WO2010056642A2

WO2010056642A2 - Methods for genetic analysis of textiles made of gossypium barbadense and gossypium hirsutum cotton

Info

Publication number: WO2010056642A2
Application number: PCT/US2009/063814
Authority: WO
Inventors: Ming-Hwa Liang; Stephane Shu Kin So
Original assignee: Applied Dna Sciences, Inc.
Priority date: 2008-11-12
Filing date: 2009-11-10
Publication date: 2010-05-20
Also published as: US8669079B2; US20110250594A1; US20140295423A1; WO2010056642A3; US9938586B2

Abstract

Methods for distinguishing between cotton species by analyzing a sample of mature cotton fibers from raw cotton materials or from textile goods are disclosed. DNA is extracted from the mature cotton fiber sample and subjected to PCR techniques which enable the identification of the species of cotton utilized in the textile or cotton material of interest.

Description

METHODS FOR GENETIC ANALYSIS OF TEXTILES MADE OF

GOSSYPIUM BARBADENSE AND GOSSYPIUM HIRSUTUM COTTON

CROSS REFERENCE

[01] This application is simultaneously co-filed with the patent application titled "Methods for Genotyping Mature Cotton Fibers and Textiles"; the co-filed patent application being hereby incorporated by reference.

FIELD

[02] This invention pertains to methods for analyzing mature cotton fibers for the detection of genetic variations among a variety of cotton species, particularly, the genetic variations between cotton species Gossypium barbadense and Gossypium hirsutum. BACKGROUND

[03] In general, there are two major types of cotton species being cultivated throughout the world, namely Gossypium barbadense and Gossypium hirsutum. These two species are from the genus Gossypium, which comprises at least 40 different cotton species. Gossypium barbadense (sea island cotton) and

Gossypium hirsutum (upland cotton) are allotetraploids and are known as New World cotton or "American" cotton. There are striking differences in the physical characteristics of the cotton fibers produced by G. barbadense compared to the cotton fibers produced by G. hirsutum. G. barbadense produces longer cotton fibers than most other cotton species and these fibers are usually called "extra long staple" (ELS) fibers, while those of G. hirsutum are shorter and are called or defined as "upland" fibers. Textiles made of ELS fibers are considered to be of higher quality compared to textiles made with shorter fibers, like those produced by G. hirsutum cotton plants. Thus, textiles produced from ELS cotton fibers are considered more valuable in the textile marketplace.

[04] There have been many studies trying to manipulate cotton genes for fiber quality improvement (USPT 6,169,174; 7,060,874; and 6,995,256), enhanced pesticide toxin production (USPT 6,686,149; 6,140,075; and 6,057,370), and herbicide resistance (USPT 7,223,906). There have also been studies investigating the genetic polymorphism of various cotton species using PCR- based markers (J. Applied Sci. Res. 3(10)1156-1169, 2007). However, no success has been found in the categorization of cotton cultivars using genetic markers on mature cotton fibers mainly because of the lack efficient primers to amplify fragmented DNA in mature cotton fibers.

[05] Unfortunately, once cotton fibers are processed and made into yarn and/or fabrics most physical properties have been altered, and there is no reliable method to determine the origin or species of the fibers utilized to produce the yarn or textile(s). Forging clothing or producing knock-off textile items is a serious problem for the textile industry, costing manufacturers and retail stores millions and perhaps billions of dollars annually, in the United States alone. Being able to identify the species of cotton utilized in a textile item would not only be a way to authenticate an item as legitimate, but would also enable the detection of forged or counterfeit textile products.

SUMMARY [06] Methods for authenticating articles of manufacture containing cotton fibers such as garments or other textile goods are described. Higher quality cotton textile goods are typically made from Gossypium barbadense known to have Extra Long Staple (ELS) cotton fibers. Counterfeit textile goods are often made with different, inferior cotton with shorter fibers, such as Gossypium hirsutum. When the cotton species in an original textile article is known, the detection of a different species of cotton in the textile articles subsequent to manufacture will indicate that a counterfeit article has been substituted. The methods provide a means for identifying the cotton species in a textile item from a sample of mature cotton fibers taken from the textile item, and for authenticating the textile items based on the identification of the cotton species. The authentication can be carried out at different points along the supply or commerce chain between manufacture and retail sale to identify the point in the chain at which counterfeit goods are introduced.

[07] In one embodiment, the method for identifying cotton species comprises collecting cotton fibers from a sample, extracting genomic DNA from the collected cotton fibers, and amplifying the DNA with PCR-based techniques, followed by analyzing the amplified product to distinguish between a first cotton species and a second cotton species. In the illustrative embodiment the first cotton species is G. barbadense and the second cotton species is G. hirsutum. In other embodiments, the first cotton species is selected from the group consisting of G. barbadense, G. hirsutum, G. arboreum or G. herbaceum. In still other embodiments, the second cotton species is selected from the group consisting of G. barbadense, G. hirsutum, G. arboreum or G. herbaceum.

[08] Further, in some embodiments the sample is a textile item while in other embodiments the sample is raw cotton material.

[09] In certain embodiments, the amplifying further comprises using at least one set of specific primers which targets sequence polymorphism between the first cotton species and the second cotton species.

