Plant genomic DNA flanking SPT event and methods for identifying SPT event
View Patent ↗Compositions and methods related to transgenic plants comprising seed production technology are provided. Specifically, maize plants having a E6611.32.1.38 event which confers seed production technology are provided. The plant harboring the E6611.32.1.38 event at the recited chromosomal location comprises the genomic/transgene junctions described. The plant genomic DNA flanking the integrated E6611.32.1.38 event can be used to design assays that will be specific for the E6611.32.1.38 event. The characterization of the genomic insertion site of the E6611.32.1.38 event provides for an enhanced breeding efficiency and enables the use of molecular markers to track the transgene insert in the breeding populations and progeny thereof. Various methods and compositions for the identification, detection, and use of the maize E6611.32.1.38 event are provided.
1. A method for identifying event E6611.32.1.38 in a biological sample, comprising detecting a E6611.32.1.38-specific region with a probe or first primer which specifically recognizes a sequence within the T-DNA insert of SEQ ID NO: 1, and a second probe or primer which specifically recognizes a sequence within either flanking sequence of SEQ ID NO: 1.
2. The method of claim 1 , further comprising amplifying a DNA fragment from a nucleic acid present in said biological sample using a polymerase chain reaction with at least two primers, wherein said first primer recognizes a sequence within the T-DNA insert of SEQ ID NO: 1, and a second primer recognizes a sequence within either flanking sequence of SEQ ID NO: 1.
3. The method of claim 2 , wherein said first primer is selected from the group consisting of SEQ ID NOS: 7, 9, 10, 11, 12, 13, and 4, and said second primer is selected from the group consisting of SEQ ID NOS: 6, 8, 15, 16, 17, 18 and 19.
4. A method of detecting the presence of event E6611.32.1.38 or progeny thereof in a biological sample, comprising:
(a) extracting a DNA sample from said biological sample;
(b) providing a pair of DNA primers, wherein one of said primers recognizes a sequence within either flanking sequence of SEQ ID NO: 1, and the second of said primers recognizes a sequence within the T-DNA insert of SEQ ID NO: 1;
(c) providing DNA amplification reaction conditions;
(d) performing a DNA amplification reaction, thereby producing a DNA amplicon molecule; and
(e) detecting said DNA amplicon molecule, wherein the detection of said DNA amplicon molecule in said DNA amplification reaction indicates the presence of event E6611.32.1.38.
5. An isolated DNA molecule comprising any one of the amplicons produced by the method of claim 4 .
6. A method of detecting the presence of DNA corresponding to the E6611.32.1.38 event in a sample, the method comprising:
(a) contacting the sample comprising DNA with a polynucleotide probe that hybridizes under stringent hybridization conditions with DNA from event E6611.32.1.38 and does not hybridize under said stringent hybridization conditions with a non-E6611.32.1.38 plant DNA;
(b) subjecting the sample and probe to stringent hybridization conditions; and
(c) detecting hybridization of the probe to the DNA, wherein detection of hybridization indicates the presence of the E6611.32.1.38 event.
7. An isolated DNA nucleotide primer sequence comprising a sequence selected from the group consisting of: SEQ ID NOS: 3, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and the complement of each of said sequences.
8. An isolated DNA molecule comprising a junction sequence amplified by the primer pair of SEQ ID NO: 6 and 7; or SEQ ID NO: 8 and 9; or SEQ ID NO: 14 and 15.
9. A method for screening seeds for the presence of DNA corresponding to event E6611.32.1.38, comprising:
(a) extracting a DNA sample from each of said seeds;
(b) contacting each DNA sample with a polynucleotide probe that hybridizes under stringent hybridization conditions with DNA from event E6611.32.1.38 and does not hybridize under said stringent hybridization conditions with DNA from a non-E6611.32.1.38 plant;
(c) subjecting the sample and probe to stringent hybridization conditions;
(d) detecting hybridization of the probe to the DNA sample, wherein detection of hybridization indicates the presence of the E6611.32.1.38 event; and
(e) separating the seeds based on the presence or absence of the E6611.32.1.38 event.
10. A pair of isolated DNA sequences, each comprising at least ten nucleotides and which when used together in a DNA amplification procedure will produce an amplicon diagnostic for event E6611.32.1.38.
11. The pair of isolated DNA sequences of claim 10 wherein the first member of the pair is chosen from the group consisting of SEQ ID NOS: 7, 9, 10, 11, 12, 13, and 14, and said second member of the pair is selected from the group consisting of SEQ ID NOS: 6, 8, 15, 16, 17, 18 and 19.
12. The isolated DNA molecule of claim 5 , comprising an amplicon which has the sequence of SEQ ID NO: 25, 28 or 32.