Abstract:
A specific region of the maize mitochondrial genome has been found to have unique sequence in each of four different cytotypes: NA and NB (male fertile), S and C (male sterile). Specific primers allow PCR reactions to amplify, identify and measure each unique sequence. Using the method, said lots can be analyzed for contaminating cytotypes and unidentified cultivars can be typed.

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     This application claims priority from U.S. Provisional application No. 60/128,511 filed Apr. 9, 1999. 
    
    
     BACKGROUND OF THE INVENTION 
     Contamination of maize male-sterile seeds by male-fertile seeds can significantly reduce crop yields. Currently, such contamination can only be detected at the time of pollen maturation. It is then necessary to manually emasculate the maize plants resulting from this contamination. The process must be done within a short and specific time frame, within one week after pollen formation. The task is very expensive and labor-intensive. A typical cost ranges from 3,000 to 4,000 FF (french francs=US$600 to $800) per hectare. In France 60,000 hectares are used to provide sufficient maize seed for planting 3,000.000 hectares of maize production. The cost of production and land area required in the U.S. are proportionate. The price of hybrid seeds is about 20,000 FF (US$4,000) per hectare. This invention&#39;s practical application includes the use of a kit that will allow the PCR amplification of cytotype-specific mtDNA sequences. 
     SUMMARY OF THE INVENTION 
     One aspect of the invention concerns a polymerase chain reaction (PCR) method that allows one to distinguish and identify without any ambiguity, various male-fertile and male-sterile maize plants. This identification is based on the knowledge of sequences specific to the mitochondrial genome (mtDNA) of the various maize cytotypes. The method can be used to quantify contamination between the various cultivars. The invention also includes the specific sequences of maize mtDNA that have been found to be cytotype-specific. 
     A specific region in the maize mitochondrial genome has been shown to be rearranged through recombination in four different maize cytotypes: NA and NB (fertiles), S and C (steriles). PCR reactions allow the amplification of DNA fragments of different sizes which are specific to each cytotype. Unidentified cultivars can therefore be identified, and cross-contamination between cultivars quantified. 
     This invention can resolve two problems: 
     1) It will identify a maize cultivar cytoplasm whose source is questionable and may be subject to an ownership dispute. 
     2) It will identify and quantify contamination of fertile seeds within a set of sterile seeds. The quantification of contamination will allow one to estimate the eventual resulting decrease in crop yield so as to better control yields. Knowing how much contamination of fertile seeds is present within a set of male-sterile seeds is very important for seed companies since the beneficial effects of heterosis would be lost resulting in a significant decrease of crop yield. Furthermore certification and commercialization of maize seeds must meet specified quality control standards. 
     In general, the invention provides novel sequences of mtDNA which are specific for maize cytotypes NA, NB, S and C. Each of the disclosed mtDNA sequences contains one or more subsequences which are unique for the designated cytotype. A method is provided using PCR amplification of the cytotype-specific subsequences to identify the cytotype of a given sample of maize tissue, seed or flour. Further, the method makes it possible to identify the cytotype composition of a sample of mixed cytotypes, for example in seeds or flour. Certain primer sequences for amplifying cytotype specific subsequences are exemplified herein. One skilled in the art will recognize that other primers can be chosen, based upon the disclosed mtDNA sequences, and the detailed conditions for DNA extraction, amplification, and analysis can all be varied within parameters known to those skilled in the art, as may be desired or necessary, depending on the particular needs of those using the method. All such variations will be understood as falling within the scope of the invention. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1 is a diagram showing the relative location of primers employed for PCR identification of maize cytotypes NA, NB, S and C. Sequences of the designated primers is given in Table 1. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Total DNA Extraction (Using a Sample of 1,000 Kernels) 
     From Maize Seeds 
     Maize kernels were allowed to germinate in the dark at 25° C. for 5 to 7 days. The etiolated small plants were then collected and homogenized in a Waring Blendor with the extraction buffer [CH 3 COONa (sodium acetate) 100 mM pH=4.8; EDTA (disodium ethylenediamine tetra-acetate) 50 mM pH=8; NaCl 500 mM; PVP (Polyvinylpyrrolidone PM 10000) 2%; Cystein 60 mM]. SDS (sodium dodecyl sulfate) was added to the homogenate to a final concentration of 1%. Denaturation was allowed for 10 mn at 65° C. and the lysate centrifuged at 10,000 g for 10 min at room temperature. The proteins were precipitated in presence of ⅓ volume of potassium γM and the mixture incubated for 20 mn at 0° C. Precipitated proteins were eliminated by centrifugation at 10,000 g for 10 mn at 4° C. A DEAE-cellulose (Diethylaminoethyl-cellulose) suspension (¼ V) was added to the DNA-containing supernatant. DEAE-cellulose (DE 52 Whatman) was prepared in the elution buffer (NaCl 2M; Tris-HCl 10 mM pH=7.5; EDTA 1 mM) then resuspended in the wash buffer (NaCl 400 mM; Tris-HCl 10 mM pH=7.5; EDTA 1 mM). The mixture was decanted, the supernatant poured off gently and the matrix washed twice with the wash buffer to eliminate proteins, polysaccharides and metabolites not bound to the matrix. The nucleic acids were eluted with the elution buffer and precipitated with 2.5 volumes of absolute ethanol and 0.5 volume of ammonium acetate 7.5M for 30 mn at −20° C. The precipitate was centrifuged for 15 mn at 15,000 g at 4° C. and resuspended in water or TE (Tris-EDTA) 0.1 mM. RNA was eliminated with RNAse A: 50 μl (10 mg/ml) in 100 μl of DNA mixture for 30 mn at 37° C. DNA was isolated by another phenol/chloroform extraction and ethanol precipitation. The DNA pellet was resuspended in 500 μl water or TE (Tris-EDTA) 0.1 mM and quantified by readings at OD260 and agarose gel electrophoresis. 
     From Maize Flour 
     We offer an alternative method of DNA extraction in order to overcome two possible technical problems concerning space and time: to eliminate the need of space to germinate the seeds and to eliminate the five days needed to grow the seedlings. 
     The 1,000 maize seeds were ground in a mill in order to obtain a fine flour. The extraction buffer was made of Tris-HCl 200 mM pH=8.0; EDTA 70 mM; NaCl 2M. 0.5 g. of sodium bisulfite was added to 100 ml of buffer just before use. After adding SDS at 1% final concentration, the mixture was gently homogenized and incubated at 65° C. for one hour (gently vortex every 10 min). The homogenate was centrifuged at 16,000 g for 15 min at 4° C. The supernatant was used for DNA precipitation with 1 volume isopropanol. The pellet was dried out in the speedvac and resuspended in 500 μl water and mixed with an equal volume of phenol/chloroform/isoamyl alcohol (10/10/1 v/v). After a 10 min centrifugation at 13,000 g at room temperature, the upper layer was saved and 500 μl of chloroform is added; the mixture was vortexed and centrifuged for 10 mn at 13,000 g at room temperature. The upper phase was once more saved for DNA precipitation using {fraction (1/10)} volume sodium acetate 3M and 1 volume of isopropanol. After 15 mn at 4° C. followed by a 15 mn centrifugation at 13,000 at 4° C., the DNA pellet was washed with 70% ethanol and dried. This final DNA pellet was resuspended into 100 μl of nanopure water and the OD was taken at 260 nm. 
     The Amplification of the Various Maize Cytotypes mtDNA Sequences 
     Plasmid Clones Containing the Sequences Specific to Each Cytoplasm 
     The DNA sequences from four clones containing mtDNA BamH1 fragments derived from the four maize cytotypes: NA, NB, C, S have been determined. Comparison of these sequences show that each DNA fragment contains a common sequence of variable length followed by a sequence unique to each cytotype (see below). 
     SEQ ID NO:1=Maize sterile cytotype S mtDNA 
     SEQ ID NO:2=Maize sterile cytotype C mtDNA 
     SEQ ID NO:3=Maize fertile cytotype NA mtDNA 
     SEQ ID NO:4=Maize fertile cytotype NB mtDNA 
     
