# Patent

## Title
Detection of Johnsongrass and its hybrid seed

## Abstract

Methods for detecting the presence or amount of Johnsongrass or Johnsongrass hybrid genetic material in a biological sample, preferably the detection of the presence or amount of Johnsongrass or Johnsongrass hybrid seed in a seed sample, are disclosed. The methods involve detection of certain nucleic acid sequences, namely Johnsongrass detection sequences, optionally through a primer-based amplification process, such as PCR. Also disclosed are isolated polynucleotides, replication compositions and kits for carrying out the detection methods.

## Background
CROSS-REFERENCE
This utility application claims the benefit of U.S. Provisional Application Ser. No. 61/329,271, filed Apr. 29, 2010.


FIELD
The disclosure relates to molecular genetics and more specifically the detection of seed purity through molecular genetics.
BACKGROUND
Sorghum is a genus of plants consisting of multiple species of grasses. Some Sorghum species are valuable crops, being used for food, animal feed and the production of alcohol and biofuels. For example Sorghum bicolor, commonly called sorghum, is one of the most important cereal crops for subsistence farmers in arid and semi-arid portions of Africa, Asia and the Americas. The crop is essential for human life on marginal lands throughout the poorest regions of the world. In developed countries, sorghum is important as a feed crop and as a crop that can be grown on marginal lands as part of a sustainable agroecosystem.
However, other Sorghum species are not so useful. For example, Sorghum halepense, commonly called Johnsongrass, is classified as an invasive species in the United States. This species is commonly found growing in crop fields and other open areas, such as pastures, and has been known to grow so quickly as to choke out the desired crops planted in farming fields. Another weed species in the Sorghum genus is S. propinquum. This species is closely related to Johnsongrass but is diploid, larger and has a different geographic distribution. Some studies have also suggested that Johnsongrass is a descendent of S. propinquum and possibly an interspecific hybrid descendent of sorghum and S. propinquum. (See, e.g., Paterson, et al., (1995) PNAS 92:6127; Paterson and Chandler, “Risk of Gene Flow from sorghum to Johnsongrass” (1996), available at http://www.isb.vt.edu/brarg/brasym96/paterson96.htm).
Unfortunately, Johnsongrass and sorghum are able to readily hybridize. While it is possible to phenotypically distinguish a hybrid plant from the cultivar, it is nearly impossible to distinguish between their seeds. This difficulty creates a genetic purity problem in sorghum seed lots. Because of this problem, one needs to be able to assess the level of Johnsongrass contamination in a seed lot to ensure that the seed lot is as pure as required or desired. Presently, such an assessment is commonly done through grow-outs, where 40,000 individual seeds are planted and grown and the number of hybrid plants counted. This solution is labor intensive, time consuming, expensive and inefficient.
Thus, there is a need for an improved method of detecting the presence of and/or quantifying the amount of Johnsongrass seed or Johnsongrass hybrid seed in sorghum seed lots.
SUMMARY
The present disclosure is drawn to methods and compositions for an efficient way of detecting Johnsongrass or its genetic material. One aspect of the present disclosure is a method for detecting the presence or amount of Johnsongrass or Johnsongrass hybrid genetic material in a biological sample, the method comprising detecting the presence or amount of at least a portion of one or more Johnsongrass detection sequences, whereby positive detection indicates the presence or amount of Johnsongrass or Johnsongrass hybrid genetic material in the biological sample.
In certain embodiments, the one or more Johnsongrass detection sequences comprise SEQ ID NOS.: 1-47 or a nucleic acid sequence essentially similar thereto. In certain other embodiments, the detecting step comprises hybridizing an oligonucleotide probe or oligonucleotide primer to at least a portion of the one or more Johnsongrass detection sequences, preferably the oligonucleotide probe or oligonucleotide primer being hybridizable to at least a portion of one or more of SEQ ID NOS.: 1-47. The oligonucleotide primer can comprise one or more of SEQ ID NOS.: 48-135 or nucleic acid sequences essentially similar thereto while the oligonucleotide probe can comprise one or more of SEQ ID NOS.: 136-140 or nucleic acid sequences essentially similar thereto. In still further embodiments, the detecting step further comprises amplifying at least a portion of the one or more Johnsongrass detection sequences or the detecting step comprises (a) hybridizing an oligonucleotide probe to at least a portion of the one or more Johnsongrass detection sequences, wherein the oligonucleotide probe further comprises a detectable label; and (b) detecting the detectable label. Also preferred are methods wherein the oligonucleotide probe is affixed to a solid support, that solid support possibly comprising a microarray.
Another aspect of the present disclosure is a method for detecting the presence or amount of Johnsongrass seed or Johnsongrass hybrid seed in a seed sample, the sample containing nucleic acids, the method comprising:

    
    
        (a) providing a reaction mixture comprising primer pairs for amplification of at least a portion of one or more of SEQ ID NOS.: 1-47 or nucleic acid sequences essentially similar thereto;
        (b) subjecting the reaction mixture to a nucleic acid amplification process that will produce an amplification product of the one or more of SEQ ID NOS.: 1-47 or nucleic acid sequences essentially similar thereto if present in the reaction mixture; and
        (c) detecting the one or more amplification products of step (b), whereby a positive detection of amplification indicates the presence or amount of Johnsongrass seed and/or Johnsongrass hybrid seed in the seed sample.
    
    


