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|
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result sql = De novo assembly and characterization of root transcriptome using Illumina paired-end sequencing and development of cSSR markers in sweet potato (Ipomoea batatas).
De novo assembly and characterization of root transcriptome using Illumina paired-end sequencing and development of cSSR markers in sweet potato (Ipomoea batatas).char = <;code = 60
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char = 6;code = 54
char = 2;code = 50
char = .;code = 46
char = 0;code = 48
char = 2;code = 50
char = %;code = 37
char = );code = 41
char = ;code = 32
char = g;code = 103
char = e;code = 101
char = n;code = 110
char = e;code = 101
char = s;code = 115
char = ;code = 32
char = w;code = 119
char = e;code = 101
char = r;code = 114
char = e;code = 101
char = ;code = 32
char = i;code = 105
char = d;code = 100
char = e;code = 101
char = n;code = 110
char = t;code = 116
char = i;code = 105
char = f;code = 102
char = i;code = 105
char = e;code = 101
char = d;code = 100
char = .;code = 46
char = ;code = 32
char = O;code = 79
char = u;code = 117
char = t;code = 116
char = ;code = 32
char = o;code = 111
char = f;code = 102
char = ;code = 32
char = t;code = 116
char = h;code = 104
char = e;code = 101
char = s;code = 115
char = e;code = 101
char = ;code = 32
char = a;code = 97
char = n;code = 110
char = n;code = 110
char = o;code = 111
char = t;code = 116
char = a;code = 97
char = t;code = 116
char = e;code = 101
char = d;code = 100
char = ;code = 32
char = u;code = 117
char = n;code = 110
char = i;code = 105
char = g;code = 103
char = e;code = 101
char = n;code = 110
char = e;code = 101
char = s;code = 115
char = ,;code = 44
char = ;code = 32
char = 5;code = 53
char = ,;code = 44
char = 0;code = 48
char = 4;code = 52
char = 6;code = 54
char = ;code = 32
char = a;code = 97
char = n;code = 110
char = d;code = 100
char = ;code = 32
char = 1;code = 49
char = 1;code = 49
char = ,;code = 44
char = 9;code = 57
char = 8;code = 56
char = 3;code = 51
char = ;code = 32
char = u;code = 117
char = n;code = 110
char = i;code = 105
char = g;code = 103
char = e;code = 101
char = n;code = 110
char = e;code = 101
char = s;code = 115
char = ;code = 32
char = w;code = 119
char = e;code = 101
char = r;code = 114
char = e;code = 101
char = ;code = 32
char = a;code = 97
char = s;code = 115
char = s;code = 115
char = i;code = 105
char = g;code = 103
char = n;code = 110
char = e;code = 101
char = d;code = 100
char = ;code = 32
char = t;code = 116
char = o;code = 111
char = ;code = 32
char = g;code = 103
char = e;code = 101
char = n;code = 110
char = e;code = 101
char = ;code = 32
char = o;code = 111
char = n;code = 110
char = t;code = 116
char = o;code = 111
char = l;code = 108
char = o;code = 111
char = g;code = 103
char = y;code = 121
char = ;code = 32
char = a;code = 97
char = n;code = 110
char = d;code = 100
char = ;code = 32
char = c;code = 99
char = l;code = 108
char = u;code = 117
char = s;code = 115
char = t;code = 116
char = e;code = 101
char = r;code = 114
char = s;code = 115
char = ;code = 32
char = o;code = 111
char = f;code = 102
char = ;code = 32
char = o;code = 111
char = r;code = 114
char = t;code = 116
char = h;code = 104
char = o;code = 111
char = l;code = 108
char = o;code = 111
char = g;code = 103
char = o;code = 111
char = u;code = 117
char = s;code = 115
char = ;code = 32
char = g;code = 103
char = r;code = 114
char = o;code = 111
char = u;code = 117
char = p;code = 112
char = ,;code = 44
char = ;code = 32
char = r;code = 114
char = e;code = 101
char = s;code = 115
char = p;code = 112
char = e;code = 101
char = c;code = 99
char = t;code = 116
char = i;code = 105
char = v;code = 118
char = e;code = 101
char = l;code = 108
char = y;code = 121
char = .;code = 46
char = ;code = 32
char = S;code = 83
char = e;code = 101
char = a;code = 97
char = r;code = 114
char = c;code = 99
char = h;code = 104
char = i;code = 105
char = n;code = 110
char = g;code = 103
char = ;code = 32
char = a;code = 97
char = g;code = 103
char = a;code = 97
char = i;code = 105
char = n;code = 110
char = s;code = 115
char = t;code = 116
char = ;code = 32
char = t;code = 116
char = h;code = 104
char = e;code = 101
char = ;code = 32
char = K;code = 75
char = y;code = 121
char = o;code = 111
char = t;code = 116
char = o;code = 111
char = ;code = 32
char = E;code = 69
char = n;code = 110
char = c;code = 99
char = y;code = 121
char = c;code = 99
char = l;code = 108
char = o;code = 111
char = p;code = 112
char = e;code = 101
char = d;code = 100
char = i;code = 105
char = a;code = 97
char = ;code = 32
char = o;code = 111
char = f;code = 102
char = ;code = 32
char = G;code = 71
char = e;code = 101
char = n;code = 110
char = e;code = 101
char = s;code = 115
char = ;code = 32
char = a;code = 97
char = n;code = 110
char = d;code = 100
char = ;code = 32
char = G;code = 71
char = e;code = 101
char = n;code = 110
char = o;code = 111
char = m;code = 109
char = e;code = 101
char = s;code = 115
char = ;code = 32
char = P;code = 80
char = a;code = 97
char = t;code = 116
char = h;code = 104
char = w;code = 119
char = a;code = 97
char = y;code = 121
char = ;code = 32
char = d;code = 100
char = a;code = 97
char = t;code = 116
char = a;code = 97
char = b;code = 98
char = a;code = 97
char = s;code = 115
char = e;code = 101
char = ;code = 32
char = (;code = 40
char = K;code = 75
char = E;code = 69
char = G;code = 71
char = G;code = 71
char = );code = 41
char = ;code = 32
char = i;code = 105
char = n;code = 110
char = d;code = 100
char = i;code = 105
char = c;code = 99
char = a;code = 97
char = t;code = 116
char = e;code = 101
char = d;code = 100
char = ;code = 32
char = t;code = 116
char = h;code = 104
char = a;code = 97
char = t;code = 116
char = ;code = 32
char = 1;code = 49
char = 7;code = 55
char = ,;code = 44
char = 5;code = 53
char = 9;code = 57
char = 8;code = 56
char = ;code = 32
char = (;code = 40
char = 3;code = 51
char = 1;code = 49
char = .;code = 46
char = 1;code = 49
char = 4;code = 52
char = %;code = 37
char = );code = 41
char = ;code = 32
char = u;code = 117
char = n;code = 110
char = i;code = 105
char = g;code = 103
char = e;code = 101
char = n;code = 110
char = e;code = 101
char = s;code = 115
char = ;code = 32
char = w;code = 119
char = e;code = 101
char = r;code = 114
char = e;code = 101
char = ;code = 32
char = m;code = 109
char = a;code = 97
char = p;code = 112
char = p;code = 112
char = e;code = 101
char = d;code = 100
char = ;code = 32
char = t;code = 116
char = o;code = 111
char = ;code = 32
char = 1;code = 49
char = 2;code = 50
char = 4;code = 52
char = ;code = 32
char = K;code = 75
char = E;code = 69