       
         
               
               
             
               
               
               
             
           
               
                 The sequences 
                   
               
             
          
           
               
                   
                 1                                                   50 
                   
               
               
                 S 
                 CGACTCACTA TAGGGAATTC GAGCTCGGTA CCCGGGGATC CCCCCCGCAT 
               
               
                 C 
                 CGACTCACTA TAGGGAATTC GAGCTCGGTA CCCGGGGATC CCCCCCGCAT 
               
               
                 NA 
                 CGACTCACTA TAGGGAATTC GAGCTCGGTA CCCGGGGATC CCCCCCGCAT 
               
               
                 NB 
                 CGACTCACTA TAGGGAATTC GAGCTCGGTA CCCGGGGATC CCCCCCGCAT 
               
               
                   
               
               
                   
                 51                                                 100 
               
               
                 S 
                 TCTGATCAGG TTCGTTACCC ATTCCGCTGT GGTCATCAAA GAAGAATAAT 
               
               
                 C 
                 TCTGATCAGG TTCGTTACCC ATTCCGCTGT GGTCATCAAA GAAGAATAAT 
               
               
                 NA 
                 TCTGATCAGG TTCGTTACCC ATTCCGCTGT GGTCATCAAA GAAGAATAAT 
               
               
                 NB 
                 TCTGATCAGG TTCGTTACCC ATTCCGCTGT GGTCATCAAA GAAGAATAAT 
               
               
                   
               
               
                   
                 101                                                150 
               
               
                 S 
                 AAAAAAGTGC TGTTGTTACA CTCAACAGAG TGAAAACGCG AGCAGCCCAA 
               
               
                 C 
                 AAAAAAGTGC TGTTGTTACA CTCAACAGAG TGAAAACGCG AGCAGCCCAA 
               
               
                 NA 
                 AAAAAAGTGC TGTTGTTACA CTCAACAGAG TGAAAACGCG AGCAGCCCAA 
               
               
                 NB 
                 AAAAAAGTGC TGTTGTTACA CTCAACAGAG TGAAAACGCG AGCAGCCCAA 
               
               
                   
               
               
                   
                 151                                                200 
               
               
                 S 
                 ACTGGGCAAC GCGGCCATAA AGCGCGAACC AACATCCGTG ATACGAAAAC 
               
               
                 C 
                 ACTGGGCAAC GCGGCCATAA AGCGCGAACC AA6ATCCGTG ATACGAAAAC 
               
               
                 NA 
                 ACTGGGCAAC GCGGCCATAA AGCGCGAACC AACATCCGTG ATACGAAAAC 
               
               
                 NB 
                 ACTGGGCAAC GCGGCCATAA AGCGCGAACC AACATCCGTG ATACGAAAAC 
               
               
                   