In certain embodiments of the disclosure, the reaction mixture of step (a) comprises primer pairs for amplification of one or more of SEQ ID NOS.: 17, 21, 31, 36 and 37 and nucleic acid sequences essentially similar thereto, preferably the primer pairs including one or more primer pairs selected from the group consisting of SEQ ID NOS.: 76 and 77; SEQ ID NOS.: 84 and 85; SEQ ID NOS.: 104 and 105; SEQ ID NOS.: 114 and 115 and SEQ ID NOS.: 116 and 117. Also preferred are methods wherein the primer pairs include SEQ ID NOS.: 76 and 77, SEQ ID NOS.: 84 and 85, SEQ ID NOS.: 104 and 105, SEQ ID NOS.: 114 and 115 and SEQ ID NOS.: 116 and 117. In certain other embodiments of the disclosure, the reaction mixture of step (a) comprises primers for the nucleic acid region SEQ ID NO.: 17, those primer pairs preferably comprising a first nucleic acid sequence comprising SEQ ID NO.: 76 and a second nucleic acid sequence comprising SEQ ID NO.: 77. In other embodiments, the reaction mixture further comprises a nucleic acid probe for one or more of the nucleic acid regions selected, the nucleic acid probes preferably comprising one or more of SEQ ID NOS.: 136-140 and the nucleic acid probes also preferably comprising a detectable label.
In other aspects, the present disclosure comprises an isolated polynucleotide essentially similar to at least 10 consecutive nucleic acid residues of SEQ ID NOS.: 48-140. In certain aspects, the isolated polynucleotide is at least 90% identical to at least 10 consecutive nucleic acid residues of SEQ ID NOS.: 48-140, preferably at least 93% identical to at least 15 consecutive nucleic acid residues of SEQ ID NOS.: 48-140. In certain embodiments, the isolated polynucleotide further comprises a detectable label.
In still other aspects, the present disclosure comprises a replication composition for use in performance of PCR, comprising (a) one or more primer pairs for amplification of at least a portion of one or more of SEQ ID NOS.: 1-47 and (b) a thermostable DNA polymerase. In certain embodiments, the one or more primer pairs of part (a) comprise primer pairs for amplification of one or more of SEQ ID NOS.: 17, 21, 31 and 36. In other embodiments, the primer pairs comprise one or more of SEQ ID NO.: 76 and SEQ ID NO.: 77; SEQ ID NO.: 84 and SEQ ID NO.: 85; SEQ ID NO.: 104 and SEQ ID NO.: 105; SEQ ID NO.: 114 and SEQ ID NO.: 115; and SEQ ID NO.: 116 and SEQ ID NO.: 117. In other embodiments, the replication composition further comprises a nucleic acid probe for one or more of the primer pairs, the nucleic acid probes selected from the group consisting of SEQ ID NOS.: 136-140.
In yet other aspects, the present disclosure comprises a kit for detection of Johnsongrass DNA in a seed sample, the kit comprising a replication composition of the present disclosure.
SUMMARY OF THE SEQUENCES
SEQ ID NO.: 1 is a nucleotide sequence present in the Johnsongrass sequence database, with a homologous sequence in the S. propinquum database, but absent from the sorghum database. SEQ ID NO.: 1 or its reverse complementary sequence contains or is hybridizable with, and can be amplified by the use of SEQ ID NO.: 48 in conjunction with SEQ ID NO.: 49.
SEQ ID NO.: 2 is a nucleotide sequence present in the Johnsongrass sequence database, with a homologous sequence in the S. propinquum database, but absent from the sorghum database. SEQ ID NO.: 2 or its reverse complementary sequence contains or is hybridizable with, and can be amplified by the use of complementary sequence contains or is hybridizable with, and can be amplified by the use of SEQ ID NO.: 50 in conjunction with SEQ ID NO.: 51.
SEQ ID NO.: 3 is a nucleotide sequence present in the Johnsongrass sequence database, with a homologous sequence in the S. propinquum database, but absent from the sorghum database. SEQ ID NO.: 3 or its reverse complementary sequence contains or is hybridizable with, and can be amplified by the use of SEQ ID NO.: 52 in conjunction with SEQ ID NO.: 53.
SEQ ID NO.: 4 is a nucleotide sequence present in the Johnsongrass sequence database, with a homologous sequence in the S. propinquum database, but absent from the sorghum database. SEQ ID NO.: 4 or its reverse complementary sequence contains or is hybridizable with, and can be amplified by the use of SEQ ID NO.: 54 in conjunction with SEQ ID NO.: 55.
SEQ ID NO.: 5 is a nucleotide sequence present in the Johnsongrass sequence database, with a homologous sequence in the S. propinquum database, but absent from the sorghum database. SEQ ID NO.:5 or its reverse complementary sequence contains or is hybridizable with, and can be amplified by the use of SEQ ID NO.: 56 in conjunction with SEQ ID NO.: 57.
SEQ ID NO.: 6 is a Johnsongrass expressed sequence tag (EST) nucleotide sequence that is absent from the sorghum and non-redundant nucleotide databases. SEQ ID NO.: 6 or its reverse complementary sequence contains or is hybridizable with and can be amplified by the use of SEQ ID NO.:58 in conjunction with SEQ ID NO.: 59.
SEQ ID NO.: 7 is a Johnsongrass EST nucleotide sequence that is absent from the sorghum and non-redundant nucleotide databases.
SEQ ID NO.: 8 is a Johnsongrass EST nucleotide sequence that is absent from the sorghum and non-redundant nucleotide databases.
SEQ ID NO.: 9 is a Johnsongrass EST nucleotide sequence that is absent from the sorghum and non-redundant nucleotide databases. SEQ ID NO.: 9 or its reverse complementary sequence contains or is hybridizable with, and can be amplified by the use of SEQ ID NO.: 60 in conjunction with SEQ ID NO.: 61.
SEQ ID NO.: 10 is a Johnsongrass EST nucleotide sequence that is absent from the sorghum and non-redundant nucleotide databases. SEQ ID NO.: 10 or its reverse complementary sequence contains or is hybridizable with, and can be amplified by the use of SEQ ID NO.: 62 in conjunction with SEQ ID NO.: 63.
SEQ ID NO.: 11 is a Johnsongrass EST nucleotide sequence that is absent from the sorghum and non-redundant nucleotide databases. SEQ ID NO.: 11 or its reverse complementary sequence contains or is hybridizable with, and can be amplified by the use of SEQ ID NO.: 64 in conjunction with SEQ ID NO.: 65.
SEQ ID NO.: 12 is a Johnsongrass EST nucleotide sequence that is absent from the sorghum and non-redundant nucleotide databases. SEQ ID NO.: 12 or its reverse complementary sequence contains or is hybridizable with, and can be amplified by the use of SEQ ID NO.: 66 in conjunction with SEQ ID NO.: 67.
SEQ ID NO.: 13 is a Johnsongrass EST nucleotide sequence that is absent from the sorghum and non-redundant nucleotide databases. SEQ ID NO.: 13 or its reverse complementary sequence contains or is hybridizable with, and can be amplified by the use of SEQ ID NO.: 68 in conjunction with SEQ ID NO.: 69.
SEQ ID NO.: 14 is a Johnsongrass EST nucleotide sequence that is absent from the sorghum and non-redundant nucleotide databases. SEQ ID NO.: 14 or its reverse complementary sequence contains or is hybridizable with, and can be amplified by the use of SEQ ID NO.: 70 in conjunction with SEQ ID NO.: 71.
SEQ ID NO.: 15 is a Johnsongrass EST nucleotide sequence that is absent from the sorghum and non-redundant nucleotide databases. SEQ ID NO.: 15 or its reverse complementary sequence contains or is hybridizable with, and can be amplified by the use of SEQ ID NO.: 72 in conjunction with SEQ ID NO.: 73.
SEQ ID NO.: 16 is a Johnsongrass EST nucleotide sequence that is absent from the sorghum and non-redundant nucleotide databases. SEQ ID NO.: 16 or its reverse complementary sequence contains or is hybridizable with, and can be amplified by the use of SEQ ID NO.: 74 in conjunction with SEQ ID NO.: 75.
SEQ ID NO.: 17 is a Johnsongrass EST nucleotide sequence that is absent from the sorghum and non-redundant nucleotide databases. SEQ ID NO.: 17 or its reverse complementary sequence contains or is hybridizable with, and can be amplified by the use of SEQ ID NO.: 76 in conjunction with SEQ ID NO.: 77, and also contains a binding site for probe SEQ ID NO.: 136.
SEQ ID NO.: 18 is a Johnsongrass EST nucleotide sequence that is absent from the sorghum and non-redundant nucleotide databases. SEQ ID NO.: 18 or its reverse complementary sequence contains or is hybridizable with, and can be amplified by the use of SEQ ID NO.: 78 in conjunction with SEQ ID NO.: 79.
SEQ ID NO.: 19 is a Johnsongrass EST nucleotide sequence that is absent from the sorghum and non-redundant nucleotide databases. SEQ ID NO.: 19 or its reverse complementary sequence contains or is hybridizable with, and can be amplified by the use of SEQ ID NO.: 80 in conjunction with SEQ ID NO.: 81.
SEQ ID NO.: 20 is an S. propinquum marker nucleotide sequence that is absent from the sorghum and non-redundant nucleotide databases. SEQ ID NO.: 20 or its reverse complementary sequence contains or is hybridizable with, and can be amplified by the use of SEQ ID NO.: 82 in conjunction with SEQ ID NO.: 83.
SEQ ID NO.: 21 is an S. propinquum marker nucleotide sequence that is absent from the sorghum and non-redundant nucleotide databases. SEQ ID NO.: 21 or its reverse complementary sequence contains or is hybridizable with, and can be amplified by the use of SEQ ID NO.: 84 in conjunction with SEQ ID NO.: 85, and also contains a binding site for probe SEQ ID NO.: 137.
SEQ ID NO.: 22 is an S. propinquum marker nucleotide sequence that is absent from the sorghum and non-redundant nucleotide databases. SEQ ID NO.: 22 or its reverse complementary sequence contains or is hybridizable with, and can be amplified by the use of SEQ ID NO.: 86 in conjunction with SEQ ID NO.: 87.
SEQ ID NO.: 23 is an S. propinquum marker nucleotide sequence that is absent from the sorghum and non-redundant nucleotide databases. SEQ ID NO.: 23 or its reverse complementary sequence contains or is hybridizable with, and can be amplified by the use of SEQ ID NO.: 88 in conjunction with SEQ ID NO.: 89.
SEQ ID NO.: 24 is an S. propinquum marker nucleotide sequence that is absent from the sorghum and non-redundant nucleotide databases. SEQ ID NO.: 24 or its reverse complementary sequence contains or is hybridizable with, and can be amplified by the use of SEQ ID NO.: 90 in conjunction with SEQ ID NO.: 91.
SEQ ID NO.: 25 is an S. propinquum marker nucleotide sequence that is absent from the sorghum and non-redundant nucleotide databases. SEQ ID NO.: 25 or its reverse complementary sequence contains or is hybridizable with, and can be amplified by the use of SEQ ID NO.: 92 in conjunction with SEQ ID NO.: 93.
SEQ ID NO.: 26 is an S. propinquum marker nucleotide sequence that is absent from the sorghum and non-redundant nucleotide databases. SEQ ID NO.: 26 or its reverse complementary sequence contains or is hybridizable with, and can be amplified by the use of SEQ ID NO.: 94 in conjunction with SEQ ID NO.: 95.
SEQ ID NO.: 27 is an S. propinquum marker nucleotide sequence that is absent from the sorghum and non-redundant nucleotide databases. SEQ ID NO.: 27 or its reverse complementary sequence contains or is hybridizable with, and can be amplified by the use of SEQ ID NO.: 96 in conjunction with SEQ ID NO.: 97.
SEQ ID NO.: 28 is an S. propinquum marker nucleotide sequence that is absent from the sorghum and non-redundant nucleotide databases. SEQ ID NO.: 28 or its reverse complementary sequence contains or is hybridizable with, and can be amplified by the use of SEQ ID NO.: 98 in conjunction with SEQ ID NO.: 99.
SEQ ID NO.: 29 is an S. propinquum marker nucleotide sequence that is absent from the sorghum and non-redundant nucleotide databases. SEQ ID NO.: 29 or its reverse complementary sequence contains or is hybridizable with, and can be amplified by the use of SEQ ID NO.: 100 in conjunction with SEQ ID NO.: 101.
SEQ ID NO.: 30 is an S. propinquum marker nucleotide sequence that is absent from the sorghum and non-redundant nucleotide databases. SEQ ID NO.: 30 or its reverse complementary sequence contains or is hybridizable with, and can be amplified by the use of SEQ ID NO.: 102 in conjunction with SEQ ID NO.: 103.
SEQ ID NO.: 31 is an S. propinquum marker nucleotide sequence that is absent from the sorghum and non-redundant nucleotide databases. SEQ ID NO.: 31 or its reverse complementary sequence contains or is hybridizable with, and can be amplified by the use of SEQ ID NO.: 104 in conjunction with SEQ ID NO.: 105, and also contains a binding site for probe SEQ ID NO.: 138.
SEQ ID NO.: 32 is an S. propinquum marker nucleotide sequence that is absent from the sorghum and non-redundant nucleotide databases. SEQ ID NO.: 32 or its reverse complementary sequence contains or is hybridizable with, and can be amplified by the use of SEQ ID NO.: 106 in conjunction with SEQ ID NO.: 107.
SEQ ID NO.: 33 is an S. propinquum marker nucleotide sequence that is absent from the sorghum and non-redundant nucleotide databases. SEQ ID NO.: 33 or its reverse complementary sequence contains or is hybridizable with, and can be amplified by the use of SEQ ID NO.: 108 in conjunction with SEQ ID NO.: 109.
SEQ ID NO.: 34 is an S. propinquum marker nucleotide sequence that is absent from the sorghum and non-redundant nucleotide databases. SEQ ID NO.: 34 or its reverse complementary sequence contains or is hybridizable with, and can be amplified by the use of SEQ ID NO.: 110 in conjunction with SEQ ID NO.: 111.
SEQ ID NO.: 35 is an S. propinquum marker nucleotide sequence that is absent from the sorghum and non-redundant nucleotide databases. SEQ ID NO.: 35 or its reverse complementary sequence contains or is hybridizable with, and can be amplified by the use of SEQ ID NO.: 112 in conjunction with SEQ ID NO.: 113.
SEQ ID NO.: 36 is an S. propinquum marker nucleotide sequence that is absent from the sorghum and non-redundant nucleotide databases. SEQ ID NO.: 36 or its reverse complementary sequence contains or is hybridizable with, and can be amplified by the use of SEQ ID NO.: 114 in conjunction with SEQ ID NO.: 115, and also contains a binding site for probe SEQ ID NO.: 139.
SEQ ID NO.: 37 is an S. propinquum marker nucleotide sequence that is absent from the sorghum and non-redundant nucleotide databases. SEQ ID NO.: 37 or its reverse complementary sequence contains or is hybridizable with, and can be amplified by the use of SEQ ID NO.: 116 in conjunction with SEQ ID NO.: 117, and also contains a binding site for probe SEQ ID NO.: 140.
SEQ ID NO.: 38 is an S. propinquum marker nucleotide sequence that is absent from the sorghum and non-redundant nucleotide databases. SEQ ID NO.: 38 or its reverse complementary sequence contains or is hybridizable with, and can be amplified by the use of SEQ ID NO.: 118 in conjunction with SEQ ID NO.: 119 or SEQ ID NO.: 120 in conjunction with SEQ ID NO.: 121.
SEQ ID NO.: 39 is an S. propinquum marker nucleotide sequence that is absent from the sorghum and non-redundant nucleotide databases.
SEQ ID NO.: 40 is an S. propinquum marker nucleotide sequence that is absent from the sorghum and non-redundant nucleotide databases. SEQ ID NO.: 40 or its reverse complementary sequence contains or is hybridizable with, and can be amplified by the use of SEQ ID NO.: 122 in conjunction with SEQ ID NO.: 123.
SEQ ID NO.: 41 is a nucleotide sequence present in the Johnsongrass sequence database, with a homologous sequence in the S. propinquum database, but absent from the sorghum database. SEQ ID NO.: 41 or its reverse complementary sequence contains or is hybridizable with, and can be amplified by the use of SEQ ID NO.: 124 in conjunction with SEQ ID NO.: 125.
SEQ ID NO.: 42 is a nucleotide sequence present in the Johnsongrass sequence database, with a homologous sequence in the S. propinquum database, but absent from the sorghum database. SEQ ID NO.: 42 or its reverse complementary sequence contains or is hybridizable with, and can be amplified by the use of SEQ ID NO.: 126 in conjunction with SEQ ID NO.: 127.
SEQ ID NO.: 43 is a nucleotide sequence present in the Johnsongrass sequence database, with a homologous sequence in the S. propinquum database, but absent from the sorghum database. SEQ ID NO.: 43 or its reverse complementary sequence contains or is hybridizable with, and can be amplified by the use of SEQ ID NO.: 128 in conjunction with SEQ ID NO.: 129.
SEQ ID NO.: 44 is a nucleotide sequence present in the Johnsongrass sequence database, with a homologous sequence in the S. propinquum database, but absent from the sorghum database. SEQ ID NO.: 44 or its reverse complementary sequence contains or is hybridizable with, and can be amplified by the use of SEQ ID NO.: 130 in conjunction with SEQ ID NO.: 131.
