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.

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′→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′→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′→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′→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′→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′→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′→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′→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′→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′→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′→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′→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′→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′→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′→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′→3′ direction, has a (cac) motif, is present in the PDK — 20s1496731g002 — 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′→3′ direction, has a (cca) motif, is present in the PDK — 20s1419261g003 — 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′→3′ direction, has a (cag) motif, is present in the PDK — 20s1569281g001 — 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′→3′ direction, has a (ccagtg) motif, is present in the PDK — 20s1327401g002 — 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.