               
               
                   
                 201                                                250 
               
               
                 S 
                 CAAAATCAGA ATGAGGAAGA AAAGGATATC TTTAGGTAAG TCATGAAAAT 
               
               
                 C 
                 CAAAATCAGA ATGAGGAAGA AAAGGATATC TTTAGGTAAG TCATGAAAAT 
               
               
                 NA 
                 CAAAATCAGA ATGAGGAAGA AAAGGATATC TTTAGGTAAG TCATGAAAAT 
               
               
                 NB 
                 CAAAATCAGA ATGAGGAAGA AAAGGATATC TTTAGGTAAG TCATGAAAAT 
               
               
                   
               
               
                   
                 251                                                300 
               
               
                 S 
                 GAAAACCCAA TAAGAACTTA CCCAATAATT TAGAACAGAT CACTTCCAAA 
               
               
                 C 
                 GAAAACCCAA TAAGAACTTA CCCAATAATT TAGAACAGAT CACTTCCAAA 
               
               
                 NA 
                 GAAAACCCAA TAAGAACTTA CCCAATAATT TAGAACAGAT CACTTCCAAA 
               
               
                 NB 
                 GAAAACCCAA TAAGAACTTA CCCAATAATT TAGAACAGAT CACTTCCAAA 
               
               
                   
               
               
                   
                 301                                                350 
               
               
                 S 
                 CGAGACATGA TGAGCTAAAG TCAAAATAAA ATGCAAGAGA AATTCAAATT 
               
               
                 C 
                 CGAGACATGA TGAGCTAAAG TCAAAATAAA ATGCAAGAGA AATTCAAATT 
               
               
                 NA 
                 CGAGACATGA TGAGCTAAAG TCAAAATAAA ATGCAAGAGA AATTCAAATT 
               
               
                 NB 
                 CGAGACATGA TGAGCTAAAG TCAAAATAAA ATGCAAGAGA AATTCAAATT 
               
               
                   
               
               
                   
                 351                                                400 
               
               
                 S 
                 ATGTAAGGCT TAGAAAAAAG TCCTGCGTAG AGTCCACGGG GAGCCTTTTT 
               
               
                 C 
                 ATGTAAGGCT TAGAAAAAAG TCCTGCGTAG AGTCCACGGG GAGCCTTTTT 
               
               
                 NA 
                 ATGTAAGGCT TAGAAAAAAG TCCTGCGTAG AGTCCACGGG GAGCCTTTTT 
               
               
                 NB 
                 ATGTAAGGCT TAGAAAAAAG TCCTGCGTAG AGTCCACGGG GAGCCTTTTT 
               
               
                   
               
               
                   
                 401                                                450 
               
               
                 S 
                 CGATTTGATC TCTAGTCAAA CTAAGACTGG AGCTTGAAGC CACTACTCTA 
               
               
                 C 
                 CGATTTGATC TCTAGTCAAA CTAAGACTGG AGCTTGAAGC CACTACTCTA 
               
               
                 NA 
                 CGATTTGATC TCTAGTCAAA CTAAGACTGG AGCTTGAAGC CACTACTCTA 
               
               
                 NB 
                 CGATTTGATC TCTAGTCAAA CTAAGACTGG AGCTTGAAGC CACTACTCTA 
               
               
                   
               
               
                   
                 451                                                500 
               
               
                 S 
                 GAACCGGAAG AAGCCTAACT TCTGCATTTT GAGTAGATTT TCTGACTATC 
               
               
                 C 
                 GAACCGGAAG AAGCCTAACT TCTGCATTTT GAGTAGATTT TCTGACTATC 
               
               
                 NA 
                 GAACCGGAAG AAGCCTAACT TCTGCATTTT GAGTAGATTT TCTGACTATC 
               
               
                 NB 
                 GAACCGGAAG AAGCCTAACT TCTGCATTTT GAGTAGATTT TCTGACTATC 
               
               
                   
               
               
                   
                 501                        * common oligo direct   550 
               
               
                 S 
                 ATTTGCCTTG AATTACAATA CGATA ATCTT CGATTGGTAG AAGTGAGTCC   
               
               
                 C 
                 ATTTGCCTTG AATTACAATA CGATA ATCTT CGATTGGTAG AAGTGAGTCC   
               
               
                 NA 
                 ATTTGCCTTG AATTACAATA CGATA ATCTT CGATTGGTAG AAGTGAGTCC   
               
               
                 NB 
                 ATTTGCCTTG AATTACAATA CGATA ATCTT CGATTGGTAG AAGTGAGTCC   
               
               
                   
               
               
                   
                 551*                                               600 
               
               
                 S 
                   G GATCTCCCT CTTTTCCACT AAACCCGCCA CTCCTACTTG TACTTTATTT 
               
               
                 C 
                   G GATCTCCCT CTTTTCCACT AAACCCGCCA CTCCTACTTG TACTTTATTT 
               
               
                 NA 
                   G GATCTCCCT CTTTTCCACT AAACCCGCCA CTCCTACTTG TACTTTATTT 
               
               
                 NB 
                   G GATCTCCCT CTTTTCCACT AAACCCGCCA CTCCTACTTG TACTTTATTT 
               
               
                   
               
               
