FR2711143A1 - Process for the identification of a fruit genus, species or variety; test kit, oligonucleotide and hybridisation probe for this indentification - Google Patents

Abstract

The invention relates to a process for the identification of a fruit genus, species or variety. It relates to a process which comprises a) the amplification of DNA sequences in nucleic acids present in the tissue or the juice by means of a polymerase chain reaction, b) the comparison of the DNA obtained with that which is obtained from a tissue or juice of fruits of a known genus, species or variety, and c) the examination of the presence of DNA elements from a known genus, species or variety based on the presence of tissues or juice derived from this genus, this species or this variety. The nucleic acids are analysed by means of a hybridisation probe in which the observation of hybridisation of the nucleic acids of fruits with a given probe is compared with that of the nucleic acid of known genus, species or variety, and the comparison is used for the determination of the probable identity of the fruit. Application to the analysis of the source of fruits.

Description

 The present invention relates to a process for identifying the origin of tissues and extracts derived from fruits, and in particular fruit juices which may be in liquid form, pure or diluted, pulps, purees, wines, jams and yoghurts, and other fruit products intended for human consumption, and it also relates to nucleic acid initiators and probes intended for use in this process as well as test kits containing them.

 Fraud on fruit juices is a very important problem, as unscrupulous producers use spoilage materials to increase their profit. This kind of fraud makes it necessary to carry out authenticity tests on these juices and on fruit purees and pulps used for the production of food products, such as wines, jams and yogurts. It is also necessary to perform tests when producing young plants to verify the genetic makeup of trees and shrubs.

 The problems vary from state to state. For example, in the United States of America, citrus products "Ambersweet" are sold as products under the name "oranges" but, according to European regulations, this sale is not authorized. The main retailers of juice-based products must therefore have authenticity tests, carried out either by themselves or by their suppliers. In addition, different fruit products are subject to different import regulations, so identification of origin is necessary for customs authorities.

 Analytical chemists have developed a variety of testing techniques, some of which are very sophisticated. For example, it is known to characterize a variety of tissues using a "genetic fingerprint", during an operation in which nucleic acids are isolated from the material to be analyzed and subjected to detection and identification. Usually, one of two techniques is used depending on whether canned sequences or markers are present in the material being tested. In particular, methods based on iil (s) probes and methods based on the polymerase chain reaction (PCR) are used. A whole range of commercial nucleic acid (S) probes ensuring the binding of conserved sequences to 5p (s) have been developed for the detection of bacterial pathogenic organisms. Examples are the colorimetric probe ("Genetrak") and chemiluminescent genetic probe ("Eugenetics") systems for Salmonella and Listeria monocytogenes. The use of such probes for human typing and identification is well documented in the technique of genetic probes.

 Amplification of the target sequences conserved by the PCR reaction before detection of the amplified PCR product is another powerful technology recently developed and which has proved invaluable in the clinical and legal sciences. The scope of PCR is considerable in other industries, including the food, agricultural and water production industries. This technique allows the creation of quantities of target sequences of the order of the microgram from quantities of the order of the nanogram in a time of 3 to 4 h. In the case of an analysis of microbial pathogens, considerable time savings are obtained by using the PCR method in place of the conventional culture methods which require days to give the same result.

 The inventors have now determined that nucleic acids can be isolated from a variety of fruits, and in particular fruit juices, that is, fluids derived from whole fruits and sold with or without other tissue. In addition, they determined that deoxyribonucleic acid (DNA) could be probed or subjected to the PCR process so that fruit source characterization could be performed, and they also determined specific probe sequences and initiators suitable for this characterization.

 The application of these techniques to fruit juices allows rapid detection of fraud without the need for a sample of fruit tissue derived therefrom.

 The invention relates to a method of identifying a genus, a species or a variety of a source of fruit tissue or juice derived from such tissue, comprising (a) amplifying sequences of DNA in nucleic acids present in tissue or juice using a polymerase chain reaction and / or nucleic acid analysis, (b) the comparison of the DNA obtained with that which is obtained from a tissue or fruit juice of a known genus, species or variety, and (c) determining the presence of DNA elements of a known genus, species or variety based on the presence of tissue or juices derived from this genus, species or variety.