char = G;code = 71
char = G;code = 71
char = ;code = 32
char = p;code = 112
char = a;code = 97
char = t;code = 116
char = h;code = 104
char = w;code = 119
char = a;code = 97
char = y;code = 121
char = s;code = 115
char = ,;code = 44
char = ;code = 32
char = a;code = 97
char = n;code = 110
char = d;code = 100
char = ;code = 32
char = 1;code = 49
char = 1;code = 49
char = ,;code = 44
char = 0;code = 48
char = 5;code = 53
char = 6;code = 54
char = ;code = 32
char = w;code = 119
char = e;code = 101
char = r;code = 114
char = e;code = 101
char = ;code = 32
char = a;code = 97
char = s;code = 115
char = s;code = 115
char = i;code = 105
char = g;code = 103
char = n;code = 110
char = e;code = 101
char = d;code = 100
char = ;code = 32
char = t;code = 116
char = o;code = 111
char = ;code = 32
char = m;code = 109
char = e;code = 101
char = t;code = 116
char = a;code = 97
char = b;code = 98
char = o;code = 111
char = l;code = 108
char = i;code = 105
char = c;code = 99
char = ;code = 32
char = p;code = 112
char = a;code = 97
char = t;code = 116
char = h;code = 104
char = w;code = 119
char = a;code = 97
char = y;code = 121
char = s;code = 115
char = ,;code = 44
char = ;code = 32
char = w;code = 119
char = h;code = 104
char = i;code = 105
char = c;code = 99
char = h;code = 104
char = ;code = 32
char = w;code = 119
char = e;code = 101
char = r;code = 114
char = e;code = 101
char = ;code = 32
char = w;code = 119
char = e;code = 101
char = l;code = 108
char = l;code = 108
char = ;code = 32
char = r;code = 114
char = e;code = 101
char = p;code = 112
char = r;code = 114
char = e;code = 101
char = s;code = 115
char = e;code = 101
char = n;code = 110
char = t;code = 116
char = e;code = 101
char = d;code = 100
char = ;code = 32
char = b;code = 98
char = y;code = 121
char = ;code = 32
char = c;code = 99
char = a;code = 97
char = r;code = 114
char = b;code = 98
char = o;code = 111
char = h;code = 104
char = y;code = 121
char = d;code = 100
char = r;code = 114
char = a;code = 97
char = t;code = 116
char = e;code = 101
char = ;code = 32
char = m;code = 109
char = e;code = 101
char = t;code = 116
char = a;code = 97
char = b;code = 98
char = o;code = 111
char = l;code = 108
char = i;code = 105
char = s;code = 115
char = m;code = 109
char = ;code = 32
char = a;code = 97
char = n;code = 110
char = d;code = 100
char = ;code = 32
char = b;code = 98
char = i;code = 105
char = o;code = 111
char = s;code = 115
char = y;code = 121
char = n;code = 110
char = t;code = 116
char = h;code = 104
char = e;code = 101
char = s;code = 115
char = i;code = 105
char = s;code = 115
char = ;code = 32
char = o;code = 111
char = f;code = 102
char = ;code = 32
char = s;code = 115
char = e;code = 101
char = c;code = 99
char = o;code = 111
char = n;code = 110
char = d;code = 100
char = a;code = 97
char = r;code = 114
char = y;code = 121
char = ;code = 32
char = m;code = 109
char = e;code = 101
char = t;code = 116
char = a;code = 97
char = b;code = 98
char = o;code = 111
char = l;code = 108
char = i;code = 105
char = t;code = 116
char = e;code = 101
char = .;code = 46
char = ;code = 32
char = I;code = 73
char = n;code = 110
char = ;code = 32
char = a;code = 97
char = d;code = 100
char = d;code = 100
char = i;code = 105
char = t;code = 116
char = i;code = 105
char = o;code = 111
char = n;code = 110
char = ,;code = 44
char = ;code = 32
char = 4;code = 52
char = ,;code = 44
char = 1;code = 49
char = 1;code = 49
char = 4;code = 52
char = ;code = 32
char = c;code = 99
char = D;code = 68
char = N;code = 78
char = A;code = 65
char = ;code = 32
char = S;code = 83
char = S;code = 83
char = R;code = 82
char = s;code = 115
char = ;code = 32
char = (;code = 40
char = c;code = 99
char = S;code = 83
char = S;code = 83
char = R;code = 82
char = s;code = 115
char = );code = 41
char = ;code = 32
char = w;code = 119
char = e;code = 101
char = r;code = 114
char = e;code = 101
char = ;code = 32
char = i;code = 105
char = d;code = 100
char = e;code = 101
char = n;code = 110
char = t;code = 116
char = i;code = 105
char = f;code = 102
char = i;code = 105
char = e;code = 101
char = d;code = 100
char = ;code = 32
char = a;code = 97
char = s;code = 115
char = ;code = 32
char = p;code = 112
char = o;code = 111
char = t;code = 116
char = e;code = 101
char = n;code = 110
char = t;code = 116
char = i;code = 105
char = a;code = 97
char = l;code = 108
char = ;code = 32
char = m;code = 109
char = o;code = 111
char = l;code = 108
char = e;code = 101
char = c;code = 99
char = u;code = 117
char = l;code = 108
char = a;code = 97
char = r;code = 114
char = ;code = 32
char = m;code = 109
char = a;code = 97
char = r;code = 114
char = k;code = 107
char = e;code = 101
char = r;code = 114
char = s;code = 115
char = ;code = 32
char = i;code = 105
char = n;code = 110
char = ;code = 32
char = o;code = 111
char = u;code = 117
char = r;code = 114
char = ;code = 32
char = u;code = 117
char = n;code = 110
char = i;code = 105
char = g;code = 103
char = e;code = 101
char = n;code = 110
char = e;code = 101
char = s;code = 115
char = .;code = 46
char = ;code = 32
char = O;code = 79
char = n;code = 110
char = e;code = 101
char = ;code = 32
char = h;code = 104
char = u;code = 117
char = n;code = 110
char = d;code = 100
char = r;code = 114
char = e;code = 101
char = d;code = 100
char = ;code = 32
char = p;code = 112
char = a;code = 97
char = i;code = 105
char = r;code = 114
char = s;code = 115
char = ;code = 32
char = o;code = 111
char = f;code = 102
char = ;code = 32
char = P;code = 80
char = C;code = 67
char = R;code = 82
char = ;code = 32
char = p;code = 112
char = r;code = 114
char = i;code = 105
char = m;code = 109
char = e;code = 101
char = r;code = 114
char = s;code = 115
char = ;code = 32
char = w;code = 119
char = e;code = 101
char = r;code = 114
char = e;code = 101
char = ;code = 32
char = d;code = 100
char = e;code = 101
char = s;code = 115
char = i;code = 105
char = g;code = 103
char = n;code = 110
char = e;code = 101
char = d;code = 100
char = ;code = 32
char = a;code = 97
char = n;code = 110
char = d;code = 100
char = ;code = 32
char = u;code = 117
char = s;code = 115
char = e;code = 101
char = d;code = 100
char = ;code = 32
char = f;code = 102
char = o;code = 111
char = r;code = 114
char = ;code = 32
char = v;code = 118
char = a;code = 97
char = l;code = 108
char = i;code = 105
char = d;code = 100
char = a;code = 97
char = t;code = 116
char = i;code = 105
char = o;code = 111
char = n;code = 110
char = ;code = 32
char = o;code = 111
char = f;code = 102
char = ;code = 32
char = t;code = 116
char = h;code = 104
char = e;code = 101
char = ;code = 32
char = a;code = 97
char = m;code = 109
char = p;code = 112
char = l;code = 108
char = i;code = 105
char = f;code = 102
