CONSERVATISM ON THE RUN (& Other Special Topics)

5. The method of claim 4, wherein the AHAS gene is located on an A, B, or D genome of a polyploidy genome. 15. The method of claim 13, wherein the whole plant comprising the one or more exogenous sequences integrated within the AHAS locus of the plant genome is further modified to incorporate an additional exogenous sequence. For example, conventional plant transformation methods deliver exogenous DNA that integrates into the genome at random locations. Thus, there remains a need for compositions and methods for the identification, selection and rapid advancement of stable targeted integration into precise locations within a plant genome, including simultaneous modification of multiple alleles across different genomes of polyploid plants, for establishing stable, heritable genetic modifications in a plant and its progeny. Recently, methods and compositions for targeted cleavage of genomic DNA have been described. The present disclosure is in the field of genomic engineering, particularly in the integration of exogenous sequences into plants, including simultaneous genomic editing of multiple alleles over multiple genomes, including in polyploid plants.

The methods and compositions described herein enable both parallel and sequential transgene stacking in plant genomes at precisely the same genomic location, including simultaneous editing of multiple alleles across multiple genomes of polyploid plant species. Thus, in one aspect, disclosed herein are methods and compositions for precise, genomic modification (e.g., transgene stacking) at one or more endogenous alleles of a plant gene. The present disclosure provides methods and compositions for precision transformation, gene targeting, targeted genomic modification and protein expression in plants. In addition, the methods and compositions of the invention allow for exogenous transgenic selectable marker-free selection and/or genomic modification of an endogenous gene in which the genomic modification produces a mutation in the endogenous gene such that the endogenous gene produces a product that results in an herbicide tolerant plant (e.g., by virtue of exploiting known mutations in an endogenous gene such as known mutations in AHAS gene that confer tolerance to Group B herbicides, or ALS inhibitor herbicides such as imidazolinone or sulfonylurea).

The strategy facilitates generation of plants that have one or more transgenes (or one or more genes of interest (GOI), wherein the transgenes do not include transgenic selectable marker genes) precisely positioned at an endogenous plant locus, for example, at one or more AHAS paralogs. In particular, the present disclosure describes a novel, transgenic marker-free private sex cam strategy for integrating an exogenous sequence and to stack traits that exploit differential selection at an endogenous locus (e.g., the acetohydroxyacid synthase (AHAS) locus) in plant genomes. 16. The method of claim 13, wherein the one or more exogenous sequence does not encode a transgenic selectable marker. Type IIS restriction enzyme and one or more zinc finger binding domains, which may or may not be engineered. Cleavage can occur through the use of specific nucleases such as engineered zinc finger nucleases (ZFN), transcription-activator like effector nucleases (TALENs), or using the CRISPR/Cas system with an engineered crRNA/tracr RNA (`single guide RNA`) to guide specific cleavage. To meet the challenge of increasing global demand for food production, many effective approaches to improving agricultural productivity (e.g., enhanced yield or engineered pest resistance) rely on either mutation breeding or introduction of novel genes into the genomes of crop species by transformation.

Also provided are cells (e.g., seeds), cell lines, organisms (e.g., plants), etc. comprising these transgene-stacked and/or simultaneously-modified alleles. See, e.g., Halpin (2005) Plant Biotechnology Journal 3:141-155. In addition, polyploidy, where the organism has two or more duplicated (autoploidy) or related (alloploid) paired sets of chromosomes, occurs more often in plant species than in animals. In addition, Moehle et al. In addition, many agriculturally important plants of the genus Brassica are also allotetraploids. This webcam sex is available in many sites for a minimal cost; also, there are websites that provide free sex webcams. Many members come just for that – having a passionate sex chat with other members and getting it off. A beach is a public place where every type and granny anal sex every age of people come to enjoy their spare times and make them stress free. Some girls are just like to show themselves for the camera and other people. After signing up people fill out their profile and are then able to message, search and communicate with other users. 4. The method of claim 1, wherein the one or more exogenous sequences are incorporated simultaneously or sequentially into the AHAS locus.

10. The method of any claim 1, wherein the one or more exogenous sequences encode a transgene or produce an RNA molecule. 8. The method of any of claim 1, wherein the site-specific nuclease is selected from the group consisting of a zinc finger nuclease, a TAL effector domain nuclease, a homing endonuclease, and a single guide RNA of a CRISPR/Cas nuclease. 11. The method of claim 10, wherein the transgene encodes a protein selected from the group consisting of a protein that increases crop yield, a protein conferring disease resistance, a protein that increases growth, a protein conferring insect resistance, a protein conferring herbicide tolerance, and combinations thereof. 2. The method of claim 1, wherein the one or more exogenous sequences are selected from the group consisting of a donor polynucleotide, a transgene, or any combination thereof. The best views are to be had on the right side of the bridge. YesCams: The Best Free Amateur Cam Website! Since this website is actually an online adult community, there are a lot of great things to do here. Transgene (or trait) stacking has great potential for production of plants, but has proven difficult.

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