                   
                 601                                                650 
               
               
                 S 
                 TCCACCTGGC TAGCTTCAAT TGCTTAGTAG GGAGTTTCCA TCCTTGACTT 
               
               
                 C 
                 TCCACCTGGC TAGCTTCAAT TGCTTAGTAG GGAGTTTCCA TCCTTGACTT 
               
               
                 NA 
                 TCCACCTGGC TAGCTTCAAT TGCTTAGTAG GGAGTTTCCA TCCTTGACTT 
               
               
                 NB 
                 TCCACCTGGC TAGCTTCAAT TGCTTAGTAG GGAGTTTCCA TCCTTGACTT 
               
               
                   
               
               
                   
                 651                                                700 
               
               
                 S 
                 TCGCGGTTCT TTCTTTGCTT TCTTCAGTTG TGTTGAGGAG CTATCTTTTG 
               
               
                 C 
                 TCGCGGTTCT TTCTTTGCTT TCTTCAGTTG TGTTGAGGAG CTATCTTTTG 
               
               
                 NA 
                 TCGCGGTTCT TTCTTTGCTT TCTTCAGTTG TGTTGAGGAG CTATCTTTTG 
               
               
                 NB 
                 TCGCGGTTCT TTCTTTGCTT TCTTCAGTTG TGTTGAGGAG CTATCTTTTG 
               
               
                   
               
               
                   
                 701                                                750 
               
               
                 S 
                 AACCCAACTA ACTCTTAAAT CCGAACTTTC CCTTGCTTAC GCCCCTAGAA 
               
               
                 C 
                 AACCCAACTA ACTCTTAAAT CCGAACTTTC CCTGCAGCTT TAGGGCTTCT 
               
               
                 NA 
                 AACCCAACTA ACTCTTAAAT CCGAACTTTC CCTGCAGCTT TAGGGCTTCT 
               
               
                 NB 
                 AACCCAACTA ACTCTTAAAT CCGAACTTTC CCTGCAGCTT TAGGGCTTCT 
               
               
                   
                                End of common for S * 
               
               
                   
               
               
                   
                 751                                                800 
               
               
                 S 
                 ACTCCGTGAA AAAACCACCT CGTTTTTCCA TAAATAAAGG ATTCCTAATA 
               
               
                 C 
                 AGTGAGGAGA GGTGATCTCT AGTTTCTCAT TCCGATAGCC ATCTCGATTA 
               
               
                 NA 
                 AGTGAGGAGA GGTGATCTCT AGTTTCTCAT TCCGATAGCC ATCTCGATTA 
               
               
                 NB 
                 AGTGAGGAGA GGTGATCTCT AGTTTCTCAT TCCGATAGCC ATCTCGATTA 
               
               
                   
               
               
                   
                 801                                                850 
               
               
                 S 
                 AAGTTTTTAC ATATCATATC CCAGCGGTAT TTTTATAATA ATTTTTGCAT 
               
               
                 C 
                 ACTGAGTTGA CTTGTGAGCT CCCCTAACCC GGCAGGCAGG CCAAAGAATG 
               
               
                 NA 
                 ACTGAGTTGA CTTGTGAGCT CCCCTAACCC GGCAGGCAGG CCAAAGAATG 
               
               
                 NB 
                 ACTGAGTTGA CTTGTGAGCT CCCCTAACCC GGCAGGCAGG CCAAAGAATG 
               
               
                   
               
               
                   
                 851                                                900 
               
               
                 S 
                 AAATAAAGTA TAATTTCCAC AAAATAACAC TTTTAGGCGC CCACTTGAAA 
               
               
                 C 
                 AAGGGCAGTG AAAATGGTTT CTTCTTCTGG CTAATTCCGA TACGAATACC 
               
               
                 NA 
                 AAGGGCAGTG AAAATGGTTT CTTCTTCTGG CTAATTCCGA TACGAATACC 
               
               
                 NB 
                 AAGGGCAGTG AAAATGGTTT CTTCTTCTGG CTAATTCCGA TACGAATACC 
               
               
                   
               
               
                   
                   * oligo S reverse         *                      950 
               
               
                 S 
                 TT CAACTTAA CGTTGGACCT TGACCAG GCC CTCTCTATTT CCAGAATT 
               
               
                 C 
                 AAAAACAGCT TACTTCCGTT CGTGTCCTCG GAAATTGCAA TTCATTCACA 
               
               
                 NA 
                 AAAAACAGCT TACTTCCGTT CGTGTCCTCG GAAATTGGAT TACTTATGAG 
               
               
                 NB 
                 AAAAACAGCT TACTTCCGTT CGTGTCCTCG GAAATTGGAT TACTTATGAG 
               
               
                   
                                       End of common for C * 
               
               
                   
               
               
                   
                 951                   *  oligo c reverse         *1000 
               
               
                 C 
                 TCTGCTCCTA TCTATTCTAT  GCTTGCTTAC CAATCGGGAC TTGCAA TTCC 
               
               
                 NA 
                 TTTCTTCGGT GCAAAAGTAG GCAAGTCCAT TTTCTCGTGT TTTTCGTCCT 
               
               
                 NB 
                 TTTCTTCGGT GCAAAAGTAG GCAAGTCCAT TTTATCGTGT TTTTCGTCCT 
               
               
                   
               
               
                   
                 1001                                              1050 
               
               
                 C 
                 TATCACCCGC CGGAGGCAGA TTACTTAATC TGAAAGCGTT ATTTCACATT 
               
               
                 NA 
                 CAAGACCTGA TTTTCCAAAG AGCATTTCAT ATAGCTGCAA AAGTTGCTCA 
               
               
                 NB 
                 CAAGACCTGA TTTTCCAAAG AAAATAGCCT GCCAAAGAGC TAGCTATAGA 
               
               
                   