[10] In some embodiments, the genomic DNA extracted from the cotton fibers originating from the textile in question comprises chloroplast DNA and in other embodiments it comprises nuclear DNA. [11] In certain embodiments, the targeted sequences of the first cotton species and the second cotton species have different detectable physical characteristics.

[12] In some embodiments, the detectable physical characteristic of the targeted sequence in most embodiments is a difference in sequence length for the first cotton species compared to the second cotton species. [13] In other embodiments, the detectable physical characteristic of the targeted sequence is a different sequence composition for the first and second cotton species. [14] In most embodiments the analyzing of the amplified products comprises detecting the size of the amplified products by capillary electrophoresis. In other embodiments gel electrophoresis may be utilized instead of capillary electrophoresis. [15] One embodiment of the method for verifying a textile article comprises collecting cotton fibers from a textile item, extracting genomic DNA from the cotton fibers collected, amplifying said DNA with PCR-based techniques, analyzing the amplified product to distinguish between a first cotton species and a second cotton species, identifying the cotton fibers from the textile item as belonging to the first cotton species or to the second cotton species, and determining from the identified cotton species if the textile article is authentic. In most embodiments for verifying a textile article, the first cotton species is G. barbadense and the second cotton species is G. hirsutum. In some embodiments for verifying a textile article, the amplifying further comprises using at least one set of specific primers which target sequence polymorphism between the first cotton species and the second cotton species. Also, the targeted sequences of the first cotton species and the second cotton species have different detectable physical characteristics. The detectable physical characteristic can be a difference in length of the targeted sequence when comparing the first and second cotton species to one another. The detectable physical characteristic can also be a slight variation in sequence composition. [16] In one embodiment of a kit useful for carrying out the methods of the invention, the kit comprises a sample collection tube for placing mature cotton fibers obtained from the textile item, a DNA extraction solution, at least one set of specific primers specific for a target sequence found in a first cotton species and a second cotton species, as well as a PCR amplification buffer solution. [17] In some embodiments the kit for determining the cotton species of a sample or item further comprises a PCR instrument. [18] In yet other embodiments, the kit may further be found comprising an internal control for comparing the size of the amplified product. [19] In certain embodiments, the kit comprises a capillary electrophoresis device. [20] All patents and publications identified herein are incorporated herein by reference in their entirety. BRIEF DESCRIPTION OF THE FIGURES

[21] Figure 1 is a diagram showing the comparison of a genetic variable region of two different cotton.

[22] Figure 2 is a flow chart of one embodiment of the methods for identifying the cotton species of mature cotton fibers.

[23] Figure 3 is a flow chart of one embodiment of the methods for authenticating an article.

[24] Figure 4 is a photograph of an electrophoresis gel of DNA extracted from mature cotton fibers of known cultivars. [25] Figure 5 is an electrogram of a capillary electrophoresis analysis of DNA extracted from cotton fabrics of known materials; Sample 1 is made of ELS cottons and Sample 2 is made of upland cottons.

DESCRIPTION Definitions

[26] Unless otherwise stated, the following terms used in this Application, including the specification and claims, have the definitions given below. It must be noted that, as used in the specification and the appended claims, the singular forms "a", "an," and "the" include plural referents unless the context clearly dictates otherwise.

[27] Optional" or "optionally" means that the subsequently described event or circumstance may occur but need not occur, and that the description includes instances where the event or circumstance occurs and instances in which it does not.

[28] The terms "those defined above" and "those defined herein" when referring to a variable incorporates by reference the broad definition of the variable as well as preferred, more preferred and most preferred definitions, if any.

[29] The term "ELS" means "extra long staple" cotton fibers. For example, those fibers produced from (3. barbadense are ELS cotton fibers. [30] The term "Upland fiber" defines cotton fibers which are shorter than ELS cotton fibers. For example, upland fibers are produced from G. hirsutum.

[31] The term "genomic DNA" means the full complement of DNA contained in the genome of a cell or organism. This includes nuclear DNA as well as DNA stored within organelles, such as the mitochondrial genome (mDNA) and the chloroplast genome (cpDNA). [32] The term "variable region" means a genetic region of similar cotton species which has sequence variation between comparing species. The variation may be for example, a difference in sequence length within a genetic region, or single nucleotide changes within in specific genetic region. [33] The term "primer" means a nucleotide with a specific nucleotide sequence which is sufficiently complimentary to a particular sequence of a target DNA molecule, such that the primer specifically hybridizes to the target DNA sequence. [34] The term "probe" refers to a binding component which binds preferentially to one or more targets (e.g., antigenic epitopes, polynucleotide sequences, macromolecular receptors) with an affinity sufficient to permit discrimination of labeled probe bound to target from nonspecifically bound labeled probe (i.e., background).

[35] The term "probe polynucleotide" means a polynucleotide that specifically hybridizes to a predetermined target polynucleotide.