char = i;code = 105
char = c;code = 99
char = a;code = 97
char = t;code = 116
char = i;code = 105
char = o;code = 111
char = n;code = 110
char = ;code = 32
char = a;code = 97
char = n;code = 110
char = d;code = 100
char = ;code = 32
char = a;code = 97
char = s;code = 115
char = s;code = 115
char = e;code = 101
char = s;code = 115
char = s;code = 115
char = m;code = 109
char = e;code = 101
char = n;code = 110
char = t;code = 116
char = ;code = 32
char = o;code = 111
char = f;code = 102
char = ;code = 32
char = t;code = 116
char = h;code = 104
char = e;code = 101
char = ;code = 32
char = p;code = 112
char = o;code = 111
char = l;code = 108
char = y;code = 121
char = m;code = 109
char = o;code = 111
char = r;code = 114
char = p;code = 112
char = h;code = 104
char = i;code = 105
char = s;code = 115
char = m;code = 109
char = ;code = 32
char = i;code = 105
char = n;code = 110
char = ;code = 32
char = g;code = 103
char = e;code = 101
char = n;code = 110
char = o;code = 111
char = m;code = 109
char = i;code = 105
char = c;code = 99
char = ;code = 32
char = D;code = 68
char = N;code = 78
char = A;code = 65
char = ;code = 32
char = p;code = 112
char = o;code = 111
char = o;code = 111
char = l;code = 108
char = s;code = 115
char = .;code = 46
char = ;code = 32
char = T;code = 84
char = h;code = 104
char = e;code = 101
char = ;code = 32
char = r;code = 114
char = e;code = 101
char = s;code = 115
char = u;code = 117
char = l;code = 108
char = t;code = 116
char = ;code = 32
char = r;code = 114
char = e;code = 101
char = v;code = 118
char = e;code = 101
char = a;code = 97
char = l;code = 108
char = e;code = 101
char = d;code = 100
char = ;code = 32
char = t;code = 116
char = h;code = 104
char = a;code = 97
char = t;code = 116
char = ;code = 32
char = 9;code = 57
char = 2;code = 50
char = ;code = 32
char = p;code = 112
char = r;code = 114
char = i;code = 105
char = m;code = 109
char = e;code = 101
char = r;code = 114
char = ;code = 32
char = p;code = 112
char = a;code = 97
char = i;code = 105
char = r;code = 114
char = s;code = 115
char = ;code = 32
char = w;code = 119
char = e;code = 101
char = r;code = 114
char = e;code = 101
char = ;code = 32
char = s;code = 115
char = u;code = 117
char = c;code = 99
char = c;code = 99
char = e;code = 101
char = s;code = 115
char = s;code = 115
char = f;code = 102
char = u;code = 117
char = l;code = 108
char = l;code = 108
char = y;code = 121
char = ;code = 32
char = a;code = 97
char = m;code = 109
char = p;code = 112
char = l;code = 108
char = i;code = 105
char = f;code = 102
char = i;code = 105
char = e;code = 101
char = d;code = 100
char = ;code = 32
char = i;code = 105
char = n;code = 110
char = ;code = 32
char = i;code = 105
char = n;code = 110
char = i;code = 105
char = t;code = 116
char = i;code = 105
char = a;code = 97
char = l;code = 108
char = ;code = 32
char = s;code = 115
char = c;code = 99
char = r;code = 114
char = e;code = 101
char = e;code = 101
char = n;code = 110
char = i;code = 105
char = n;code = 110
char = g;code = 103
char = ;code = 32
char = t;code = 116
char = e;code = 101
char = s;code = 115
char = t;code = 116
char = s;code = 115
char = .;code = 46
char = <;code = 60
char = b;code = 98
char = r;code = 114
char = /;code = 47
char = >;code = 62
char = <;code = 60
char = b;code = 98
char = r;code = 114
char = /;code = 47
char = >;code = 62
char = <;code = 60
char = b;code = 98
char = >;code = 62
char = C;code = 67
char = O;code = 79
char = N;code = 78
char = C;code = 67
char = L;code = 76
char = U;code = 85
char = S;code = 83
char = I;code = 73
char = O;code = 79
char = N;code = 78
char = ;code = 32
char = :;code = 58
char = ;code = 32
char = <;code = 60
char = /;code = 47
char = b;code = 98
char = >;code = 62
char = T;code = 84
char = h;code = 104
char = i;code = 105
char = s;code = 115
char = ;code = 32
char = s;code = 115
char = t;code = 116
char = u;code = 117
char = d;code = 100
char = y;code = 121
char = ;code = 32
char = g;code = 103
char = e;code = 101
char = n;code = 110
char = e;code = 101
char = r;code = 114
char = a;code = 97
char = t;code = 116
char = e;code = 101
char = d;code = 100
char = ;code = 32
char = a;code = 97
char = ;code = 32
char = s;code = 115
char = u;code = 117
char = b;code = 98
char = s;code = 115
char = t;code = 116
char = a;code = 97
char = n;code = 110
char = t;code = 116
char = i;code = 105
char = a;code = 97
char = l;code = 108
char = ;code = 32
char = f;code = 102
char = r;code = 114
char = a;code = 97
char = c;code = 99
char = t;code = 116
char = i;code = 105
char = o;code = 111
char = n;code = 110
char = ;code = 32
char = o;code = 111
char = f;code = 102
char = ;code = 32
char = s;code = 115
char = w;code = 119
char = e;code = 101
char = e;code = 101
char = t;code = 116
char = ;code = 32
char = p;code = 112
char = o;code = 111
char = t;code = 116
char = a;code = 97
char = t;code = 116
char = o;code = 111
char = ;code = 32
char = t;code = 116
char = r;code = 114
char = a;code = 97
char = n;code = 110
char = s;code = 115
char = c;code = 99
char = r;code = 114
char = i;code = 105
char = p;code = 112
char = t;code = 116
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result sql = BACKGROUND : The tuberous root of sweet potato is an important agricultural and biological organ. There are not sufficient transcriptomic and genomic data in public databases for understanding of the molecular mechanism underlying the tuberous root formation and development. Thus, high throughput transcriptome sequencing is needed to generate enormous transcript sequences from sweet potato root for gene discovery and molecular marker development.
RESULTS : In this study, more than 59 million sequencing reads were generated using Illumina paired-end sequencing technology. De novo assembly yielded 56,516 unigenes with an average length of 581 bp. Based on sequence similarity search with known proteins, a total of 35,051 (62.02%) genes were identified. Out of these annotated unigenes, 5,046 and 11,983 unigenes were assigned to gene ontology and clusters of orthologous group, respectively. Searching against the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG) indicated that 17,598 (31.14%) unigenes were mapped to 124 KEGG pathways, and 11,056 were assigned to metabolic pathways, which were well represented by carbohydrate metabolism and biosynthesis of secondary metabolite. In addition, 4,114 cDNA SSRs (cSSRs) were identified as potential molecular markers in our unigenes. One hundred pairs of PCR primers were designed and used for validation of the amplification and assessment of the polymorphism in genomic DNA pools. The result revealed that 92 primer pairs were successfully amplified in initial screening tests.