 The invention is based on the inventors' determination that, although the nucleic acid concentrations present in many juices are insufficient to allow direct application of a DNA probe, they are sufficient to allow the application of techniques based on the PCR process to obtain a sufficient quantity of DNA. In addition, they found that this nucleic acid was characteristic of the fruit, and that the strain DNA did not appear in samples of descendants in amounts that can be detected. The inventors have also determined the sequence of several PCR initiators giving distinct profiles when they are applied to the nucleic acid of fruit or fruit tissue.

 The DNA profile is very advantageously obtained by applying the PCR technique to the amplification of the DNA sequences present in the DNA of the fruits, so that characteristic DNA products are obtained and can be compared to known products so that they can be identified. For example, the inventors have found, by comparison of the DNA profiles of oranges and grapefruits, which are fruits of the same kind, that there are clear differences and that their mixtures have an aspect of combinatorial design.

 It was found in particular that the application of the statistical amplification of polymorphic DNA (RAPD) to the extracted DNA allowed the satisfactory characterization of fruit juices and tissues with sufficient differentiation to allow the identification of the origin of the constituents of mixtures. The use of the preferred technique allows an easy distinction between different species of harvested fruit and, with less frequency, between different varieties of fruit, for example varieties of oranges, grapefruits and apples. The inventors determined that the strain did not affect the DNA profile of the progeny and thus that the genes did not move between cells, and thus demonstrated the reliability of the technique applied to such "double genome" plants. ".

 The preferred method of the invention includes a procedure for extracting high quality DNA from an intact fruit or processed fruit juice. This procedure involves freezing tissue which gives a solid which is then mechanically transformed into a powder which is extracted by a solvent capable of taking DNA. The freezing step comprises sufficient freezing of the tissue in liquid nitrogen and the mechanical powdering is advantageously carried out by grinding. Solvent extraction is advantageously carried out by known methods using cetyltrimethylammonium bromide (CTAB) and / or phenol-chloroform. The use of these techniques allows obtaining DNA suitable for the formation of profiles for fruits of very diverse varieties, such as Navel Valencia and satsuma oranges, red apples and Granny Smith, lemons, limes, grapefruit, bananas, kiwi fruit, passion fruit, papaya, peaches, grapefruit, grapes and plums.

The use of such extraction techniques gives variable DNA yields between different genera and species of fruit (see Table 1), but it is found that all of them are sufficient for the preferred process.
RAPD.

TABLE 1
Fruit Country of origin DNA yield, ug / g
Apple
France Red 0.14
Granny Smith France 0.98
Lemon South Africa 1.71
Argentine lemon 0.54
EUA file 3.92
Grapefruit South Africa 1.09
West Indian banana 0.27
Kiwi New Zealand 5.58
Fruit of the
passion Kenya 9.48
Papaya Brazil 4.20
Fishing Italy 2.71
Pineapple Costa Rica 49.92
Plum England 1.46
Mango Kenya 5.50
Melon Galia Israel 7.82
Orange
Navel N1-625 South Africa 3.70
N5-J1207 Morocco 3.64
Juice Morocco 0.128 / cm3
Juice conc. Morocco 6.16
Processed juice Morocco 0.094 / cm3
N6-1207.1 Spain 3.83
N6-1207.2 Spain 1.25
The invention also relates to specific initiator sequences which have been determined to give satisfactory profiles for the identification of the genus, species or variety of fruits, as well as target sequences enabling PCR amplification at 1 'end 5' of sequences corresponding to the following identification sequences 1 to 6
Ident. that is. 1: GGTCTAGAGG
2 2: ACTCCAGTCC
3 3: TGGAGGGCTC
4 4: AGACGGCCCT
5 5: GTCCCAGCAG
6 6: TGTCGGTCGT
Thus, the initiators identified by these sequences can vary from sequences complementary to the target sequences at the 5 'end of the target, but also allow hybridization at the 3' end. Such initiator sequences include the sequences having the sequence identification numbers 7 to 12 which follow and are preferably formed from these sequences.

Ident. that is. 7: CCTCTAGACC 8 8: GGACTGGAGT
"9: GAGCCCTCCA
10: AGGGCCGTCT
11: CTGCTGGGAC
12: ACGACCGACA
It is possible, with these initiators, to characterize and thus to identify a large number of fruit tissues and more precisely fruit juices, as indicated in Table 2 which follows.

The preferred technique used for obtaining RAPD profiles is that which is conventional for PCR analysis techniques, that is to say as described by
Mazurier et al (1990), Nucleic Acid Res. 18. 6531-6535 and
Mazurier and Werners (1992), Res. Microbiol. 14. 260-262.