[36] The term "oligomer" refers to a chemical entity that contains a plurality of monomers. As used herein, the terms "oligomer" and "polymer" are used interchangeably. Examples of oligomers and polymers include polydeoxyribonucleotides (DNA), polyribonucleotides (RNA), other polynucleotides which are C-glycosides of a purine or pyrimidine base, polypeptides (proteins), polysaccharides (starches, or polysugars), and other chemical entities that contain repeating units of like chemical structure. [37] The term "PCR" refers to polymerase chain reaction. This refers to any technology where a nucleotide sequence is amplified via temperature cycling techniques in the presence of a nucleotide polymerase, preferably a DNA polymerase. This includes but is not limited to real-time PCR technology, reverse transcriptase- PCR, and standard PCR methods. [38] The term "nucleic acid" means a polymer composed of nucleotides, e.g. deoxyribonucleotides or ribonucleotides, or compounds produced synthetically which can hybridize with naturally occurring nucleic acids in a sequence specific manner analogous to that of two naturally occurring nucleic acids, i.e., can participate in hybridization reactions, e.g., cooperative interactions through Pi electron stacking and hydrogen bonds, such as Watson-Crick base pairing interactions, Wobble interactions, etc.

[39] The terms "ribonucleic acid" and "RNA" as used herein mean a polymer composed of ribonucleotides. [40] The terms "deoxyribonucleic acid" and "DNA" as used herein mean a polymer composed of deoxyribonucleotides.

[41] The term "polynucleotide" or "nucleotide" refer to single or double stranded polymer composed of nucleotide monomers of generally greater than

50 nucleotides in length. [42] The term "monomer" as used herein refers to a chemical entity that can be covalently linked to one or more other such entities to form an oligomer.

Examples of "monomers" include nucleotides, amino acids, saccharides, peptides, and the like.

[43] The term "identifiable sequence" or "detectable sequence" means a nucleotide sequence which can by detected by hybridization and/or PCR technology by a primer or probe designed for specific interaction with the target nucleotide sequence to be identified. The interaction of the target nucleotide sequence with the specific probe or primer can be detected by optical and/or visual means to determine the presence of the target nucleotide sequence. [44] In general, the term "textile" may be used to refer to fibers, yarns, or fabrics. More particularly, the term "textile" as used herein refers to raw cotton, ginned cotton, cotton fibers, cotton yams, cotton fabrics, yarn that is blended with cotton, fabric that is blended with cotton, or any combination thereof. [45] The term "mature cotton fiber" as used herein refers to a cotton fiber having a lumen wall that separates the secondary wall (consisting of cellulose) from the lumen that has naturally collapsed. The lumen is the hollow canal that runs the length of the fiber and is filled with living protoplast during the growth period; after the fiber matures and the boll opens the protoplast dries up and the lumen will naturally collapse and leave a large central void in each fiber. [46] The present specification describes methods for identifying and verifying the cotton species of mature cotton fibers. The methods enable cotton species identification of raw cotton bales for import/export purposes, identification of the origin of a particular cotton in raw or manufactured form, as well as identifying the cotton species in yarn and manufactured garments. In particular, the invention relates to verifying the type/species of cotton used in the manufacturing of an article comprising cotton. Most of the cotton textiles produced are made from two different cotton species, G. barbadense and G. hirsutum. [47] The methods allow a user to genetically identify textiles made of G. barbadense and G. hirsutum cotton fibers. The process involves extracting DNA from mature cotton fibers or textiles and amplifing DNA using specific primers and the Polymerase Chain Reaction (PCR) method. Specific primers targeting regions exhibiting sequence variation or sequence polymorphism between G. barbadense and G. hirsutum were used to distinguish between species. [48] There are subtle genetic variations in the genetic makeup of the two cotton species. The methods provide genetic tests which definitively identify fibers and textiles as being made of either G. barbadense or G. hirsutum cotton. The methods utilize at least one variable sequence region found within the cotton genomes of interest, which allows the cotton species to be distinguished from one another. In general, to identify cotton fibers originated from either G. barbadense or G. hirsutum, endogenous genetic markers based on the genetic variation between these two species were utilized, as depicted in Figure 1.

[49] Figure 1 is a diagram showing the comparison of a genetic variable region of an Extra Long Staple (ELS) cotton species (e.g. G. barbadense) to a Non-ELS cotton species (e.g. G. hirsutum). A variable region can differ in either DNA length or DNA sequence, while the non-variable regions have identical DNA sequences between the species being compared. Thus, the variable region can be utilized as an endogenous marker to distinguish between cotton species. By targeting regions of the DNA genome(s), which have sequence variations such as the region depicted in FIG.1 , the mature cotton fibers can be identified as either G. barbadense or G. hirsutum by the length or size of the target sequence. The method allows for cotton sequence-specific simple sequence repeats

(SSRs) associated primers for cotton species differentiation. This allows the use of SSRs primers to detect endogenous markers with varying DNA length polymorphisms between the different species. [50] In other embodiments, the methods utilize SNP or haplotyping to distinguish different cotton species. Primers are designed to amplify DNA fragments containing SNP/haplotypes and then the amplified DNA fragments are analyzed by sequencing. SNP analysis kits are available, such as the TagMan™ kit by ABI, or SNP analysis can be carried by a synthesis method, such as Pyrosequencing™.

