Blue aleurone and other segregation systems

Abstract

The present invention relates to materials and methods for creating and maintaining a cereal plant line for the production of a hybrid cereal plant, that include for example, and not limitation, using the BLue Aleurone (BLA) system.

1 - 231 . (canceled) 
     
     
         232 . A method for manufacturing a cereal plant line, seed, or part thereof, for the production of a hybrid cereal plant line, comprising:
 a) crossing a first cereal plant homozygous for a male fertility gene mutation comprising a disomic alien addition chromosome carrying a dominant male fertility restoration gene and at least one selection marker gene with a second cereal plant homozygous for a male fertility gene mutation and for a homoeologous pairing suppressor gene mutation;   b) harvesting, selecting, and planting at least one seed produced in step a) homozygous for a male fertility gene mutation comprising a monosomic alien chromosome carrying a dominant male fertility restoration gene and at least one selection marker gene and a single copy of the homoeologous pairing suppressor gene mutation;   c) self-fertilizing a cereal plant produced in step b);   d) harvesting, selecting, and planting at least one seed produced in step c) homozygous for a male fertility gene mutation and for the homoeologous pairing suppressor gene mutation comprising a euploid number of chromosomes and the monosomic alien addition chromosome;   e) self-fertilizing a cereal plant produced in step d);   f) harvesting at least four seeds from step e);   g) counting the number of the seeds of step f) from a first group expressing the at least one selection marker and a second group not expressing the at least one selection marker in order to determine the segregation ratio; and   h) keeping the seeds of step f) if the ratio of the number of seeds of first group:second group tends to about 3:1 and discarding the seeds of step f) if the ratio of the number of seeds of first group:second group tends to about 1:3,   
       wherein the male fertility restorer gene is from  Triticum boeoticum  or  Triticum monococcum  and comprises a nucleic acid sequence selected from the group consisting of:
 (i) a nucleic acid sequence as set forth in SEQ ID NO: 1, 6, 7, 8, or 10, or fragments or variants thereof that produce functional amino acid sequences; 
 (ii) a nucleic acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence as set forth in SEQ ID NO: 1, 6, 7, 8, or 10, or fragments thereof that produce functional amino acid sequences; 
 (iii) a nucleic acid sequence having a coding sequence as set forth in SEQ ID NO: 2, 4, 9, 11, or 14, or fragments or variants thereof that produce functional amino acid sequences; 
 (iv) a nucleic acid sequence having a coding sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence as set forth in SEQ ID NO: 2, 4, 9, 11, or 14, or fragments thereof that produce functional amino acid sequences; 
 (v) a nucleic acid sequence encoding an amino acid sequence as set forth in SEQ ID NO: 3, 5, 15, 42, or 43, or fragments or variants thereof that produce functional amino acid sequences; and 
 (vi) a nucleic acid sequence encoding an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 3, 5, 15, 42, or 43, or fragments thereof, and 
 
       wherein the selection marker gene is a blue aleurone gene, preferably wherein the blue aleurone gene is from  Agropyron  elongatum,  Agropyron trichophorum , or  Triticum monococcum , and more preferably wherein the blue aleurone gene comprises a nucleic acid sequence selected from the group consisting of:
 (i) a nucleic acid sequence having a coding sequence of SEQ ID NO: 44 or 12, or fragments or variants thereof that produce functional amino acid sequences; 
 (ii) a nucleic acid sequence having a coding sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 44 or 12, or fragments thereof that produce functional amino acid sequences; 
 (iii) a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO: 45 or 13, or fragments or variants thereof that produce functional amino acid sequences; and 
 (iv) a nucleic acid sequence encoding an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 45 or 13, or fragments thereof. 
 
     
     
         233 . A cereal plant or part thereof, wherein the cereal plant is obtained from a method of  claim 232 , and wherein the cereal plant does not comprise a mis-division of the alien addition chromosome and/or the cereal plant does not comprise a breakage of the alien addition chromosome. 
     
