) motif, it is present in the
PDK.sub.--20 s1419261g003.sub.--1 scaffold (described in
http://qatar-weill.cornell.edu/research/datepalmGenome/download.html),
and it is flanked, in 5', by the nucleotide sequence SEQ ID NO: 33
and, in 3', by the nucleotide sequence SEQ ID NO: 34.
[0135] The mPdIRD33 marker is characterized in that, in the
5'.fwdarw.3' direction, it has a (cag) motif, it is present in the
PDK.sub.--20 s1569281g001.sub.--1 scaffold (described in
http://qatar-weill.cornell.edu/research/datepalmGenome/download.html),
and it is flanked, in 5', by the nucleotide sequence SEQ ID NO: 35
and, in 3', by the nucleotide sequence SEQ ID NO: 36.
[0136] The mPdIRD40 marker is characterized in that, in the
5'.fwdarw.3' direction, it has a (ccagtg) motif, it is present in
the PDK.sub.--20 s1327401g002.sub.--1 scaffold (described in
http://qatar-weill.cornell.edu/research/datepalmGenome/download.html),
and it is flanked, in 5', by the nucleotide sequence SEQ ID NO: 37
and, in 3', by the nucleotide sequence SEQ ID NO: 38.
II--Use of the Sets of Markers for Identifying Date Palm Genotypes
and/or Date Palm Cultivars
[0137] As the present inventors have demonstrated, the sets of
molecular markers described above can be used for identifying date
palm genotypes. Generally, an identification method according to
the invention is carried out by detecting, using a set of markers,
an SSR polymorphism in the genome of a date palm individual, and
comprises, for each of the mini- or microsatellite markers of the
set, the amplification of a region of the genome of the date palm
to be tested, the region comprising the SSR sequence.
Date Palm Samples
[0138] In a method according to the invention, the step consisting
in amplifying a region (or portion) of the genome of the date palm
comprising a repeat SSR motif is carried out on a sample of the
date palm to be tested, and preferably on genomic DNA extracted
from the sample of the date palm. The term "genomic DNA" is
intended to mean herein the DNA of the nuclear genome (i.e. the
genomic DNA contained in the nucleus) and of the chloroplast genome
(i.e. the genomic DNA contained in the chloroplasts).
[0139] Any date palm sample containing genomic DNA can therefore be
used in the implementation of the present invention. For example,
the genomic DNA can be extracted from date palm protoplasts,
calluses, embryos, leaves, stipes, roots, shoots or cuttings.
[0140] The methods for extracting DNA from biological tissues are
well known in the art (see, for example, Sambrook et al.,
"Molecular Cloning--A Laboratory Manual", Cold Spring Harbor
Laboratory Press, 1989). There are also numerous commercially
available kits (for example from BD Biosciences Clontech (Palo
Alto, Calif.), Epicentre Technologies (Madison, Wis.), Gentra
Systems, Inc. (Minneapolis, Minn.), MicroProbe Corp. (Bothell,
Wash.), Organon Teknika (Durham, N.C.) and Qiagen Inc. (Valencia,
Calif.)) which can be used to extract the DNA from date palm
samples.
Amplification of the Microsatellite Sequence
[0141] As those skilled in the art will recognize, in the
implementation of the invention, the amplification of a portion of
the date palm genome comprising a microsatellite sequence according
to the invention can be carried out by any appropriate technique
known in the art, since the technique used is not a limiting factor
of the invention.
[0142] Preferably, in a method according to the invention, the
genomic DNA amplification reactions are carried out by PCR
(Polymerase Chain Reaction) amplification (Mullis and Faloona,
Methods Enzymol., 1987, 155: 355-350) which offers the advantage of
analyzing the molecular markers in a short time while using low DNA
concentrations. The flanking regions of the microsatellites serve
as primers during the PCR. Given that these regions are conserved,
a pair of primers specific for these flanking regions specifically
amplifies only this microsatellite (and not another).
[0143] The various amplicons (or amplification products) generated
from a given microsatellite region have characteristic and
reproducible sizes. The variation in the sizes of the PCR products
is caused by differences in the number of repeats of the motif of
the microsatellite. These sizes are therefore indicative of the
length (if the date palm sample is homozygous) or the lengths (if
the date palm sample is heterozygous) of the microsatellite
sequence in the individual.
[0144] Those skilled in the art know how to select the optimal
conditions (temperature, time and number of cycles, pH, and
concentration of the reagents) for performing a PCR amplification
("PCR Protocols: A Guide to Methods and Applications", M. A. Innis
(Ed.), 1990, Academic Press: New York; "PCR Strategies", M. A.
Innis (Ed.), 1995, Academic Press: New York; "Polymerase chain
reaction: basic principles and automation in PCR: A Practical
Approach", McPherson et al. (Eds.), 1991, IRL Press: Oxford; R. K.
Saiki et al., Nature, 1986, 324: 163-166).
Primers and Probes for the Amplification of a Portion of the Date
Palm Genome Comprising an SSR Sequence
[0145] On the basis of the nucleotide sequence of a scaffold or
other genomic sequence in which a microsatellite of a set of
molecular markers of the invention is located and of the position
of this microsatellite in this scaffold or this sequence, those
skilled in the art know how to design SSR primers suitable for the
amplification of a portion of the date palm genome which comprises
the microsatellite sequence.