CONCLUSION : This study generated a substantial fraction of sweet potato transcript sequences, which can be used to discover novel genes associated with tuberous root formation and development and will also make it possible to construct high density microarrays for further characterization of gene expression profiles during these processes. Thousands of cSSR markers identified in the present study can enrich molecular markers and will facilitate marker-assisted selection in sweet potato breeding. Overall, these sequences and markers will provide valuable resources for the sweet potato community. Additionally, these results also suggested that transcriptome analysis based on Illumina paired-end sequencing is a powerful tool for gene discovery and molecular marker development for non-model species, especially those with large and complex genome.
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char = e;code = 101
char = v;code = 118
char = e;code = 101
char = l;code = 108
char = o;code = 111
char = p;code = 112
char = m;code = 109
char = e;code = 101
char = n;code = 110
char = t;code = 116
char = ;code = 32
char = o;code = 111
char = f;code = 102
char = ;code = 32
char = c;code = 99
char = S;code = 83
char = S;code = 83
char = R;code = 82
char = ;code = 32
char = m;code = 109
char = a;code = 97
char = r;code = 114
char = k;code = 107
char = e;code = 101
char = r;code = 114
char = s;code = 115
char = ;code = 32
char = i;code = 105
char = n;code = 110
char = ;code = 32
char = s;code = 115
char = w;code = 119
char = e;code = 101
char = e;code = 101
char = t;code = 116
char = ;code = 32
char = p;code = 112
char = o;code = 111
char = t;code = 116
char = a;code = 97
char = t;code = 116
char = o;code = 111
char = ;code = 32
char = (;code = 40
char = I;code = 73
char = p;code = 112
char = o;code = 111
char = m;code = 109
char = o;code = 111
char = e;code = 101
char = a;code = 97
char = ;code = 32
char = b;code = 98
char = a;code = 97
char = t;code = 116
char = a;code = 97
char = t;code = 116
char = a;code = 97
char = s;code = 115
char = );code = 41
char = .;code = 46
result sql = De novo assembly and characterization of root transcriptome using Illumina paired-end sequencing and development of cSSR markers in sweet potato (Ipomoea batatas).
char = b;code = 98
char = >;code = 62
char = B;code = 66
char = A;code = 65
char = C;code = 67
char = K;code = 75
char = G;code = 71
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char = O;code = 79
char = U;code = 85
char = N;code = 78
char = D;code = 68
char = ;code = 32
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char = /;code = 47
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char = >;code = 62
char = T;code = 84
char = h;code = 104
char = e;code = 101
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char = t;code = 116
char = u;code = 117
char = b;code = 98
char = e;code = 101
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char = s;code = 115
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char = r;code = 114
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char = o;code = 111
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char = o;code = 111
char = f;code = 102
char = ;code = 32
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char = ;code = 32
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char = o;code = 111
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char = t;code = 116
char = o;code = 111
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char = u;code = 117
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char = a;code = 97
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char = i;code = 105
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char = T;code = 84
char = h;code = 104
char = e;code = 101
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char = e;code = 101
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char = a;code = 97
char = r;code = 114
char = e;code = 101
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char = ;code = 32
char = s;code = 115
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char = r;code = 114
char = a;code = 97
char = n;code = 110
char = s;code = 115
char = c;code = 99
char = r;code = 114
char = i;code = 105
char = p;code = 112
char = t;code = 116
char = o;code = 111
char = m;code = 109
char = i;code = 105
char = c;code = 99
char = ;code = 32
char = a;code = 97
char = n;code = 110
char = d;code = 100
char = ;code = 32
char = g;code = 103
char = e;code = 101
char = n;code = 110
char = o;code = 111
char = m;code = 109
char = i;code = 105
char = c;code = 99
char = ;code = 32
char = d;code = 100
char = a;code = 97
char = t;code = 116
char = a;code = 97
char = ;code = 32
char = i;code = 105
char = n;code = 110
char = ;code = 32
char = p;code = 112
char = u;code = 117
char = b;code = 98
char = l;code = 108
char = i;code = 105
char = c;code = 99
char = ;code = 32
char = d;code = 100
char = a;code = 97
char = t;code = 116
char = a;code = 97
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char = a;code = 97
char = s;code = 115
char = e;code = 101
char = s;code = 115
char = ;code = 32
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char = u;code = 117
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char = t;code = 116
char = a;code = 97
char = n;code = 110
char = d;code = 100
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char = n;code = 110
char = g;code = 103
char = ;code = 32
char = o;code = 111
char = f;code = 102
char = ;code = 32
char = t;code = 116
char = h;code = 104
char = e;code = 101
char = ;code = 32
char = m;code = 109
char = o;code = 111
char = l;code = 108
char = e;code = 101
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char = u;code = 117
char = l;code = 108
char = a;code = 97
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char = c;code = 99
char = h;code = 104
char = a;code = 97
char = n;code = 110
char = i;code = 105
char = s;code = 115
char = m;code = 109
char = ;code = 32
char = u;code = 117
char = n;code = 110
char = d;code = 100
char = e;code = 101
char = r;code = 114
char = l;code = 108
char = y;code = 121
char = i;code = 105
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char = t;code = 116
char = h;code = 104
char = e;code = 101
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char = r;code = 114
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char = o;code = 111
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char = a;code = 97
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char = a;code = 97
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char = d;code = 100
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char = e;code = 101
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char = p;code = 112
char = m;code = 109
char = e;code = 101
char = n;code = 110
char = t;code = 116
char = .;code = 46
char = ;code = 32
char = T;code = 84
char = h;code = 104
char = u;code = 117
char = s;code = 115
char = ,;code = 44
char = ;code = 32
char = h;code = 104
char = i;code = 105
char = g;code = 103
char = h;code = 104
char = ;code = 32
char = t;code = 116
char = h;code = 104
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char = g;code = 103
char = h;code = 104
char = p;code = 112
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char = t;code = 116
char = r;code = 114
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char = p;code = 112
char = t;code = 116
char = o;code = 111
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char = ;code = 32
char = s;code = 115
char = e;code = 101
char = q;code = 113
char = u;code = 117
char = e;code = 101
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char = c;code = 99
char = i;code = 105
char = n;code = 110
char = g;code = 103
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char = i;code = 105
char = s;code = 115
char = ;code = 32
char = n;code = 110
char = e;code = 101
char = e;code = 101
char = d;code = 100
char = e;code = 101
char = d;code = 100
char = ;code = 32
char = t;code = 116
char = o;code = 111
char = ;code = 32
char = g;code = 103