SEQ ID NO.: 45 is a nucleotide sequence present in the Johnsongrass sequence database, with a homologous sequence in the S. propinquum database, but absent from the sorghum database. SEQ ID NO.: 45 or its reverse complementary sequence contains or is hybridizable with, and can be amplified by the use of SEQ ID NO.: 132 in conjunction with SEQ ID NO.: 133.
SEQ ID NO.: 46 is a nucleotide sequence present in the Johnsongrass sequence database, with a homologous sequence in the S. propinquum database, but absent from the sorghum database. SEQ ID NO.: 46 or its reverse complementary sequence contains or is hybridizable with, and can be amplified by the use of SEQ ID NO.: 134 in conjunction with SEQ ID NO.: 135.
SEQ ID NO.: 47 is a nucleotide sequence present in the Johnsongrass sequence database, with a homologous sequence in the S. propinquum database, but absent from the sorghum database.
SEQ ID NOS.: 48 and 49 are the nucleotide sequences of regions of the Johnsongrass genome, each capable of being used as a probe or primer, either alone or in combination, in detection of the presence of SEQ ID NO.: 1 or its complementary sequence and the subsequent detection of the presence of Johnsongrass.
SEQ ID NOS.: 50 and 51 are the nucleotide sequences of regions of the Johnsongrass genome, each capable of being used as a probe or primer, either alone or in combination, in detection of the presence of SEQ ID NO.: 2 or its complementary sequence and the subsequent detection of the presence of Johnsongrass.
SEQ ID NOS.: 52 and 53 are the nucleotide sequences of regions of the Johnsongrass genome, each capable of being used as a probe or primer, either alone or in combination, in detection of the presence of SEQ ID NO.: 3 or its complementary sequence and the subsequent detection of the presence of Johnsongrass.
SEQ ID NOS.: 54 and 55 are the nucleotide sequences of regions of the Johnsongrass genome, each capable of being used as a probe or primer, either alone or in combination, in detection of the presence of SEQ ID NO.: 4 or its complementary sequence and the subsequent detection of the presence of Johnsongrass.
SEQ ID NOS.: 56 and 57 are the nucleotide sequences of regions of the Johnsongrass genome, each capable of being used as a probe or primer, either alone or in combination, in detection of the presence of SEQ ID NO.: 5 or its complementary sequence and the subsequent detection of the presence of Johnsongrass.
SEQ ID NOS.: 58 and 59 are the nucleotide sequences of regions of the Johnsongrass genome, each capable of being used as a probe or primer, either alone or in combination, in detection of the presence of SEQ ID NO.: 6 or its complementary sequence and the subsequent detection of the presence of Johnsongrass.
SEQ ID NOS.: 60 and 61 are the nucleotide sequences of regions of the Johnsongrass genome, each capable of being used as a probe or primer, either alone or in combination, in detection of the presence of SEQ ID NO.: 9 or its complementary sequence and the subsequent detection of the presence of Johnsongrass.
SEQ ID NOS.: 62 and 63 are the nucleotide sequences of regions of the Johnsongrass genome, each capable of being used as a probe or primer, either alone or in combination, in detection of the presence of SEQ ID NO.: 10 or its complementary sequence and the subsequent detection of the presence of Johnsongrass.
SEQ ID NOS.: 64 and 65 are the nucleotide sequences of regions of the Johnsongrass genome, each capable of being used as a probe or primer, either alone or in combination, in detection of the presence of SEQ ID NO.: 11 or its complementary sequence and the subsequent detection of the presence of Johnsongrass.
SEQ ID NOS.: 66 and 67 are the nucleotide sequences of regions of the Johnsongrass genome, each capable of being used as a probe or primer, either alone or in combination, in detection of the presence of SEQ ID NO.: 12 or its complementary sequence and the subsequent detection of the presence of Johnsongrass.
SEQ ID NOS.: 68 and 69 are the nucleotide sequences of regions of the Johnsongrass genome, each capable of being used as a probe or primer, either alone or in combination, in detection of the presence of SEQ ID NO.: 13 or its complementary sequence and the subsequent detection of the presence of Johnsongrass.
SEQ ID NOS.: 70 and 71 are the nucleotide sequences of regions of the Johnsongrass genome, each capable of being used as a probe or primer, either alone or in combination, in detection of the presence of SEQ ID NO.: 14 or its complementary sequence and the subsequent detection of the presence of Johnsongrass.
SEQ ID NOS.: 72 and 73 are the nucleotide sequences of regions of the Johnsongrass genome, each capable of being used as a probe or primer, either alone or in combination, in detection of the presence of SEQ ID NO.: 15 or its complementary sequence and the subsequent detection of the presence of Johnsongrass.
SEQ ID NOS.: 74 and 75 are the nucleotide sequences of regions of the Johnsongrass genome, each capable of being used as a probe or primer, either alone or in combination, in detection of the presence of SEQ ID NO.: 16 or its complementary sequence and the subsequent detection of the presence of Johnsongrass.
SEQ ID NOS.: 76 and 77 are the nucleotide sequences of regions of the Johnsongrass genome, each capable of being used as a probe or primer, either alone or in combination, in detection of the presence of SEQ ID NO.: 17 or its complementary sequence and the subsequent detection of the presence of Johnsongrass.
SEQ ID NOS.: 78 and 79 are the nucleotide sequences of regions of the Johnsongrass genome, each capable of being used as a probe or primer, either alone or in combination, in detection of the presence of SEQ ID NO.: 18 or its complementary sequence and the subsequent detection of the presence of Johnsongrass.
SEQ ID NOS.: 80 and 81 are the nucleotide sequences of regions of the Johnsongrass genome, each capable of being used as a probe or primer, either alone or in combination, in detection of the presence of SEQ ID NO.: 19 or its complementary sequence and the subsequent detection of the presence of Johnsongrass.
SEQ ID NOS.: 82 and 83 are the nucleotide sequences of regions of the Johnsongrass genome, each capable of being used as a probe or primer, either alone or in combination, in detection of the presence of SEQ ID NO.: 20 or its complementary sequence and the subsequent detection of the presence of Johnsongrass.
SEQ ID NOS.: 84 and 85 are the nucleotide sequences of regions of the Johnsongrass genome, each capable of being used as a probe or primer, either alone or in combination, in detection of the presence of SEQ ID NO.: 21 or its complementary sequence and the subsequent detection of the presence of Johnsongrass.
SEQ ID NOS.: 86 and 87 are the nucleotide sequences of regions of the Johnsongrass genome, each capable of being used as a probe or primer, either alone or in combination, in detection of the presence of SEQ ID NO.: 22 or its complementary sequence and the subsequent detection of the presence of Johnsongrass.
SEQ ID NOS.: 88 and 89 are the nucleotide sequences of regions of the Johnsongrass genome, each capable of being used as a probe or primer, either alone or in combination, in detection of the presence of SEQ ID NO.: 23 or its complementary sequence and the subsequent detection of the presence of Johnsongrass.
SEQ ID NOS.: 90 and 91 are the nucleotide sequences of regions of the Johnsongrass genome, each capable of being used as a probe or primer, either alone or in combination, in detection of the presence of SEQ ID NO.: 24 or its complementary sequence and the subsequent detection of the presence of Johnsongrass.
SEQ ID NOS.: 92 and 93 are the nucleotide sequences of regions of the Johnsongrass genome, each capable of being used as a probe or primer, either alone or in combination, in detection of the presence of SEQ ID NO.: 25 or its complementary sequence and the subsequent detection of the presence of Johnsongrass.
SEQ ID NOS.: 94 and 95 are the nucleotide sequences of regions of the Johnsongrass genome, each capable of being used as a probe or primer, either alone or in combination, in detection of the presence of SEQ ID NO.: 26 or its complementary sequence and the subsequent detection of the presence of Johnsongrass.
SEQ ID NOS.: 96 and 97 are the nucleotide sequences of regions of the Johnsongrass genome, each capable of being used as a probe or primer, either alone or in combination, in detection of the presence of SEQ ID NO.: 27 or its complementary sequence and the subsequent detection of the presence of Johnsongrass.
SEQ ID NOS.: 98 and 99 are the nucleotide sequences of regions of the Johnsongrass genome, each capable of being used as a probe or primer, either alone or in combination, in detection of the presence of SEQ ID NO.: 28 or its complementary sequence and the subsequent detection of the presence of Johnsongrass.
SEQ ID NOS.: 100 and 101 are the nucleotide sequences of regions of the Johnsongrass genome, each capable of being used as a probe or primer, either alone or in combination, in detection of the presence of SEQ ID NO.: 29 or its complementary sequence and the subsequent detection of the presence of Johnsongrass.
SEQ ID NOS.: 102 and 103 are the nucleotide sequences of regions of the Johnsongrass genome, each capable of being used as a probe or primer, either alone or in combination, in detection of the presence of SEQ ID NO.: 30 or its complementary sequence and the subsequent detection of the presence of Johnsongrass.
SEQ ID NOS.: 104 and 105 are the nucleotide sequences of regions of the Johnsongrass genome, each capable of being used as a probe or primer, either alone or in combination, in detection of the presence of SEQ ID NO.: 31 or its complementary sequence and the subsequent detection of the presence of Johnsongrass.
SEQ ID NOS.: 106 and 107 are the nucleotide sequences of regions of the Johnsongrass genome, each capable of being used as a probe or primer, either alone or in combination, in detection of the presence of SEQ ID NO.: 32 or its complementary sequence and the subsequent detection of the presence of Johnsongrass.
SEQ ID NOS.: 108 and 109 are the nucleotide sequences of regions of the Johnsongrass genome, each capable of being used as a probe or primer, either alone or in combination, in detection of the presence of SEQ ID NO.: 33 or its complementary sequence and the subsequent detection of the presence of Johnsongrass.
SEQ ID NOS.: 110 and 111 are the nucleotide sequences of regions of the Johnsongrass genome, each capable of being used as a probe or primer, either alone or in combination, in detection of the presence of SEQ ID NO.: 34 or its complementary sequence and the subsequent detection of the presence of Johnsongrass.
SEQ ID NOS.: 112 and 113 are the nucleotide sequences of regions of the Johnsongrass genome, each capable of being used as a probe or primer, either alone or in combination, in detection of the presence of SEQ ID NO.: 35 or its complementary sequence and the subsequent detection of the presence of Johnsongrass.
SEQ ID NOS.: 114 and 115 are the nucleotide sequences of regions of the Johnsongrass genome, each capable of being used as a probe or primer, either alone or in combination, in detection of the presence of SEQ ID NO.: 36 or its complementary sequence and the subsequent detection of the presence of Johnsongrass.
SEQ ID NOS.: 116 and 117 are the nucleotide sequences of regions of the Johnsongrass genome, each capable of being used as a probe or primer, either alone or in combination, in detection of the presence of SEQ ID NO.: 37 or its complementary sequence and the subsequent detection of the presence of Johnsongrass.
SEQ ID NOS.: 118, 119, 120 and 121 are the nucleotide sequences of regions of the Johnsongrass genome, each capable of being used as a probe or primer, either alone or in combination, in detection of the presence of SEQ ID NO.: 38 or its complementary sequence and the subsequent detection of the presence of Johnsongrass.
SEQ ID NOS.: 122 and 123 are the nucleotide sequences of regions of the Johnsongrass genome, each capable of being used as a probe or primer, either alone or in combination, in detection of the presence of SEQ ID NO.: 40 or its complementary sequence and the subsequent detection of the presence of Johnsongrass.
SEQ ID NOS.: 124 and 125 are the nucleotide sequences of regions of the Johnsongrass genome, each capable of being used as a probe or primer, either alone or in combination, in detection of the presence of SEQ ID NO.: 41 or its complementary sequence and the subsequent detection of the presence of Johnsongrass.
SEQ ID NOS.: 126 and 127 are the nucleotide sequences of regions of the Johnsongrass genome, each capable of being used as a probe or primer, either alone or in combination, in detection of the presence of SEQ ID NO.: 42 or its complementary sequence and the subsequent detection of the presence of Johnsongrass.
SEQ ID NOS.: 128 and 129 are the nucleotide sequences of regions of the Johnsongrass genome, each capable of being used as a probe or primer, either alone or in combination, in detection of the presence of SEQ ID NO.: 43 or its complementary sequence and the subsequent detection of the presence of Johnsongrass.
SEQ ID NOS.: 130 and 131 are the nucleotide sequences of regions of the Johnsongrass genome, each capable of being used as a probe or primer, either alone or in combination, in detection of the presence of SEQ ID NO.: 44 or its complementary sequence and the subsequent detection of the presence of Johnsongrass.
SEQ ID NOS.: 132 and 133 are the nucleotide sequences of regions of the Johnsongrass genome, each capable of being used as a probe or primer, either alone or in combination, in detection of the presence of SEQ ID NO.: 45 or its complementary sequence and the subsequent detection of the presence of Johnsongrass.
SEQ ID NOS.: 134 and 135 are the nucleotide sequences of regions of the Johnsongrass genome, each capable of being used as a probe or primer, either alone or in combination, in detection of the presence of SEQ ID NO.: 46 or its complementary sequence and the subsequent detection of the presence of Johnsongrass.
SEQ ID NO.: 136 is the nucleotide sequence of a region of the Johnsongrass genome capable of being used as a probe or primer in detection of the presence of SEQ ID NO.: 17 and subsequent detection of the presence of Johnsongrass. This sequence is preferably used as a probe that will hybridize to and allow detection of SEQ ID NO.: 17.
SEQ ID NO.: 137 is the nucleotide sequence of a region of the Johnsongrass genome capable of being used as a probe or primer in detection of the presence of SEQ ID NO.: 21 and subsequent detection of the presence of Johnsongrass. This sequence is preferably used as a probe that will hybridize to and allow detection of SEQ ID NO.: 21.
SEQ ID NO.: 138 is the nucleotide sequence of a region of the Johnsongrass genome capable of being used as a probe or primer in detection of the presence of SEQ ID NO.: 31 and subsequent detection of the presence of Johnsongrass. This sequence is preferably used as a probe that will hybridize to and allow detection of SEQ ID NO.: 31.
SEQ ID NO.: 139 is the nucleotide sequence of a region of the Johnsongrass genome capable of being used as a probe or primer in detection of the presence of SEQ ID NO.: 36 and subsequent detection of the presence of Johnsongrass. This sequence is preferably used as a probe that will hybridize to and allow detection of SEQ ID NO.: 36.
SEQ ID NO.: 140 is the nucleotide sequence of a region of the Johnsongrass genome capable of being used as a probe or primer in detection of the presence of SEQ ID NO.: 37 and subsequent detection of the presence of Johnsongrass. This sequence is preferably used as a probe that will hybridize to and allow detection of SEQ ID NO.: 37.
The sequences conform with 37 C.F.R. §§1.821-1.825 (“Requirements for patent applications Containing Nucleotide Sequences and/or Amino Acid Sequence Disclosures—the Sequence Rules”) and are consistent with World Intellectual Property Organization (WIPO) Standard ST.25 (1998) and the sequence listing requirements of the EPO and PCT (Rules 5.2 and 49.5(a-bis) and Section 208 and Annex C of the Administrative Instructions). The symbols and format used for nucleotide sequence data comply with the rules set forth in 37 C.F.R. §1.822.