[0146] The terms "primer" and "PCR primer" are used here
interchangeably and denote an oligonucleotide which is capable of
acting as a starting point for the synthesis of an amplification
product, when it is placed under appropriate amplification
conditions (for example, salt concentration, temperature and pH) in
the presence of nucleotides and of a nucleic acid polymerization
agent (for example a DNA polymerase). A primer according to the
invention comprises an oligonucleotide advantageously containing
between 5 and 50 nucleotides, generally between 15 and 50
nucleotides, preferably between 20 and 35 nucleotides, and even
more preferably between 20 and 25 nucleotides (for example, 20, 21,
22, 23, 24 or 25 nucleotides).
[0147] The term "SSR primer" refers to a primer which is specific
for a region flanking or adjacent to the microsatellite region and
which, in combination with another SSR primer, is capable of
specifically amplifying the microsatellite region. The
amplification is generally carried out using a pair of SSR primers
comprising a forward (or "sense") primer and a reverse (or
"antisense") primer which each hybridize on one of the two strands
of the genomic DNA.
[0148] Preferably, a forward SSR primer according to the invention
comprises, or consists of, a sequence of 15 to 50 consecutive
nucleotides, preferably of 20 to 35 consecutive nucleotides, and
even more preferably of 20 to 25 consecutive nucleotides, of the
sequence of at most 1000 nucleotides flanking, in 5', the
microsatellite region according to the invention. Preferably, a
reverse SSR primer according to the invention comprises, or
consists of, a sequence of 15 to 50 consecutive nucleotides,
preferably of 20 to 35 consecutive nucleotides, and even more
preferably of 20 to 25 consecutive nucleotides, of the sequence
complementary to the sequence of at most 1000 nucleotides flanking,
in 3', the microsatellite region according to the invention.
[0149] The term "sequence complementary to" a given nucleotide
sequence is intended to mean a sequence which forms, by
hybridization, a stable duplex with said nucleotide sequence. The
term "sequence complementary to" denotes both the complementary
sequence presented in the 3'.fwdarw.5' direction and the
complementary sequence presented in the 5'.fwdarw.3' direction
(i.e. the reverse complementary sequence). The term "hybridization"
is intended to mean herein the head-to-tail association of two
single-stranded polynucleotides by Watson-Crick pairings (A-T,
G-C). In certain cases, the hybridization is perfect, i.e. the
sequences are totally complementary. Thus, for example, the term
"the sequence complementary to the sequence SEQ ID NO: 1" is the
nucleotide sequence which is perfectly or totally complementary to
SEQ ID NO: 1.
[0150] Thus, in the implementation of a method of the invention,
the amplification of a region of the genome of the date palm
comprising the sequence of the microsatellite having the (ga) motif
of the mPdCIR010 marker is preferably carried out using a pair of
SSR primers comprising: [0151] a forward SSR primer of sequence SEQ
ID NO: 1 (ACCCCGGACGTGAGGTG), and [0152] a reverse SSR primer of
sequence SEQ ID NO: 2 (CGTCGATCTCCTCCT TTGTCTC).
[0153] The amplification of a region of the genome of the date palm
comprising the sequence of the microsatellite having the (ga) motif
of the mPdCIR015 marker is preferably carried out using a pair of
SSR primers comprising: [0154] a forward SSR primer of sequence SEQ
ID NO: 3 (AGCTGGCTCCTCCCTTCTTA), and [0155] a reverse SSR primer of
sequence SEQ ID NO: 4 (GCTCGGTTGGACTTGTTCT).
[0156] The amplification of a region of the genome of the date palm
comprising the sequence of the microsatellite having the (ga) motif
of the mPdCIR016 marker is preferably carried out using a pair of
SSR primers comprising: [0157] a forward SSR primer of sequence SEQ
ID NO: 5 (AGCGGGAAATGAAAAGGTAT), and [0158] a reverse SSR primer of
sequence SEQ ID NO: 6 (ATGAAAACGTGCCAAATGTC).
[0159] The amplification of a region of the genome of the date palm
comprising the sequence of the microsatellite having the (ga) motif
of the mPdCIR025 marker is preferably carried out using a pair of
SSR primers comprising: [0160] a forward SSR primer of sequence SEQ
ID NO: 7 (GCACGAGAAGGCTTATAGT), and [0161] a reverse SSR primer of
sequence SEQ ID NO: 8 (CCCCTCATTAGGATTCTAC).
[0162] The amplification of a region of the genome of the date palm
comprising the sequence of the microsatellite having the (ga) motif
of the mPdCIR032 marker is preferably carried out using a pair of
SSR primers comprising: [0163] a forward SSR primer of sequence SEQ
ID NO: 9 (CAAATCTTTGCCGTGAG), and [0164] a reverse SSR primer of
sequence SEQ ID NO: 10 (GGTGTGGAGTAATCATGTAGTAG).
[0165] The amplification of a region of the genome of the date palm
comprising the sequence of the microsatellite having the (ga) motif
of the mPdCIR035 marker is preferably carried out using a pair of
SSR primers comprising: [0166] a forward SSR primer of sequence SEQ
ID NO: 11 (ACAAACGGCGATGGGATTAC), and [0167] a reverse SSR primer
of sequence SEQ ID NO: 12 (CCGCAGCTCACCTCTTCTAT).
[0168] The amplification of a region of the genome of the date palm
comprising the sequence of the microsatellite having the (ga) motif
of the mPdCIR057 marker is preferably carried out using a pair of
SSR primers comprising: [0169] a forward SSR primer of sequence SEQ
ID NO: 13 (AAGCAGCAGCCCTTCCGTAG), and [0170] a reverse SSR primer
of sequence SEQ ID NO: 14 (GTTCTCACTCGCCCAAAAATAC).