char = e;code = 101
char = n;code = 110
char = e;code = 101
char = r;code = 114
char = a;code = 97
char = t;code = 116
char = e;code = 101
char = ;code = 32
char = e;code = 101
char = n;code = 110
char = o;code = 111
char = r;code = 114
char = m;code = 109
char = o;code = 111
char = u;code = 117
char = s;code = 115
char = ;code = 32
char = t;code = 116
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char = a;code = 97
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char = s;code = 115
char = c;code = 99
char = r;code = 114
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char = p;code = 112
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char = ;code = 32
char = s;code = 115
char = e;code = 101
char = q;code = 113
char = u;code = 117
char = e;code = 101
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char = c;code = 99
char = e;code = 101
char = s;code = 115
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char = o;code = 111
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char = s;code = 115
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char = p;code = 112
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char = o;code = 111
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char = r;code = 114
char = o;code = 111
char = o;code = 111
char = t;code = 116
char = ;code = 32
char = f;code = 102
char = o;code = 111
char = r;code = 114
char = ;code = 32
char = g;code = 103
char = e;code = 101
char = n;code = 110
char = e;code = 101
char = ;code = 32
char = d;code = 100
char = i;code = 105
char = s;code = 115
char = c;code = 99
char = o;code = 111
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char = e;code = 101
char = r;code = 114
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char = d;code = 100
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char = l;code = 108
char = a;code = 97
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char = e;code = 101
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char = /;code = 47
char = >;code = 62
char = <;code = 60
char = b;code = 98
char = r;code = 114
char = /;code = 47
char = >;code = 62
char = <;code = 60
char = b;code = 98
char = >;code = 62
char = R;code = 82
char = E;code = 69
char = S;code = 83
char = U;code = 85
char = L;code = 76
char = T;code = 84
char = S;code = 83
char = ;code = 32
char = :;code = 58
char = ;code = 32
char = <;code = 60
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char = s;code = 115
char = t;code = 116
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char = h;code = 104
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char = c;code = 99
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char = g;code = 103
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char = e;code = 101
char = a;code = 97
char = d;code = 100
char = s;code = 115
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char = w;code = 119
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char = e;code = 101
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char = g;code = 103
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char = e;code = 101
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char = a;code = 97
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char = e;code = 101
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char = I;code = 73
char = l;code = 108
char = l;code = 108
char = u;code = 117
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char = n;code = 110
char = g;code = 103
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char = t;code = 116
char = e;code = 101
char = c;code = 99
char = h;code = 104
char = n;code = 110
char = o;code = 111
char = l;code = 108
char = o;code = 111
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char = .;code = 46
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char = D;code = 68
char = e;code = 101
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char = o;code = 111
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char = y;code = 121
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char = a;code = 97
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char = ;code = 32
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char = f;code = 102
char = ;code = 32
char = 5;code = 53
char = 8;code = 56
char = 1;code = 49
char = ;code = 32
char = b;code = 98
char = p;code = 112
char = .;code = 46
char = ;code = 32
char = B;code = 66
char = a;code = 97
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char = e;code = 101
char = d;code = 100
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char = o;code = 111
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char = r;code = 114
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char = t;code = 116
char = y;code = 121
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char = e;code = 101
char = a;code = 97
char = r;code = 114
char = c;code = 99
char = h;code = 104
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char = w;code = 119
char = i;code = 105
char = t;code = 116
char = h;code = 104
char = ;code = 32
char = k;code = 107
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char = o;code = 111
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char = n;code = 110
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char = i;code = 105
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char = s;code = 115
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char = ;code = 32
char = a;code = 97
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char = a;code = 97
char = l;code = 108
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char = o;code = 111
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char = o;code = 111
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char = t;code = 116
char = o;code = 111
char = ;code = 32
char = g;code = 103
char = e;code = 101
char = n;code = 110
char = e;code = 101
char = ;code = 32
char = o;code = 111
char = n;code = 110
char = t;code = 116
char = o;code = 111
char = l;code = 108
char = o;code = 111
char = g;code = 103
char = y;code = 121
char = ;code = 32
char = a;code = 97
char = n;code = 110
char = d;code = 100
char = ;code = 32
char = c;code = 99
char = l;code = 108
char = u;code = 117
char = s;code = 115
char = t;code = 116
char = e;code = 101
char = r;code = 114
char = s;code = 115
char = ;code = 32
char = o;code = 111
char = f;code = 102
char = ;code = 32
char = o;code = 111
char = r;code = 114
char = t;code = 116
char = h;code = 104
char = o;code = 111
char = l;code = 108
char = o;code = 111
char = g;code = 103
char = o;code = 111
char = u;code = 117
char = s;code = 115
char = ;code = 32
char = g;code = 103
char = r;code = 114
char = o;code = 111
char = u;code = 117
char = p;code = 112
char = ,;code = 44
char = ;code = 32
char = r;code = 114
char = e;code = 101
char = s;code = 115
char = p;code = 112
char = e;code = 101
char = c;code = 99
char = t;code = 116
char = i;code = 105
char = v;code = 118
char = e;code = 101
char = l;code = 108
char = y;code = 121
char = .;code = 46
char = ;code = 32
char = S;code = 83
char = e;code = 101
char = a;code = 97
char = r;code = 114
char = c;code = 99
char = h;code = 104
char = i;code = 105
char = n;code = 110
char = g;code = 103
char = ;code = 32
char = a;code = 97
char = g;code = 103
char = a;code = 97
char = i;code = 105
char = n;code = 110
char = s;code = 115
char = t;code = 116
char = ;code = 32
char = t;code = 116
char = h;code = 104
char = e;code = 101
char = ;code = 32
char = K;code = 75
char = y;code = 121
char = o;code = 111
char = t;code = 116
char = o;code = 111
char = ;code = 32
char = E;code = 69
char = n;code = 110
char = c;code = 99
char = y;code = 121
char = c;code = 99
char = l;code = 108
char = o;code = 111
char = p;code = 112
char = e;code = 101
char = d;code = 100
char = i;code = 105
char = a;code = 97
char = ;code = 32
char = o;code = 111
char = f;code = 102
char = ;code = 32
char = G;code = 71
char = e;code = 101
char = n;code = 110
char = e;code = 101
char = s;code = 115
char = ;code = 32
char = a;code = 97
char = n;code = 110
char = d;code = 100
