Soybean ROD1 gene sequences and uses thereof
The present invention relates to soybean ROD1 nucleic acid sequences and proteins and the use thereof to create plants with increased levels of C18:1 and reduced levels of saturated fatty acids in the seeds.
1. A method for determining the presence or absence of a knock-out allele of a ROD1 gene in a biological sample, comprising
providing genomic DNA from said biological sample, and
analyzing said genomic DNA for the presence of at least one molecular marker linked to said knock-out allele of the ROD1 gene by
amplifying at least a portion of said genomic DNA in a polymerase chain reaction (PCR) assay, wherein the portion comprises the molecular marker, or
contacting the genomic DNA with a nucleic acid probe, under high stringency conditions that allow hybridization of the probe with a portion of the genomic DNA encoding the molecular marker;
wherein said knock-out allele of the ROD1 gene encodes a protein having at least 90% sequence identity to SEQ ID No. 3 or SEQ ID No. 6, and
wherein the molecular marker is a stop codon or a splice site mutation in said knock-out allele of the ROD1 gene,
wherein the at least one molecular marker comprises a stop codon mutation at position 2947-2949, 3049-3051, 3088-3090, 3495-3497, 3513-3515, 3519-3521, 3600-3602, 3615-3617, 4410-4412, 4416-4418, 4428-4430, and/or 4455-4457 of SEQ ID NO: 1, or at position 3013-3015, 3103-3105, 3142-3144, 3530-3532, 3548-3550, 3554-3556, 3635-3637, 4077-4079, 4191-4193, 4197-4199, 4209-4211, 4236-4238, and/or 4380-4382 of SEQ ID NO: 4, or
wherein the at least one molecular marker comprises a splice site mutation at position 3264, 3265, 3479, 3480, 3617, 3618, 4298, and/or 4299 of SEQ ID NO: 1, or position 3318, 3319, 3514, 3515, 3652, 3653, 4079, and/or 4080 of SEQ ID NO: 4.
2. The method of claim 1 , wherein the at least one molecular marker comprises a stop codon mutation at position 2947-2949, 3049-3051, 3088-3090, 3495-3497, 3513-3515, 3519-3521, 3600-3602, 3615-3617, 4410-4412, 4416-4418, 4428-4430, and/or 4455-4457 of SEQ ID NO: 1.
3. The method of claim 1 , wherein the at least one molecular marker comprises a stop codon mutation at position 3013-3015, 3103-3105, 3142-3144, 3530-3532, 3548-3550, 3554-3556, 3635-3637, 4077-4079, 4191-4193, 4197-4199, 4209-4211, 4236-4238, and/or 4380-4382 of SEQ ID NO: 4.
4. The method of claim 1 , wherein the at least one molecular marker comprises a splice site mutation at position 3264, 3265, 3479, 3480, 3617, 3618 4298, and/or 4299 of SEQ ID NO: 1.
5. The method of claim 1 , wherein the at least one molecular marker comprises a splice site mutation at position 3318, 3319, 3514, 3515, 3652, 3653, 4079, and/or 4080 of SEQ ID NO: 4.
6. The method of claim 1 , wherein the PCR assay is performed using a pair of primers configured to specifically amplify said knock-out allele of a ROD1gene.
7. The method of claim 6 , wherein each primer is capable of specifically binding to
a sequence within the 5′ or 3′ flanking region of the knock-out allele of the ROD1 gene,
a sequence of the knock-out allele of the ROD1 gene which encodes the molecular marker, or
a sequence spanning the joining region between the 3′ or 5′ flanking region, and the region encoding the molecular marker, of the knock-out allele of the ROD1 gene;
wherein the knock-out allele of the ROD1 gene comprises a mutant allele having a stop codon mutation at position 2947-2949, 3049-3051, 3088-3090, 3495-3497, 3513-3515, 3519-3521, 3600-3602, 3615-3617, 4410-4412, 4416-4418, 4428-4430, and/or 4455-4457 of SEQ ID NO: 1.
8. The method of claim 6 , wherein each primer is capable of specifically binding to
a sequence within the 5′ or 3′ flanking region of the knock-out allele of the ROD1 gene,
a sequence of the knock-out allele of the ROD1 gene which encodes the molecular marker, or
a sequence spanning the joining region between the 3′ or 5′ flanking region, and the region encoding the molecular marker, of the knock-out allele of the ROD1 gene;
wherein the knock-out allele of the ROD1 gene comprises a mutant allele having a splice site mutation at position 3264, 3265, 3479, 3480, 3617, 3618 4298, and/or 4299 of SEQ ID NO: 1.
9. The method of claim 6 , wherein each primer is capable of specifically binding to
a sequence within the 5′ or 3′ flanking region of the knock-out allele of the ROD1 gene,
a sequence of the knock-out allele of the ROD1 gene which encodes the molecular marker, or
a sequence spanning the joining region between the 3′ or 5′ flanking region, and the region encoding the molecular marker, of the knock-out allele of the ROD1 gene;
wherein the knock-out allele of the ROD1 gene comprises a mutant allele having a stop codon mutation at position 3013-3015, 3103-3105, 3142-3144, 3530-3532, 3548-3550, 3554-3556, 3635-3637, 4077-4079, 4191-4193, 4197-4199, 4209-4211, 4236-4238, and/or 4380-4382 of SEQ ID NO: 4.
10. The method of claim 6 , wherein each primer is capable of specifically binding to
a sequence within the 5′ or 3′ flanking region of the knock-out allele of the ROD1 gene,
a sequence of the knock-out allele of the ROD1 gene which encodes the molecular marker, or
a sequence spanning the joining region between the 3′ or 5′ flanking region, and the region encoding the molecular marker, of the knock-out allele of the ROD1 gene;
wherein the knock-out allele of the ROD1 gene comprises a mutant allele having a splice site mutation at position 3318, 3319, 3514, 3515, 3652, 3653, 4079, and/or 4080 of SEQ ID NO: 4.