[0171] The amplification of a region of the genome of the date palm
comprising the sequence of the microsatellite having the (ga) motif
of the mPdCIR063 marker is preferably carried out using a pair of
SSR primers comprising: [0172] a forward SSR primer of sequence SEQ
ID NO: 15 (CTTTTATGTGGTCTGAGAGA), and [0173] a reverse SSR primer
of sequence SEQ ID NO: 16 (TCTCTGATCTTGGGTTCTGT).
[0174] The amplification of a region of the genome of the date palm
comprising the sequence of the microsatellite having the (ga) motif
of the mPdCIR078 marker is preferably carried out using a pair of
SSR primers comprising: [0175] a forward SSR primer of sequence SEQ
ID NO: 17 (TGGATTTCCATTGTGAG), and [0176] a reverse SSR primer of
sequence SEQ ID NO: 18 (CCCGAAGAGACGCTATT).
[0177] The amplification of a region of the genome of the date palm
comprising the sequence of the microsatellite having the (ga) motif
of the mPdCIR085 marker is preferably carried out using a pair of
SSR primers comprising: [0178] a forward SSR primer of sequence SEQ
ID NO: 19 (GAGAGAGGGTGGTGTTATT), and [0179] a reverse SSR primer of
sequence SEQ ID NO: 20 (TTCATCCAGAACCACAGTA).
[0180] The amplification of a region of the genome of the date palm
comprising the sequence of the minisatellite having the
(ctaactactata) motif of the CpfM12 marker is preferably carried out
using a pair of SSR primers comprising: [0181] a forward SSR primer
of sequence SEQ ID NO: 21 (CCGCCCACGATGAAGTAATGTA), and [0182] a
reverse SSR primer of sequence SEQ ID NO: 22
(GTCACGGGTTCAAATCCTGTCTC).
[0183] The amplification of a region of the genome of the date palm
comprising the sequence of the microsatellite having the (gt+ga)
motif of the PdCUC3-ssr1 marker is preferably carried out using a
pair of SSR primers comprising: [0184] a forward SSR primer of
sequence SEQ ID NO: 23 (CGTGGACTCATGACTCGCATGTCC), and [0185] a
reverse SSR primer of sequence SEQ ID NO: 24
(GGTCCTTGCCGGTGGCCTTC).
[0186] The amplification of a region of the genome of the date palm
comprising the sequence of the microsatellite having the (ga) motif
of the PdCUC3-ssr2 marker is preferably carried out using a pair of
SSR primers comprising: [0187] a forward SSR primer of sequence SEQ
ID NO: 25 (ACATTGCTCTTTTGCCATGGGCT), and [0188] a reverse SSR
primer of sequence SEQ ID NO: 26 (CGAGCAGGTGGGGTTCGGGT).
[0189] The amplification of a region of the genome of the date palm
comprising the sequence of the microsatellite having the (ga) motif
of the PdAG1-ssr1 marker is preferably carried out using a pair of
SSR primers comprising: [0190] a forward SSR primer of sequence SEQ
ID NO: 27 (TCTGATTTCGTTTACTTCTTAGGA), and [0191] a reverse SSR
primer of sequence SEQ ID NO: 28 (TTCATATTCAGTTGTCGGGTGTA).
[0192] The amplification of a region of the genome of the date palm
comprising the sequence of the microsatellite having the (ga) motif
of the PdAP3-ssr marker is preferably carried out using a pair of
SSR primers comprising: [0193] a forward SSR primer of sequence SEQ
ID NO: 29 (GAGAAATAGAGAGCTGTGCAAG), and [0194] a reverse SSR primer
of sequence SEQ ID NO: 30 (CTGCAGTACTCGGAGAACTTG).
[0195] The amplification of a region of the genome of the date palm
comprising the sequence of the microsatellite having the (cac)
motif of the mPdIRD13 marker is preferably carried out using a pair
of SSR primers comprising: [0196] a forward SSR primer of sequence
SEQ ID NO: 31 (GCGGAGACAGGAGATGGTAA), and [0197] a reverse SSR
primer of sequence SEQ ID NO: 32 (CTTGACTGCTTCTGCTGCTG).
[0198] The amplification of a region of the genome of the date palm
comprising the sequence of the microsatellite having the (cca)
motif of the mPdIRD31 marker is preferably carried out using a pair
of SSR primers comprising: [0199] a forward SSR primer comprising,
or consisting of, between 15 and 50 consecutive nucleotides of the
sequence of at most 1000 nucleotides flanking, in 5', position
29073 in the PDK.sub.--20 s1419261g003.sub.--1 scaffold
(http://qatar-weill.cornell.edu/research/datepalmGenome/download.html),
and [0200] a reverse SSR primer comprising, or consisting of,
between 15 and 50 consecutive nucleotides of the sequence
complementary to the sequence of at most 1000 nucleotides flanking,
in 3', position 29093 in the PDK.sub.--20 s1419261g003.sub.--1
scaffold.
[0201] The sequence of at most 1000 nucleotides may comprise any
number of nucleotides preferably between 1000 and 250, for example
1000, 900, 800, 700, 600, 500, 400, 300 or 250.
