Methods for genotyping mature cotton fibers and textiles
Methods for distinguishing between cotton cultivars of a specific 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 cultivar of a particular cotton species utilized in the textile or cotton material of interest.
1. A method for identifying cotton cultivar comprising:
collecting cotton fibers from a manufactured textile;
extracting genomic DNA from the cotton fibers collected, wherein the genomic DNA extracted from the mature cotton fibers comprises chloroplast DNA;
amplifying said chloroplast DNA with PCR-based techniques using one or more primer sets and thereby producing one or more amplicons;
generating a variable amplicon length polymorphism profile of said chloroplast DNA;
comparing the variable amplicon length polymorphism profile for the mature cotton fibers from the manufactured textile with a known variable amplicon length
and thereby identifying at least one cotton cultivar from the manufactured textile.
2. The method of claim 1 , wherein first cotton cultivar is from the species G. barbadense .
3. The method of claim 2 , wherein at least one cotton cultivar identified is from the species G.hirsutum.
4. The method of claim 1 , wherein the amplifying comprises:
using at least one set of specific primers which targets length polymorphisms between a first cotton cultivar and the second cotton cultivar.
5. The method of claim 2 , wherein the amplifying comprises:
using at least one set of specific primers targeting polymorphic sequences differing in length between each of at least three G. barbadense cultivars.
6. The method of claim 4 , wherein the polymorphic sequence of the cotton cultivars targeted by the primers differ in another detectable physical characteristic in addition to the difference in sequence length.
7. The method of claim 6 , wherein the additional detectable physical characteristic comprises a difference in a nucleotide composition.
8. The method of claim 1 , wherein the genomic DNA extracted from the mature cotton fibers comprises mitochondrial DNA.
9. The method of claim 1 , wherein the genomic DNA extracted from the mature cotton fibers comprises nuclear DNA.
10. The method of claim 1 , wherein the analyzing of the one or more amplified products comprises detecting the size of the one or more amplified products by gel electrophoresis.
11. The method of claim 1 , wherein the analyzing of the one or more amplified products comprises detecting the size of the one or more amplified products by capillary electrophoresis.
12. The method of claim 1 , wherein the analyzing of the amplified chloroplast DNA comprises detecting the size of the one or more amplicons amplified by real time PCR.
13. The method of claim 4 , wherein the first cotton cultivar and the second cotton cultivar are from a cotton species is selected from the group consisting of G. barbadense, G. hirsutum, G. arboreum , or G. herbaceum .
14. A method for verifying the authenticity of a manufactured textile comprising:
collecting mature cotton fibers from the manufactured textile;
extracting genomic DNA from the comprising chloroplast DNA from the mature cotton fibers collected;
amplifying said chloroplast DNA with PCR-based techniques using one or more primer sets producing one or more amplified products forming a variable amplicon length polymorphism profile for the mature cotton fibers from the textile article;
comparing the variable amplicon length polymorphism profile from the mature cotton fibers with one or more known variable amplicon length polymorphism profiles;
identifying the mature cotton fibers from the manufactured textile as belonging to one or more cotton cultivars; and
thereby determining whether the manufactures textile is authentic or counterfeit.
15. The method of claim 14 , wherein at least one cotton cultivar identified is from the species G. barbadense.
16. The method of claim 14 , wherein at least one of the cotton cultivars identified is from the species is G. hirsutum .
17. The method of claim 14 , wherein the amplifying further comprises;
using at least one set of specific primers which targets a sequence polymorphism between the first cotton cultivar and the second cotton cultivar.
18. The method of claim 15 , wherein amplifying further comprises:
using two or more sets of specific primers which targets sequence length polymorphisms between G. barbadense cultivars.
19. The method of claim 18 , wherein the targeted sequence of the first G. barbadense cultivar and the second G. barbadense cultivar have difference in a detectable physical characteristic in addition to the sequence length polymorphisms.
20. The method of claim 19 , wherein the difference in a detectable physical characteristic of the targeted sequences comprises a difference in a nucleotide composition.
21. The method of claim 14 , wherein the genomic DNA extracted from the cotton fibers further comprises nuclear DNA.
22. The method of claim 14 , wherein the analyzing of the amplified products comprises detecting the size of the amplified products by gel electrophoresis.
23. The method of claim 14 , wherein the analyzing of the amplified products comprises detecting the size of the amplified products by capillary electrophoresis.
24. The method of claim 1 , wherein the manufactured textile comprises raw cotton, ginned cotton, yarn blended with cotton and or a fabric blended with cotton.
25. The method of claim 1 , wherein the amplifying if said chloroplast DNA comprises using a nested set of primers.
26. The method of claim 14 , wherein the amplifying of said chloroplast DNA comprises using a nested set of primers.
27. The method of claim 26 , wherein the cotton cultivar identified is from a cotton species selected from the group consisting of G. barbadense, G hirsutum, G. arboretum , or G. herbaceum .
28. The method of claim 14 , wherein at least one of the cotton cultivars identified is G. arboretum or G. herbaceum .
29. The method of claim 14 , wherein the one or more amplified products are produced by real-time PCR.
30. The method of claim 29 , further comprising quantifying one or more of the amplified products by real-time PCR.
31. The method of claim 12 , further comprising quantifying one or more of the amplified products by real-time PCR.