 In another aspect of the invention, the latter relates to test kits comprising oligonucleotides having sequences complementary to identification sequences n 1 to 6 and comprising in particular respective identification sequences n "7 to 12, essentially comprising these last sequences.

 In another embodiment, the invention relates to test kits containing oligonucleotides as described above and having, in addition, comparative motifs of known fruits derived from one or more RAPD techniques using each of these initiators, considered together or separately.

 The oligonucleotides described above can be used as PCR initiators for RAPD analysis of tissues and fruit juices and can be used for the formation of oligonucleotide hybridization probes which are themselves useful for the analysis and characterization of acids. tissue nuclei and fruit juices by hybridization on "Southern" paper.

When used as PCR initiators, the oligonucleotides may undergo an increase in length allowing the use of more severe hybridization conditions and thus the increase in the specificity of the method. For example, the initiator of the identification sequence No. 12 can be extended so that it forms direct and reverse initiators capable of ensuring the binding of the target DNA under more severe conditions by the addition of one or more multiple bases at its 3 'end as shown by the following sequences
direct initiator sequence: (bases 1 to 10 are those of identification sequence No. 12, the initiator is enlarged by adding a desired number of bases in the order of bases 11 to 51 of the sequence which follows) 5'-ACGACCGACA AACGATGAAA ATACTCACGG CTGTGCATAA AGACGCGCAA
TATTAGAGAA GGTGGGTTGA TTTGGATTTG GCGT-3 '
reverse initiator sequence: (bases 1 to 10 are those of identification sequence No. 12, the initiator is enlarged by adding a desired number of bases in the order of bases 11 to 51 of the sequence which follows) 5'-ACGACCGACA CCAAAACCAA AGTCAAGAAC TTCGGCGCAA TAGAGCATAC
TTCCAATGAA TTTCACATAT GATCACTA-3 '
It is obvious to those skilled in the art that initiators consisting for example of 5 bases or more, advantageously 10 bases or more and preferably 15 bases or more, of these sequences can be used for the amplification of the DNA of oranges and for obtaining a fragment of approximately 1 kb (kilobase) characteristic of the orange species. The specificity of these initiators is increased by increasing their length corresponding to the specified sequences. The sequence no. 12 is known (Operon
Technologies), but the oligonucleotides derived from the rest of the sequence are new.

 Thus, these products produced with other derived initiators can themselves be used as hybridization probes on "Southern" paper. In particular, these probes comprise individual PCR products labeled with the PCR reaction of tissues derived from the fruit using PCR initiators of the sequence described above.

 A preferred set of polynucleotideoligonucleotide probes includes those derived by performing a PCR reaction on DNA derived from Florida orange-grapefruit hybrid tissue, including leaves, using the single sequence initiator having the identification number "12" to trigger the amplification. The polynucleotide, which has a length of about 1 kbp (kilopair of bases) is specific to the species "Ambersweet".

 Another preferred polynucleotide for labeling and use as a probe is a "Downstream / Bgl II" fragment which can be obtained from this 1 kilobase polynucleotide, and having a length of approximately 200 bp. The preferred 200 bp product hybridizes specifically with the orange and "Ambersweet" genome nucleic acid, but not with that of grapefruit or a lime.

 A second group of such polynucleotides obtained according to the invention is made available by using the sequence having the identification number 11 for the amplification of the DNA derived from any of the orange cultivars indicated in the present specification. . The product has a fragment of about 1 kbp to 500 bp and can be derived using "EcoRV / Bgl II". Neither this product nor the 500 bp fragment was found in DNA derived from grapefruit or a lime. Other polynucleotides can be derived by cleavage of this fragment in a manner which is also specific for the DNA of oranges.

 In another aspect, the invention relates to a method for producing a 200 bp polynucleotide according to the invention comprising the cloning of a derivative product by the PCR process of approximately 1 kbp, which can be obtained using of the sequence having the identification number 12 for the amplification of the DNA of "Ambersweet" and in particular of the leaf DNA of "Ambersweet", in a vector system, and by cleavage of the necessary product of approximately 200 bp from the product obtained by using the restriction enzymes "Downstream" and "Bgl II", then by a labeling step. The labeling is carried out by any of the conventional methods of incorporating a biological, chemical or radioactive marker, known in the art.