[0202] In one particular embodiment, a pair of primers for the
amplification of a region of the genome of the date palm comprising
the sequence of the microsatellite having the (cca) motif of the
mPdIRD31 marker comprise a forward SSR primer of sequence SEQ ID
NO: 33 (GCAGGTGGACTGCAAAATCT) and a reverse SSR primer of sequence
SEQ ID NO: 34 (CTATTGGGGTGCTGATCCAT).
[0203] The amplification of a region of the genome of the date palm
comprising the sequence of the microsatellite having the (cag)
motif of the mPdIRD33 marker is preferably carried out using a pair
of SRR primers comprising: [0204] a forward SSR primer comprising,
or consisting of, between 15 and 50 consecutive nucleotides of the
sequence of at most 1000 nucleotides flanking, in 5', position 5206
in the PDK.sub.--20 s1569281g001.sub.--1 scaffold
(http://qatar-weill.cornell.edu/research/datepalmGenome/download.html),
and [0205] a reverse SSR primer comprising, or consisting of,
between 15 and 50 consecutive nucleotides of the sequence
complementary to the sequence of at most 1000 nucleotides flanking,
in 3', position 5226 in the PDK.sub.--20 s1569281g001.sub.--1
scaffold.
[0206] The sequence of at most 1000 nucleotides may comprise any
number of nucleotides preferably between 1000 and 250, for example
1000, 900, 800, 700, 600, 500, 400, 300 or 250.
[0207] In one particular embodiment, a pair of primers for the
amplification of a region of the genome of the date palm comprising
the sequence of the microsatellite having the (cag) motif of the
mPdIRD33 marker comprises a forward SSR primer of sequence SEQ ID
NO: 35 (GGAGCATACAGTGGGTTTGC) and a reverse SSR primer of sequence
SEQ ID NO: 36 (CAGCCTGGGAATGAGGATAG).
[0208] The amplification of a region of the genome of the date palm
comprising the sequence of the microsatellite having the (ccagtg)
motif of the mPdIRD40 marker is preferably carried out using a pair
of SSR primers comprising: [0209] a forward SSR primer comprising,
or consisting of, between 15 and 50 consecutive nucleotides of the
sequence of at most 1000 nucleotides flanking, in 5', position
16193 in the PDK.sub.--20 s1327401g002.sub.--1 scaffold
(http://qatar-weill.cornell.edu/research/datepalmGenome/download.html),
and [0210] a reverse SSR primer comprising, or consisting of,
between 15 and 50 consecutive nucleotides of the sequence
complementary to the sequence of at most 1000 nucleotides flanking,
in 3', position 16216 in the PDK.sub.--20 s1327401g002.sub.--1
scaffold.
[0211] The sequence of at most 1000 nucleotides may comprise any
number of nucleotides preferably of between 1000 and 250, for
example 1000, 900, 800, 700, 600, 500, 400, 300 or 250.
[0212] In one particular embodiment, a pair of primers for the
amplification of a region of the genome of the date palm comprising
the sequence of the microsatellite having the (ccagtg) motif of the
mPdIRD40 marker comprises a forward SSR primer of sequence SEQ ID
NO: 37 (GAGAGATGCGTCAGGGAATC) and a reverse SSR primer of sequence
SEQ ID NO: 38 (CCAGAATCTTCCAAGCAAGC).
[0213] In some embodiments, a primer according to the invention is
labeled so as to enable its detection (and, consequently, that of
the amplification products or amplicons obtained by PCR). Various
types of labeling, known to those skilled in the art, can be used
(radioactive labeling, fluorescence labeling, chemiluminescence
labeling, labeling of M13 type, etc.). The marker may be integrated
into the oligonucleotide forming the primer, or can be linked to
this oligonucleotide (for example by covalent bonding). The term
"labeled primer" or "probe" is therefore intended to denote a
primer which contains, or which is linked or bonded (for example
covalently) to, a detectable marker.
[0214] The primers can be prepared by any appropriate method known
to those skilled in the art, chosen in particular from the
conventional oligonucleotide synthesis methods, such as the
solid-phase synthesis methods. The primers according to the
invention may, for example, be prepared using an oligonucleotide
synthesizer (such as those sold, for example, by Applied Biosystems
or GE Healthcare). Likewise, methods for labeling oligonucleotides
are known in the art.
[0215] The present invention relates to the primers described
herein (or any other primer which can be deduced from the
information provided) and also to the use thereof for identifying
the genotype (and/or cultivar) of the date palm by amplification of
a portion of the genome of the date palm which comprises a
microsatellite sequence according to the invention.
Amplicon Analysis
[0216] In one method for identifying the genotype of the date palm
using one of the two sets of molecular markers according to the
invention, after amplification, for each marker of the set, of a
portion of the genome of the date palm comprising the
microsatellite sequence, the amplicons are analyzed in order to
determine the genotype of the date palm tested.
[0217] The analysis of the amplicons can be carried out by any
method. The analysis generally comprises the separation of the
amplicons according to their size (and therefore to the size of the
microsatellite sequence). The separation may, for example, be
carried out by electrophoresis (a technique which makes it possible
to separate DNA fragments according to their electrical charge and
their size) on agarose gel or on polyacrylamide gel. The separation
is followed by the detection of the separated fragments by staining
with ethidium bromide or silver, or else by detection of the
detectable marker of the primer (by fluorescence, radioactivity,
etc.).
[0218] In order to determine the genotype of the date palm tested,
the sizes of the amplicons obtained can be compared with the sizes
of the amplicons obtained after amplification, carried out under
the same conditions, of the genomic DNA of a control date palm. The
term "control date palm" denotes herein a date palm of which the
genotype (origin, variety, cultivar, etc.) is known.
