Compositions and methods for producing tobacco plants and products having altered alkaloid levels

Abstract

The present disclosure provides the identification of tobacco Nic1 locus. Also provided are tobacco plants with altered total alkaloid and nicotine levels and commercially acceptable leaf grade, their development via breeding or transgenic approaches, and production of tobacco products from these tobacco plants. Also provided are compositions and methods for producing tobacco plants having novel Nic1 mutations or alleles to reduce nicotine levels. Further provided are sequence polymorphisms and molecular markers for breeding tobacco with reduced nicotine or nicotine free while maintaining tobacco leaf grade and tobacco product quality.

1 . A tobacco plant, or part thereof, comprising a Nicotine1 (Nic1) locus comprising a first allele of a first SNP marker selected from the group consisting of SEQ ID NOs: 131 to 136, and a Nicotine2 (Nic2) locus comprising a second allele of a second SNP marker selected from the group consisting of SEQ ID NOs: 137 to 142; wherein said tobacco plant produces leaves having a USDA grade index value of at least about 65% of the USDA grade index value of leaves of a control plant when grown in similar growth conditions, wherein said control plant shares an essentially identical genetic background with said tobacco plant except said Nic1 and Nic2 loci; wherein said tobacco plant comprises a nicotine level less than 2.0%; wherein said first allele of said first SNP marker comprises one or more nucleotides selected from the group consisting of
 nucleotide A at the polymorphic site of said SEQ ID Nos. 131,   nucleotide T at the polymorphic site of said SEQ ID Nos. 132,   nucleotide G at the polymorphic site of said SEQ ID Nos. 133,   nucleotide C at the polymorphic site of said SEQ ID Nos. 134,   nucleotide T at the polymorphic site of said SEQ ID Nos. 135, and   nucleotide G at the polymorphic site of said SEQ ID Nos. 136; and   wherein said second allele of said second SNP marker comprises one or more nucleotides selected from the group consisting of   nucleotide C at the polymorphic site of said SEQ ID Nos. 137,   nucleotide A at the polymorphic site of said SEQ ID Nos. 138,   nucleotide A at the polymorphic site of said SEQ ID Nos. 139,   nucleotide A at the polymorphic site of said SEQ ID Nos. 140,   nucleotide G at the polymorphic site of said SEQ ID Nos. 141, and   nucleotide C at the polymorphic site of said SEQ ID Nos. 142.   
     
     
         2 . The tobacco plant, or part thereof, of  claim 1 , wherein said tobacco plant is capable of producing leaves having a USDA grade index value of 50 or more. 
     
     
         3 . The tobacco plant, or part thereof, of  claim 1 , wherein said tobacco plant comprises nicotine at a level below 25% of the nicotine level of said control plant when grown in similar growth conditions. 
     
     
         4 . (canceled) 
     
     
         5 . (canceled) 
     
     
         6 . (canceled) 
     
     
         7 . (canceled) 
     
     
         8 . (canceled) 
     
     
         9 . (canceled) 
     
     
         10 . A population of the tobacco plants of  claim 1 . 
     
     
         11 . Cured tobacco material from said tobacco plant of  claim 1 . 
     
     
         12 . The cured tobacco material of  claim 11 , wherein said cured tobacco material comprises a lower level of nicotine compared to cured tobacco material from said control tobacco plant when grown in similar growth conditions. 
     
     
         13 . The cured tobacco material of  claim 11 , wherein said tobacco plant comprises nicotine at a level between 0.2% and 0.6%. 
     
     
         14 . The cured tobacco material of  claim 11 , wherein said cured tobacco material is made by a curing process selected from the group consisting of flue curing, air curing, fire curing, and sun curing. 
     
     
         15 . A tobacco product comprising the cured tobacco material of  claim 11 . 
     
     
         16 . The tobacco product of  claim 15 , wherein said tobacco product is selected from the group consisting of a cigarette, a cigarillo, a non-ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, shredded tobacco, and cut tobacco. 
     
     
         17 . The tobacco product of  claim 15 , wherein said tobacco product is a smokeless tobacco product. 
     
     
         18 . (canceled) 
     
     
         19 . The tobacco plant, or part thereof, of  claim 1 , wherein said tobacco plant comprises nicotine at a level below 10% of the nicotine level of said control plant when grown in similar growth conditions. 
     
     
         20 . (canceled) 
     
     
         21 . The tobacco plant, or part thereof, of  claim 1 , wherein said tobacco plant is not a flue-cured tobacco. 
     
     
         22 . The tobacco plant, or part thereof, of  claim 1 , wherein said tobacco plant is a burley tobacco. 
     
     
         23 . The tobacco plant, or part thereof, of  claim 1 , wherein said tobacco plant is an oriental tobacco. 
     
     
         24 . A tobacco seed giving rise to the tobacco plant of  claim 1 . 
     
     
         25 . The tobacco plant, or part thereof, of  claim 1 , wherein said Nic1 locus, said Nic2 locus, or both are also present in LA Burley 21. 
     
     
         26 . A method of selecting a tobacco plant, said method comprising: (a) isolating nucleic acids from a collection of tobacco germplasm; (b) assaying said nucleic acids for one or more first markers within 5 centimorgans (cM) of a first SNP marker selected from the group consisting of SEQ ID NOs. 131 to 136; and (c) selecting a tobacco plant having a low nicotine trait based on said marker assay. 
     
     
         27 . The method of  claim 26 , further comprising: assaying the nucleic acids for one or more second markers within 5 centimorgans (cM) of a second SNP marker selected from the group consisting of SEQ ID NOs. 137 to 142. 
     
     
         28 . The method of  claim 27 , wherein said one or more first markers are selected from the group consisting of SEQ ID NOs. 131 to 136, and wherein said one or more second markers are selected from the group consisting of SEQ ID NOs. 137 to 142.