                                       * end of common between NA and NB 
               
               
                   
               
               
                   
                 1051                                              1100 
               
               
                 C 
                 CGTTATTTCA TATCATATGA ATTAGGCGGA TCATACACCG ATCTCAGAAC 
               
               
                 NA 
                 TCCGATTTTC CCTCGCTTTT ATTCGTAACA TGGCGTAGTT ACTCATTGGG 
               
               
                 NB 
                 AATACAGTCA GTCGGGGTAA ACTGAAAGAA GGAAATCTAC ATTCATAGAT 
               
               
                   
               
               
                   
                 1101                   * oligo NA reverse        *1150 
               
               
                 C 
                 TAATACGCTT TCGCTTTCTC CTGACCCTCA ATCCAAAAGA AAAGGTTTGG 
               
               
                 NA 
                 TCCGCTGAGA AGAAAACAAT A GGTCGTCAC TACCTATGCT TTCATC TAGA 
               
               
                 NB 
                 AGTAGGGCAA TAGGCTCACT TACATAGTGG GGACAGCAAT AGGCTCACTT 
               
               
                   
               
               
                   
                 1151                                              1200 
               
               
                 C 
                 CGTTGCAGTT CATATGCTTC CTTGCGCTAG AAACCAATAC GCACGTACGA 
               
               
                 NA 
                 GGAAGTCAGG CTGTGATTCG TAACATGGCG TAGTTACTCA CAGGCCGTGG 
               
               
                 NB 
                 ACTTTTGAAA TAAGAGGGTC GGTCAACTCT CGGCAGATAG GGTAGTTGCT 
               
               
                   
               
               
                   
                 1201                                              1250 
               
               
                 C 
                 GGCCTACGAT TCTCTAGTTG TATTGGCAAG GGAAAGAAGA TGCTATGGGA 
               
               
                 NA 
                 ATCTCCTTTT TCTAATCAAG CAGCAGGCCG TGGGTTTTCT GGTTCTGCGC 
               
               
                 NB 
                 AAATAAAAGC AAAGAAAGAG TGAGTTGAAA GAAGAAATGT ATGTGGGTCA 
               
               
                   
               
               
                   
                 1251                                              1300 
               
               
                 C 
                 ATGTTTGGAA TGAAA 
               
               
                 NA 
                 TTCAGAAGAG GAAAGTTTCC GTGATTCGTA TCTGCTTTGA GGGCAGGTC 
               
               
                 NB 
                 CCATATAATA TATATAATAG TACCTAACTT ACTAGGTATC TCGGTCAACT 
               
               
                   
               
               
                   
                 1301                                              1350 
               
               
                 NB 
                 GCTAGCTTTT CATCCTTGGT GCAGTATTTC CACCTTCTAT TGCTTTGCTT 
               
               
                   
               
               
                   
                 1351                      * oligo NB reverse        *1400 
               
               
                 NB 
                 GCCTAGCTCT TACTACCGTT ATTT CCATCT GAGAACTAGA CGCCCGGTT C 
               
               
                   
               
               
                   
                 1401                                              1450 
               
               
                 NB 
                 AACTTAGCCA AGTTAAGGGA ACGCACTACT CTTCCCTACA AGCAACGGAT 
               
               
                   
               
               
                   
                 1451                                              1500 
               
               
                 NB 
                 TGAGGCTAGC GCGAAAGCCG GTGCGCGTTA GGCGCATCCG TTTTCTTGCT 
               
               
                   
               
               
                   
                 1501                                              1550 
               
               
                 NB 
                 TAGTAGTTAC TCACTGGGAT TCCCGCAGTG CAGCCGTTGC TTGTTGGGCT 
               
               
                   
               
               
                   
                 1551                                              1600 
               
               
                 NB 
                 ACAAAGCACT TCTTAGACGG AAGAGCGGCA AGCAAGCCTA CTCAAGTACC 
               
               
                   
               
               
                   
                 1601                                              1650 
               
               
                 NB 
                 TAGCACTCCA CAACCCATTA GTTGGAAGGC AATCAGCAAC TTAGCACACT 
               
               
                   
               
               
                   
                 1651                                              1700 
               
               
                 NB 
                 AGAGCATACA ACGACCTTTA GCAACCTTTT CTCGAGCGAT AGTCTAGTAG 
               
               
                   
               
               
                   
                 1701                                              1750 
               
               
                 NB 
                 GTCAAGCCCG AGTGGAGGCC ATCCATCCCT TTCTGATGAA ACCTTTGTTG 
               
               
                   
               
               
                   
                 1751                                        1794 
               
               
                 NB 
                 CCCTAGCTTG AATTGAAGGT TTGGCTAAGG ATGGGGATCC TCTA 
               
             
          
         
       
     
     The Polymerase Chain Reactions 
     In order to amplify the DNA, we have synthesized five oligonucleotides: one which hybridizes to the DNA sequence common to the four fragments and four others, each one specific to the unique sequence of each of the four cytotypes: NA, NB, C, S. 
     Choice of Primers 
     These five synthetic oligonucleotides have been chosen so that they allow amplification in the same experimental conditions for all reactions. The denaturation temperature is high enough to permit full hybridization at 55° C. (see below). To insure a better annealing, the last 3′ chosen as a C or a G. 
     