[51] While sequence length is the physical characteristic targeted by the endogenous marker shown in FIG. 1 , the physical characteristic used to identify the cotton fibers as G. barbadense or G. hirsutum could be differences in site specific mutations which can be detected by specific nucleotide probes designed for a specific portion of the target sequence of each cotton species of interest. By utilizing a partial difference in sequence composition between the cotton species of interest, probes can be designed to detect one cotton species and not the other. The probes can be arranged in a high throughput array setup, to analyze and authenticate a large number of samples at one time. [52] FIG. 2 is a flow chart illustrating one embodiment of a method 100 for identifying the species of mature cotton fibers. At event 110, mature cotton fibers are collected and/or obtained from a cotton source. The cotton source may be raw cotton fibers from a specific geographical location, from a cotton textile, cotton article or garment, or from a source where the species is already known. The cotton fibers may also be from anthropological textiles as well as fibers from artwork canvases or any other article where knowing the cotton species utilized for an article would be advantageous in verifying or authenticating the item. The method and kit allows for verification of articles in a manner that helps prevent forgers and counterfeiters from substituting false or counterfeit goods in place of authentic items. [53] At event 120, the method 100 further comprises extracting genomic DNA from the collected cotton fibers. In general, a variety of nucleic acid extraction solutions have been developed over the years for extracting nucleic acid sequences from a sample of interest. See, for example, Sambrook et al. (Eds.) Molecular Cloning, Cold Spring Harbor Press, 1989. Many such methods typically require, for example, a detergent-mediated step, a proteinase treatment step, a phenol and/or chloroform extraction step, and/or an alcohol precipitation step. Some nucleic acid extraction solutions may comprise an ethylene glycol- type reagent or an ethylene glycol derivative to increase the efficiency of nucleic acid extraction while other methods only use grinding and/or boiling the sample in water. Other methods, including solvent-based systems and sonication, could also be utilized in conjunction with other extraction methods. [54] In most embodiments, about 5 mg to about 30 mg of cotton fiber or cotton lint, and in certain instances between about 10 mg to about 15 mg of cotton fibers are needed for sufficient DNA to be extracted from the sample for analysis. DNA extraction protocols are* derived from standard molecular biology DNA extraction procedures, which can be easily accomplished by persons skilled in the art. [55] While extracting DNA from seeds and leaves from various cotton species is quite common for cotton genomics research, utilizing DNA from the mature cotton fibers is quite unusual and unique. The amount of DNA, as well as the integrity of the DNA isolated from mature cotton fibers, may not be adequate for researching genetic variability and relationships among a plurality of genotypes or cotton species. The use of cotton fibers as a DNA source in the present invention is, surprisingly, sufficient for identifying and distinguishing between at least two different cotton species. This is partly due to the simplicity of the genetic test preformed in the methods of the invention, when compared to the plurality of genetic techniques needed to carry out polymorphism studies on large number of various genotypes or large number of cotton species. [56] The embodiment of FIG. 2, in event 130, further comprises amplifying a target sequence or specific DNA region of the cotton DNA genome extracted from the cotton fibers in event 120. Polymerase Chain Reaction procedures enable the target sequence within the cotton species genome to be amplified or copied to a detectable quantity. In general, amplification comprises temperature cycling of the genomic DNA sample in the presence of free nucleotides, a specific set of primers for the target DNA, and a polymerase, which allows the target DNA to be copied, i.e. amplified. [57] By comparing DNA genomic sequences of the two cotton species of interest, i.e. G. barbadense (GenBank Accession No. NC__008641 ) and G. hirsutum (GenBank Accession No. NC_007944), variable regions between the two species were detected and possible genetic markers to distinguish between these two cotton species were identified in these variable sequence regions. Utilizing primer software known to those skilled in the art, primers were identified and designed for the selected variable region(s) of the two cotton DNA sequences, allowing for the amplified PCR product to be identified as being from either G. barbadense or G. hirsutum. PCR primers were designed according to known criteria and PCR procedure may be conducted in commercially available instruments. Primers were designed to distinguish between the targeted sequences of the two species by exploiting the differences in at least one physical characteristic related to the targeted sequences. The "physical characteristic" utilized to distinguish between the two cotton species being either a detectable difference in length/size of the targeted sequence or a detectable DNA sequence variation (i.e. base substitution) specific for each of cotton species being analyzed. [58] Generally the primers designed for PCR amplification in event 130 utilize simple sequence repeats (SSR) or microsatellites which are polymorphic loci present in nuclear and organellar DNA (e.g. chloroplast DNA). Identifying regions comprising SSR's enables one set of primers to amplify a target sequence in either G. barbadense or G. hirsutum, with the target sequences from one species to the other being different in length due to the presence of varying number of SSR's found in the target sequence.

[59] In some embodiments, nested set primers were utilized to achieve appropriate amplification of the targeted variable region of the two cotton species. Again, these nested set primers were identified and designed utilizing primer software and the genomic sequences of the cotton species of interest using primer design methods known to those skilled in the art of primer design.

[60] Examples of primers utilized in some of the embodiments of the methods are given below. The primer sequences given below are only exemplary and are not meant to be limiting in the scope for distinguishing or verifying cotton fibers as being either the G. barbadense or G. hirsutum cotton species. [61] Exemplary primers were designed for an intergenic region, or space, between the petN and psbM genes of genomic DNA. Utilizing a sequence insertion of approximately 51 bases found in the G. hirsutum species which is lacking in the G. barbadense species. Primer 1 (Seq. ID. No. 1 ) and Primer 2 (Seq. ID. No. 2) were designed to target as sequence of 411 base pairs in length verses a 360 base pair sequence for the G. hirsutum species and the G. barbadense species, respectively. The sequences of this set of primers are the following:

Primer 1 GTTGG GG AG AAACTG ATCCA (Seq. ID. No. 1) Primer 2 AAAAAGGATTCAAATCTCACGTC (Seq. ID. No.2) [62] While the above example utilizes a variable region within an intron, it is also possible to utilize a variable region found within an exon or gene region. The targeted marker or sequence is also not limited to a variable region found in chloroplast DNA, but may also be located in nuclear genomic DNA, as well as mitochondrial DNA.