DETAILED DESCRIPTION
The present disclosure relates in part to methods for identifying and/or quantifying Johnsongrass and/or Johnsongrass hybrid genetic material in biological samples, which methods are based on nucleic acid sequences present in Johnsongrass. In particular, methods are provided for identifying and/or quantifying Johnsongrass seed and/or Johnsongrass hybrid seed in seed samples. In one particular embodiment, the methods comprise amplifying a sequence of a nucleic acid using a polymerase chain reaction with at least two primers. In other embodiments, the methods comprise detection through the use of detectably labeled oligonucleotide probes or microarrays. Primers and probes useful for detecting nucleic acid sequences present in Johnsongrass are also provided.
Selective detection of Johnsongrass genetic material in biological sample is provided. The primers, probes, nucleotide sequences provided herein enable selective and specific detection of Johnsongrass genetic material including DNA, RNA from other closely related species. The selective and specific detection methods disclosed herein also enable quantifying the presence and the amount of Johnsongrass genetic material including Johnsongrass hybrid material in a biological sample. For example, the methods disclosed herein provide seed purity determination for sorghum samples.
The present disclosure further relates in part to certain specific nucleotide sequences present in Johnsongrass that are described herein, which can be used to develop specific identification methods for detecting Johnsongrass and/or Johnsongrass hybrid genetic material in biological samples. The disclosure also relates in part to replication compositions and kits useful for identifying Johnsongrass in biological samples, the kits comprising one or more oligonucleotide primers or probes that specifically recognize the specific Johnsongrass nucleotide sequences described herein.
The entire contents of all references cited in this disclosure are specifically incorporated by reference herein. Further, when an amount, concentration, or other value or parameter is given as either a range, preferred range, or a list of upper preferable values and lower preferable values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether ranges are separately disclosed. Where a range of numerical values is recited herein, unless otherwise stated, the range is intended to include the endpoints thereof, and all integers and fractions within the range. It is not intended that the scope of the disclosure be limited to the specific values recited when defining a range.
The terms “consisting essentially of” generally means e.g., the specified nucleic acid sequence and those that do not materially affect the basic and material characteristics of the specified nucleic acid sequence. Similarly, components or steps that do not materially affect the basic and material characteristics of the claimed invention are encompassed by the term “consisting essentially of”.
The term “sorghum” refers to Sorghum bicolor. The term “Johnsongrass” refers to Sorghum halepense. The term “Johnsongrass hybrid” refers to a plant or portion thereof, such as a seed that contains genetic elements from both Johnsongrass and another species, especially a species from the Sorghum genus. An example of a Johnsongrass hybrid is a seed that results from a cross of sorghum and Johnsongrass.
Selectively discriminates generally refers to the ability of the probes and primers disclosed herein to preferentially bind or hybridize to a genetic material of Johnsongrass or its hybrid compared to binding to genetic material from a related species, e.g., Sorghum bicolor. 
As used herein, a biological sample is any sample that contains biological material, including a sample of a seed, more than one seed, a plant, plant material or products comprising plant material. The term “plant” is intended to encompass plant tissue at any stage of maturity, as well as any cells, tissues or organs taken from or derived from any such plant, including, without limitation, any seeds, leaves, stems, flowers, roots, single cells, gametes, cell cultures, tissue cultures or protoplasts. “Plant material”, as used herein refers to material which is obtained or derived from a plant. It is to be understood that, in the context of the present disclosure, such biological samples are tested for the presence of nucleic acids, and, therefore, such samples comprise nucleic acids.
The term “Johnsongrass detection sequence” refers to a nucleic acid sequence which has been found to be present in one or more strains of Johnsongrass and/or Sorghum propinquum, but partially or completely absent from, or of relatively low homology in one or more strains of sorghum.
The terms “polynucleotide”, “polynucleotide sequence”, “nucleic acid sequence”, “nucleic acid fragment” and “oligonucleotide” are used interchangeably herein. These terms encompass nucleotide sequences and the like. A polynucleotide may be a polymer of RNA or DNA that is single- or double-stranded, that optionally contains synthetic, non-natural or altered nucleotide bases. A polynucleotide in the form of a polymer of DNA may be comprised of one or more strands of cDNA, genomic DNA, synthetic DNA or mixtures thereof.
The term “isolated” refers to materials, such as nucleic acid molecules and/or proteins, which are substantially free or otherwise removed from components that normally accompany or interact with the materials in a naturally occurring environment. Isolated polynucleotides may be purified from a host cell in which they naturally occur. Conventional nucleic acid purification methods known to skilled artisans may be used to obtain isolated polynucleotides. The term also embraces recombinant polynucleotides and chemically synthesized polynucleotides.
“Polymerase chain reaction” is abbreviated PCR. “Ligase chain reaction” is abbreviated LCR. “Strand displacement amplification” is abbreviated SDA.
The term “amplification” refers to synthesis of multiple copies of a nucleic acid sequence through a primer-directed reaction, such as PCR, LCR or SDR. The term “amplification product” refers to nucleic acid fragments produced during a primer-directed amplification reaction. The term “replication composition” refers to a composition that contains the elements necessary to perform some form of nucleic acid replication or amplification. If PCR methodology is selected, the replication composition may comprise, for example, nucleotide triphosphates, two (or more) primers with appropriate sequences, thermostable polymerase, buffers, solutes and proteins. These reagents and details describing procedures for their use in amplifying nucleic acids are provided, for example, in U.S. Pat. No. 4,683,202 (1987, Mullis, et al.) and U.S. Pat. No. 4,683,195 (1986, Mullis, et al.). If LCR methodology is selected, then the nucleic acid replication compositions may comprise, for example: a thermostable ligase (e.g., Thermus aquaticus ligase), two sets of adjacent oligonucleotides (wherein one member of each set is complementary to each of the target strands), Tris-HCl buffer, KCl, EDTA, NAD, dithiothreitol and salmon sperm DNA. See, for example, Tabor, et al., (1985) Proc. Natl. Acad. Sci. USA 82:1074-1078.
The term “primer” refers to an oligonucleotide (synthetic or occurring naturally), which is capable of acting as a point of initiation of nucleic acid synthesis or replication along a complementary strand when placed under conditions in which synthesis of a complementary strand is catalyzed by a polymerase. Typically, primers are oligonucleotides from 10 to 30 nucleic acids in length, but longer or shorter sequences can be employed. Primers may be provided in double-stranded form, though the single-stranded form is preferred. A primer can further contain a detectable label, for example a 5′ end label.
The term “probe” refers to an oligonucleotide (synthetic or occurring naturally) that is complementary (though not necessarily fully complementary) to a polynucleotide of interest and forms a duplexed structure by hybridization with at least one strand of the polynucleotide of interest. Typically, probes are oligonucleotides from 10 to 50 nucleic acids in length, but longer or shorter sequences can be employed. A probe can further contain a detectable label.
A probe can either be an independent entity or complexed with or otherwise attached to a primer, such as where a probe is connected via its 3′ terminus to a primer's 5′ terminus through a linker, which may be a nucleotide or non-nucleotide linker and which may be a non-amplifiable linker, such as a hexethylene glycol (HEG) or 18-carbon linker. In such a case, this would be termed a “primer-probe complex.” One example of such a probe-primer complex can be found in U.S. Pat. No. 6,326,145, incorporated herein by reference in its entirety, which are frequently referred to as “Scorpion probes” or “Scorpion primers.”
As used herein, the terms “label” and “detectable label” refer to a molecule capable of detection, including, but not limited to, radioactive isotopes, fluorescers, chemiluminescers, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, chromophores, dyes, metal ions, metal sols, semiconductor nanocrystals, ligands (e.g., biotin, avidin, streptavidin or haptens) and the like. A detectable label can also include a combination of a reporter and a quencher.
The term “reporter” refers to a substance or a portion thereof that is capable of exhibiting a detectable signal, which signal can be suppressed by a quencher. The detectable signal of the reporter is, for example, fluorescence in the detectable range. The term “quencher” refers to a substance or portion thereof that is capable of suppressing, reducing, inhibiting, etc., the detectable signal produced by the reporter.
As used herein, the terms “quenching” and “fluorescence energy transfer” refer to the process whereby, when a reporter and a quencher are in close proximity, and the reporter is excited by an energy source, a substantial portion of the energy of the excited state nonradiatively transfers to the quencher where it either dissipates nonradiatively or is emitted at a different emission wavelength than that of the reporter.
Preferably, the reporter may be selected from fluorescent organic dyes modified with a suitable linking group for attachment to the oligonucleotide, such as to the terminal 3′ carbon or terminal 5′ carbon. The quencher may also be selected from organic dyes, which may or may not be fluorescent, depending on the embodiment of the present disclosure. Generally, whether the quencher is fluorescent or simply releases the transferred energy from the reporter by non-radiative decay, the absorption band of the quencher should at least substantially overlap the fluorescent emission band of the reporter to optimize the quenching. Non-fluorescent quenchers or dark quenchers typically function by absorbing energy from excited reporters, but do not release the energy radioactively.
Selection of appropriate reporter-quencher pairs for particular probes may be undertaken in accordance with known techniques. Fluorescent and dark quenchers and their relevant optical properties from which exemplary reporter-quencher pairs may be selected are listed and described, for example, in Berlman, In: Handbook of Fluorescence Spectra of Aromatic Molecules, 2nd ed., Academic Press, New York, 1971, the content of which is incorporated herein by reference.
Preferred reporter-quencher pairs may be selected from xanthene dyes including fluoresceins and rhodamine dyes. Particularly preferred reporters may be selected from dyes such as SYBR green, 5-carboxyfluorescein (5-FAMTM available from Applied Biosystems of Foster City in Calif.), 6-carboxyfluorescein (6-FAM), tetrachloro-6-carboxyfluorescein (TET), 2,7-dimethoxy-4,5-dichloro-6-carboxyfluorescein, hexachloro-6-carboxyfluorescein (HEX), 6-carboxy-2′,4,7,7′-tetrachlorofluorescein (6-TETTM available from Applied Biosystems), carboxy-X-rhodamine (ROX), 6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein (6-JOETM available from Applied Biosystems), VICTM dye products available from Molecular Probes, Inc., NEDTM dye products available from available from Applied Biosystems and the like. Other preferred fluorescent compounds for use as reporters are the naphthylamines, having an amino group in the alpha or beta position. Included among such naphthylamino compounds are 1-demethylaminonaphthyl-5 sulfonate, 1-anilino-8-naphthalene sulfonate and 2-p-touidinyl-6-naphthalene sulfonate. Other dyes include 3-phenyl-7-isocyanatocoumarin; acridines such as 9-isoth iocyanatoacridine; N-(p-(2-benzoxazolyl)phenyl)maleimide; benzoxadiazoles; stilbenes; pyrenes and the like.
Particularly preferred quenchers may be selected from 6-carboxy-tetramethyl-rhodamine, 4-(4-dimethylaminophenylazo)benzoic acid (DABYL), tetramethylrhodamine (TAMRA), BHQ-0TM BHQ-1 TM, BHQ-2TM and BHQ-3TM, each of which are available from
Biosearch Technologies, Inc. of Novato in Calif., QSY-7TM, QSY-9TM, QSY21TM and QSY35TM, each of which are available from Molecular Probes, Inc. and the like.
One preferred example of a probe that contains a reporter and a quencher is a probe that is to be used in a 5′-exonuclease assay, such as the Taqman® real-time PCR technique. In this context, the oligonucleotide probe will have a reporter attached at or near its 5′ or 3′ end and a quencher attached at or near the other end such that the quencher is able to quench any fluorescence of the reporter. The probe will also contain sufficient number of phosphodiester linkages adjacent to its 5′ end so that the 5′ to 3′ nuclease activity employed during the amplification reaction can efficiently degrade the bound probe to separate the reporters and quenchers, thereby allowing the reporter to fluoresce.
Yet another example of a probe that contains a reporter and quencher is a Molecular Beacon type probe, which contains a probe region flanked by self-complementary regions that allow the probe to form a stem-loop structure when unbound from the probe's target sequence. Such probes typically have a reporter attached at or near one terminus and a quencher attached at or near the other terminus such that the quencher is in sufficiently close proximity to the reporter to cause quenching when the probe is in its unbound, and thus stem-loop, form.
The term “replication inhibitor moiety” refers to any atom, molecule or chemical group that is attached to the 3′ terminal hydroxyl group of an oligonucleotide that will block the initiation of chain extension for replication of a nucleic acid strand. Examples include, but are not limited to 3′-deoxynucleotides (e.g., cordycepin), dideoxynucleotides, phosphate, ligands (e.g., biotin and dinitrophenol), reporter molecules (e.g., fluorescein and rhodamine), carbon chains (e.g., propanol), a mismatched nucleotide or polynucleotide or peptide nucleic acid units. The term “non-participatory” refers to the lack of participation of a probe or primer in a reaction for the amplification of a nucleic acid molecule. Specifically a non-participatory probe or primer is one that will not serve as a substrate for, or be extended by, a DNA or RNA polymerase. A “non-participatory probe” is inherently incapable of being chain extended by a polymerase. It may or may not have a replication inhibitor moiety.