       
         
               
             
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Sequences of the primers 
               
             
          
           
               
                   
                   
                   
                   
                 SEQ 
               
               
                   
                   
                   
                   
                 ID 
               
               
                 Oligo 
                 Sequence 
                 O 
                 P 
                 NO. 
               
               
                   
               
               
                 OLIGO-NA 
                 GATGAAAGCA TAGGTAGTGA CGACC 
                 R 
                 1122- 
                 5 
               
               
                   
                   
                   
                 1146 
               
               
                 OLIGO-NB 
                 AACCQGGCGT CTAGTTCTCA GATGG 
                 R 
                 1375- 
                 6 
               
               
                   
                   
                   
                 1399 
               
               
                 OLIGO-S 
                 CTGGTCAAGG TCCAACGTTA AGTTG 
                 R 
                  904- 
                 7 
               
               
                   
                   
                   
                  927 
               
               
                 OLIGO-C 
                 TTGCAAGTCC CGATTGGTAA GCAAGC 
                 R 
                  971- 
                 8 
               
               
                   
                   
                   
                  996 
               
               
                 OLIGO-com 
                 ATCTTCGATT GGTAGAAGTG AGTCCG 
                 D 
                  526- 
                 9 
               
               
                   
                   
                   
                  551 
               
               
                   
               
               
                 The sequences of these oligonucleotides are as follow:  
               
               
                 O = Orientation  [D = direct; R - reverse] 
               
               
                 P = Position    [base pair number from the above sequence] 
               
             
          
         
       
     
     Experimental Procedure 
     a) To find if a set of sterile seeds are contaminated with one or both the fertile cytotypes, the following reaction was set up. 
     The reactions were done in a final volume of 50 μl 
     200 ng DNA 
     5 μl NTP 2 mM 
     5 μl enzyme buffer 
     0.5 μl AmpliTaq polymerase (2.5 U) 
     4 μl MgCl 2  20 mM 
     4 μl oligo com. 
     4 μl oligo NA 
     4 μl oligo NB 
     4 μl DNA @ 50 ng/μl 
     19.5 μl H 2 O 
     The PCR cycles 
     Denaturation: 1 mn 95° C. 
     Amplification: 40 sec. at 95° C.; 40 sec. at 55° C.; 40 sec. at 72° C. 
     30 cycles of amplification 
     b) Results 
     The amplification products were visualized on a 1.2% agarose gel stained with ethidium bromide. The size of the DNA fragments flanked by the 2 oligonucleotides were: fertile maize type NB: 873 bp; fertile maize type NA: 620 bp; male sterile maize type C: 470 bp; male sterile type S: 401 bp. A single PCR was enough to identify the presence of fertile maize contamination within a male-sterile sample. 
     Application of this method using total DNA extracted from maize seedlings or flour has shown that it is possible to characterize each maize cytotype in a mixture of cytotypes without any ambiguity. Some experiments have been done by mixing decreasing amount of DNA from one cytotype with the other cytotypes which have shown that it is possible to detect the specific fragment without any ambiguity, in a ratio 1 to 10,000. This corresponds to a contamination of one seed among 10,000 seeds. This ratio can be substantially lowered by optimizing the PCR method. The method was also applied in a real environment with mixed maize seeds from which the amount of contamination was determined. Quantification of the contamination was assessed by recording the number of PCR cycles needed to obtain the same amount of DNA amplified with different amount of DNA, then extrapolating to the origin (PCR cycle number=0). Therefore this invention allows the identification of the four maize cytotypes, and the quantification of each cytotype within a mixture. 
     c) Control Reactions 
     1) To avoid amplification of foreign DNA traces that might be present in the buffers, the same PCR reactions must be conducted without DNA extracted from the maize kernels. 
     2) Control of DNA synthesis. If the PCR test is negative, one must be sure that non-amplification is real and not the result of a faulty synthesis. The same PCR reactions must be done in the presence of sterile-specific oligos in order to assure that the polymerase is working. 
     Today&#39;s methods do not allow one to identify in a precise manner the different maize cytotypes. The identification is obtained by performing crosses that require more than one generation. The identification is therefore time consuming, tedious and costly. The method of the invention is molecular-based PCR method leading to a quick and precise result. The method also allows one to quantify the amount of each cytotype in a mixture, which is not possible by doing traditional crosses. 
     Time needed to realize those tests is two days from the time the raw material (flour or seedlings) is available. It takes one week if the maize kernels need to be germinated in the dark. 
     BIBLIOGRAPHY 
     a) Fauron, C. M. R. and Havlik, M., The maize mitochondrial genome of the normal type and the cytoplasmic male sterile type T have very different organization,  Curr. Genet.,  15, 149, 1989. 
     b) Sangaré, A., Weil, J. H., Grienenberger, J. M., Fauron, C. and Lonsdale, D., Localization and organization of tRNA genes on the mitochondrial genomes of fertile and male-sterile lines of maize,  Mol. Gen. Genet.,  223, 224, 1990. 
     