[63] The method 100 illustrated in FIG. 2 further comprises analyzing the PCR product(s) for a specific physical characteristic related to only one of the cotton species of interest in event 140. When the identifying physical characteristic is the length or size of the PCR product produced in event 130, techniques which allow for efficient size differentiation between the PCR products produced by one cotton species over another are utilized. Gel electrophoresis, both agarose and polyacrylamide gels, depending on the size range of the PCR product(s) produced, are capable of distinguishing the varying sizes of the PCR product produced by either DNA from mature cotton fibers of G. barbadense or G. hirsutum. For example, when utilizing primers 1 and 2 for PCR amplification, a 1.5% agarose gel, stained with DNA binding dye ethidium bromide is sufficient to resolve the size differences of the PCR products produced by G. barbadense or G. hirsutum. Running standard size marker controls on a gel with the PCR products produced from the methods enables the length (e.g. number of base pairs) of the PCR product to be determined. [64] In other embodiments, where the physical characteristic of the PCR products being analyzed in event 140 is size/length, a capillary electrophoresis device such as an AB1310 genetic analyzer can be utilized to determine the size of the PCR product(s) produced. For capillary electrophoresis, the length of the DNA amplicon produced by PCR is determined by extrapolating size data from an internal control ran with the sample of known size. In general, the size resolution of the capillary electrophoresis is capable of distinguishing between amplicons which differ by only about 1 base pair. The sensitivity of capillary electrophoresis enables in some embodiments the use of primers which target variable regions of the cotton species where the difference between the size of the G. barbadense amplicons and the G. hirsutum amplicons is less than about 10 base pairs but greater than about 1 base pair, and in some embodiments the size difference can be less than about 6 base pairs but greater than about 3 base pairs. Example 2 below shows the results of utilizing a target sequence which varies only by 6 base pairs between the two species.

[65] In event 150, the species of the analyzed cotton fibers is determined. This determination can be made by comparing the identified length of the PCR products to the theoretical and actual known size of the targeted and amplified sequence for G. barbadense or G. hirsutum. The PCR products from known G. barbadense or G. hirsutum DNA may also be run on a gel during electrophoresis to aid in identifying the species from which the sample originated. Once the PCR product length as been determined by either gel electrophoresis, capillary electrophoresis or other similar DNA sizing techniques, the analyzed sample can be identified and confirmed as a particular cotton species. [66] In other embodiments, the physical characteristic being analyzed is not length but a partial DNA sequence specific to a particular species. In this situation, PCR amplification may be performed in the presence of a non-primer detectable probe which specifically binds the PCR amplification product produced by one species and not the other. PCR primers are designed according to known criteria and PCR may be conducted in commercially available instruments. The probe is preferably a DNA oligonucleotide specifically designed to bind to the amplified PCR product. The probe preferably has a 5' reporter dye and a downstream 3' quencher dye covalently bonded to the probe which allows fluorescent resonance energy transfer. Suitable fluorescent reporter dyes include 6-carboxy-fluorescein (FAM), tetrachloro-β-carboxy-fluorescein (TET), 2,7-dimethoxy-4,5-dichloro-6-carboxy-fluorescein (JOE) and hexachloro- 6-carboxy-fluorescein (HEX). A suitable quencher dye is (4-(dimethy(amino)- phenyl)-azo)-benzoic acid (Dabcyl). These dyes are commercially available from Perkin-Elmer, Philadelphia, Pa. Detection of the PCR amplification product may occur at each PCR amplification cycle. At any given cycle during the PCR amplification, the amount of PCR product is proportional to the initial number of template copies. The number of template copies is detectable by fluorescence of the reporter dye. When the probe is intact, the reporter dye is in proximity to the quencher dye which suppresses the reporter fluorescence. During PCR, the DNA polymerase cleaves the probe in the 5'-3' direction separating the reporter dye from the quencher dye increasing the fluorescence of the reporter dye which is no longer in proximity to the quencher dye. The increase in fluorescence is measured and is directly proportional to the amplification during PCR. This detection system is now commercially available as the TaqMan® PCR system from Perkin-Elmer, which allows real time PCR detection. [67] A probe specific for the G. barbadense PCR product may be analyzed in the same or a separate PCR sample tube as the probe specific for the G. hirsutum PCR product. Utilizing real-time PCR, the signal from a probe corresponding to a specific cotton species will identify which cotton species has been collected. [68] Figure 3 is a flow chart illustrating generally a method 200 for authenticating or identifying an article as being manufactured with a specified cotton species. Usually verifying or authenticating an article occurs after the article has been introduced into a supply chain. Frequently, counterfeiters and forgers have the best access to articles after they are being shipped from the manufacturer/producer to wholesale or retail outlets or locations. With the methods described herein, a producer can track and authenticate articles or goods, allowing for better monitoring of when and where counterfeit goods are being replaced with forgeries or otherwise altered. When the article has entered a supply chain, a manufacturer or an authorized individual can collect a sample from the article at any desired point along the supply chain for authentication purposes. On other occasions, manufacturers deliberately replace higher quality raw materials (cotton fibers) with lower quality ones, in order to lower costs. However, by doing this, manufacturers not only violate labeling laws and licensing agreements, but also damage consumer confidence regarding brand name products.