Moreover, the skilled artisan recognizes that substantially similar nucleic acid sequences encompassed by this invention are also defined by their ability to hybridize (under moderately stringent conditions, e.g., 0.5×SSC, 0.1% SDS, 60° C.) with the sequences exemplified herein, or to any portion of the nucleotide sequences disclosed herein and which are functionally equivalent to any of the nucleic acid sequences disclosed herein. Stringency conditions can be adjusted to screen for moderately similar fragments, such as homologous sequences from distantly related organisms, to highly similar fragments, such as genes that duplicate functional enzymes from closely related organisms. Post-hybridization washes determine stringency conditions.
The term “selectively hybridizes” includes reference to hybridization, under stringent hybridization conditions, of a nucleic acid sequence to a specified nucleic acid target sequence to a detectably greater degree (e.g., at least 2-fold over background) than its hybridization to non-target nucleic acid sequences and to the substantial exclusion of non-target nucleic acids. Selectively hybridizing sequences typically have about at least 80% sequence identity, or 90% sequence identity, up to and including 100% sequence identity (i.e., fully complementary) with each other.
The term “stringent conditions” or “stringent hybridization conditions” includes reference to conditions under which a probe will selectively hybridize to its target sequence. Stringent conditions are sequence-dependent and will be different in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences can be identified which are 100% complementary to the probe (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing). Generally, a probe is less than about 1000 nucleotides in length, optionally less than 500 nucleotides in length.
Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C., and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55° C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60 to 65° C.
Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. For DNA-DNA hybrids, the Tm can be approximated from the equation of Meinkoth et al., Anal. Biochem. 138:267-284 (1984): Tm=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L; where M is the molarity of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. The Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. Tm is reduced by about 1° C. for each 1% of mismatching; thus, Tm, hybridization and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with ≧90% identity are sought, the Tm can be decreased 10° C. Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions can utilize a hybridization and/or wash at 1, 2, 3 or 4° C. lower than the thermal melting point (Tm); moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9 or 10° C. lower than the thermal melting point (Tm); low stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15 or 20° C. lower than the thermal melting point (Tm). Using the equation, hybridization and wash compositions, and desired Tm, those of ordinary skill will understand that variations in the stringency of hybridization and/or wash solutions are inherently described. If the desired degree of mismatching results in a Tm of less than 45° C. (aqueous solution) or 32° C. (formamide solution) it is preferred to increase the SSC concentration so that a higher temperature can be used. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2 “Overview of principles of hybridization and the strategy of nucleic acid probe assays”, Elsevier, N.Y. (1993) and Current Protocols in Molecular Biology, Chapter 2, Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995). Hybridization and/or wash conditions can be applied for at least 10, 30, 60, 90, 120 or 240 minutes.
For hybridizations with shorter nucleic acids, i.e., oligonucleotides, the position of mismatches becomes more important and the length of the oligonucleotide determines its specificity (see, Sambrook, et al., supra, 11.7-11.8). In one preferred embodiment the length for a hybridizable nucleic acid, i.e., an oligonucleotide primer or probe, is at least about 10 consecutive nucleotides. In other embodiments a preferred minimum length for a hybridizable nucleic acid is at least about 11 nucleotides, at least about 12 nucleotides, at least about 13 nucleotides, at least about 14 nucleotides, at least about 15 nucleotides, at least about 16 nucleotides, at least about 17 nucleotides, at least about 18 nucleotides, at least about 19 nucleotides, at least about 20 nucleotides; at least about 21 nucleotides, at least about 22 nucleotides, at least about 23 nucleotides, at least about 24 nucleotides, at least about 25 nucleotides, at least about 26 nucleotides, at least about 27 nucleotides, at least about 28 nucleotides, at least about 29 nucleotides, at least about 30 nucleotides, at least about 35 nucleotides, at least about 40 nucleotides, at least about 45 nucleotides, at least about 50 nucleotides, at least about 55 nucleotides, at least about 60 nucleotides, at least about 70 nucleotides, at least about 80 nucleotides, at least about 90 nucleotides, at least about 100 nucleotides, at least about 120 nucleotides, at least about 140 nucleotides, at least about 160 nucleotides, at least about 180 nucleotides or at least about 200 nucleotides. The nucleotides mentioned herein may be substantially consecutive. Furthermore, the skilled artisan will recognize that the temperature and wash solution salt concentration may be adjusted as necessary according to factors such as length of the probe.
As used herein, “sequence identity” with regard to nucleotide sequences (DNA or RNA), refers to the number of positions with identical nucleotides divided by the number of nucleotides in the shorter of the two sequences. The alignment of the two nucleotide sequences can be performed, for example, by the Wilbur and Lipmann algorithm (Wilbur and Lipmann, (1983) Proc. Natl. Acad. Sci. USA 80:726) using a window-size of 20 nucleotides, a word length of 4 nucleotides and a gap penalty of 4. Computer-assisted analysis and interpretation of sequence data, including sequence alignment as described above, can, e.g., be conveniently performed using the programs of the IntelligeneticsTM Suite (Intelligenetics Inc., Calif.) or the sequence analysis software package of the
Genetics Computer Group (GCG, University of Wisconsin Biotechnology center). Sequences are indicated as “essentially similar” when they have a sequence identity of at least about 75%, particularly at least about 80%, more particularly at least about 85%, still more particularly about 90%, even more particularly about 91%, still more particularly about 92%, even more particularly about 93%, still more particularly about 94%, even more particularly about 95%, still more particularly about 96%, even more particularly about 97%, still more particularly about 98%, even more particularly about 99%, and especially about 100%. When RNA sequences are said to be essentially similar or have a certain degree of sequence identity with DNA sequences, thymidine (T) in the DNA sequence is considered equal to uracil (U) in the RNA sequence. “Complementary to” as used herein refers to the complementarity between the A and T(U), and G and C nucleotides in nucleotide sequences.
A “kit” as used herein refers to a set of reagents for the purpose of performing the method of the disclosure, more particularly, the identification of Johnsongrass in biological samples. A preferred embodiment of the kit of the disclosure comprises at least one or two specific primers, as described above. Optionally, the kit can further comprise any other reagent described herein in the PCR protocol. Alternatively, according to another embodiment of this disclosure, the kit can comprise a specific probe, as described above, which specifically hybridizes with nucleic acids of biological samples to identify the presence of Johnsongrass. Optionally, the kit can further comprise any other reagent (such as but not limited to hybridizing buffer or label) for identification of Johnsongrass in biological samples, using the specific probe.
Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described by, for example, Sambrook, et al., supra and by Ausubel, et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-Interscience (1987). Standard PCR protocols are described in the art, such as in “PCR Applications Manual” (Roche Molecular Biochemicals, 2nd Edition, 1999). It is understood, however, that a number of parameters in the PCR protocol may need to be adjusted to specific laboratory conditions and may be modified slightly to obtain similar results. For instance, use of a different method for preparation of DNA may require adjustment of, for instance, the amount of primers, polymerase and annealing conditions used. Similarly, the selection of other primers may dictate other optimal conditions for the PCR protocol. These adjustments will, however, be apparent to a person skilled in the art and are furthermore detailed in current PCR application manuals such as the one cited above.
Johnsongrass Detection Sequences
As discussed above, Johnsongrass is closely related to sorghum and is likely an interspecific hybrid of sorghum and S. propinquum. Methods and compositions disclosed herein provide detection of Johnsongrass or Johnsongrass hybrid material, even in a background of sorghum, through the detection of the presence of one or more Johnsongrass detection sequences or nucleic acid sequences present in one or more varieties of Johnsongrass and/or S. propinquum but absent from or of relatively low homology in one or more varieties of sorghum.
Identification of appropriate Johnsongrass detection sequences can be achieved through in silico analysis and comparison of databases of sorghum, Johnsongrass, and S. propinquum nucleic acid sequences. Examples of suitable Johnsongrass detection sequences include SEQ ID NOS.:1-47 and nucleic acid sequences essentially similar thereto. In particular, nucleic acid sequences 75% identical, 80% identical, 85% identical, 90% identical, 91% identical, 92% identical, 93% identical, 94% identical, 95% identical, 96% identical, 97% identical, 98% identical, 99% identical or 100% identical to SEQ ID NOS.: 1-47 or a portion thereof are useful in the present disclosure.
Following the determination of appropriate Johnsongrass detection sequences, detection of Johnsongrass according to the present disclosure can be accomplished by any method capable of positively detecting the presence of one or more Johnsongrass detection sequences, or some portion thereof, in a biological sample. Importantly, the detection method need not detect the entire Johnsongrass detection sequence, but rather only needs to be directed toward a portion of sufficient length to positively identify the desired Johnsongrass detection sequence. To that end, detection will preferably be directed toward a portion of the Johnsongrass detection sequence at least about 10 nucleic acids in length, at least about 11 nucleic acids in length, at least about 12 nucleic acids in length, at least about 13 nucleic acids in length, at least about 14 nucleic acids in length, at least about 15 nucleic acids in length, at least about 16 nucleic acids in length, at least about 17 nucleic acids in length, at least about 18 nucleic acids in length, at least about 19 nucleic acids in length, at least about 20 nucleic acids in length, at least about 21 nucleic acids in length, at least about 22 nucleic acids in length, at least about 23 nucleic acids in length, at least about 24 nucleic acids in length, at least about 25 nucleic acids in length, at least about 26 nucleic acids in length, at least about 27 nucleic acids in length, at least about 28 nucleic acids in length, at least about 29 nucleic acids in length, at least about 30 nucleic acids in length, at least about 35 nucleic acids in length, at least about 40 nucleic acids in length, at least about 45 nucleic acids in length, at least about 50 nucleic acids in length, at least about 55 nucleic acids in length, at least about 60 nucleic acids in length, at least about 65 nucleic acids in length, at least about 70 nucleic acids in length, at least about 75 nucleic acids in length, at least about 80 nucleic acids in length, at least about 85 nucleic acids in length, at least about 90 nucleic acids in length, at least about 95 nucleic acids in length, at least about 100 nucleic acids in length, at least about 110 nucleic acids in length, at least about 120 nucleic acids in length, at least about 130 nucleic acids in length, at least about 140 nucleic acids in length, at least about 150 nucleic acids in length, at least about 160 nucleic acids in length, at least about 170 nucleic acids in length, at least about 180 nucleic acids in length, at least about 190 nucleic acids in length, at least about 200 nucleic acids in length, at least about 225 nucleic acids in length, at least about 250 nucleic acids in length, at least about 275 nucleic acids in length, at least about 300 nucleic acids in length, at least about 350 nucleic acids in length, at least about 400 nucleic acids in length, at least about 450 nucleic acids in length, at least about 500 nucleic acids in length, at least about 600 nucleic acids in length, at least about 700 nucleic acids in length, at least about 800 nucleic acids in length, at least about 900 nucleic acids in length, at least about 1000 nucleic acids in length or about the full length of the Johnsongrass detection sequence identified.
While examples of suitable methods for detecting these regions are included herein, it is to be understood that the disclosure is not limited to the methods described. Rather any suitable method can be employed to detect these Johnsongrass detection sequences and, subsequently, Johnsongrass or Johnsongrass hybrid material.
Oligonucleotides
Several suitable methods of detecting Johnsongrass detection sequences involve the use of oligonucleotide primers and/or probes. For example, oligonucleotide primers capable of hybridizing to Johnsongrass detection sequences can be employed in a PCR reaction. As discussed above, these primers need not be fully complementary to the target sequence to be hybridizable thereto and thus useful in the present disclosure. The oligonucleotide primers of the present disclosure can also contain a detectable label, for example a 5′ end label. SEQ ID NOS.: 48-135 and nucleic acid sequences essentially similar thereto are examples of oligonucleotide primers useful in the present disclosure. In particular, oligonucleotide primers 75% identical, 80% identical, 85% identical, 90% identical, 91% identical, 92% identical, 93% identical, 94% identical, 95% identical, 96% identical, 97% identical, 98% identical, 99% identical or 100% identical to SEQ ID NOS.: 48-135 or a portion thereof are useful in the present disclosure. Further, hybridization using oligonucleotide probes is commonly employed for detection of target sequences and the methodologies are generally known to one skilled in the art. Again, these probes need not be fully complementary to the target sequence to be hybridizable thereto, and thus useful in the present disclosure.
A nucleic acid probe sequence can also optionally be employed with the primer sequence pairs of the present disclosure in an amplification based detection technique, such as in the 3′-exonuclease assay.
