Development of simple sequence repeat (ssr) core primer group based on whole genome sequence of pomegranate and application thereof

Abstract

The development of a simple sequence repeat (SSR) core primer group based on the whole genome sequence of pomegranate and the applications thereof are disclosed. The primer group includes 11 primer pairs: PG080, PG130, PG139, PG152, PG153, PG140, PG098, PG070, PG077, PG090, and PG093. The SSR core primer group of the present disclosure has the advantages such as high polymorphism, good repeatability, stable amplification, and clear and easy to score bands, and is applicable to the fields of pomegranate variety identification, DNA fingerprinting construction, genetic diversity assessment and phylogenetic study, and the like, providing a new tool for pomegranate molecular marker-assisted selection and having an excellent application prospect.

What is claimed is: 
     
         1 . A Simple Sequence Repeat (SSR) core primer group based on a whole genome sequence of pomegranate, the SSR core primer group comprising:
 11 primer pairs comprising PG080, PG130, PG139, PG152, PG153, PG140, PG098, PG070, PG077, PG090, and PG093;   wherein nucleotide sequences of each of the 11 primer pairs are sequentially shown as follows:   
       
         
           
                 
                 
                 
                 
               
                     
                 
                     
                   Primer 
                   Forward primer 
                   Reverse primer 
                 
                     
                 
                     
                   PG080 
                   ctgactgttgcaga 
                   aggaggtgaaacaa 
                 
                     
                     
                   gagtaggctg 
                   cgaatagctg 
                 
                     
                 
                     
                   PG130 
                   ctcatatggcgatt 
                   aagttcgataaatt 
                 
                     
                     
                   ctctgtcctt 
                   gcactggtgg 
                 
                     
                 
                     
                   PG139 
                   gtttccttccctca 
                   agtgggattttacc 
                 
                     
                     
                   acccaaaa 
                   aagtcgaaca 
                 
                     
                 
                     
                   PG152 
                   catcagaatcgtcc 
                   cagagagaagaaga 
                 
                     
                     
                   ccttgtg 
                   gagaccgagc 
                 
                     
                 
                     
                   PG153 
                   gtgtttgatgctcc 
                   gccttcaacggtct 
                 
                     
                     
                   catttcattt 
                   ttcttcttct 
                 
                     
                 
                     
                   PG140 
                   caagaaagtgtgtg 
                   ccaaaccttacccc 
                 
                     
                     
                   agcgattgat 
                   tctctctctc 
                 
                     
                 
                     
                   PG098 
                   tgccttcttaagga 
                   ctaacctcatgcac 
                 
                     
                     
                   cttcaccaac 
                   ttgtcatcca 
                 
                     
                 
                     
                   PG070 
                   cacctctgcttcag 
                   caactcaacacaat 
                 
                     
                     
                   caaacaaata 
                   atccaaccca 
                 
                     
                 
                     
                   PG077 
                   gtcagtctcctcct 
                   agacgaagcacctg 
                 
                     
                     
                   tcttcaatgg 
                   agaaggaat 
                 
                     
                 
                     
                   PG090 
                   attcttttatacta 
                   atgtcatgagagga 
                 
                     
                     
                   accaaaatttgcga 
                   cccacaaa 
                 
                     
                 
                     
                   PG093 
                   cgtcaataggacgt 
                   gatgacgtggcaga 
                 
                     
                     
                   ccctgagata 
                   gtaagagagc 
                 
                     
                 
             
                
                
                
               
               
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         2 . The SSR core primer group of  claim 1 , wherein the 11 primer pairs are used for pomegranate variety identification. 
     
     
         3 . The SSR core primer group of  claim 2 , wherein:
 the 11 primer pairs are carried out a capillary electrophoresis with fluorescence detection, according to results of amplification loci detection by the capillary electrophoresis;   when a number of differential loci between two varieties is greater than or equal to 3, the two varieties are determined to be different varieties; and   when the number of differential loci between the two varieties is less than 3, the two varieties are determined to be substantially similar or the same variety.   
     
     
         4 . The SSR core primer group of  claim 1 , wherein the 11 primer pairs are used for pomegranate DNA fingerprinting construction. 
     
     
         5 . The SSR core primer group of  claim 1 , wherein the 11 primer pairs are used for pomegranate genetic diversity assessment and phylogenetic study. 
     
     
         6 . A method for developing a Simple Sequence Repeat (SSR) core primer group based on a whole genome sequence of pomegranate, the method comprising:
 (1) extracting genomic deoxyribonucleic acid (DNA) of pomegranate, wherein a hexadecyltrimethylammonium bromide (CTAB) method is utilized to extract DNA, the extracted DNA is added with 50 μL of 0.1M Tris-EDTA (TE) buffer for dissolving overnight and then stored at −20° C. until use;   (2) determining SSR loci with different repeat units within a range of the whole genome, wherein the SSR search criteria are 11 repeat units for dinucleotide repeats, 8 repeat units for trinucleotide repeats, 6 repeat units for tetranucleotide repeats, 5 repeat units for pentanucleotide repeats, and 4 repeat units for hexanucleotide repeats;   (3) designing 45 SSR primer pairs, wherein from the obtained SSR loci, 5 SSR loci are randomly selected on each chromosome, the SSR primers are designed by Primer 3.0 using flanking sequences of SSRs, parameters for designing the SSR primers include:
 a length of polymerase chain reaction (PCR) products is in a range of 100-350 bp; 
 a melting temperature (Tm) is between 50-70° C., ensuing a difference in Tm value between two SSR primers does not exceed 4° C.; 
 a guanine-cytosine (GC) % content is between 40-65%; 
 a length of the SSR primers is in a range of 18-28 bp; 
 wherein the conserved flanking sequences and the SSR loci used for the SSR primer design are at least 20-23 bases apart to ensure the specificity of the SSR primers; 
   (4) screening the 45 SSR primer pairs to select 11 primer pairs with stable amplification, clear bands, and high polymorphism, wherein:   PCR amplification is conducted in 20 μL of reaction mixture containing 1.0 ng of DNA, 0.4 μM of forward primers, 0.4 μM of reverse primers, 4 mM of MgCl 2 , 400 μM of dNTPs, 1.0 U of Taq-DNA polymerase, and ddH 2 O to the total volume of 20 μL;   touchdown PCR is carried out under the following conditions:
 5 minutes at 95° C.; 
 11 cycles, with a decrease of 0.8° C. in the melting temperature after each cycle {30 s at 95° C.; 30 s at 65° C.; 50 s at 72° C.}; 
 22 cycles {30 s at 95° C.; 30 s at 55° C.; 50 s at 72° C.}; and 
 a final extension of 8 minutes at 72° C.; 
   fragment sizes of the PCR products are determined by capillary electrophoresis.