Map-based genome mining method for identifying regulatory loci controlling the level of gene transcripts and products

Abstract

The invention pertains to a method for identifying one or more regions within a genome of an organism of interest that mediate the expression of one or more genes of interest The method comprises identifying a first and a second organism of interest, the first organism of interest is characterized by exhibiting a measurable response to an environmental stimulus, or otherwise exhibiting a phenotype associated with differential gene expression associated with a process of interest. The second organism of interest is characterized by lacking or not exhibit as strong a response to the stimulus as that observed within the first organism of interest, or it exhibits a different phenotype compared with that of the first organism of interest, wherein the different phenotype is associated with the process of interest or it exhibits a phenotype of interest that segregates when compared with the first organism of interest, or a combination thereof. The first and second organisms of interest are crossed to produce a population of segregated progeny and RNA is extracted from each of the segregated progeny. The level of gene expression for one or more genes of interest that are associated with the response to an environmental stimulus, or process of interest is quantified. A linkage map of the segregated progeny using one or more markers is prepared, and the relationship between said one or more markers on the linkage map and the gene expression of the one or more genes of interest is determined and one or more quantitative trail loci (QTL) identified. This method also pertains to identifying one or more QTLs associated with one or more genes of interest in segregated progeny that are subjected a desired environmental stimulus. Furthermore, this method may be used for the identification of one or more QTL corresponding to a transcription factor or any factor controlling the expression of one or more genes of interest, and the one or more genes located at the one or more QTL may be isolated and characterized.

The embodiments of the invention in which an exclusive property of privilege is claimed are defined as follows:  
     
         1 . A method for identifying one or more regions within a genome of an organism of interest that mediate the expression of one or more genes of interest comprising: 
 i) identifying a first organism of interest characterized in that said first organism of interest exhibits a measurable response to an environmental stimulus, or otherwise exhibits a phenotype associated with differential gene expression associated with a process of interest;    ii) identifying a second organism of interest characterized in that said second organism of interest: 
 a) lacks or does not exhibit as strong a response to said stimulus as that of said first organism of interest;  
 b) exhibits a different phenotype compared with that of said first organism of interest, said different phenotype associated with said process of interest;  
 c) exhibits a phenotype of interest that segregates in a population resulting from a cross with said first organism of interest;  
 d) or a combination of two or more of a), b) c) and d);  
   iii) crossing said first and second organisms of interest to produce a population of segregated progeny;    iv) extracting RNA from each of said segregated progeny and quantifying the level of gene expression of one or more genes of interest, said one or more genes of interest associated with said response to an environmental stimulus, or said process of interest;    v) preparing a linkage map of said segregated progeny using one or more markers;    vi) determining a relationship between said one or more markers on said linkage map and said gene expression of said one or more genes of interest and identifying one or more quantitative trait loci (QTL).    
     
     
         2 . The method of  claim 1 , wherein after said step of crossing (step iii)), and prior to said step of extracting RNA, said segregated progeny are subjected a desired environmental stimulus or are characterized as being at a specific developmental stage.  
     
     
         3 . The method of  claim 1 , wherein said method is used for the identification of one or more QTL corresponding to a transcription factor or any factor controlling the expression of said one or more genes of interest.  
     
     
         4 . The method of  claim 1 , wherein after said step of determining (step vi)), one or mores genes located at said one or more QTL are isolated and characterized.  
     
     
         5 . The method of  claim 1 , wherein in said steps of identifying (steps i) and ii)), said environmental stimulus, or said process of interest is selected from the group consisting of: 
 a biotic stress;    an abiotic stress;    an alteration in mineral nutrition;    an alteration in light quality or flux;    a response arising from an addition of one or more exogenous chemicals;    any stress that leads to an alteration in levels of endogenous chemicals within said plant,    a developmental, chemical or environmental control of gene expression during embryogenesis;    organogenesis;    senescence;    maturation;    dormancy;    germination;    circadian, ultradian and other internal rhythms;    harvestable yield;    seed set;    photosynthate translocation;    an alteration in sink and source capacities within said plant;    an alteration of leaf area index;    an alteration of root shoot ratio;    an alteration in nutritive value of harvested material;    an alteration in plant morphology;    a change in rate of cell cycle;    an alteration in rate of cell differentiation;    an alteration in cell size;    an alteration in plant life cycle, and    genomic rearrangements.    
     
     
         6 . The method of  claim 1 , wherein in said step of determining (step vi)), a marker is identified at said QTL.  
     
     
         7 . A use of said marker of  claim 6  for tracing progeny of an organism.  
     