[69] The method 200 comprises, at event 210, collecting mature cotton fibers from an article to be authenticated. Collecting a sample may involve scraping, cutting or dissolving a portion of the article to obtain a cotton fiber sample for analysis. The collecting of the sample may be carried out, for example, by cutting the article to remove mature cotton fibers from the article. The sample collecting in other embodiments may be achieved by tweezing, scraping, or abrading the article with appropriate sampling tools configured to remove a sufficient amount of cotton fibers or cotton lint for analysis. Collecting fiber samples may, for example, occur at any point along the supply or commerce chain where there is concern about or risk of introduction of counterfeit articles. [70] The method 200 for authenticating an article further comprises, in event 220, obtaining or extracting genomic DNA from the collected cotton fibers. Extraction, isolation or purification of the genomic DNA obtained in the sample may be required. A variety of nucleic acid extraction solutions have been developed over the years for extracting nucleic acid sequences from a sample of interest. See, for example, Sambrook et al. (Eds.) "Molecular Cloning", (1989) Cold Spring Harbor Press. Many such methods typically require one or more steps of, for example, a detergent-mediated step, a proteinase treatment step, a phenol and/or chloroform extraction step, and/or an alcohol precipitation step. Some nucleic acid extraction solutions may comprise an ethylene glycol-type reagent or an ethylene glycol derivative to increase the efficiency of nucleic acid extraction, while other methods use only grinding and/or boiling the sample in water. Other methods, including solvent-based systems and sonication, could also be utilized in conjunction with other extraction methods. [71] The authentication method 200 further comprises, in event 230, amplifying a specific region of the extracted genomic DNA by PCR based techniques. The procedures and techniques for PCR amplification of a targeted sequence are similar to those described above in the methods illustrated in FIG.2. There are numerous cultivars of both G, barbadense and G. hirsutum, which are utilized in the manufacturing of cotton textiles worldwide. The primers designed and selected in many embodiments enable a user to distinguish between the two species, G. barbadense or G. hirsutum independent of the type of cultivar or strain of the species. In other embodiments primers, may be selected and used for distinguishing other cotton species, such as G. arboreum and G. herbaceum. [72] The method 200 for authenticating an article further comprises, in event 240, analyzing the PCR product for a specific physical characteristic, such as length of the PCR product or a specific DNA sequence associated with a specific cotton species (e.g. G. barbadense or G. hirsutum). Analyzing the PCR product produced is generally the same as described above in the methods illustrated in FIG.2. Numerous ELS cultivars have been shown to produce the identical or very similar length PCR products, which are easily identifiable as G. barbadense and are distinguishable from various Upland cultivars of G. hirsutum. Example 1 below describes the identification results of various cultivars in more detail. When the aim is to authenticate a cotton textile as either being G. barbadense or G. hirsutum cotton, a simplistic analysis is preferred. In many instances, a textile shipment maybe on a stringent time table and a quick but accurate test is required. Utilizing primers which recognize a difference in length of a target sequence between the two species fulfills the desire for a quick and reliable verification test.

[73] In event 250, the results of the analysis of the collected sample are reviewed and a query or determination is made as to whether or not a physical characteristic (i.e. known PCR product length) for a particular species was detected in the sample. If the physical characteristic associated with a particular cotton species nucleic acid is not found or not detected in the collected sample of the textile article at event 250, the conclusion at event 270 from the analysis is that the article is not made from that particular cotton species and may have been tampered with or substituted. If the physical characteristic associated with the cotton species is detected in the sample at event 250, then the article is verified in event 260 as being authentic. [74] If a determination is made in event 270 that an article is not authentic, a different, earlier point in the supply or commerce chain may be selected and events 210 through 250 may be repeated. Thus an article from an earlier point in the supply chain would be selected, cotton fibers collected, and analyzed. If it is again determined that the article is not authentic or has been otherwise tampered with, then events 210-250 may be repeated with an article selected from yet an earlier point in the supply chain. In this manner, the time and/or location of tampering or counterfeit substitution of the textile article may be located. [75] Kits for verifying cotton fibers and authenticating articles of interest incorporate the methods described herein. The kits may comprise, for example, a container of the nucleic acid extraction buffer, and a sample tube for holding a collected mature cotton fiber sample of the item or article to be authenticated. The kits may further comprise at least one primer set configured to produce amplified PCR fragments from both ELS and Upland genomic DNA. The kits may include a collection tool for taking a sample of the labeled article for transfer to the sample tube. The kits may also further comprise a portable electrophoretic device (e.g. gel apparatus or capillary electrophoresis system) for analyzing PCR products. The kits may also include an internal control for fragment size comparison for capillary analysis. [76] By way of example and not limitation, the collection tool of the kit may comprise a blade or scissors for cutting a piece of the article, or the like. The sample tube of the kit may include a sealable vial or Eppendorf tube, and may contain solvent or solution for extraction of the nucleic acids (e.g. DNA) from the sample taken from the textile article.

