Cry1f and cry1ac transgenic cotton lines and event-specific identification thereof

Abstract

This invention relates to plant breeding and the protection of plants from insects. More specifically, this invention includes novel transformation events of cotton plants comprising one or more polynucleotide sequences, as described herein, inserted into specific site(s) within the genome of a cotton cell. In highly preferred embodiments, said polynucleotide sequences encode “stacked” Cry1F and Cry1Ac lepidopteran insect inhibitory proteins. However, the subject invention includes plants having single cry1F or cry1Ac events, as described herein. Additionally, the invention is related to cotton plants derived from that transformation event and to assays for detecting the presence of the event in a sample. More specifically, the present invention provides DNA and related assays for detecting the presence of certain insect-resistance events in cotton. The assays are based on the DNA sequences of recombinant constructs inserted into the cotton genome and of the genomic sequences flanking the insertion sites. These sequences are unique. Based on these insert and border sequences, event-specific primers were generated. PCR analysis demonstrated that these cotton lines can be identified in different cotton genotypes by analysis of the PCR amplicons generated with these event-specific primer sets. Thus, these and other related procedures can be used to uniquely identify these cotton lines. Kits and conditions useful in conducting the assays are also provided. These materials and methods can also be used to assist breeding programs to further develop traits in cotton.

1 . A transgenic cotton plant comprising an insert that disrupts a genomic sequence selected from the group consisting of
 a. a first DNA sequence comprising a 5′ end comprising nucleotides 1-2074 of SEQ ID NO:1, a first interior segment comprising SEQ ID NO:15, and a 3′ end comprising nucleotides 12,749-15,490 of SEQ ID NO:1, and   b. a second DNA sequence comprising a 5′ end comprising nucleotides 1-527 of SEQ ID NO:2, a second interior segment comprising SEQ ID NO:16, and a 3′ end comprising nucleotides 8,901-9,382 of SEQ ID NO:2.   
     
     
         2 . A cotton plant comprising at least one insert that disrupts a genomic sequence selected from the group consisting of
 a. a first DNA sequence comprising a 5′ end comprising nucleotides 1-2074 of SEQ ID NO:1, and a 3′ end comprising nucleotides 12,749-15,490 of SEQ ID NO:1; and   b. a second DNA sequence comprising a 5′ end comprising nucleotides 1-527 of SEQ ID NO:2, and a 3′ end comprising nucleotides 8,901-9,382 of SEQ ID NO:2.   
     
     
         3 . The plant of  claim 1  wherein said insert is a non-cry1F and non-cry1Ac insert. 
     
     
         4 . The plant of  claim 2  wherein said insert is a non-cry1F and non-cry1Ac insert. 
     
     
         5 . A method for targeted homologous recombination, said method comprising targeting a genomic cotton sequence for insertion of a transgene genetic insert, and introducing said transgene genetic insert into said genomic cotton sequence, wherein said genomic sequence is selected from the group consisting of
 a. a first DNA sequence comprising a 5′ end comprising nucleotides 1-2074 of SEQ ID NO:1, and a 3′ end comprising nucleotides 12,749-15,490 of SEQ ID NO:1; and   b. a second DNA sequence comprising a 5′ end comprising nucleotides 1-527 of SEQ ID NO:2, and a 3′ end comprising nucleotides 8,901-9,382 of SEQ ID NO:2.   
     
     
         6 . A seed of the plant of  claim 1 , wherein said seed comprises said insert. 
     
     
         7 . A seed of the plant of  claim 2 , wherein said seed comprises said insert.