     
         8 . A use of said marker of  claim 6  for determining hybridity of an organism.  
     
     
         9 . A use of said marker of  claim 6  for identifying a variation of linked phenotypic and/or expression trait.  
     
     
         10 . A use of said marker of  claim 6  for construction of a genetic map.  
     
     
         11 . A use of said marker of  claim 6  for identifying individual progeny from a cross.  
     
     
         12 . A use of said marker of  claim 6 , wherein said marker is a DNA marker, and said DNA marker is used to isolating a genomic DNA sequence surrounding a gene-coding or non-coding DNA sequence.  
     
     
         13 . A use of said marker of  claim 12 , wherein said genomic sequence is a promoter or a regulatory sequence.  
     
     
         14 . A use of said marker of  claim 6  for marker-assisted selection, map-based cloning, hybrid certification, fingerprinting, genotyping, as an allele specific marker, or a combination thererof.  
     
     
         15 . A use of said marker of  claim 6  in plants.  
     
     
         16 . A method for identifying a regulatory gene that mediates expression of a gene of interest comprising: 
 i) identifying one or more Quantitative Trait Loci (QTLs) that explain a significant proportion of variation of expression of one or more genes of interest;    ii) mapping one or more regulatory genes;    iii) determining whether said one or more regulatory genes maps with said one or more QTLs;    iv) isolating one or more regulatory genes within said one or more QTLs; and    v) sequencing one or more regulatory genes isloated in step iv).    
     
     
         17 . A method for identifying a regulatory gene that mediates expression of a gene of interest comprising: 
 i) identifying one or more Quantitative Trait Loci (QTLs) that explain a significant proportion of variation of expression of one or more genes of interest; and    ii) isolating one or more regulatory genes within said one or more QTLs.    iii) sequencing the regulatory gene.    
     
     
         18 . A method for identifying a regulatory sequence that mediates expression of a gene of interest comprising: 
 i) identifying one or more Quantitative Trait Loci (QTLs) that explain a significant proportion of variation of expression of one or more genes of interest;    ii) mapping one or more regulatory genes;    iii) determining whether said one or more regulatory genes maps with said one or more QTLs;    iv) isolating one or more regulatory genes wihtin said one or more QTLs; and    v) sequencing one or more regulatory genes, including the regulatory sequences, of said one or more regulatory genes isolated in step iv).    
     
     
         19 . A method for identifying a regulatory sequence that mediates expression of a gene of interest comprising: 
 i) identifying one or more Quantitative Trait Loci (QTLs) that explain a significant proportion of variation of expression of one or more genes of interest; and    ii) isolating one or more regulatory genes within said one or more QTLs.    iii) sequencing the regulatory genes including the regulatory sequences.    
     
     
         20 . A method of mediating a response of an organism of interest to an environmental stimulus comprising transforming said organism of interest with a regulatory gene identified using the method of  claim 16 .  
     
     
         21 . A method of mediating a response of an organism of interest to an environmental stimulus comprising transforming said organism of interest with a regulatory gene identified using the method of  claim 17 .  
     
     
         22 . A method of mediating development of an organism of interest comprising transforming said organism of interest with a regulatory gene identified using the method of  claim 16 .  
     
     
         23 . A method of mediating development of an organism of interest comprising transforming said organism of interest with a regulatory gene identified using the method of  claim 17 .  
     
     
         24 . A method of mediating a response of an organism of interest to an environmental stimulus comprising transforming said organism of interest with a regulatory sequence identified using the method of  claim 18  fused to a gene of interest.  
     
     
         25 . A method of mediating a response of an organism of interest to an environmental stimulus comprising transforming said organism of interest with a regulatory sequence identified using the method of  claim 19  fused to a gene of interest.  
     
     
         26 . A method of mediating development of an organism of interest comprising transforming said organism of interest with a regulatory sequence identified using the method of  claim 18  fused to a gene of interest.  
     
     
         27 . A method of mediating development of an organism of interest comprising transforming said organism of interest with a regulatory sequence identified using the method of  claim 19  fused to a gene of interest.  
     
     
         28 . The method of  claim 16 , wherein said step of isolating comprises obtaining said regulatory gene using positional cloning.  
     
     
         29 . The method of  claim 17 , wherein said step of isolating comprises obtaining said regulatory gene using positional cloning.  
     
     
         30 . The method of  claim 18 , wherein said step of isolating comprises obtaining said regulatory sequence using positional cloning.  
     
     
         31 . The method of  claim 19 , wherein said step of isolating comprises obtaining said regulatory sequence using positional cloning.