6-30-2015
Sequences of nested PCR products from Soybean leaf/cotyledon extracted RNAs
BLAST of sequences show 98% identity with SVNV-M sequence. A stretch of sequence is
highlighted to show the identity between seed transmitted leaf positives and DL4 (field
isolate, standard)
1. Water control sequenced with M primers (mentioned in the manuscript)
No good sequence data, as expected
2. #27-2 (positive) using M primers
TGANNGAGTTAAAATGCGGTTATGATACTTTCCATCAGTATATAGGATTAGAGCCTGTTTCAGATTACTTTGCAGA
TGAAATTGATGATGGAACTATGTTTGTGAAAAAAGACTTCAAAATCGGTAAAATAAATCTCATTGTCGATTTACCAT
CAGAACTCTTTCAAACTATAGCAAAAAAACCAAAAGTGGCAATGGTTGCCTCAGCTTGCAAAGGTTGTTTAAGCTG
TAGTTCAGGAATAAACTGTGATTTAGAGTTCACATCTGACAGCATCTTTTCTTGCAGAATAAGCTGGGAGCATTGC
ACATCTGAACCAGAACAGATAGCTATGAAAAAAGGGACTAATGTTTTCAAGTTAAACATGTTTTGCTCTAAAGATC
CAACTCTTACAAAGATTATCCTTGTACCTTCAGATGATGAGTCCATGGCTTTAGAGTTTGAAACAAAAAATGTGGA
AATCTCTGACGAAAAANCCCCNC
3. #31-2 (negative) using M primers
No good sequence data, as expected
4. #34-1 (positive) using M primers
NAGNGAGTNAAAATGCGGTTATGATACCTTCCATCAGTATATAGGATTAGAGCCTGTTTCAGATTACTTTGCAGAT
GAAATTGATGATGGAACTATGTTTGTAAAAAAAGACTTCAAAATCGGCAAAATAAATCTCATTGTCGATTTACCAT
CAGAACTCTTTCAAACTATAGCAAAAAAACCAAAAGTGGCAATGGTTGCCTCAGCTTGCAAAGGTTGTTTAAGCTG
TAGTTCAGGAATAAACTGTGATTTAGAGTTCACATCTGACAGCATCTTTTCTTGCAGAATAAGCTGGGAGCATTGC
ACATCTGAACCAGAACAGATAGCTATGAAAAAAGGGACTAATGTTTTCAAGTTAAACATGTTTTGCTCTAAAGATC
CAACTCTTACAAAGATTATCCTTGTACCTTCAGATGATGAGTCTATGGCTTTAGAGTTTGAAACAAAAAATGTGGA
AATCTCTGACCGAAAANCCCCNNC
5. #34-2 (positive) using M primers
NGANTGAGTTAAAATGCGGTTATGATACTTTCCATCAGTATATAGGATTAGAGCCTGTTTCAGATTACTTTGCAGA
TGAAATTGATGATGGAACTATGTTTGTGAAAAAAGACTTCAAAATCGGTAAAATAAATCTCATTGTCGATTTACCAT
CAGAACTCTTTCAAACTATAGCAAAAAAACCAAAAGTGGCAATGGTTGCCTCAGCTTGCAAAGGTTGTTTAAGCTG
TAGTTCAGGAATAAACTGTGATTTAGAGTTCACATCTGACAGCATCTTTTCTTGCAGAATAAGCTGGGAGCATTGC
ACATCTGAACCAGAACAGATAGCTATGAAAAAAGGGACTAATGTTTTCAAGTTAAACATGTTTTGCTCTAAAGATC
CAACTCTTACAAAGATTATCCTTGTACCTTCAGATGATGAGTCCATGGCTTTAGAGTTTGAAACAAAAAATGTGGA
AATCTCTGACCCGAAAAACCCAT
6. # DL4 (field isolate; positive control) using M primers
NGGANNGAGTTAAAATGCGGTTATGATACTTTCCATCAGGTATATAGGATTAGAGCCTGTTTCAGATTACTTTGCA
GATGAAATTGATGATGGAACTATGTTTGTGAAAAAAGACTTCAAAATCGGTAAAATAAATCTCATTGTCGATTTAC
CATCAGAACTCTTTCAAACTATAGCAAAAAAACCAAAAGTGGCAATGGTTGCCTCAGCTTGCAAAGGTTGTTTAAG
CTGTAGTTCAGGAATAAACTGTGATTTAGAGTTCACATCTGACAGCATCTTTTCTTGCAGAATAAGCTGGGAGCAT
TGCACATCTGAACCAGAACAGATAGCTATGAAAAAAGGGACTAATGTTTTCAAGTTAAACATGTTTTGCTCTAAAG