[0219] The step of comparing the sizes of the amplicons obtained
from the sample of date palm tested may comprise the use of several
control date palms. In particular, the step of comparing the sizes
of the amplicons obtained, for the set of 19 or the set of 7
markers, from the date palm sample may comprise comparison with the
sizes of the amplicons obtained, for the same set of markers, from
the genotypes used by the inventors, and grouped together in the
database PhoenixDB http://phoenixdb.mpl.ird.fr (login: phoenix;
password: s7RSFV).
[0220] In order to determine the genotype of the date palm tested,
the amplicons may, after separation according to their size, be
sequenced in order to determine the number(s) of repeats of the SSR
motif in the genome of the date palm tested. The DNA fragment
sequencing methods are known in the art. In particular, it is
possible to use automatic sequencers such as those available from
Beckman, Applied BioSystems, or LiCor Biosciences.
Identification of the Genotype and/or the Cultivar of the Date Palm
Tested
[0221] As indicated above, the set of 19 markers makes it possible
to analyze 100% of the date palm genotypes. The date palms tested
using a method according to the invention may originate from any
region of the world, in particular from Israel, Libya, Tunisia,
Mauritania, Algeria, Pakistan, Iraq, Sudan, Cape Verde, Spain,
Niger, Morocco, Italy, Oman, Djibouti, Iran, India, Thailand or
Vietnam.
[0222] In particular, the set of 19 markers makes it possible to
identify the genotypes and varieties of date palms of the species
Phoenix dactylifera L. chosen from Aqdool, Taj, Lemsi male,
Edaghed, Halwa, Halwaya, Gadri (Yellow Seed), Ashrasi, Badrayah,
Gadri, Kentichi, Mech Degla, Benosh, Besser Helou, Khmag, Laloo,
Awreeq, Dguel Mghas, Amreer, Dokhar, Arechti, Deglet Jdir,
Barakawi, Elche, Outaghsait, Bufea, Adam Goas, Deglet Wlad Mahmoud,
Tunisian Ghars, Gadri, Jonah, Akerbouche, Baydir, Tinjdel, Abbad,
Ali pun chohara, Timakur, Moroccan Bou Feggous, Algerian Bou
Feggous (Fagous), Thorry, Tunisian Alig, Peggy Ann (close to
Thorry), Algerian Kenta, La Confitera, Medjoul, Back Cross Medjool
4th G, Romana I, Bidh-Hmam, Tunisian Kenta, Chikh, Chikh (Hamuri),
Chikh (Mhammed), Hamraya III, Algerian Alig, Alig (Bu'Rus), Alig
(Ftimi), Tamazouart, Deglet Gurara, Lagou, Bayd Hmam, Bitamoda,
Bent Cherk, Bent Cherk (Cherka), Bukhannus, Tinasser, Degla Baida,
Gondaila, Cheddakh, Deglet Hassan, Timedjwel, Takhoudrayt, Tunisian
Bou Feggous, Bessir, Bouskri, Deglet Bey, Deglet Bey (Menakher),
Sekani, Talees, Dogna, Tinhamour, Taghiyat, Tunisian Hmara, Gasbi,
Me'udiya, Tadmamt, Tindukan, Aman, Takerboucht, Tazizawt, Ouarglia,
Aghares, Goundi, Peli Sundar, Khara Basra, Amir Hajj, Mobai (Den
wala), Mobai (Dost Muhammadi Wala), Mobai, Horra, Rachna, Fasli,
Shakri, Zaidi (Danda), Zaidi (Halimi), Zaidi (Masri), Zaidi (not
Karblain), Zaidi, Zaidi, Dhakki, Dhakki (Congon), Khalas, Seib,
Naghal (Gajjar/Gujjar), Naghal, Lolo, Bahlani, Gabeeni Mobai,
Romana II, Dokhar, Adam Alkag, Aseel (Angor), Aseel, Aseel (Dhakki
Zard), Aseel (Khurma), Chohara, Qasab, Badmi, Barhi, Hardasht,
Hardasht (Jwana), Jeremiah, Khastawi, Tadala, Medina Kaylanya,
Braim, Makran, Mcharret, Taghera, Saidi, Tati, Timrisa, Deglet
Nour, Deglet Nour mere des Pa, Adam Hror, Eve, D'quel El Hadj,
Banekhluf, Bukezzine, Ahmar, Rukkan, Shado, Gulistan, Khurmo,
Halawi (Champa Kali), Halawi (Gulistan), Halawi, Halawi (Hillawi),
Halawi (Neelum), Halawi (Pela Dora), Halawi (Zohdi), Khupra, Adam
Timu, Choti Sandori, Chataya, Tawadan, Yellow Lulu, Bouldjib,
Aghaliane, Ain Zbib, Tazerzist, Deglet Jito, Adam Bchir, Ma'tug,
Ajwa, Chapshoq, Khalass Oman, Zerin, Surkh Begum Jungi, Gurbago,
Karblain, Karblain (Ko-harba), Sajho Wali, Kozan Abad, Basra,
Tabarzal, Pashpag, Pasht Kharoch, Black Bou Sthammi, Shamran,
Otakanr, Kesba, Kesba (Sokrya), Zardo, Deglet Mech'a, Begum Jungi
II, Kokna, Zard Begum Jungi, Dhaidi wali, Bounaringa, Hawwa wali,
Khalass, Akhrot, Fard (Dhandari), Fard (Farz), Fard, Ajoujil,
Qantar, Jaman, Ko-harba, Goknah, Azizaou (Arab name: Adam Zrak),
Azizaou (Arab name: Amazigh), Tafeziwin, Adam Deglet Noir, Timliha,
Adam Esof, Dguel Sidi Khlil, Adam Araw, Gadri (Red), Ain Zbib,