[77] The kit may comprise primers and/or probes as well as solutions appropriate for PCR analysis. The kit may further comprise a small PCR instrument for analysis of the extracted nucleic acids from the mature cotton fibers. [78] The capillary electrophoresis device of the kit may comprise an internal control for detecting the fragiment size of the amplified PCR product(s). [79] The illustrative kits thus provide a convenient, portable system for practicing the methods of the invention. [80] Illustrative methods for authenticating textile articles utilizing mature cotton fibers are provided in the following Examples.

EXAMPLES

[81] The following preparations and examples are given to enable those skilled in the art to more clearly understand and to practice the present methods. They should not be considered as limiting the scope of the invention, but merely as being illustrative and representative thereof.

EXAMPLE I [82] Differentiation of ELS and upland cotton fibers. Mature cotton fibers of 25 known cultivars from various regions around the world were tested with cotton specific SSRs primers, among which 15 samples are ELS (G. barbadense) and 10 samples are Upland G. hirsutum cotton.

Table 1. List of known cotton cultivars. Samples are raw mature cotton fibers.

[83] For DNA extraction, 10-15mg of mature cotton fibers were weighed and put into 1.5ml Eppendorf tubes. DNA extraction protocols are derived from standard molecular biology DNA extraction procedures, which can be easily accomplished by people skilled in the art. Sample tubes were then vortexed and centrifuged briefly to be used as PCR templates. For DNA amplification, a PCR master mix containing 10ul of amplification buffer, 0.5ul of 1OuM forward and reverse primers, 5ul of water, and 4ul of DNA extracts were put into 0.2ml thin wall PCR tubes and run for 40 cycles for DNA amplification. PCR anneal temperature and duration times were adjusted according to individual primer sets, and anyone familiar in the art of PCR will be able to design their own scheme. The primer set was designed in such way that ELS cotton DNA produced 360bp amplicons and the Upland cotton sample(s) produced 411 bp amplicons. The primer set comprised of Primer 1 and Primer 2, Primer 1 (Seq. ID. No. 1 ) having the sequence of GTTGGGGAGAAACTGATCCA, and Primer 2 (Seq. ID. No. 2) having the sequence of AAAAAGGATTCAAATCTCACGTC. These primers are only exemplary to demonstrate the instant invention.

[84] Those with ordinary skill in the art of designing primer sets would be able to produce a plurality of different primer sets other than those described above, which can distinguish between the two types of cotton analyzed here, given the genomic sequences of G. barbadense (GenBank Accession No. NC_008641) and G. hirsutum (GenBank Accession No. NC_007944). These specific primers are merely exemplary. [85] The PCR products were then analyzed by gel electrophoresis with a 1.5% agarose gel and stained with DNA binding dye ethidium bromide. Lengths of DNA fragments were calculated by extrapolating size data from standard molecular markers run on the same gel. Figure 4 is a photograph of an agarose gel comprising electrophoretic results of the 25 samples described in Table 1. The gel electrophoresis photograph shows that G. barbadense samples #1-15 produced the same sized PCR DNA fragments (360 bp), while the PCR fragments produced by G. hirsutum samples #16-25 are of a larger size (411 bp). DNA fragment lengths were calculated by extrapolating size data from molecular marker run on the same gel.

[86] This example demonstrates that ELS cotton fibers can be identified and authenticated as not being upland cotton fibers and visa versa, by standard gel electrophoresis techniques.

EXAMPLE Il

[87] Differentiation of ELS and upland cotton fabrics. Fabrics made of either ELS or Upland cotton fibers were tested for genetic identification. For DNA extraction, 10-15mg of fabrics were weighted and put into 1.5ml Eppendorf tubes. DNA extraction protocols are derived from standard molecular biology DNA extraction procedures, which can be accomplished by those skilled in the art. Sample tubes were then vortexed and centrifuged briefly to be used as PCR templates. For DNA amplification, a PCR master mix containing 10ul of amplification buffer, 0.5ul of 1OuM forward and reverse primers, 5ul of water, and 4ul of DNA extracts were put into 0.2ml thin wall PCR tubes and run for 40 cycles for DNA amplification. PCR annealing temperature and duration times were adjusted according to individual primer sets, and any one familiar in the art of PCR will be able to design their own scheme. The primer set was designed to take advantage of a 5 base pair deletion in a variable region between the psbZ- trnG intergenic space in the G. hirsutum genome versus the G. barbadense genome, in such way that ELS (G. barbadense) cotton DNA produced 237bp amplicons and the Upland (C-J. hirsutum) produced 232bp amplicons. [88] After PCR amplification, the PCR products were analyzed by capillary electrophoresis using an ABI 310 genetic analyzer. DNA fragment sizes were calculated by extrapolating size data from utilizing an internal control included with the capillary electophoretic sample(s). [89] As the capillary electrophoresis electrogram shows, Sample #1's fragment is 237bp in length, and Sample #1 is made of ELS cotton fibers; while Sample #2's fragment is 232bp in length, and Sample #2 is made of Upland cotton fibers. [90] Figure 4 is an electrogram of a capillary electrophoresis analysis of PCR products produced from DNA extracted from cotton fibers collected from textile materials where the cotton species utilized to manufacture the item was known. Sample #1 , fibers from a red cloth are identified and confirmed as ELS cotton; Sample #2, the T shirt cotton fibers, were determined to be from Upland cotton. The theoretical and detectable? size for the amplified product from ELS cotton is 237bp and the amplified product from upland cotton is 232bp. This example demonstrates that cotton textile articles can be identified as made from ELS or Upland cotton species.