char = ;code = 32
char = G;code = 71
char = e;code = 101
char = n;code = 110
char = o;code = 111
char = m;code = 109
char = e;code = 101
char = s;code = 115
char = ;code = 32
char = P;code = 80
char = a;code = 97
char = t;code = 116
char = h;code = 104
char = w;code = 119
char = a;code = 97
char = y;code = 121
char = ;code = 32
char = d;code = 100
char = a;code = 97
char = t;code = 116
char = a;code = 97
char = b;code = 98
char = a;code = 97
char = s;code = 115
char = e;code = 101
char = ;code = 32
char = (;code = 40
char = K;code = 75
char = E;code = 69
char = G;code = 71
char = G;code = 71
char = );code = 41
char = ;code = 32
char = i;code = 105
char = n;code = 110
char = d;code = 100
char = i;code = 105
char = c;code = 99
char = a;code = 97
char = t;code = 116
char = e;code = 101
char = d;code = 100
char = ;code = 32
char = t;code = 116
char = h;code = 104
char = a;code = 97
char = t;code = 116
char = ;code = 32
char = 1;code = 49
char = 7;code = 55
char = ,;code = 44
char = 5;code = 53
char = 9;code = 57
char = 8;code = 56
char = ;code = 32
char = (;code = 40
char = 3;code = 51
char = 1;code = 49
char = .;code = 46
char = 1;code = 49
char = 4;code = 52
char = %;code = 37
char = );code = 41
char = ;code = 32
char = u;code = 117
char = n;code = 110
char = i;code = 105
char = g;code = 103
char = e;code = 101
char = n;code = 110
char = e;code = 101
char = s;code = 115
char = ;code = 32
char = w;code = 119
char = e;code = 101
char = r;code = 114
char = e;code = 101
char = ;code = 32
char = m;code = 109
char = a;code = 97
char = p;code = 112
char = p;code = 112
char = e;code = 101
char = d;code = 100
char = ;code = 32
char = t;code = 116
char = o;code = 111
char = ;code = 32
char = 1;code = 49
char = 2;code = 50
char = 4;code = 52
char = ;code = 32
char = K;code = 75
char = E;code = 69
char = G;code = 71
char = G;code = 71
char = ;code = 32
char = p;code = 112
char = a;code = 97
char = t;code = 116
char = h;code = 104
char = w;code = 119
char = a;code = 97
char = y;code = 121
char = s;code = 115
char = ,;code = 44
char = ;code = 32
char = a;code = 97
char = n;code = 110
char = d;code = 100
char = ;code = 32
char = 1;code = 49
char = 1;code = 49
char = ,;code = 44
char = 0;code = 48
char = 5;code = 53
char = 6;code = 54
char = ;code = 32
char = w;code = 119
char = e;code = 101
char = r;code = 114
char = e;code = 101
char = ;code = 32
char = a;code = 97
char = s;code = 115
char = s;code = 115
char = i;code = 105
char = g;code = 103
char = n;code = 110
char = e;code = 101
char = d;code = 100
char = ;code = 32
char = t;code = 116
char = o;code = 111
char = ;code = 32
char = m;code = 109
char = e;code = 101
char = t;code = 116
char = a;code = 97
char = b;code = 98
char = o;code = 111
char = l;code = 108
char = i;code = 105
char = c;code = 99
char = ;code = 32
char = p;code = 112
char = a;code = 97
char = t;code = 116
char = h;code = 104
char = w;code = 119
char = a;code = 97
char = y;code = 121
char = s;code = 115
char = ,;code = 44
char = ;code = 32
char = w;code = 119
char = h;code = 104
char = i;code = 105
char = c;code = 99
char = h;code = 104
char = ;code = 32
char = w;code = 119
char = e;code = 101
char = r;code = 114
char = e;code = 101
char = ;code = 32
char = w;code = 119
char = e;code = 101
char = l;code = 108
char = l;code = 108
char = ;code = 32
char = r;code = 114
char = e;code = 101
char = p;code = 112
char = r;code = 114
char = e;code = 101
char = s;code = 115
char = e;code = 101
char = n;code = 110
char = t;code = 116
char = e;code = 101
char = d;code = 100
char = ;code = 32
char = b;code = 98
char = y;code = 121
char = ;code = 32
char = c;code = 99
char = a;code = 97
char = r;code = 114
char = b;code = 98
char = o;code = 111
char = h;code = 104
char = y;code = 121
char = d;code = 100
char = r;code = 114
char = a;code = 97
char = t;code = 116
char = e;code = 101
char = ;code = 32
char = m;code = 109
char = e;code = 101
char = t;code = 116
char = a;code = 97
char = b;code = 98
char = o;code = 111
char = l;code = 108
char = i;code = 105
char = s;code = 115
char = m;code = 109
char = ;code = 32
char = a;code = 97
char = n;code = 110
char = d;code = 100
char = ;code = 32
char = b;code = 98
char = i;code = 105
char = o;code = 111
char = s;code = 115
char = y;code = 121
char = n;code = 110
char = t;code = 116
char = h;code = 104
char = e;code = 101
char = s;code = 115
char = i;code = 105
char = s;code = 115
char = ;code = 32
char = o;code = 111
char = f;code = 102
char = ;code = 32
char = s;code = 115
char = e;code = 101
char = c;code = 99
char = o;code = 111
char = n;code = 110
char = d;code = 100
char = a;code = 97
char = r;code = 114
char = y;code = 121
char = ;code = 32
char = m;code = 109
char = e;code = 101
char = t;code = 116
char = a;code = 97
char = b;code = 98
char = o;code = 111
char = l;code = 108
char = i;code = 105
char = t;code = 116
char = e;code = 101
char = .;code = 46
char = ;code = 32
char = I;code = 73
char = n;code = 110
char = ;code = 32
char = a;code = 97
char = d;code = 100
char = d;code = 100
char = i;code = 105
char = t;code = 116
char = i;code = 105
char = o;code = 111
char = n;code = 110
char = ,;code = 44
char = ;code = 32
char = 4;code = 52
char = ,;code = 44
char = 1;code = 49
char = 1;code = 49
char = 4;code = 52
char = ;code = 32
char = c;code = 99
char = D;code = 68
char = N;code = 78
char = A;code = 65
char = ;code = 32
char = S;code = 83
char = S;code = 83
char = R;code = 82
char = s;code = 115
char = ;code = 32
char = (;code = 40
char = c;code = 99
char = S;code = 83
char = S;code = 83
char = R;code = 82
char = s;code = 115
char = );code = 41
char = ;code = 32
char = w;code = 119
char = e;code = 101
char = r;code = 114
char = e;code = 101
char = ;code = 32
char = i;code = 105
char = d;code = 100
char = e;code = 101
char = n;code = 110
char = t;code = 116
char = i;code = 105
char = f;code = 102
char = i;code = 105
char = e;code = 101
char = d;code = 100
char = ;code = 32
char = a;code = 97
char = s;code = 115
char = ;code = 32
char = p;code = 112
char = o;code = 111
char = t;code = 116
char = e;code = 101
char = n;code = 110
char = t;code = 116
char = i;code = 105
char = a;code = 97
char = l;code = 108
char = ;code = 32
char = m;code = 109
char = o;code = 111
char = l;code = 108
char = e;code = 101
char = c;code = 99
char = u;code = 117
char = l;code = 108
char = a;code = 97
char = r;code = 114
char = ;code = 32
char = m;code = 109
char = a;code = 97
char = r;code = 114
char = k;code = 107
char = e;code = 101
char = r;code = 114
char = s;code = 115
char = ;code = 32
char = i;code = 105
char = n;code = 110
char = ;code = 32
char = o;code = 111
char = u;code = 117
char = r;code = 114
char = ;code = 32
char = u;code = 117
char = n;code = 110
char = i;code = 105
char = g;code = 103
char = e;code = 101
char = n;code = 110
char = e;code = 101
char = s;code = 115
char = .;code = 46
char = ;code = 32
char = O;code = 79
char = n;code = 110
char = e;code = 101
char = ;code = 32
char = h;code = 104
char = u;code = 117
char = n;code = 110
char = d;code = 100
char = r;code = 114
char = e;code = 101
char = d;code = 100
char = ;code = 32
char = p;code = 112
char = a;code = 97
char = i;code = 105
char = r;code = 114
char = s;code = 115
char = ;code = 32
char = o;code = 111
char = f;code = 102
char = ;code = 32
char = P;code = 80
char = C;code = 67
char = R;code = 82
char = ;code = 32
char = p;code = 112
char = r;code = 114
char = i;code = 105
char = m;code = 109
char = e;code = 101
char = r;code = 114
char = s;code = 115
char = ;code = 32
char = w;code = 119
char = e;code = 101
char = r;code = 114
char = e;code = 101
char = ;code = 32
char = d;code = 100
char = e;code = 101
char = s;code = 115
char = i;code = 105
char = g;code = 103
char = n;code = 110
char = e;code = 101
char = d;code = 100
char = ;code = 32
char = a;code = 97
char = n;code = 110
char = d;code = 100
char = ;code = 32
char = u;code = 117
char = s;code = 115
char = e;code = 101
char = d;code = 100
char = ;code = 32
char = f;code = 102
char = o;code = 111
char = r;code = 114
char = ;code = 32
char = v;code = 118
char = a;code = 97
char = l;code = 108