       
         
           
             9 
           
           
             1 
             948 
             DNA 
             Maize 
           
            1
cgactcacta tagggaattc gagctcggta cccggggatc ccccccgcat tctgatcagg     60
ttcgttaccc attccgctgt ggtcatcaaa gaagaataat aaaaaagtgc tgttgttaca    120
ctcaacagag tgaaaacgcg agcagcccaa actgggcaac gcggccataa agcgcgaacc    180
aacatccgtg atacgaaaac caaaatcaga atgaggaaga aaaggatatc tttaggtaag    240
tcatgaaaat gaaaacccaa taagaactta cccaataatt tagaacagat cacttccaaa    300
cgagacatga tgagctaaag tcaaaataaa atgcaagaga aattcaaatt atgtaaggct    360
tagaaaaaag tcctgcgtag agtccacggg gagccttttt cgatttgatc tctagtcaaa    420
ctaagactgg agcttgaagc cactactcta gaaccggaag aagcctaact tctgcatttt    480
gagtagattt tctgactatc atttgccttg aattacaata cgataatctt cgattggtag    540
aagtgagtcc ggatctccct cttttccact aaacccgcca ctcctacttg tactttattt    600
tccacctggc tagcttcaat tgcttagtag ggagtttcca tccttgactt tcgcggttct    660
ttctttgctt tcttcagttg tgttgaggag ctatcttttg aacccaacta actcttaaat    720
ccgaactttc ccttgcttac gcccctagaa actccgtgaa aaaaccacct cgtttttcca    780
taaataaagg attcctaata aagtttttac atatcatatc ccagcggtat ttttataata    840
atttttgcat aaataaagta taatttccac aaaataacac ttttaggcgc ccacttgaaa    900
ttcaacttaa cgttggacct tgaccaggcc ctctctattt ccagaatt                 948
 
           
             2 
             1265 
             DNA 
             Maize 
           
            2
cgactcacta tagggaattc gagctcggta cccggggatc ccccccgcat tctgatcagg     60
ttcgttaccc attccgctgt ggtcatcaaa gaagaataat aaaaaagtgc tgttgttaca    120
ctcaacagag tgaaaacgcg agcagcccaa actgggcaac gcggccataa agcgcgaacc    180
aacatccgtg atacgaaaac caaaatcaga atgaggaaga aaaggatatc tttaggtaag    240
tcatgaaaat gaaaacccaa taagaactta cccaataatt tagaacagat cacttccaaa    300
cgagacatga tgagctaaag tcaaaataaa atgcaagaga aattcaaatt atgtaaggct    360
tagaaaaaag tcctgcgtag agtccacggg gagccttttt cgatttgatc tctagtcaaa    420
ctaagactgg agcttgaagc cactactcta gaaccggaag aagcctaact tctgcatttt    480
gagtagattt tctgactatc atttgccttg aattacaata cgataatctt cgattggtag    540
aagtgagtcc ggatctccct cttttccact aaacccgcca ctcctacttg tactttattt    600
tccacctggc tagcttcaat tgcttagtag ggagtttcca tccttgactt tcgcggttct    660
ttctttgctt tcttcagttg tgttgaggag ctatcttttg aacccaacta actcttaaat    720
ccgaactttc cctgcagctt tagggcttct agtgaggaga ggtgatctct agtttctcat    780
tccgatagcc atctcgatta actgagttga cttgtgagct cccctaaccc ggcaggcagg    840
ccaaagaatg aagggcagtg aaaatggttt cttcttctgg ctaattccga tacgaatacc    900
aaaaacagct tacttccgtt cgtgtcctcg gaaattgcaa ttcattcaca tctgctccta    960
tctattctat gcttgcttac caatcgggac ttgcaattcc tatcacccgc cggaggcaga   1020
ttacttaatc tgaaagcgtt atttcacatt cgttatttca tatcatatga attaggcgga   1080
tcatacaccg atctcagaac taatacgctt tcgctttctc ctgaccctca atccaaaaga   1140
aaaggtttgg cgttgcagtt catatgcttc cttgcgctag aaaccaatac gcacgtacga   1200
ggcctacgat tctctagttg tattggcaag ggaaagaaga tgctatggga atgtttggaa   1260
tgaaa                                                               1265
 
           
             3 
             1299 
             DNA 
             Maize 
           
            3
cgactcacta tagggaattc gagctcggta cccggggatc ccccccgcat tctgatcagg     60
ttcgttaccc attccgctgt ggtcatcaaa gaagaataat aaaaaagtgc tgttgttaca    120
ctcaacagag tgaaaacgcg agcagcccaa actgggcaac gcggccataa agcgcgaacc    180
aacatccgtg atacgaaaac caaaatcaga atgaggaaga aaaggatatc tttaggtaag    240
tcatgaaaat gaaaacccaa taagaactta cccaataatt tagaacagat cacttccaaa    300
cgagacatga tgagctaaag tcaaaataaa atgcaagaga aattcaaatt atgtaaggct    360
tagaaaaaag tcctgcgtag agtccacggg gagccttttt cgatttgatc tctagtcaaa    420
ctaagactgg agcttgaagc cactactcta gaaccggaag aagcctaact tctgcatttt    480
gagtagattt tctgactatc atttgccttg aattacaata cgataatctt cgattggtag    540
aagtgagtcc ggatctccct cttttccact aaacccgcca ctcctacttg tactttattt    600
tccacctggc tagcttcaat tgcttagtag ggagtttcca tccttgactt tcgcggttct    660
ttctttgctt tcttcagttg tgttgaggag ctatcttttg aacccaacta actcttaaat    720
ccgaactttc cctgcagctt tagggcttct agtgaggaga ggtgatctct agtttctcat    780
tccgatagcc atctcgatta actgagttga cttgtgagct cccctaaccc ggcaggcagg    840
ccaaagaatg aagggcagtg aaaatggttt cttcttctgg ctaattccga tacgaatacc    900
aaaaacagct tacttccgtt cgtgtcctcg gaaattggat tacttatgag tttcttcggt    960
gcaaaagtag gcaagtccat tttctcgtgt ttttcgtcct caagacctga ttttccaaag   1020
agcatttcat atagctgcaa aagttgctca tccgattttc cctcgctttt attcgtaaca   1080
tggcgtagtt actcattggg tccgctgaga agaaaacaat aggtcgtcac tacctatgct   1140
ttcatctaga ggaagtcagg ctgtgattcg taacatggcg tagttactca caggccgtgg   1200
atctcctttt tctaatcaag cagcaggccg tgggttttct ggttctgcgc ttcagaagag   1260
gaaagtttcc gtgattcgta tctgctttga gggcaggtc                          1299
 