Oligonucleotides of the present disclosure could also contain both primer and probe regions, and thus be employed as a primer-probe complex in an appropriate assay, such as a Scorpion probe assay. Such primer-probe complexes commonly contain a non-amplifiable linker that connects the 3′ terminus of the probe region to the 5′ terminus of the primer region. This non-amplifiable linker stops extension of a complementary strand from proceeding into the probe region of the primer-probe complex. Examples of such non-amplifiable linkages include hexethylene glycol (HEG) and, preferably, 18-carbon linkers. Primer-probe complexes of the present disclosure can also contain a self-complementary region that allows the primer-probe complex to form a stem-loop structure when the probe is unbound from its target DNA, which may be useful, for example, in bringing the reporter and quencher into sufficiently close proximity to one another to cause the reporter signal to be quenched. In some instances, a blocking oligonucleotide can be employed with a primer-probe complex, which blocking oligonucleotide is capable of hybridizing to the probe region of the primer-probe complex when the probe region is unbound from its target DNA. If the reporter is attached to the primer-probe complex and the quencher is attached to the blocking oligonucleotide, this can bring the reporter and quencher into sufficiently close proximity to one another to allow quenching to occur.
Oligonucleotide probes of the present disclosure can also be affixed to a solid support, such as a glass microarray slide.
Assay Methods
Detection of the Johnsongrass detection sequences, and subsequent detection of the presence or amount of Johnsongrass or Johnsongrass generic material, may be accomplished in any suitable manner. Preferred methods are primer-directed amplification methods and nucleic acid hybridization methods.
Primer-Directed Amplification Assay Methods
A variety of primer-directed nucleic acid amplification methods are known in the art which can be employed in the present disclosure, including thermal cycling methods (e.g., PCR, RT-PCR and LCR), as well as isothermal methods and strand displacement amplification (SDA). The preferred method is PCR. In one preferred embodiment, one or more primer pairs selected from SEQ ID NOS.: 48-135 and nucleic acid sequences essentially similar thereto may be used as primers in PCR amplification for the detection of one or more of SEQ ID NOS.: 1-47 and nucleic acid sequences essentially similar thereto.
A skilled person will understand that any generally acceptable thermal cycling conditions may be used for successfully detecting Johnsongrass detection sequences using oligonucleotides of the instant disclosure, and, depending on the sample to be tested and other laboratory conditions, routine optimization for the cycling conditions may be necessary to achieve optimal sensitivity and specificity.
Any suitable nucleic acid replication composition in any format can be used in the present disclosure. A typical replication composition for PCR amplification may comprise, for example, dATP, dCTP, dGTP, dTTP, target specific primers and a suitable polymerase. If the replication composition is in liquid form, suitable buffers known in the art may be used (see, e.g., Sambrook, J., et al., supra). Alternatively, if the replication composition is contained in a tablet form, then typical tabletization reagents may be included, such as stabilizers and binding agents.
A preferred replication composition of the instant disclosure comprises (a) at least one primer pair selected from SEQ ID NOS.: 48-135 and nucleic acid sequences essentially similar thereto, and (b) thermostable DNA polymerase. Another preferred replication composition of the present disclosure comprises (a) one or more primer pairs selected from SEQ ID NOS.: 76 and 77, SEQ ID NOS.: 84 and 85, SEQ ID NOS.: 104 and 105, SEQ ID NOS.: 114 and 115 and SEQ ID NOS.: 116 and 117 and nucleic acid sequences essentially similar thereto and (b) a thermostable DNA polymerase. Yet another preferred composition is a replication composition comprising (a) one or more primer pairs selected from SEQ ID NOS.: 76 and 77, SEQ ID NOS.: 84 and 85, SEQ ID NOS.: 104 and 105, SEQ ID NOS.: 114 and 115, and SEQ ID NOS.: 116 and 117 and nucleic acid sequences essentially similar thereto; (b) a thermostable DNA polymerase and (c) one or more probes selected from SEQ ID NOS.: 136-140 and nucleic acid sequences essentially similar thereto, the probes comprising a detectable label. Preferably the detectable label comprises a reporter capable of emitting a detectable signal and a quencher capable of substantially quenching the reporter and preventing the emission of the detectable signal when the reporter and quencher are in sufficiently close proximity to one another. A preferred kit of the instant disclosure comprises any one of the above replication compositions.
In some instances, an internal positive control can be included in the reaction. The internal positive control can include control template nucleic acids (e.g., DNA or RNA), control primers and control nucleic acid probe. The control reaction is useful to validate the amplification reaction. Amplification of the control DNA occurs within the same reaction tube as the sample that is being tested and therefore indicates a successful amplification reaction when samples are target negative, i.e., no target amplification product is produced. Control DNA will be of appropriate size and base composition to permit amplification in a primer-directed amplification reaction. The control template DNA sequence may be obtained from essentially any source, but must be reproducibly amplified under the same conditions that permit the amplification of the target amplification product.
Primer-directed amplification products of Johnsongrass detection sequences, such as SEQ ID NOS.: 1-47 and nucleic acid sequences essentially similar thereto, can be analyzed using various methods, including both heterogeneous detection methods and homogenous detection methods.
Homogenous detection may be employed to carry out “real-time” primer-directed nucleic acid amplification and detection, using primer pairs of the instant disclosure (e.g., real-time PCR and real-time RT-PCR). Preferred real-time methods are set forth in U.S. Pat. Nos. 6,171,785, 5,994,056, 6,326,145, 5,804,375, 5,538,848, 5,487,972 and 5,210,015, each of which is hereby incorporated by reference in its entirety.
A preferred real-time detection method is the 5′-exonuclease detection method, as set forth in U.S. Pat. Nos. 5,804,375, 5,538,848, 5,487,972 and 5,210,015, each of which is hereby incorporated by reference in its entirety. One example of such a method is the Taqman real time PCR assay. In the 5′-exonuclease detection assay, a modified probe is employed during PCR which binds intermediate to or between the two members of the amplification primer pair.
It should be understood that the present disclosure could be operated using a combination of these techniques, such as by having a Scorpion probe directed to one target region and a Taqman® probe directed to a second target region. It should also be understood that the disclosure is not limited to the above-described techniques. Rather, one skilled in the art would recognize that other techniques for detecting amplification as known in the art could also be used. For example, techniques such as PCR-based quantitative sequence detection (QSD) may be performed using nucleic acid probes which, when present in the single-stranded state in solution, are configured such that the reporter and quencher are sufficiently close to substantially quench the reporter's emission. However, upon hybridization of the intact reporter-quencher nucleic acid probe with the amplified target nucleic acid sequence, the reporter and quenchers become sufficiently distant from each other. As a result, the quenching is substantially abated causing an increase in the fluorescence emission detected.
Nucleic Acid Hybridization Methods
In addition to primer-directed amplification assay methods, nucleic acid hybridization assay methods can be employed in the present disclosure for detection of Johnsongrass detection sequences. The basic components of a nucleic acid hybridization test include a probe, a sample suspected of containing Johnsongrass material, and a specific hybridization method. Typically, the probe length can vary from as few as 10 bases to the full length of the Johnsongrass detection sequence. Only part of the probe molecule need be complementary to the nucleic acid sequence to be detected. In addition, the complementarity between the probe and the target sequence need not be perfect. Hybridization does occur between imperfectly complementary molecules with the result that a certain fraction of the bases in the hybridized region are not paired with the proper complementary base. Examples of probes useful in nucleic acid hybridization methods in the present disclosure are any of SEQ ID NOS.: 48-140 and nucleic acid sequences essentially similar thereto.
Hybridization methods are well known in the art. Typically, the probe and sample must be mixed under conditions which will permit nucleic acid hybridization. This involves contacting the probe and sample in the presence of an inorganic or organic salt under the proper concentration and temperature conditions. The probe and sample nucleic acids must be in contact for a long enough time that any possible hybridization between the probe and sample nucleic acid may occur. The concentration of probe or target in the mixture will determine the time necessary for hybridization to occur. The higher the probe or target concentration, the shorter the hybridization incubation time needed.
Various hybridization solutions can be employed. Typically, these comprise from about 20 to 60% volume, preferably 30%, of a polar organic solvent. A common hybridization solution employs about 30-50% v/v formamide, about 0.15 to 1M sodium chloride, about 0.05 to 0.1M buffers, such as sodium citrate, Tris-HCl, PIPES or HEPES (pH range about 6-9), about 0.05 to 0.2% detergent, such as sodium dodecylsulfate or between 0.5-20 mM EDTA, FICOLL (Pharmacia Inc.) (about 300-500 kilodaltons), polyvinylpyrrolidone (about 250-500 kdal) and serum albumin. Also included in the typical hybridization solution will be unlabeled carrier nucleic acids from about 0.1 to 5 mg/mL, fragmented nucleic DNA (e.g., calf thymus or salmon sperm DNA or yeast RNA) and optionally from about 0.5 to 2% wt/vol glycine. Other additives may also be included, such as volume exclusion agents which include a variety of polar water-soluble or swellable agents (e.g., polyethylene glycol), anionic polymers (e.g., polyacrylate or polymethylacrylate) and anionic saccharidic polymers (e.g., dextran sulfate).
Nucleic acid hybridization is adaptable to a variety of assay formats. One of the most suitable is the sandwich assay format. The sandwich assay is particularly adaptable to hybridization under non-denaturing conditions. A primary component of a sandwich-type assay is a solid support. The solid support has adsorbed to it or covalently coupled to it immobilized nucleic acid probe that is unlabeled and complementary to one portion of the DNA sequence.
The sandwich assay may be encompassed in an assay kit. This kit would include a first component for the collection of samples suspected of contamination and buffers for the disbursement and lysis of the sample. A second component would include media in either dry or liquid form for the hybridization of target and probe polynucleotides, as well as for the removal of undesirable and nonduplexed forms by washing. A third component includes a solid support (e.g., dipstick) upon which is fixed (or to which is conjugated) unlabeled nucleic acid probe(s) that is (are) complementary to one or more Johnsongrass detection sequences, such as SEQ ID NOS.: 1-47 and nucleic acid sequences essentially similar thereto. A fourth component would contain labeled probe that is complementary to a second and different region of the same DNA strand to which the immobilized, unlabeled nucleic acid probe of the third component is hybridized.
Another preferred nucleic acid hybridization method involves the use of DNA or oligonucleotide microarrays. In this technique, oligonucleotide probes, such as one or more of SEQ ID NOS.: 48-140 and nucleic acid sequences essentially similar thereto, are attached to a solid support, such as a glass slide. Labeled nucleic acids derived from the sample of interest are then hybridized to the oligonucleotides attached to the solid support. Following washing of unbound nucleic acids, the slide is visualized, with a positive detection of label indicating the presence of the target for which each oligonucleotide is directed. In a preferred method, more than one, and preferably all, of the oligonucleotides of SEQ ID NOS.: 48-140 are attached to the solid support such that detection of more than one, and preferably all, of SEQ ID NOS.: 1-47 can be simultaneously monitored and detected.
EXAMPLES
The present disclosure is further defined in the following Examples. It should be understood that these Examples, while indicating preferred embodiments of the disclosure, are given by way of illustration only.
Unless otherwise stated, all recombinant DNA techniques are carried out according to standard protocols as described, for example, in Sambrook, et al., (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, NY and in Volumes 1 and 2 of Ausubel, et al., (1994) Current Protocols in Molecular Biology, Current Protocols, USA. Standard materials and methods for plant molecular work are described, for example, in Plant Molecular Biology Labfax (1993) by Croy published by BIOS Scientific Publications Ltd (UK) and Blackwell Scientific Publications, UK.
Example 1
In Silico Identification of Candidate Johnsongrass Detection Sequences
Candidate Johnsongrass detection sequences were identified. To begin the candidate identification process, a database of Johnsongrass and Sorghum propinquum sequences was derived. The database consisted of a set of Johnsongrass sequences, consisting of 1965 expressed sequence tags (ESTs) and 191 restriction fragment length polymorphism (RFLP) marker sequences, and a set of S. propinquum sequences, consisting of 20,881 ESTs, 23,703 genome survey sequences (GSSs) and 223 marker sequences, all of which were obtained from the genBank database of the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/) as of Nov. 11, 2007.
Each of these sets of sequences was aligned against the public v1.0 release (Sbi1 assembly) of the Sorghum bicolor genome (http://genome.jgi-psf.org/Sorbi1/Sorbi1.home.html) using BLASTN (Altschul, et al., 1997) at expected value cutoff of e-10, 70% identity and 70 nt overlap. Sequences that were found to be present in Johnsongrass and/or S. propinquum but absent from the sorghum database were retained and further screened against the NCBI non-redundant nucleotide database (nt) with the above parameters to further identify those sequences likely to be unique to Johnsongrass and S. propinquum. The GSSs and ESTs thus identified were then assembled into longer sequences using the PHRAP assembly program (Version 0.990329; http://www.phrap.org/phredphrap/phrap.html) at default parameters. The Johnsongrass and S. propinquum sequences were then further aligned against each other with the BLASTN algorithm to identify homologue markers with high specificity. This process identified a set of candidate Johnsongrass detection sequences that were subsequently used for laboratory validation.
Example 2
PCR Validation of Candidate Sequences
Primer Design:
Forty-three of the candidate Johnsongrass detection sequences identified through the in silico analysis of Example 1 were selected for laboratory validation via PCR assays. First, the Primer3 software program was used to design PCR amplification primers for the 43 candidate sequences using the default software settings. Table 1 lists the 44 primer pairs that were designed (one of the Johnsongrass detection sequences had two distinct primer sets designed for it).