char = i;code = 105
char = d;code = 100
char = a;code = 97
char = t;code = 116
char = i;code = 105
char = o;code = 111
char = n;code = 110
char = ;code = 32
char = o;code = 111
char = f;code = 102
char = ;code = 32
char = t;code = 116
char = h;code = 104
char = e;code = 101
char = ;code = 32
char = a;code = 97
char = m;code = 109
char = p;code = 112
char = l;code = 108
char = i;code = 105
char = f;code = 102
char = i;code = 105
char = c;code = 99
char = a;code = 97
char = t;code = 116
char = i;code = 105
char = o;code = 111
char = n;code = 110
char = ;code = 32
char = a;code = 97
char = n;code = 110
char = d;code = 100
char = ;code = 32
char = a;code = 97
char = s;code = 115
char = s;code = 115
char = e;code = 101
char = s;code = 115
char = s;code = 115
char = m;code = 109
char = e;code = 101
char = n;code = 110
char = t;code = 116
char = ;code = 32
char = o;code = 111
char = f;code = 102
char = ;code = 32
char = t;code = 116
char = h;code = 104
char = e;code = 101
char = ;code = 32
char = p;code = 112
char = o;code = 111
char = l;code = 108
char = y;code = 121
char = m;code = 109
char = o;code = 111
char = r;code = 114
char = p;code = 112
char = h;code = 104
char = i;code = 105
char = s;code = 115
char = m;code = 109
char = ;code = 32
char = i;code = 105
char = n;code = 110
char = ;code = 32
char = g;code = 103
char = e;code = 101
char = n;code = 110
char = o;code = 111
char = m;code = 109
char = i;code = 105
char = c;code = 99
char = ;code = 32
char = D;code = 68
char = N;code = 78
char = A;code = 65
char = ;code = 32
char = p;code = 112
char = o;code = 111
char = o;code = 111
char = l;code = 108
char = s;code = 115
char = .;code = 46
char = ;code = 32
char = T;code = 84
char = h;code = 104
char = e;code = 101
char = ;code = 32
char = r;code = 114
char = e;code = 101
char = s;code = 115
char = u;code = 117
char = l;code = 108
char = t;code = 116
char = ;code = 32
char = r;code = 114
char = e;code = 101
char = v;code = 118
char = e;code = 101
char = a;code = 97
char = l;code = 108
char = e;code = 101
char = d;code = 100
char = ;code = 32
char = t;code = 116
char = h;code = 104
char = a;code = 97
char = t;code = 116
char = ;code = 32
char = 9;code = 57
char = 2;code = 50
char = ;code = 32
char = p;code = 112
char = r;code = 114
char = i;code = 105
char = m;code = 109
char = e;code = 101
char = r;code = 114
char = ;code = 32
char = p;code = 112
char = a;code = 97
char = i;code = 105
char = r;code = 114
char = s;code = 115
char = ;code = 32
char = w;code = 119
char = e;code = 101
char = r;code = 114
char = e;code = 101
char = ;code = 32
char = s;code = 115
char = u;code = 117
char = c;code = 99
char = c;code = 99
char = e;code = 101
char = s;code = 115
char = s;code = 115
char = f;code = 102
char = u;code = 117
char = l;code = 108
char = l;code = 108
char = y;code = 121
char = ;code = 32
char = a;code = 97
char = m;code = 109
char = p;code = 112
char = l;code = 108
char = i;code = 105
char = f;code = 102
char = i;code = 105
char = e;code = 101
char = d;code = 100
char = ;code = 32
char = i;code = 105
char = n;code = 110
char = ;code = 32
char = i;code = 105
char = n;code = 110
char = i;code = 105
char = t;code = 116
char = i;code = 105
char = a;code = 97
char = l;code = 108
char = ;code = 32
char = s;code = 115
char = c;code = 99
char = r;code = 114
char = e;code = 101
char = e;code = 101
char = n;code = 110
char = i;code = 105
char = n;code = 110
char = g;code = 103
char = ;code = 32
char = t;code = 116
char = e;code = 101
char = s;code = 115
char = t;code = 116
char = s;code = 115
char = .;code = 46
char = <;code = 60
char = b;code = 98
char = r;code = 114
char = /;code = 47
char = >;code = 62
char = <;code = 60
char = b;code = 98
char = r;code = 114
char = /;code = 47
char = >;code = 62
char = <;code = 60
char = b;code = 98
char = >;code = 62
char = C;code = 67
char = O;code = 79
char = N;code = 78
char = C;code = 67
char = L;code = 76
char = U;code = 85
char = S;code = 83
char = I;code = 73
char = O;code = 79
char = N;code = 78
char = ;code = 32
char = :;code = 58
char = ;code = 32
char = <;code = 60
char = /;code = 47
char = b;code = 98
char = >;code = 62
char = T;code = 84
char = h;code = 104
char = i;code = 105
char = s;code = 115
char = ;code = 32
char = s;code = 115
char = t;code = 116
char = u;code = 117
char = d;code = 100
char = y;code = 121
char = ;code = 32
char = g;code = 103
char = e;code = 101
char = n;code = 110
char = e;code = 101
char = r;code = 114
char = a;code = 97
char = t;code = 116
char = e;code = 101
char = d;code = 100
char = ;code = 32
char = a;code = 97
char = ;code = 32
char = s;code = 115
char = u;code = 117
char = b;code = 98
char = s;code = 115
char = t;code = 116
char = a;code = 97
char = n;code = 110
char = t;code = 116
char = i;code = 105
char = a;code = 97
char = l;code = 108
char = ;code = 32
char = f;code = 102
char = r;code = 114
char = a;code = 97
char = c;code = 99
char = t;code = 116
char = i;code = 105
char = o;code = 111
char = n;code = 110
char = ;code = 32
char = o;code = 111
char = f;code = 102
char = ;code = 32
char = s;code = 115
char = w;code = 119
char = e;code = 101
char = e;code = 101
char = t;code = 116
char = ;code = 32
char = p;code = 112
char = o;code = 111
char = t;code = 116
char = a;code = 97
char = t;code = 116
char = o;code = 111
char = ;code = 32
char = t;code = 116
char = r;code = 114
char = a;code = 97
char = n;code = 110
char = s;code = 115
char = c;code = 99
char = r;code = 114
char = i;code = 105
char = p;code = 112
char = t;code = 116
char = ;code = 32
char = s;code = 115
char = e;code = 101
char = q;code = 113
char = u;code = 117
char = e;code = 101
char = n;code = 110
char = c;code = 99
char = e;code = 101
char = s;code = 115
char = ,;code = 44
char = ;code = 32
char = w;code = 119
char = h;code = 104
char = i;code = 105
char = c;code = 99
char = h;code = 104
char = ;code = 32
char = c;code = 99
char = a;code = 97
char = n;code = 110
char = ;code = 32
char = b;code = 98
char = e;code = 101
char = ;code = 32
char = u;code = 117
char = s;code = 115
char = e;code = 101
char = d;code = 100
char = ;code = 32
char = t;code = 116
char = o;code = 111
char = ;code = 32
char = d;code = 100
char = i;code = 105
char = s;code = 115
char = c;code = 99
char = o;code = 111
char = v;code = 118
char = e;code = 101
char = r;code = 114
char = ;code = 32
char = n;code = 110
char = o;code = 111
char = v;code = 118
char = e;code = 101
char = l;code = 108
char = ;code = 32
char = g;code = 103
char = e;code = 101
char = n;code = 110
char = e;code = 101
char = s;code = 115
char = ;code = 32
char = a;code = 97
char = s;code = 115
char = s;code = 115
char = o;code = 111
char = c;code = 99
char = i;code = 105
char = a;code = 97
char = t;code = 116
char = e;code = 101
char = d;code = 100
char = ;code = 32
char = w;code = 119
char = i;code = 105
char = t;code = 116
char = h;code = 104
char = ;code = 32
char = t;code = 116
char = u;code = 117
char = b;code = 98
char = e;code = 101
char = r;code = 114
char = o;code = 111
char = u;code = 117
char = s;code = 115
char = ;code = 32
char = r;code = 114
char = o;code = 111
char = o;code = 111
char = t;code = 116
char = ;code = 32
char = f;code = 102
char = o;code = 111
char = r;code = 114
char = m;code = 109
char = a;code = 97
char = t;code = 116
char = i;code = 105
char = o;code = 111
char = n;code = 110
char = ;code = 32
char = a;code = 97
char = n;code = 110
char = d;code = 100
char = ;code = 32
char = d;code = 100
char = e;code = 101
char = v;code = 118
char = e;code = 101
char = l;code = 108
char = o;code = 111
char = p;code = 112
char = m;code = 109
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result sql = BACKGROUND : The tuberous root of sweet potato is an important agricultural and biological organ. There are not sufficient transcriptomic and genomic data in public databases for understanding of the molecular mechanism underlying the tuberous root formation and development. Thus, high throughput transcriptome sequencing is needed to generate enormous transcript sequences from sweet potato root for gene discovery and molecular marker development.