     
         234 . A method for manufacturing a cereal plant line homozygous for a male fertility gene mutation comprising at least one homoeologous alien addition chromosome, the method comprising
 a) crossing a cereal plant comprising at least one homoeologous alien addition chromosome with a cereal plant nullosomic for said genome to which the homoeologous chrosomome relates genetically;   b) harvesting and selecting seeds comprising the alien chromosome and generating a plant from said seeds;   c) crossing the plant of b) with a cereal plant;   d) harvesting and selecting seeds comprising the alien chromosome and not comprising any monosomic chromosome, preferably by use of qPCR and/or flow cytometry, and generating a plant from said seeds;   e) optionally, backcrossing the plant of d) with a cereal plant, and harvesting and selecting seeds comprising the alien chromosome from said cross(es);   f) crossing the plant of d) or e) with a cereal plant homozygous for a male fertility gene mutation;   g) harvesting and selecting seeds comprising the alien chromosome and generating a plant from said seeds;   h) selfing the plant of g), harvesting and selecting seeds comprising the alien chromosome; and   i) generating plants from the seeds of h) and selecting a cereal plant homozygous for a male fertility gene mutation which comprises the at least one homoeologous alien addition chromosome, wherein the at least one homoeologous alien addition chromosome is translocated to at least one homoeologous chromosome pair, wherein the pair consisting of a first and second chromosome, the first chromosome is native to the cereal plant and the second chromosome comprises the alien chromosome or fragment thereof comprising a dominant male fertility restorer gene and at least one selection marker gene, and
 wherein the male fertility restorer gene is from  Triticum boeoticum  or  Triticum monococcum  and comprises a nucleic acid sequence selected from the group consisting of: 
   (i) a nucleic acid sequence as set forth in SEQ ID NO: 1, 6, 7, 8, or 10, or fragments or variants thereof that produce functional amino acid sequences;   (ii) a nucleic acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence as set forth in SEQ ID NO: 1, 6, 7, 8, or 10, or fragments thereof that produce functional amino acid sequences;   (iii) a nucleic acid sequence having a coding sequence as set forth in SEQ ID NO: 2, 4, 9, 11, or 14, or fragments or variants thereof that produce functional amino acid sequences;   (iv) a nucleic acid sequence having a coding sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence as set forth in SEQ ID NO: 2, 4, 9, 11, or 14, or fragments thereof that produce functional amino acid sequences;   (v) a nucleic acid sequence encoding an amino acid sequence as set forth in SEQ ID NO: 3, 5, 15, 42, or 43, or fragments or variants thereof that produce functional amino acid sequences; and   (vi) a nucleic acid sequence encoding an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 3, 5, 15, 42, or 43, or fragments thereof, and
 wherein the selection marker gene is a blue aleurone gene, preferably wherein the blue aleurone gene is from  Agropyron  elongatum,  Agropyron trichophorum , or  Triticum monococcum , and more preferably wherein the blue aleurone gene comprises a nucleic acid sequence selected from the group consisting of: 
   (i) a nucleic acid sequence having a coding sequence of SEQ ID NO: 44 or 12, or fragments or variants thereof that produce functional amino acid sequences;   (ii) a nucleic acid sequence having a coding sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 44 or 12, or fragments thereof that produce functional amino acid sequences;   (iii) a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO: 45 or 13, or fragments or variants thereof that produce functional amino acid sequences; and   (iv) a nucleic acid sequence encoding an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 45 or 13, or fragments thereof.   
     
     
         235 . The method of  claim 234 , wherein the method further comprises:
 j) selfing the plant selected in step i) for obtaining
 I) a cereal plant homozygous for a male fertility gene mutation which comprises the at least one homoeologous alien addition chromosome heterozygously, 
 II) a cereal plant homozygous for a male fertility gene mutation which comprises the at least one homoeologous alien addition chromosome homozygously, 
 III) a cereal plant homozygous for a male fertility gene mutation which does not comprise the at least one homoeologous alien addition chromosome. 
   
     
     
         236 . A cereal plant or part thereof produced by the method of  claim 234 , wherein the seed, progeny, or part thereof comprises the dominant male fertility restorer gene and the selection marker gene on the same side of the centromere of the second chromosome.