ATCCAACTCTTACAAAGATTATCCTTGTACCTTCAGATGATGAGTCCATGGCTTTAGAGTTTGAAACAAAAAATGTG
GAAATCTCTGACCCAGAAAACCATCATAGATCNCNATGATGATGCCTTCAATGAAGAAGTTGAATATGATA
#34-1 (positive) using M primers (PCR done on 6/26/15)
CGAGNGAGTNAAAATGCGGTTATGATACTTTCCATCAGTATATAGGATTAGAGCCTGTTTCAGATTACTTTGCAGA
TGAAATTGATGATGGAACTATGTTTGTGAAAAAAGACTTCAAAATCGGTAAAATAAATCTCATTGTCGATTTACCAT
CAGAACTCTTTCAAACTATAGCAAAAAAACCAAAAGTGGCAATGGTTGCCTCAGCTTGCAAAGGTTGTTTAAGCTG
TAGTTCAGGAATAAACTGTGATTTAGAGTTCACATCTGACAGCATCTTTTCTTGCAGAATAAGCTGGGAGCATTGC
ACATCTGAACCAGAACAGATAGCTATGAAAAAAGGGACTAATGTTTTCAAGTTAAACATGTTTTGCTCTAAAGATC
CAACTCTTACAAAGATTATCCTTGTACCTTCAGATGATGAGTCCATGGCTTTAGAGTTTGAAACAAAAAATGTGGA
AATCTCTGACCCGAAAACCCATC
7. #82-1 (positive, Carol also identified this one as positive) using M primers
NGTGAGTAAAATGCGGTTATGATACTTTCCATCAGTATATAGGATTAGAGCCTGTTTCAGATTACTTTGCAGATGA
AATTGATGATGGAACTATGTTTGTGAAAAAAGACTTCAAAATCGGTAAAATAAATCTCATTGTCGATTTACCATCA
GAACTCTTTCAAACTATAGCAAAAAAACCAAAAGTGGCAATGGTTGCCTCAGCTTGCAAAGGTTGTTTAAGCTGTA
GTTCAGGAATAAACTGTGATTTAGAGTTCACATCTGACAGCATCTTTTCTTGCAGAATAAGCTGGGAGCATTGCAC
ATCTGAACCAGAACAGATAGCTATGAAAAAAGGGACTAATGTTTTCAAGTTAAACATGTTTTGCTCTAAAGATCCA
ACTCTTACAAAGATTATCCTTGTACCTTCAGATGATGAGTCCATGGCTTTAGAGTTTGAAACAAAAAATGTGGAAA
TCTCTGACCCGAAAANCCCATCAA
8. #82-1 (positive, Carol also identified this one as positive) using genomic (RboH1 reverse)
primer obtained from Medhi’s lab
CAAGNNCATAGGTTCTTATAGCTTTGTGCTGGGGCTCCGTTAGGGNCCTTTGATGAGGATCTTTGGATATCTAATT
CTGGAAAGACCAAATAATGTCAACATTTATAGATGATTTTGTAAAGCTAAGAACAAAGAGTTGCTATTCACCTTGA
TTTTGAAGGATGGTTATAGGTTGAATTTGGTGCTCTAGTTTCCATCCTCATAAGGTTTCCCTTTCTTGGTTGAGCATT
GTGAGGCTCACAAACCTGCATTTCTAAGTATTAGTACAACTAGATGTTCAATAATTACCATATTTATATGAACCTTT
GACTTACTAAATACATAGGATTGATTGAGTTCGCAGTTCAGACATTAACATCACCTGTGCAAATGTGTTTTTAAGCT
CTGTAGTCCAATCTCCCAAGGTTCGTATATGAACACTCAAGTAGTCATCTCCAGGTGCAGAGGTGATGGAGAAAG
GATGGGAATCCCTTA
Conclusions:
1. We have 4 positives out of 48 RNA samples. They are: #27-2, 34-1, 34-2 and 82-1.
Nested PCR products (cDNAs were synthesized from RNA isolates using specific M
primers and superscript III) of all these samples show nice sequence data with 98%
identity compared to known M gene sequence (TN isolate).
2. #82-1 turned out positive (visible bands on gel) with both M primer (Ranjit) and N
primer (Carol).
3. PCR product of #82-1 cDNA (Carol; random primer) using genomic primer (from
Medhi’s lab) does not match with known SVNV RNA sequences.