Ououchtt, Tinhud, Tawragha, Tissibi, Ighes N'wagada, Tilemsu, Kasho
wali, Desi, Halini, Hilali, Hussaini, Hussaini (Jan-sohar), Zard
Kharoch, Bahram, Naz Tabakki, Saylani, Lasora, Khadrawy, Bdmalki,
Bdmalki (Sarmadti), Maktoom, Kharoch, Jhajhri, Roghni, Gharas,
Surkh, Noor Muhammadi Much, Mathusaleh, Aghammu, Mozati, Surkh
Dhakki, Bent Qbala, Nasser Oussaleh, Bawa'adhim, Timliha, Tifazwin,
Tanslit, Hmira, Taghera, Tgaza, Asemmat, Adam Bulla, Hamraya I,
Be-Rehmi, Tinrigh, Khadri, Tazarzayet, Ebrea, Savi Khaji, Andekly,
Begum Jungi, Litima, Harthan (Aharthan), Harthan (Oumazer), Klair,
Timjouhart, red Lulu, Ouzamig, Tachlilat, Dimolo, Tawraghet,
Tazougart, Tinicine, Hamraya II, Angou (=Algerian Ghars), Ghars,
Tanteboucht, Tanteboucht (El Kayed), Abdel Azzaz, Bacheir,
Tantabecht, Zoq El Moggar, Taramount, Bandid, El Gachouche, D'quel
M'rass, Takermoust, Tizouyadj, Zaghraya, Ratti Khajji, Begun and
Nisri, Dfor Lgot.
[0223] This set of markers also makes it possible to identify the
genotypes of palms of the species Phoenix canariensi, Phoenix
atlantica, Phoenix acaulis, Phoenix loureiroi, Phoenix
andamanensis, Phoenix pusilla, Phoenix reclinata, Phoenix
caespitosa, Phoenix roebelenii, Phoenix paludosa, Phoenix
sylvestris and Phoenix theophrasti.
[0224] The set of 7 markers according to the invention makes it
possible to analyze 99% of the date palm cultivars. In particular,
the set of 7 markers makes it possible to identify the cultivars of
Phoenix dactylifera L. chosen from the cultivars originating from
Algeria, such as the cultivars Adam Hror, Aghares, Ajoujil,
Akerbouche, Bent Qbala, Chikh, Degla Baida, Deglet Jdir, Dfor Lgot,
Dguel Mghas, Takerboucht, Takermoust, Tanslit, Tanteboucht; Tati,
Tgaza, Thorry, Timjouhart, Tinasser, Tissibi and Zaghraya; the
cultivars originating from Arabia, such as the cultivars Ajwa and
Khalass; the cultivars originating from Spain, such as the cultivar
La Confitera; the cultivars originating from Iraq, such as the
cultivars Ashrasi, Badmi, Bahram, Barhi, Basra, Bdmalki, Benosh,
Halawi, Khadrawi, Khara Basra, Khastawi, Maktoom, Saylani and
Zaidi; the cultivars originating from Italy, such as the cultivars
Ebrea and Romana; the cultivars originating from Libya, such as the
cultivars Amreer, Aqdool, Awreeq, Khmag, Taghiyat and Talees; the
cultivars originating from Morocco, such as the cultivars Black Bou
Sthammi, Moroccan Bou Feggous, Bouskri and Medjoul; the cultivars
originating from Mauritania, such as the cultivars Ahmar, Edaghed
and Sekani; the cultivars originating from Oman, such as the
cultivars Bounaringa, Fard, Hilali and Naghal; the cultivars
originating from Pakistan, such as the cultivars Ali puri chohara,
Aseel, Begun, Dhakki, Halini, Hardasht, Hussaini, Karblain, Kozan
Abad, Mobai, Mozati, Nisri, Pashpag, Rukkan, Sajho Wali, Shakri,
Shamran and Wahn wali; the cultivars originating from Sudan, such
as the cultivars Barakawi, Bitamoda, Dogna and Gondaila; the
cultivars originating from Tunisia, such as the cultivars Algerian
Alig, Tunisian Alig, Aman, Angou (=Algerian Ghars), Arechti, Besser
Helou, Bessir, Bidh-Hmam, Tunisian Bou Feggous, Cheddakh, Deglet
Bey, Deglet Nour, Gasbi, Tunisian Ghars, Goundi, Tunisian Hmara,
Horra, Tunisian Kenta, Kentichi, Lagou and Tazerzist.
III--Kits for Identifying the Genotype of the Date Palm
[0225] The present invention also relates to kits comprising
material of use for implementing a method according to the
invention. In particular, the present invention relates to kits for
identifying the genotype of the date palm and/or identifying the
cultivar of the date palm, containing material which makes it
possible to detect, in the genome of the date palm, the
polymorphism of 19 microsatellite sequences according to the
invention or of 7 microsatellite sequences according to the
invention.
[0226] Generally, a kit according to the invention comprises at
least one set of pairs of SSR primers described herein enabling the
amplification of each of the (19 or 7) markers of a set of the
invention. A kit according to the invention can be designed so as
to be used with a particular amplification technique, in particular
a PCR technique.
