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Development of novel germplasm using segregates from transgenic crosses

a technology of transgenic crosses and germplasm, applied in the field of plant breeding, can solve the problems of notoriously sensitive to the concentration and purity of the starting template, and requires substantial time and effort to achieve, and achieves the effects of improving the breeding germplasm, reducing the risk of infection, and improving the quality of the germplasm

Active Publication Date: 2011-06-07
MONSANTO TECH LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0014]For evaluating and breeding traits that confer glyphosate tolerance either by themselves, or in vector stacks with other traits of value to producers, the use of DNA technology, in particular PCR zygosity technology based on flanking sequences for the 40-3-2 event will increase the efficiency of the selection process, and a breeder's ability to build upon the existing germplasm base that already contains event 40-3-2 as well as other useful traits. This is currently the only method that can demonstrate the absence of all identified genetic elements of the 40-3-2 event in breeding populations in a relatively inexpensive manner.

Problems solved by technology

Nevertheless, it is notoriously sensitive to the concentration and purity of the starting template DNA, among other variables.
Screening for loss of a transgene by genetic segregation in progeny is another method that is widely known but requires substantial time and effort to achieve.

Method used

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  • Development of novel germplasm using segregates from transgenic crosses
  • Development of novel germplasm using segregates from transgenic crosses

Examples

Experimental program
Comparison scheme
Effect test

example 1

f 40-3-2 lacking inserted DNA

[0049]An elite soybean cultivar comprising ROUNDUP READY® event 40-3-2 is crossed with a conventional (non-transgenic) cultivar, and segregation of traits and the transgene is followed by PCR zygosity testing. The presence of 5′ or 3′ junction sequences may be followed as shown schematically in FIG. 1. The PCR assay may be either singleplex, or multiplex. An internal quantitation control may be included. In the case of the 40-3-2 event, the inserted DNA consists of, in the 5′ to 3′ direction, a functional insert, an intervening (rearranged) genomic DNA, a non-functional 72 bp fragment of the gene encoding CP4 EPSPS, and additional genomic sequence. Referring to FIG. 1, use of primer set A-B-C on template DNA derived from progeny of a cross of soybean event 40-3-2 with another plant, for instance, allows one to positively differentiate between progeny that are homozygous for the inserted DNA, homozygous for the inserted DNA, or lack the inserted DNA. PCR ...

example 2

progeny of 40-3-2 that comprose a different glyphosate tolerance transgene, and do not contain the functional 40-3-2 insertion

[0055]An elite soybean cultivar comprising ROUNDUP READY® event 40-3-2 may be crossed with a soybean cultivar comprising a different transgenic event that confers glyphosate tolerance, and segregation of the two transgenic inserts and additional agronomic traits is followed. The PCR-based zygosity test for sequences specific to event 40-3-2 is performed as described in Example 1. Similar zygosity testing may optionally be performed to identify progeny homozygous or hemizygous for the other transgenic event or events. Progeny are screened by the PCR-based zygosity test for loss of the 40-3-2 insertion, and selected for glyphosate resistance and other traits of interest.

[0056]The methods used to identify heterozygous from homozygous progeny containing 40-3-2 insertion DNA are described in a zygosity assay for which examples of conditions are described in Table ...

example 3

n of a GA21 event-containing corn line - segregant analysis

[0061]The present invention may be applied to corn breeding. An inbred corn line comprising event GA21 was crossed to another inbred line comprising event NK603, and segregation of progeny was followed in order to efficiently identify progeny that lack sequences associated with the GA21 event, comprise the NK603 event, and exhibit other DNA markers of the original parent line that comprises GA21.

[0062]Conversion of the GA21 line was performed by means of a multi-tiered marker-assisted breeding approach of event-specific PCR-based assays, linked-marker PCR and Southern blot analysis, to confirm the presence of event NK603-related DNA sequences and absence of event GA21-related DNA sequences. Following multiple backcrosses, we confirmed the presence of the NK603 event, and of DNA markers associated with the genetic background and agronomic qualities of the original parent that comprises GA21.

[0063]The recurrent parent corn inb...

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Abstract

This invention provides a method for the development of novel plant germplasm using segregates from a transgenic line combined with PCR-based zygosity testing and, optionally, Southern blot analysis.

Description

[0001]This application claims benefit of U.S. Provisional application Ser. No. 60 / 703,917 filed Jul. 29, 2005, the entire contents of which are incorporated by reference herein.FIELD OF THE INVENTION[0002]The invention in the field of plant breeding provides a method for the development of plant germplasm using segregates from a transgenic line. A method of zygosity testing to evaluate for the absence of a transgene is also disclosed.BACKGROUND OF THE INVENTION[0003]Widely planted crop germplasm often represents the most elite lines containing a combination of the yield, agronomic, and pest and disease resistance traits most desired by growers. Because of its combination of elite traits, this germplasm may serve to generate commercially available seed, and may also be used as a source or future plant breeding efforts. In some cases, this germplasm may often comprise a transgenic trait in addition to the elite traits it exhibits. Thus, there exists a need in the field of plant breedi...

Claims

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Application Information

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Patent Type & Authority Patents(United States)
IPC IPC(8): A01H5/00C12N15/82C12N5/04
CPCC12N15/8209A01H1/02A01H1/04C12N15/8275
Inventor ARNEVIK, CINDY L.DOBERT, RAYMONDZENG, QINGYILISTELLO, JENNIFERHECK, GREGORY R.SOTERES, JOHNWU, KUNSHENG
Owner MONSANTO TECH LLC
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