 The method, the oligonucleotides and the kits according to the invention are now described purely by way of illustration with reference to the examples which follow. Other embodiments within the scope of the invention are obvious to a person skilled in the art on reading these examples.

Examples
Example 1: Procedure for isolating DNA from commercially available fruit juices.

 Initial attempts to isolate DNA from 100 cm3 of commercial fruit juice were made, but the optical density values (at 260 nm) were negative and no DNA genome bands could be visualized after agar gel electrophoresis. Isolation was then attempted using 400 cc of fruit juice, and positive optical density values were obtained, but the genome DNA could not be observed after gel electrophoresis. The products obtained by the PCR technique were such that, although the DNA could not be observed on the agar gels, the quantity available was sufficient for amplification. Such a volume condition obviously depends on the origin and previous treatment of the juice.

3
The pH of 400 cm of commercial fruit juice was adjusted to 7.0 with 8.33N sodium hydroxide, and the juice was centrifuged at 10,000 g to obtain a solid phase and d 'one phase which floats, and the two phases have been separated. DNA was isolated from the supernatant phase by the addition of 3M ammonium acetate and isopropanol intended to cause precipitation. Centrifugation at 10,000 g gave a precipitated DNA pellet which was treated with a solution of a cationic detergent (cetyltri methylammonium bromide, CTAB) at a concentration of 0.8% w / v so that contamination by the polysaccharides is reduced and the inhibition of the enzymes involved in the cloning procedures is avoided. Chloroform was added to the treated DNA, and the mixture, which separated into two phases, was centrifuged. The aqueous phase containing the DNA was removed and the DNA was precipitated with isopropanol. A DNA pellet was obtained by centrifugation and this pellet was resuspended in medium.
TE (Tris-HCl, Na2EDTA).

A phenol and chloroform treatment was used to denature the proteins which may have been present, which formed a layer at the interface of the organic and aqueous phases, and the operation was repeated until no proteins remained. the interface. A "Sevag" treatment (chloroform / isoamyl alcohol) was carried out by adding isoamyl alcohol intended to prevent foaming of the mixture during the subsequent swirling intended to facilitate the separation of the two phases. RNA ribonucleic acid was removed using
RNAse, then phenol chloroform and "Sevag" extraction was carried out, as before, the DNA was precipitated with isopropanol and 3N ammonium acetate, and the pellet was centrifuged. DNA, and the resulting pellet was washed with 70% ethanol. The final pellet was vacuum dried and resuspended in sterile distilled water.

EXAMPLE 2 Extraction of DNA from a Fruit Tissue
30 g of the fruit tissue were frozen in liquid nitrogen and ground into a powder for the extraction of DNA. "TES" extraction buffer (Tris-HCl,
Na2EDTA and SDS) to the material that thawed to cause cell destruction, resulting in reduced nuclease activity. 1M PAH (phenanthroline hydrate) reducing agents were added and
1M DTT (dithiotreitol) to inactivate proteins, and phenol was added to cause their denaturation. The mixture was subjected to centrifugation and the resulting aqueous phase was treated in the same manner as the "floating" aqueous phase produced after the initial NaOH treatment of Example 1.

Example 3 RAPD Analysis of DNA Derived from Tissues and Fruit Juices
The RADP technique was implemented with six DNA initiators comprising the sequences of identification numbers 7 to 12 respectively (Operon
Technologies) with separate amplifications, the initiators acting at both ends of the target DNA to be analyzed. Amplifications were carried out in a volume
3 total of 25 mm container
2.5 mm3 of a x10 buffer (devoid of Mg +)
3
0.1 mm dNTP mixture (100 mM each)
2.0 mm3 of 25 mM MgCi2
3
5.0 mm from a single oligonucleotide initiator
at 10 bases (3 ng / mm) 3 3
2.5 mm genome template DNA (10 ng
0.2 mm Taq DNA polymerase (1 unit)
3
12.7 mm sterile distilled water.

With the exception of the genome matrix, the initiator and Taq DNA polymerase, all the above materials constitute the basic buffer solution prepared in volume and stored at -20 "C. All the amplifications were carried out with an apparatus "Perkin-Elmer" thermal cycle control with the following temperature program
1 cycle including 3 min at 94 "C
1 min at 36 "C
2 min at 72 "C
44 cycles including 1 min at 94 "C
1 min at 36 "C
2 min at 72 "C
1 final cycle including 10 min at 72 "C
999 min at 18 "C
The amplification products were analyzed by electrophoresis on 1.4% agar gel at 150 V (constant voltage) for 2 h and using staining ethidium bromide to visualize the profiles obtained.