[91] While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process step or steps, to the objective spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

Claims

CLAIMSWhat is claimed is:

1. A method for identifying cotton species comprising: collecting cotton fibers from a sample; extracting genomic DNA from the cotton fibers collected; amplifying said DNA with PCR-based techniques; and analyzing the amplified product to distinguish between a first cotton species and a second cotton species.

2. The method of claim 1 wherein the first cotton species is G. barbadense.

3. The method of claim 2 wherein the second cotton species is G. hirsutum.

4. The method of claim 1 wherein the sample is a textile item selected from a group consisting of raw cotton, ginned cotton, yarn blended with cotton, and fabric blended with cotton.

5. The method of claim 1 wherein the amplifying further comprises using at least one set of specific primers which target sequence polymorphism between the first cotton species and the second cotton species.

6. The method of claim 3 wherein amplifying further comprises using at least one set of specific primers which target sequence polymorphism between G. barbadense and G. hirsutum.

7. The method of claim 5 wherein the targeted sequence of the first cotton species and the second cotton species have different detectable physical characteristics.

8. The method of claim 7 wherein the detectable physical characteristic of the targeted sequence is a different sequence length for the first cotton species compared the second cotton species.

9. The method of claim 1 wherein the genomic DNA extracted from the cotton fibers comprises chloroplast DNA.

10. The method of claim 1 wherein the genomic DNA extracted from the cotton fibers comprises mitochondrial DNA.

11. The method of claim 1 wherein the genomic DNA extracted from the cotton fibers comprises nuclear DNA.

12. The method of claim 6, wherein the detectable physical characteristic of the targeted sequence is a different sequence composition.

13. The method of claim 6 wherein the detectable physical characteristic of the targeted sequence is a different sequence length.

14. The method of claim 13 wherein analyzing the amplified product to distinguish between a first cotton species and a second cotton species further comprises the use of a hybridizing probe specific for the target sequence of either the first or second cotton species.

15. The method of claim 13 wherein the analyzing of the amplified products comprises detecting the size of the amplified products by gel electrophoresis.

16. The method of claim 13 wherein the analyzing of the amplified products comprises detecting the size of the amplified products by capillary electrophoresis.

17. The method of claim 13 wherein the analyzing of the amplified products comprises detecting the size of the amplified products by real-time PCR.

18. The method of claim 1 where the first cotton species is selected from the group consisting of G. barbadense, G. hirsutum, G. arboreum or G. herbaceum.

19. The method of claim 1 where the second cotton species is selected from the group consisting of G. barbadense, G. hirsutum, G. arboreum or G. herbaceum.

20. A method for verifying a textile article comprising: collecting cotton fibers from a textile item; extracting genomic DNA from the cotton fibers collected; amplifying said DNA with PCR-based techniques; analyzing the amplified product to distinguish between a first cotton species and a second cotton species; identifying the cotton fibers from the textile item as belonging to the first cotton species or to the second cotton species; and determining from the identified cotton species if the textile article is authentic.

21. The method of claim 20 wherein the genomic DNA extracted from the cotton fibers comprises nuclear DNA.

22. The method of claim 20 wherein the first cotton species is G. barbadense.

23. The method of claim 22 wherein the second cotton species is G. hirsutum.

24. The method of claim 20 wherein the amplifying further comprises using at least one set of specific primers which target sequence polymorphism between the first cotton species and the second cotton species.

25. The method of claim 23 wherein amplifying further comprises using at least one set of specific primers which target sequence polymorphism between G. barbadense and G. hirsutum.

26. The method of claim 25 wherein the targeted sequence of the first cotton species and the second cotton species have different detectable physical characteristics.

27. The method of claim 25 wherein the detectable physical characteristic of the targeted sequence is a different sequence composition.

28. The method of claim 26 wherein the detectable physical characteristic of the targeted sequence is a different sequence length for the first cotton species compared the second cotton species.

29. The method of claim 20 wherein the genomic DNA extracted from the cotton fibers comprises chloroplast DNA.

30. The method of claim 25 wherein the detectable physical characteristic of the targeted sequence is a different sequence length.

31. The method of claim 30 wherein analyzing the amplified product to distinguish between a first cotton species and a second cotton species further comprises the use of a hybridizing probe specific for the target sequence of either the first or second cotton species.

32. The method of claim 30 wherein the analyzing of the amplified products comprises detecting the size of the amplified products by gel electrophoresis.

33. The method of claim 30 wherein the analyzing of the amplified products comprises detecting the size of the amplified products by capillary electrophoresis.

34. A kit for determining the cotton species utilized in a textile item, comprising: a sample collection tube for placing mature cotton fibers obtained from the textile item; a DNA extraction solution; at least one set of specific primers to distinguish between a first cotton species and a second cotton species; and a PCR amplification buffer solution.

35. The kit of claim 34 further comprising an PCR instrument.

36. The kit of claim 34 further comprising an internal control for comparing the size of the amplified product.

37. The kit of claim 36 further comprising a capillary electrophoresis device.