[0227] In particular, in some embodiments of the invention, the kit
comprises the following pairs of primers: [0228] a forward SSR
primer of sequence SEQ ID NO: 1 and a reverse SSR primer of
sequence SEQ ID NO: 2; [0229] a forward SSR primer of sequence SEQ
ID NO: 3 and a reverse SSR primer of sequence SEQ ID NO: 4; [0230]
a forward SSR primer of sequence SEQ ID NO: 5 and a reverse SSR
primer of sequence SEQ ID NO: 6; [0231] a forward SSR primer of
sequence SEQ ID NO: 7 and a reverse SSR primer of sequence SEQ ID
NO: 8; [0232] a forward SSR primer of sequence SEQ ID NO: 9 and a
reverse SSR primer of sequence SEQ ID NO: 10; [0233] a forward SSR
primer of sequence SEQ ID NO: 11 and a reverse SSR primer of
sequence SEQ ID NO: 12; [0234] a forward SSR primer of sequence SEQ
ID NO: 13 and a reverse SSR primer of sequence SEQ ID NO: 14;
[0235] a forward SSR primer of sequence SEQ ID NO: 15 and a reverse
SSR primer of sequence SEQ ID NO: 16; [0236] a forward SSR primer
of sequence SEQ ID NO: 17 and a reverse SSR primer of sequence SEQ
ID NO: 18; [0237] a forward SSR primer of sequence SEQ ID NO: 19
and a reverse SSR primer of sequence SEQ ID NO: 20; [0238] a
forward SSR primer of sequence SEQ ID NO: 21 and a reverse primer
of sequence SEQ ID NO: 22; [0239] a forward SSR primer of sequence
SEQ ID NO: 23 and a reverse SSR primer of sequence SEQ ID NO: 24;
[0240] a forward SSR primer of sequence SEQ ID NO: 25 and a reverse
SSR primer of sequence SEQ ID NO: 26; [0241] a forward primer of
sequence SEQ ID NO: 27 and a reverse SSR primer of sequence SEQ ID
NO: 28; [0242] a forward SSR primer of sequence SEQ ID NO: 29 and a
reverse SSR primer of sequence SEQ ID NO: 30; [0243] a forward SSR
primer of sequence SEQ ID NO: 31 and a reverse SSR primer of
sequence SEQ ID NO: 32; [0244] a forward SSR primer of sequence SEQ
ID NO: 33 and a reverse SSR primer of sequence SEQ ID NO: 34;
[0245] a forward SSR primer of sequence SEQ ID NO: 35 and a reverse
SSR primer of sequence SEQ ID NO: 36; and [0246] a forward SSR
primer of sequence SEQ ID NO: 37 and a reverse SSR primer of
sequence SEQ ID NO: 38.
[0247] In other embodiments of the invention, the kit comprises the
following pairs of primers: [0248] a forward SSR primer of sequence
SEQ ID NO: 17 and a reverse SSR primer of sequence SEQ ID NO: 18;
[0249] a forward SSR primer of sequence SEQ ID NO: 21 and a reverse
SSR primer of sequence SEQ ID NO: 22; [0250] a forward SSR primer
of sequence SEQ ID NO: 25 and a reverse SSR primer of sequence SEQ
ID NO: 26; [0251] a forward SSR primer of sequence SEQ ID NO: 29
and a reverse SSR primer of sequence SEQ ID NO: 30; [0252] a
forward SSR primer of sequence SEQ ID NO: 33 and a reverse SSR
primer of sequence SEQ ID NO: 34; [0253] a forward SSR primer of
sequence SEQ ID NO: 35 and a reverse SSR primer of sequence SEQ ID
NO: 36; and [0254] a forward SSR primer of sequence SEQ ID NO: 37
and a reverse SSR primer of sequence SEQ ID NO: 38.
[0255] It is understood that a kit may comprise the two sets of
pairs of primers.
[0256] A kit according to the invention may also comprise reagents
or solutions for extracting the genomic DNA from the date palm
sample, PCR amplification reagents or solutions, reagents or
solutions for separating amplicons according to their size,
sequencing reagents or solutions, and/or detection means. Protocols
for using these reagents and/or solutions may be included in the
kit.
[0257] The various components of the kit may be provided in solid
form (for example in lyophilized form) or in liquid form. A kit may
optionally comprise a container containing each of the reagents or
solutions, and/or containers for carrying out certain steps of the
method of the invention.
[0258] A kit according to the invention may also comprise
instructions for carrying out the method of the invention for
detecting, in the date palm genome, the SSR polymorphism of each of
the 19 or of the 7 markers of a set according to the invention. The
instructions for carrying out a method according to the invention
may comprise instructions for the extraction of genomic DNA from
date palm samples, instructions regarding the PCR amplification
conditions, instructions regarding the analysis of the amplicons
obtained and/or instructions for interpreting the results.
[0259] A kit according to the invention may also comprise an
information sheet in the form stipulated by a governmental agency
regulating the preparation, sale and use of biological
products.
[0260] Unless otherwise defined, all the technical and scientific
terms used herein have the same meaning as that commonly understood
by an ordinary specialist in the field to which this invention
belongs. Likewise, all the publications and patent applications,
all the patents and any other references mentioned herein are
incorporated by way of reference.
Example
[0261] The following example describes some embodiments of the
present invention. However, it is understood that the example is
provided only by way of illustration and does not in any way limit
the scope of the invention.