 A series of tissues and fruit juices were used for the implementation of the PCR techniques in the presence of each of the separate oligonucleotide initiators described above, with sequences made up of those which have the identification numbers 7 to 12.

The combinations of initiators and DNA gave either no product (NP), or analogous product profiles (+) for all the DNAs tested, or distinct product profiles (-). The results of these techniques
PCR are shown in Table 2 below. The spaces indicate that the particular initiator has not been applied.

Thus, the compositions giving the result (-) are suitable for the identification of the specific DNA and thus of the tissue-particular fruit juice in a RAPD application.

TABLE 2 N ° initiator sequence 7 8 9 10
FRUIT
Red apple NP
Granny smith
South African Lemon NP +
Argentine Lemon NP
Lime
NP + Grapefruit
Banana
Orange Valencia V3-805 - +
Navel N5-1207 ++
"N5-PJ1207 ++
Processed juice
Navel N5-J1207 +
Juice
Navel N5-CJ1207 ++
Concentrated juice
In addition to these results, the oligonucleotide initiator having the sequence of identification number 10 was applied to PCR techniques on tissues of a number of varieties of Navel Valencia and satsuma oranges.

All the results, except for accession N2-625, gave the same profile by the following accessions: N1-625,
N2-625, N3-805, N3-625, N4-805, N5-1207, N6-1207-1,
N6-1207-2, V1-625, V2-805, V3-805, V4-1016, V5-805,
V6-1016, V7-1016, VL-1207, S1-1207.

 It was noted that all tissue and juice samples could give a profile by using either of the initiators used. Some initiators have given similar profiles to neighboring species, for example the sequences having the identification numbers 8 and 9. Differences could be determined between different accessions of Navel oranges and between red apples and Granny Smith, as indicated by the sequence with the identification number 7.

 The initiators having the sequences of identification numbers 11 and 12 were particularly useful for distinguishing the orange species from other fruits or from the orange "Ambersweet", and grapefruit hybrids from the orange varieties .

Example 4: Preparation of an oligonucleotide probe capable of distinguishing oranges
A DNA sequence initiator of identification number 12 was used for the amplification of a DNA fragment from leaves of "Ambersweet" derived from genome DNA using the PCR technique. product fragment having a length of approximately 1 kb. The product underwent further amplification in 16 cycles and was analyzed on agar gel, and a 1 kb polymorphic band was cut and purified using the "Geneclean Kit" (Bio 101 Inc.,
USA). The amount of DNA produced was then determined.

The 1 kb fragment was cloned into a vector system of plasmid pGEM-T ("Promega") and single and double restriction enzyme digests were established for the relative localization of the enzyme sites present in the vector. The enzymes chosen "Downstream" and "Bgl II" did not affect the native sites of the vector. The oligonucleotide product of "Downstream / Bgl II" fragment of about 200 kbp was extracted from the rest of the material, purified and labeled for use as a probe by conventional methods. It has been found, by using the Southern hybridization / transfer analysis, that this probe allows the hybridization of DNA from the orange genome, but not from grapefruit or lime.

Example 5: Use of the RAPD technique to distinguish between grapes
The process of Example 3 was repeated on both varieties of black and white grapes using a number of initiators, including those of the sequences having the identification numbers 9, 10 and 17 .

The use of an initiator with identification number 9 gave identical RAPD diagrams for the two varieties while a initiator with identification number 12 gave different diagrams for black and white grapes and an initiator for number identification 10 gave bands only with black grapes.

Example 6: Use of the RAPD technique for the distinction of an interspecific hybrid "Sweetie" of grapefruit
The initiator having the identification number 8 was used for the production of RAPD DNA diagrams from DNA samples of an interspecific hybrid "Sweetie" and grapefruit. Several bands were produced with "Sweetie" DNA and did not appear with grapefruit DNA as a template.

 It is understood that the invention has only been described and claimed as a preferred example and that it is possible to provide any equivalence in its constituent elements without going beyond its ambit.