           
             4 
             1794 
             DNA 
             Maize 
           
            4
cgactcacta tagggaattc gagctcggta cccggggatc ccccccgcat tctgatcagg     60
ttcgttaccc attccgctgt ggtcatcaaa gaagaataat aaaaaagtgc tgttgttaca    120
ctcaacagag tgaaaacgcg agcagcccaa actgggcaac gcggccataa agcgcgaacc    180
aacatccgtg atacgaaaac caaaatcaga atgaggaaga aaaggatatc tttaggtaag    240
tcatgaaaat gaaaacccaa taagaactta cccaataatt tagaacagat cacttccaaa    300
cgagacatga tgagctaaag tcaaaataaa atgcaagaga aattcaaatt atgtaaggct    360
tagaaaaaag tcctgcgtag agtccacggg gagccttttt cgatttgatc tctagtcaaa    420
ctaagactgg agcttgaagc cactactcta gaaccggaag aagcctaact tctgcatttt    480
gagtagattt tctgactatc atttgccttg aattacaata cgataatctt cgattggtag    540
aagtgagtcc ggatctccct cttttccact aaacccgcca ctcctacttg tactttattt    600
tccacctggc tagcttcaat tgcttagtag ggagtttcca tccttgactt tcgcggttct    660
ttctttgctt tcttcagttg tgttgaggag ctatcttttg aacccaacta actcttaaat    720
ccgaactttc cctgcagctt tagggcttct agtgaggaga ggtgatctct agtttctcat    780
tccgatagcc atctcgatta actgagttga cttgtgagct cccctaaccc ggcaggcagg    840
ccaaagaatg aagggcagtg aaaatggttt cttcttctgg ctaattccga tacgaatacc    900
aaaaacagct tacttccgtt cgtgtcctcg gaaattggat tacttatgag tttcttcggt    960
gcaaaagtag gcaagtccat tttatcgtgt ttttcgtcct caagacctga ttttccaaag   1020
aaaatagcct gccaaagagc tagctataga aatacagtca gtcggggtaa actgaaagaa   1080
ggaaatctac attcatagat agtagggcaa taggctcact tacatagtgg ggacagcaat   1140
aggctcactt acttttgaaa taagagggtc ggtcaactct cggcagatag ggtagttgct   1200
aaataaaagc aaagaaagag tgagttgaaa gaagaaatgt atgtgggtca ccatataata   1260
tatataatag tacctaactt actaggtatc tcggtcaact gctagctttt catccttggt   1320
gcagtatttc caccttctat tgctttgctt gcctagctct tactaccgtt atttccatct   1380
gagaactaga cgcccggttc aacttagcca agttaaggga acgcactact cttccctaca   1440
agcaacggat tgaggctagc gcgaaagccg gtgcgcgtta ggcgcatccg ttttcttgct   1500
tagtagttac tcactgggat tcccgcagtg cagccgttgc ttgttgggct acaaagcact   1560
tcttagacgg aagagcggca agcaagccta ctcaagtacc tagcactcca caacccatta   1620
gttggaaggc aatcagcaac ttagcacact agagcataca acgaccttta gcaacctttt   1680
ctcgagcgat agtctagtag gtcaagcccg agtggaggcc atccatccct ttctgatgaa   1740
acctttgttg ccctagcttg aattgaaggt ttggctaagg atggggatcc tcta         1794
 
           
             5 
             25 
             DNA 
             Maize 
             
               misc_feature 
               (1)..(25) 
               Oligonucleotide primer 
             
           
            5
gatgaaagca taggtagtga cgacc                                           25
 
           
             6 
             25 
             DNA 
             Maize 
             
               misc_feature 
               (1)..(25) 
               Oligonucleotide primer 
             
           
            6
aaccgggcgt ctagttctca gatgg                                           25
 
           
             7 
             25 
             DNA 
             Maize 
             
               misc_feature 
               (1)..(25) 
               Oligonucleotide primer 
             
           
            7
ctggtcaagg tccaacgtta agttg                                           25
 
           
             8 
             26 
             DNA 
             Maize 
             
               misc_feature 
               (1)..(26) 
               Oligonucleotide primer 
             
           
            8
ttgcaagtcc cgattggtaa gcaagc                                          26
 
           
             9 
             26 
             DNA 
             Maize 
             
               misc_feature 
               (1)..(26) 
               Oligonucleotide primer 
             
           
            9
atcttcgatt ggtagaagtg agtccg                                          26