TABLE 1







List of primers surveyed










Primer
Target
Left (Forward) Primer
Right (Reverse) Primer













Set
SEQ ID
Name
SEQ ID
Sequence
SEQ ID
Sequence





A
 1
Contig7
 48
AATCAAGCCATTGGTTCCTG
 49
AAGCCAAGGAAGAGGTGGAT


 


B
 2
Contig39
 50
GGGATGGACATGGTGAAATC
 51
GAAGTCGATGACGACCACCT


 


C
 3
Contig44
 52
GATGGATGGTGACACAGCAC
 53
TTGAGTCAGCCAGTCAGTCG


 


D
 4
Contig46
 54
ATAAAAACGGCCTCCTTGCT
 55
TGCTCTTCACACAACCAAGC


 


E
 5
Contig51
 56
GCGAGGTCGAGAAGATGAAG
 57
TGGATCAGTTGGTCTTGCAG


 


F
 6
Gr2.1
 58
TCCGTCAACTTCCACCTTTC
 59
AGGGTTGACTCAGGGAGGTT


 


G
 9
Gr2.4
 60
CCAGCGATAGTGCACACATT
 61
CTCTCTCTCGCGCTCTGTCT


 


H
10
Gr2.5
 62
CTAACCTCACCGCTTTAGGG
 63
CCAGCGGGAGAGAGAAAAA


 


I
11
Gr2.6
 64
GAGTGCGTTCAGAGGTTCAA
 65
CTTGACTGGGTTTGCCATTG


 


J
12
Gr2.7
 66
TGTGAAATTCCATGGCAAAA
 67
ATAGGCTCCGAGTGCTACGA


 


K
13
Gr2.8
 68
TGAATGATGAAGGCAATGGA
 69
TGCATTCGGGACTTTACACA


 


L
14
Gr2.9
 70
CATTGGTTTTGCGTATCGTG
 71
GACCGACCGTCTTTAACCAA


 


M
15
Gr2.10
 72
TGAATGATGAAGGCAATGGA
 73
AGAGCCGTTGATGAGCCTAA


 


N
16
Gr2.11
 74
GGGCTGTGTCCATGATCTTT
 75
AGGGCATCCAGACAAACAAC


 


O
17
Gr2.12
 76
GATGTTGGCTTCACCGTTTT
 77
TATTTCGCAGGACGACCTTT


 


P
18
Gr2.13
 78
GCACGAGCCACCTATCTGTC
 79
AGAGGGCACGGTGAGGTTAG


 


Q
19
Gr2.14
 80
GTCGACGAATTCCAGTAGCC
 81
GCAAGCTTGAAGTGGGAACT


 


R
20
Gr4.1
 82
ATGCACCACTGATTTGGTGA
 83
TTCACAACCAGTGACCACAAA


 


S
21
Gr4.2
 84
TAGCTGCTACCACGCACACT
 85
GGCCTTGTACGTTGTTGGAC


 


T
22
Gr4.3
 86
TTTTATGCCCACACCTCCAT
 87
GTGTGGCGTACTTCAGACCA


 


U
23
Gr4.4
 88
ATCTGTATGGGAACCGTGCT
 89
TGTCTTGGAGGGGTGGTTAG


 


V
24
Gr4.5
 90
TGCACGTTGCTTCCTTTGTA
 91
AGAGTGCTCGGTCACTCACA


 


W
25
Gr4.6
 92
TTTGGGCTCCACATGTCATA
 93
GTACGGGGATGTTTTGGTTG


 


X
26
Gr4.7
 94
GGTGATTAGCCCGTTTTTCA
 95
ATAAGATGCGCCCATAGCAC


 


Y
27
Gr4.8
 96
GCACAGCCAAAGCATACAGA
 97
AATGGGCCCACTGATAACAA


 


Z
28
Gr4.9
 98
AGCTGAGGGGGAGATAGACC
 99
AAATTCTGCCCACCAGATTG


 


AA
29
Gr4.10
100
ACCAGCGAGAAGAGCCACTA
101
GTGGATGGTGAAGGAGTCGT


 


AB
30
Gr4.11
102
CCTCGCACTTGTGCTCATTA
103
TGTGCAATGAAGCCATGTTT


 


AC
31
Gr4.12
104
TCCCTGTGACACTGCAAGAG
105
CCCCGGACTTCAACAGTCTA


 


AD
32
Gr4.13
106
TGACAGCCACTCGAGAACAG
107
GCCAACAATCACAGCAATGT


 


AE
33
Gr4.14
108
ACAGGGTGACTGGAATGGAG
109
AGGCATTCTGGTGTCTGTCC


 


AF
34
Gr4.15
110
GATCAAGGGGAGATCGTCAA
111
CCGATGCCTTGAAAATGACT


 


AG
35
Gr4.16
112
CTGACACATGTCCTGGTTGG
113
ATGGCATCAAGATCCTCTGG


 


AH
36
Gr4.17
114
ATTTTGCGCATGGATATGGT
115
GACGGTGAGGCCTGTTATGT


 


AI
37
Gr4.18
116
GAAGTTTTCCGCCTTCCTCT
117
TTGCCAAACTGCTTCACAAG


 


AJ
38
Gr4.19
118
CTGTCACCGGAGAAGAGGAG
119
GGAGGTCGAAGAGAGTGACG


 


AK
38
Gr4.19
120
CTCGACAGGGGAGTTCTCAG
121
CGGCCAACTCATTATCTCGT


 


AL
40
Gr4.21
122
GCACGAGGCTAACACAAACA
123
GACAACATTCTCCGCTTGGT


 


AM
41
pRC
124
TTGTTCATCAAGGCAGCAAG
125
CGCAGTCGACATGGTTTATG


 


AN
42
pSHR
126
CTTAATTGGCAGGGTGCAGT
127
GACATCTACAACGCCGTCCT


 


AO
43
Rhiz1-12
128
ATAAGTTGGCATTGGCTTCG
129
CAGCGAACTCAACAATGCAC


 


AP
44
Rhiz1-27
130
CTCGTGTTGTTGCTCGTGTT
131
CGGAGAATAGCTCCATTCCA


 


AQ
45
Rhiz1-36
132
GTGGAGTGGGTCCTCAAGAA
133
GTATCACACACGCCCACTTG


 


AR
46
Rhiz1-5
134
CCCTCAAGACCCTGTTGTGT
135
GTTGTAAAGCCAGGCCACAT









Sorghum and Johnsongrass Samples:

Following primer design, the 44 primer sets listed in Table 1 were used in validation experiments against multiple samples of sorghum and Johnsongrass. The samples tested included 36 sorghum samples from Pioneer Hi-Bred's germplasm and 18 Johnsongrass extensions, four of which were collected from Pioneer Hi-Bred's fields in Texas (designated JG1-4) and the remainder originating from a variety of other geographical locations were provided by John Erpelding of the USDA-ARS, Tropical Agriculture Research Station in Puerto Rico. Table 2 lists the samples of various sorghum varieties and Johnsongrass extensions that were used in this study.







TABLE 2







Samples of sorghum and Johnsongrass surveyed









Sorghum
Johnsongrass
source













PHY50FW
EDH7
JG1
Plainview, TX


leaf





PHY50FW
FPA66
JG2
Plainview, TX


SS





PHY50FW
NTC31
JG3
Plainview, TX


bulkf





ShumSS
ZYL24
JG4
Plainview, TX


F1





ShumSS
8M429W
Grif14527
(USDA)


F2





ShumSS
CAJ14W
Grif14530
(USDA)


F3





ABH23G
ENZ27W
Grif14531
(USDA)


AGK1G
FEH14W
Grif16339
(USDA)


HVC2G
IC0010W
PI185458
(USDA)


KCK8G
IFU870W
PI201766
(USDA)


MPH30G
IPU970W
PI209217
(USDA)


QL35G
IZY39W
PI228364
(USDA)


QTA51G
MEY16W
PI271615
(USDA)


EC78GW
YGC87W
PI302160
(USDA)


YGY5GW
IS3620C
PI302268
(USDA)


BTX623
MFY55W
PI302277
(USDA)


SS6B@
P9990GW
PI302281
(USDA)


YYJ6G
YYU28W
Sorghum
Puerto Rico




halepense
(USDA)









Sample Preparation:

Sorghum samples were received in the form of genomic DNA from Pioneer Hi-Bred Research Center in Georgetown, Canada. The sorghum DNAs were isolated from 2 cm×2 cm (for fully developed plants) or 3 cm×3 cm (for young plants) pieces of leaf tissue as follows: The tissues were freeze dried overnight and then ground by placing stainless steel balls in the sample tube and shaking the tube in a paint shaker for 90 seconds. 250 μl of the dried, ground tissue was taken to a new tube and combined with 900 μl of extraction buffer containing 2% hexadecyltrimethyl ammonium bromide, 1.4M NaCl, 100 mM Tris-Cl pH 8.0, 20 mM EDTA, 0.5% sodium bisulfate and 1% β-mercaptoethanol. The samples were incubated at 65° C. for 40 minutes and then the DNA was isolated via a standard extraction method utilizing chloroform/isoamyl alcohol (24:1 v:v), isopropanol, and centrifugation. The DNA was washed in 70% ethanol, dried, and then resuspended in water containing 50 μg/ml RNase.
Johnsongrass varieties were received as seed. To prepare leaf DNA samples, seeds were grown in flats in a growth room until plants were mature enough for the leaves to be sampled. Leaves were sampled by cutting vegetative tissue equivalent to approximately eight leaf punches (1-2 inch long leaf strips). Leaf samples were frozen and then lyophilized over night in a freeze-drier. Dry tissue was ground to powder on a Genogrinder at 1500 strokes/min for one minute prior to DNA isolation. DNA isolation was performed using a NucleoSpin DNA isolation kit (Macherey-Nagel) following the manufacturer's recommended protocol. To prepare seed DNA samples, seed was ground into flour and then DNA was isolated from that material using a NucleoSpin DNA isolation kit (Macherey-Nagel) following the manufacturer's recommended protocol.
PCR Screening:
Each of the 44 primer sets was initially screened against sorghum PHY50FW and one or two of three different possible Johnsongrass samples (JG1 leaf, JG1 seed, and JG4 leaf). Genomic DNA of the samples was used as template in PCR reactions. PCR amplifications were performed on Mx3000 and Mx3005 (Stratagene) thermocyclers. Reactions were set up using 50 ng Genomic DNA, 4 μM of each primer, Stratagene 2X Brilliant master mix and 0.4 μl ROX reference dye in a total reaction volume of 25 μl. The theromocycle profile included an enzyme activation step at 95° C. for 10 minutes; 40 cycles of denaturation at 95° C. for 30 sec, annealing at 60° C. for 60 sec, elongation at 72° C. for 30 sec and a post-cycling final elongation at 72° C. for 7 min. In each run, for every genomic DNA sample used, there was also an endogenic gene control, Sorgoleone 1 (SOR1, Yang, et al., 2004) amplification performed to ensure the amplicability of the DNA. Positive amplification was determined using SYBR Green melt curve analysis as well as visualization via 2% agarose gel electrophoresis. The results of this screen are shown in Table 3, below. Primer sets that gave positive amplification results from Johnsongrass DNA but not from sorghum DNA were subsequently used in a second round of screening.