Claims (18)

 1. A method of identifying a genus, a species or a variety of a source of fruit tissue or juice derived from such tissue, characterized in that it comprises (a) amplification of DNA sequences in nucleic acids present in tissue or juice using a polymerase chain reaction, (b) comparing the DNA obtained with that obtained from tissue or juice fruits of a known genus, species or variety, and (c) determining the presence of DNA elements of a known genus, species or variety based on the presence of tissues or juices derived from that genus , this species or this variety.  2. Method according to claim 1, characterized in that the nucleic acids are analyzed using a nucleic probe for hybridization, in which the appearance of hybridization of fruit nucleic acids with a given probe is compared with that of the known genus, species or variety, and the comparison is used to determine the likely identity of the fruit.  3. Method according to one of claims 1 and 2, characterized in that the statistical amplification of polymorphic DNA (RAPD) is applied to DNA of tissues or juice, and the profiles obtained are used for comparison from DNA to that of known profiles.  4. Method according to any one of the preceding claims, characterized in that, before amplification and analysis, the nucleic acid is extracted from the fruit tissues by freezing the tissues in solid form, the solid matter being mechanically transformed into powder and the powder undergoing extraction with a solvent capable of collecting DNA.  5. Method according to claim 4, characterized in that the freezing step comprises freezing the tissue in liquid nitrogen, and the mechanical powdering step comprises a grinding step.  6. Method according to any one of claims 1 to 5, characterized in that, after amplification and analysis, the nucleic acid is extracted from the fruit juice by precipitation, and undergoes treatment with cetyltrimethylammonium bromide (CTAB) or with phenol-chloroform or both.  7. Method according to any one of claims 3 to 6, characterized in that the RAPD technique is implemented with one or more initiators targeted for triggering the PCR amplification at the 5 ′ end of one of the DNA sequences  Ident. seq. 1: GGTCTAGAGG  "2: ACTCCAGTCC  "3: TGGAGGGCTC  "4: AGACGGCCCT  5 5: GTCCCAGCAG  6 6: TGTCGGTCGT  8. Method according to any one of claims 3 to 7, characterized in that the RAPD technique is implemented with one or more initiators comprising any one of the sequences having the identification numbers 7 to 12:  Ident. seq. 7: CCTCTAGACC  "8: GGACTGGAGT  "9: GAGCCCTCCA  10: AGGGCCGTCT  11: CTGCTGGGAC  12: ACGACCGACA  9. Test kit intended to be used for the identification of the genus, species or variety of tissues or fruit juice, characterized in that it contains an oligonucleotide comprising the sequence of one of the numbers of identifications 7 to 12.  10. Test kit according to claim 9, characterized in that it further comprises comparative diagrams of known fruits derived from one or more operations RAPD using respectively each of the oligonucleotides used as initiators, alone or in combinations.  11. Oligonucleotide comprising at least five bases of sequence 5'-ACGACCGACA AACGATGAAA ATACTCACGG CTGTGCATAA AGACGCGCAA  TATTAGAGAA GGTGGGTTGA TTTGGATTTG GCGT-3 '.  12. Oligonucleotide comprising at least five bases of sequence 5'-ACGACCGACA CCAAAACCAA AGTCAAGAAC TTCGGCGCAA TAGAGCATAC  TTCCAATGAA TTTCACATAT GATCACTA-3 '  13. Oligonucleotide according to one of claims 11 and 12, characterized in that it comprises at least fifteen bases of the given sequence.  14. Hybridization probe, with a polynucleotide or an oligonucleotide, characterized in that it is obtained by PCR amplification of nucleic acid derived from tissues or fruit juice, using one or more oligonucleotides having the identification numbers 7 to 12 as initiators, and by purification or isolation of the product.  15. Probe according to claim 14, characterized in that it is obtained from orange leaves of Florida "Ambersweet", only using a primer with the identification number sequence 12 for triggering amplification.  16. Probe according to claim 15, characterized in that it is obtained by exposure of the polynucleotide of claim 14 to restriction endonucleases "Aval / Bgl II" and by isolation of a fragment of the products having a length of approximately 200 base pairs (bp).  17. Probe according to claim 16, characterized in that it is obtained from an orange cultivar, using only a sequence initiator having the identification number 11 for triggering the amplification.  18. Probe according to claim 14, characterized in that it can be obtained by exposure of the polynucleotide of claim 17 to restriction endonucleases "EcoRV" and "Bgl II" and by isolation of a fragment of the products, having a length of approximately 500 base pairs (bp).