RESULTS : In this study, more than 59 million sequencing reads were generated using Illumina paired-end sequencing technology. De novo assembly yielded 56,516 unigenes with an average length of 581 bp. Based on sequence similarity search with known proteins, a total of 35,051 (62.02%) genes were identified. Out of these annotated unigenes, 5,046 and 11,983 unigenes were assigned to gene ontology and clusters of orthologous group, respectively. Searching against the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG) indicated that 17,598 (31.14%) unigenes were mapped to 124 KEGG pathways, and 11,056 were assigned to metabolic pathways, which were well represented by carbohydrate metabolism and biosynthesis of secondary metabolite. In addition, 4,114 cDNA SSRs (cSSRs) were identified as potential molecular markers in our unigenes. One hundred pairs of PCR primers were designed and used for validation of the amplification and assessment of the polymorphism in genomic DNA pools. The result revealed that 92 primer pairs were successfully amplified in initial screening tests.
CONCLUSION : This study generated a substantial fraction of sweet potato transcript sequences, which can be used to discover novel genes associated with tuberous root formation and development and will also make it possible to construct high density microarrays for further characterization of gene expression profiles during these processes. Thousands of cSSR markers identified in the present study can enrich molecular markers and will facilitate marker-assisted selection in sweet potato breeding. Overall, these sequences and markers will provide valuable resources for the sweet potato community. Additionally, these results also suggested that transcriptome analysis based on Illumina paired-end sequencing is a powerful tool for gene discovery and molecular marker development for non-model species, especially those with large and complex genome.
Publication: BMC Genomics 2010 ;11:726. :doi: 10.1186/1471-2164-11-726.
Publication Date: 2010
Study Author(s): Wang, Zhangying;Fang, Boping;Chen, Jingyi;Zhang, Xiongjian;Luo, Zhongxia;Huang, Lifei;Chen, Xinliang;Li, Yujun;
Institution: Crops Research Institute, Guangdong Academy of Agricultural Sciences, Guangzhou, 510640 PR China.
Shortcut link to this study:
Publication Date: 2010
Study Author(s): Wang, Zhangying;Fang, Boping;Chen, Jingyi;Zhang, Xiongjian;Luo, Zhongxia;Huang, Lifei;Chen, Xinliang;Li, Yujun;
Institution: Crops Research Institute, Guangdong Academy of Agricultural Sciences, Guangzhou, 510640 PR China.
Shortcut link to this study:
BACKGROUND : The tuberous root of sweet potato is an important agricultural and biological organ. There are not sufficient transcriptomic and genomic data in public databases for understanding of the molecular mechanism underlying the tuberous root formation and development. Thus, high throughput transcriptome sequencing is needed to generate enormous transcript sequences from sweet potato root for gene discovery and molecular marker development.
RESULTS : In this study, more than 59 million sequencing reads were generated using Illumina paired-end sequencing technology. De novo assembly yielded 56,516 unigenes with an average length of 581 bp. Based on sequence similarity search with known proteins, a total of 35,051 (62.02%) genes were identified. Out of these annotated unigenes, 5,046 and 11,983 unigenes were assigned to gene ontology and clusters of orthologous group, respectively. Searching against the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG) indicated that 17,598 (31.14%) unigenes were mapped to 124 KEGG pathways, and 11,056 were assigned to metabolic pathways, which were well represented by carbohydrate metabolism and biosynthesis of secondary metabolite. In addition, 4,114 cDNA SSRs (cSSRs) were identified as potential molecular markers in our unigenes. One hundred pairs of PCR primers were designed and used for validation of the amplification and assessment of the polymorphism in genomic DNA pools. The result revealed that 92 primer pairs were successfully amplified in initial screening tests.
CONCLUSION : This study generated a substantial fraction of sweet potato transcript sequences, which can be used to discover novel genes associated with tuberous root formation and development and will also make it possible to construct high density microarrays for further characterization of gene expression profiles during these processes. Thousands of cSSR markers identified in the present study can enrich molecular markers and will facilitate marker-assisted selection in sweet potato breeding. Overall, these sequences and markers will provide valuable resources for the sweet potato community. Additionally, these results also suggested that transcriptome analysis based on Illumina paired-end sequencing is a powerful tool for gene discovery and molecular marker development for non-model species, especially those with large and complex genome.
RESULTS : In this study, more than 59 million sequencing reads were generated using Illumina paired-end sequencing technology. De novo assembly yielded 56,516 unigenes with an average length of 581 bp. Based on sequence similarity search with known proteins, a total of 35,051 (62.02%) genes were identified. Out of these annotated unigenes, 5,046 and 11,983 unigenes were assigned to gene ontology and clusters of orthologous group, respectively. Searching against the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG) indicated that 17,598 (31.14%) unigenes were mapped to 124 KEGG pathways, and 11,056 were assigned to metabolic pathways, which were well represented by carbohydrate metabolism and biosynthesis of secondary metabolite. In addition, 4,114 cDNA SSRs (cSSRs) were identified as potential molecular markers in our unigenes. One hundred pairs of PCR primers were designed and used for validation of the amplification and assessment of the polymorphism in genomic DNA pools. The result revealed that 92 primer pairs were successfully amplified in initial screening tests.
CONCLUSION : This study generated a substantial fraction of sweet potato transcript sequences, which can be used to discover novel genes associated with tuberous root formation and development and will also make it possible to construct high density microarrays for further characterization of gene expression profiles during these processes. Thousands of cSSR markers identified in the present study can enrich molecular markers and will facilitate marker-assisted selection in sweet potato breeding. Overall, these sequences and markers will provide valuable resources for the sweet potato community. Additionally, these results also suggested that transcriptome analysis based on Illumina paired-end sequencing is a powerful tool for gene discovery and molecular marker development for non-model species, especially those with large and complex genome.
PMID: 21182800
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