Identification of the Sets of Molecular Markers of Microsatellite
Type
Materials and Methods
[0262] Plant Material.
[0263] A collection of 550 Phoenix dactylifera cultivars collected
in Tunisia, Algeria, Morocco, Pakistan, Egypt and Oman was
assembled. Samples of P. canariensis, P. loureiroi, P. reclinata
and P. roebelenii were also collected. In this sample, a
subcollection of 100 reference varieties were selected on the basis
of their significance in the principal Phoenix-growing
countries.
[0264] DNA Extraction.
[0265] Each sample consisting of leaves was lyophilized for 72
hours by means of an Alpha1-4LD Plus lyophilizer (Fisher
Scientific, France). The lyophilized leaves were ground with the
TissueLyser System (Qiagen, USA) and then the extraction was
carried out with the Dneasy plant kit (Qiagen, USA) according to
the maker's protocol.
[0266] The DNA obtained was assayed with the Tecan GENios.TM.
spectrofluorimeter (Tecan, Switzerland). The concentrations of all
the samples were adjusted to 10 ng/.mu.l for the rest of the
manipulation.
[0267] Genetic Analyses.
[0268] The genetic analyses (number of alleles, heterozygosity
observed and expected, Fis, Wright fixation index) were carried out
using the Genetix 4.05 software (Belkhir et al., 2004, Genetix
4.05, software on Windows.TM. for population genetics. Genome,
Populations, Interactions Laboratory, CNRS UMR 5171, University of
Montpellier II, Montpellier, France).
Description of the Microsatellite Markers
[0269] A new optimized collection of microsatellites was defined by
the exploitation of microsatellite markers described in the
literature or identified from the available genomic resources.
[0270] The mPdCIR078, mPdCIR015, mPdCIR016, mPdCIR032, mPdCIR035,
mPdCIR057, mPdCIR085, mPdCIR010, mPdCIR025 and mPdCIR063 markers
are (GA)n dinucleotide loci, which have been described by Billotte
et al. (Molecular Ecology Notes, 2004, 4: 256-258).
[0271] The mPdIRD031, mPdIRD033, mPdIRD040 and mPdIRD013 markers
are microsatellite tandem repeats of 3 or 6 base pairs that the
present inventors identified in silico in coding sequences of the
sequence of the genome of the date palm (Al-Dous et al., Nature
Biotechnology, 2011, 29:
521-527--http://qatar-weill.cornell.edu/research/datepalmGenome/download.-
html). These 4 loci were selected among 47 for their amplification
capacity and the polymorphism that they generate.
[0272] The PdCUC3-ssr1CUC and PdCUC3-ssr2 markers are dinucleotide
sequences identified in CUC genes (Adam et al., Molecular Biology
and Evolution, 2010, 28: 1439-1454).
[0273] The PdAG1-ssr1 and PdAP3-ssr1 markers are dinucleotide
sequences identified by the present inventors respectively in the
AG1 and AP3 genes (not published).
[0274] The CpfM12 marker is a minisatellite marker of the
chloroplast genome (Henderson et al., Conservation Genetics, 2006,
7: 213-223).
[0275] On the basis of these markers, the inventors identified 2
sets according to the objective pursued: a set of 19 markers which
makes it possible to identify 100% of the genotypes, and a reduced
set of 7 loci for cultivar certification. For the latter, an
identification key was defined which makes it possible to identify
99% of the genotypes (FIG. 1).
Sequence CWU 1
1
38117DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 1accccggacg tgaggtg 17222DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
2cgtcgatctc ctcctttgtc tc 22320DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 3agctggctcc tcccttctta
20419DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 4gctcggttgg acttgttct 19520DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
5agcgggaaat gaaaaggtat 20620DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 6atgaaaacgt gccaaatgtc
20719DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 7gcacgagaag gcttatagt 19819DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
8cccctcatta ggattctac 19917DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 9caaatctttg ccgtgag
171023DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 10ggtgtggagt aatcatgtag tag 231120DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
11acaaacggcg atgggattac 201220DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 12ccgcagctca cctcttctat
201320DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 13aagcagcagc ccttccgtag 201422DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
14gttctcactc gcccaaaaat ac 221520DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 15cttttatgtg gtctgagaga
201620DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 16tctctgatct tgggttctgt 201717DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
17tggatttcca ttgtgag 171817DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 18cccgaagaga cgctatt
171919DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 19gagagagggt ggtgttatt 192019DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
20ttcatccaga accacagta 192122DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 21ccgcccacga tgaagtaatg ta
222223DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 22gtcacgggtt caaatcctgt ctc 232324DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
23cgtggactca tgactcgcat gtcc 242420DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
24ggtccttgcc ggtggccttc 202523DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 25acattgctct tttgccatgg gct
232620DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 26cgagcaggtg gggttcgggt 202724DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
27tctgatttcg tttacttctt agga 242823DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
28ttcatattca gttgtcgggt gta 232922DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 29gagaaataga gagctgtgca ag
223021DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 30ctgcagtact cggagaactt g 213120DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
31gcggagacag gagatggtaa 203220DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 32cttgactgct tctgctgctg
203320DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 33gcaggtggac tgcaaaatct 203420DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
34ctattggggt gctgatccat 203520DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 35ggagcataca gtgggtttgc
203620DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 36cagcctggga atgaggatag 203720DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
37gagagatgcg tcagggaatc 203820DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 38ccagaatctt ccaagcaagc 20
* * * * *
References