TABLE 4.
Genetic variation of the tunicaminyluracil biosynthetic gene cluster in sequenced Rathayibacter toxicus, R. iranicus, and R. agropyri strains, and their predicted impact to protein function.
| Gene | Gene size (bp)a | No. of strains | No. of haplotypes | No. (%) of polymorphic sites | No. of INDELs (bp)b | No. of non-synonymous mutationsc | Type of non-synonymous mutation | Predicted mutation toleratedd |
| R. toxicus | ||||||||
| tunA | 993 | 26 | 2 | 21(2.1%) | – | 8 | Missense | Neutral |
| tunB | 1014 | 26 | 3 | 4(0.4%) | – | 1 | Missense | Neutral |
| tunC | 1002 | 26 | 2 | 8(0.8%) | – | 2 | Missense | Neutral |
| tunD | 1365 | 26 | 3 | 5(0.4%) | 1 (6 bp) | 3 | Nonsense/Missense | Neutral |
| tunE | 699 | 26 | 2 | 11(1.6%) | 1 (6 bp) | 4 | Missense | Neutral |
| tunF | 963 | 26 | 2 | 0(0.0%) | – | 0 | – | – |
| tunG | 579 | 26 | 2 | 1(0.2%) | – | 0 | – | – |
| tunH | 1548 | 26 | 2 | 6(0.4%) | – | 4 | Missense | Neutral |
| tunI | 993 | 26 | 2 | 6(0.6%) | – | 5 | Missense | Neutral |
| tunJ | 789 | 26 | 2 | 0(0.0%) | – | 0 | – | – |
| tunK | 291 | 26 | 2 | 0(0.0%) | – | 0 | – | – |
| tunL | 744 | 26 | 2 | 1(0.1%) | – | 1 | Missense | Neutral |
| tunO | 675 | 26 | 2 | 10(1.5%) | – | 6 | Missense | nd |
| tunP | 1224 | 26 | 2 | 1(0.1%) | – | 1 | Missense | nd |
| R. iranicus | ||||||||
| tunA | 978 | 6 | 2 | 41(4.3%) | 1 (15 bp) | 12 | Missense | Neutral |
| tunB | 1014 | 6 | 3 | 42(4.1%) | – | 12 | Missense | Neutral∗ |
| tunC | 1002 | 6 | 3 | 54(5.4%) | – | 20 | Missense | Neutral |
| tunD | 1362 | 6 | 3 | 51(3.7%) | – | 20 | Missense | Neutral |
| tunE | 693 | 6 | 3 | 16(2.3%) | – | 2 | Missense | Neutral |
| tunF | 939 | 6 | 3 | 30(3.2%) | – | 11 | Missense | Neutral |
| tunH | 1530 | 6 | 3 | 76(5.0%) | – | 36 | Missense | Neutral |
| tunI | 963 | 6 | 4 | 31(3.2%) | – | 14 | Missense | Neutral |
| tunJ | 786 | 6 | 3 | 27(3.4%) | – | 7 | Missense | Neutral |
| tunK | 291 | 6 | 2 | 24(8.2%) | – | 11 | Missense | Neutral |
| tunL | 732 | 6 | 3 | 25(3.4%) | – | 15 | Missense | Neutral |
| tunP | 1224 | 6 | 3 | 32(2.6%) | – | 8 | Missense | nd |
| tunQ | 1056 | 6 | 2 | 31(2.9%) | – | 15 | Missense | nd |
| tunR | 675 | 6 | 3 | 20(3.0%) | – | 11 | Missense | nd |
| tunS | 369 | 6 | 2 | 41(11.1%) | – | 26 | Missense | nd |
| tunT | 213 | 6 | 2 | 5(2.3%) | 1 (3 bp) | 3 | Nonsense/Missense | nd |
| R. agropyri | ||||||||
| tunA | 963 | 8 | 5 | 23(2.4%) | – | 7 | Missense | Neutral |
| tunB | 1014 | 8 | 4 | 25(2.5%) | – | 7 | Missense | Deleterious |
| tunC | 996 | 8 | 4 | 21(2.1%) | – | 7 | Missense | Neutral |
| tunD | 1362 | 8 | 6 | 29(2.1%) | – | 13 | Missense | Deleterious |
| tunE | 693 | 8 | 6 | 14(2.0%) | – | 4 | Missense | Neutral |
| tunF | 939 | 8 | 5 | 25(2.7%) | – | 13 | Missense | Neutral |
| tunH | 1530 | 8 | 5 | 52(3.4%) | – | 22 | Missense | Neutral∗ |
| tunI | 963 | 8 | 6 | 38(3.9%) | – | 15 | Missense | Neutral |
| tunJ | 786 | 8 | 4 | 16(2.0%) | – | 3 | Missense | Neutral |
| tunK | 291 | 8 | 4 | 8(2.7%) | – | 5 | Missense | Neutral |
| tunL | 720 | 8 | 5 | 15(2.1%) | 1 (3 bp) | 8 | Missense | Neutral |
| tunP | 1227 | 8 | 6 | 22(1.8%) | – | 9 | Missense | nd |
| tunQ | 1056 | 8 | 6 | 24(2.3%) | – | 12 | Missense | nd |
| tunR | 675 | 8 | 6 | 50(7.4%) | 2 (1 bp) | 33 | Nonsense/Missense | nd |
aThe gene size based on the reference strains R. toxicus FH79 (Sechler et al., 2017), R. iranicus FH6, and R. agropyri CA4 (Schroeder et al., 2018). bThe number of unique INDELs (insertion/deletion) within sequenced fragments. Each INDEL was treated as a single polymorphic site. cThe number of non-synonymous mutations does not include INDELs. dThe functional effect of missense mutations, among the TGC-conserved Rathayibacter genes (tunA-tunL), were predicted in silico with PROVEAN (Protein Variation Effect Analyzer) and SIFT (Sorting Intolerant From Tolerant) (Choi and Chan, 2015; Vaser et al., 2016). ∗, a missense mutation was predicted to be deleterious with PROVEAN, but tolerated with SIFT; nd, not determined due to the lack of adequate reference sequences.