TABLE 3







Results of first round screening of potential Johnsongrass detection sequences












Primer
Target
Sorghum
Johnsongrass
Johnsongrass
Johnsongrass













set
SEQ ID
Name
PHY50FW
JG1 Leaf
JG1 Seed
JG4 Leaf
















A
1
Contig7
Y
Y




B
2
Contig39
Y
Y


C
3
Contig44
Y
Y


D
4
Contig46
Y
Y


E
5
Contig51
Y
Y


F
6
Gr2.1
N

N


G
9
Gr2.4
N

N


H
10
Gr2.5
N

N


I
11
Gr2.6
Y

Y


J
12
Gr2.7
N

N


K
13
Gr2.8
N

N


L
14
Gr2.9
Y

Y


M
15
Gr2.10
N

N


N
16
Gr2.11
N

Y


O
17
Gr2.12
N

Y


P
18
Gr2.13
N

Y


Q
19
Gr2.14
Y/N

Y


R
20
Gr4.1
Y

Y


S
21
Gr4.2
N

Y


T
22
Gr4.3
Y

Y


U
23
Gr4.4
Y

Y


V
24
Gr4.5
Y

Y


W
25
Gr4.6
Y

Y
Y


X
26
Gr4.7
N

N


Y
27
Gr4.8
Y

Y


Z
28
Gr4.9
Y

Y
Y


AA
29
Gr4.10
Y

Y
Y


AB
30
Gr4.11
N

Y
Y


AC
31
Gr4.12
N

Y


AD
32
Gr4.13
Y

Y
Y


AE
33
Gr4.14
Y

Y
Y


AF
34
Gr4.15
Y

Y
Y


AG
35
Gr4.16
Y

Y
Y


AH
36
Gr4.17
N

Y
Y


AI
37
Gr4.18
N

Y
Y


AJ
38
Gr4.19
N

Y
N


AK
38
Gr4.19
Y

Y
Y


AL
40
Gr4.21
N

N
N


AM
41
pRC
Y
Y


AN
42
pSHR
Y
Y


AO
43
Rhiz1-12
Y
Y


AP
44
Rhiz1-27
Y
Y


AQ
45
Rhiz1-36
Y
Y









In the second part of the survey, nine primer sets were selected for screening across all of the 54 sorghum and Johnsongrass samples. The primer sets selected were primer sets N, O, P, S, AB, AC, AH, Al and AJ, which are directed toward Johnsongrass detection sequences Gr2.11, Gr2.12, Gr2.13, Gr4.2, Gr4.11, Gr4.12, Gr4.17, Gr4.18 and Gr4.19, respectively. PCR amplifications were performed as described above and positive amplification results were again determined using SYBR Green melt curve analysis as well as visualization via 2% agarose gel electrophoresis. The results of this screen are shown in Table 4, below.







TABLE 4







Results of screen of nine select primer sets against


54 samples of Johnsongrass and sorghum
















Sorghum or
Primer
Primer
Primer
Primer
Primer
Primer
Primer
Primer
Primer


Johnsongrass
set N
set O
set P
set S
set AB
set AC
set AH
set AI
set AJ


sample
(Gr2.11)
(Gr2.12)
(Gr2.13)
(Gr4.2)
(Gr4.11)
(Gr4.12)
(Gr4.17)
(Gr4.18)
(Gr4.19)





JG1
+
+
+
+
+
+
+
+
+/−


JG2
+
+
+
+
+
+
+
+
+


JG3
−
+
−
+
−
+
+
+
−


JG4
+
+
+
+
+
−
+
+
−


Grif14527
+
+
+
+
+
+
+
+
+


Grif14530
−
+
−
+
+/−
+
+
+
+/−


Grif14531
+
+
+
+
+
+
+
+
−


Grif16339
−
+
+
+
+
+
+
+
−


PI185458
−
+
+
+
+
+
+
+
−


PI201766
+
+
+
+
+/−
+
+
+
−


PI209217
+
+
−
+
+
+
+
+
−


PI228364
+
+
−
+
+/−
+
+
+/−
+/−


PI271615
−
+
+
+
+
+
+
+
+


PI302160
+
+
+
+
−
+
+
+
+


PI302268
+
+
−
+
+
+
+
+
+


PI302277
+
+
+
+/−
−
+
−
+
−


PI302281
+
+
+
+
+/−
+
−
+
−


S. halepense
+
+
+
+
+
+
+
+
+


PHY50FWleaf
−
−
−
−
−
−
−
−
−


PHY50FWSS
−
−
−
−
−
−
−
−
−


PHY50FWbulkf
−
−
−
+/−
−
−
−
−
−


ShumSS F1
−
−
−
−
−
−
−
−
−


ShumSS F2
−
+/−
−
−
−
−
−
−
−


ShumSS F3
−
−
−
−
−
−
−
−
−


ABH23G
−
−
−
−
−
−
−
+/−
−


AGK1G
−
−
−
+/−
−
−
−
−
−


HVC2G
−
−
−
−
−
−
−
−
−


KCK8G
−
−
−
+/−
−
−
−
−
−


MPH30G
−
−
−
−
−
−
−
−
−


QL35G
−
−
+
−
−
−
−
−
−


QTA51G
−
−
−
+/−
−
−
−
−
−


EC78GW
−
+/−
−
+/−
−
−
−
+/−
−


YGY5GW
−
−
−
−
−
−
−
−
−


BTX623
−
−
−
−
−
−
−
−
−


SS6B@
−
−
−
−
−
−
−
−
−


YYJ6G
−
−
−
−
−
−
−
−
−


EDH7
−
−
+
−
−
−
+/−
−
−


FPA66
−
−
−
−
−
−
−
−
−


NTC31
−
−
−
−
−
−
−
−
−


ZYL24
−
−
+
−
−
−
−
−
−


8M429W
−
−
−
−
−
−
−
−
−


CAJ14W
−
−
−
+/−
−
−
−
+/−
−


ENZ27W
−
−
+/−
−
−
−
−
−
−


FEH14W
−
−
−
−
−
−
−
−
−


IC0010W
−
−
+
−
−
−
−
+/−
−


IFU870W
−
−
+
+/−
−
−
−
−
−


IPU970W
−
−
+
−
−
−
−
−
−


IZY39W
−
−
+/−
−
−
−
−
−
−


MEY16W
−
−
−
−
−
−
−
+/−
−


YGC87W
−
−
+
−
−
−
−
−
−


IS3620C
−
−
−
−
−
−
−
−
−


MFY55W
−
−
−
−
−
−
−
−
−


P9990GW
−
−
−
+/−
−
−
−
−
−


YYU28W
−
−
+
+/−
−
−
−
−
−









Example 3
Real-Time PCR Trial
Probe Design:
Five of the Johnsongrass detection sequences identified through the in silico analysis of Example 1 and tested through the PCR screening of Example 2 were selected for Taqman® Real-Time PCR probe design. The Primer3 software program was again used to design the Taqman® probes using the default software settings. Each probe was designed with a FAM reporter attached to its 5′ end and a BHQ1 quencher attached to its 3′ end. Table 5 lists the five probe sequences that were designed.







TABLE 5







Probes directed toward specific


Johnsongrass detection sequences









Probe
Target











SEQ
SEQ




ID NO.
ID NO.
Name
Probe Sequence





136
17
Gr2.12
Fam-CAAGACATCCAGCGTTTCCT-





BHQ1


 


137
21
Gr4.2
Fam-GATCGATCGATCGAGCAAGT-





BHQ1


 


138
31
Gr4.12
Fam-TCTCAAAGGGAAAACGATGG-





BHQ1


 


139
36
Gr4.17
Fam-CATACTGCGAGCAGACCAAC-





BHQ1


 


140
37
Gr4.18
Fam-TGTTTAGGCGAAGGGAAATG-





BHQ1









Real-Time PCR:

To test the ability of a real-time PCR technique to detect Johnsongrass DNA in a background of sorghum DNA, probe SEQ ID NO.: 136 with a FAM-BHQ1 reporter-quencher pair attached, which is a real-time PCR probe directed toward Johnsongrass detection sequence Gr2.12 (SEQ ID NO.: 17), was used in conjunction with the above-discussed primers directed toward that same Johnsongrass detection sequence (Primer set O) in a real-time PCR trial. Each reaction performed contained 50-100 ng genomic
DNA, 9 μM of each primer from primer set O, 4 μM of the SEQ ID NO.: 136 probe and Roche master mix in a total reaction volume of 25 μl. Further, each reaction was a multiplex reaction including primers (4 μM each of forward and reverse) and a probe (4 μM) directed toward a Johnsongrass/sorghum positive endogenic control gene, ADHexon4—3, to ensure the amplification was working properly. The amplifications were performed on Mx3000 and Mx3005 (Stratagene) thermocyclers using a theromocycle profile that included an enzyme activation step at 95° C. for 10 minutes followed by 40 cycles of denaturation at 95° C. for 10 sec, annealing at 60° C. for 60 sec and elongation at 72° C. for 30 sec.
The genomic DNA employed in each PCR reaction was sorghum YS8400 genomic DNA with varying amounts of Johnsongrass JG1 DNA spiked therein, the ratios of Johnsongrass JG1:sorghum YS8400 DNA ranging from 1:1000 to 1:10,000. In addition, pure sorghum YS8400 DNA and pure Johnsongrass JG1 DNA were used in negative and positive control reactions, respectively. In total 12 separate reaction mixtures were tested, each one being carried out either in triplicate or sextuplicate. The results of this study can be found in Table 6.







TABLE 6







Real-time PCR trial of Johnsongrass detection sequence Gr.2.12 (SEQ ID No.: 17)









Test Mixture




















S1
S2
S3
S4
S5
S6
S7
S8
S9
S10
S11
S12/S13









JG1:Sorghum DNA Ratio






























Pure
Pure



1:1000
1:2000
1:3000
1:4000
1:5000
1:6000
1:7000
1:8000
1:9000
1:10,000
Sorghum
Johnsongrass























Rep. 1
+
+
+
+
+
+
+
−
+
+
−
+


Rep. 2
+
+
+
+
−
+
+
+
+
+
−
+


Rep. 3
+
+
+
+
+
+
+
−
+
−
−
+


Rep. 4
+
+
+








+


Rep. 5
+
+
+








+


Rep. 6
+
+
+








+









As these results indicate, using the Gr2.12 (SEQ ID NO.: 17) Johnsongrass detection sequence, in this example, the real-time PCR method is able to successfully detect Johnsongrass DNA in a background of sorghum DNA, even at a 1:10,000 ratio.

Example 4
Seed Lot Testing
Tests were also performed to determine the ability of real-time PCR analysis of Johnsongrass detection sequences to detect Johnsongrass contamination in a sorghum seed lot. Three separate sorghum seed lots were tested: 84G62-2101, 84G62-N284 and 85G47-M042. For each of the three seed lots, 20 jars of seeds, each jar containing 6,000 seeds, were examined by real time PCR. For each jar, the seeds were first ground to flour and then the DNA from the seed flour was isolated using a NucleoSpin DNA isolation kit (Macherey-Nagel) as per the manufacturer's recommended protocol. The real-time PCR reactions were performed as described in Example 3 above, again using the probe
(SEQ ID NO.: 136) directed toward Johnsongrass detection sequence Gr2.12 (SEQ ID NO.: 17). Further, in addition to the experimental samples, a negative control reaction (pure sorghum YS8400 flour) and several positive control reactions (pure Johnsongrass JG3 seed pools; spiked Johnsongrass seed into a sorghum seed jar at a 1:6000 ratio; and spiked Johnsongrass DNA into a sorghum DNA sample at a 1:3000 ratio) were performed.
In this experimental trial, the real time PCR technique was able to positively identify Johnsongrass contamination (positive Johnsongrass amplification) in two of the 20 jars tested for sorghum lot number 84G62-N284, while the other two sorghum lots tested were found to be negative. This shows the potential usefulness of this method and the identified Johnsongrass detection sequences for detecting Johnsongrass contamination in sorghum seed lots.
The present disclosure is not limited to the embodiments described and exemplified above, but is capable of variation and modification within the scope of the appended claims.

## Claims

1. A method of determining quality of Sorghum bicolor seed production, the method comprising:
a. providing a biological sample comprising Sorghum bicolor seeds, wherein the biological sample is suspected of also containing Johnsongrass or Johnsongrass hybrid genetic material;
b. providing the primer pair of SEQ ID NOS.: 76 and 77 or the probe of SEQ ID NO: 136 that selectively binds to the Johnsongrass or Johnsongrass hybrid genetic material to discriminate against related genetic material;
c. performing a hybridization or an amplification reaction wherein the primer pair of SEQ ID NOS.: 76 and 77 or probe of SEQ ID NO: 136 binds to Johnsongrass or Johnsongrass hybrid genetic material and does not substantially bind to genetic material from Sorghum bicolor; and
d. determining the quality of Sorghum bicolor seed production based on the presence or the amount of Johnsongrass or Johnsongrass hybrid genetic material.

2. The method of claim 1, wherein the genetic material is DNA.