Table 1.
Tilletia species, their host and geographical origin and their suppliers.
Fig 1.
Alignment of the mt genomes of T. indica isolates, Ps2 (KX394364), F11 (DQ993184) and T. walkeri isolate, TJ23 (EF536375) showed identical gene order.
Gene sizes are drawn to relative lengths and the arrows indicate the direction of transcription. The reverse arrow indicates transcription from the complementary strand. The black shaded and white unshaded boxes indicate presence and absence respectively of the corresponding presence/absence variation (PAV), labelled PAV1, PAV2, PAV3, PAV4 and PAV5 in the genomes.
Table 2.
Sequences of primer pairs for the amplification of the five PAV elements; PAV1, PAV2, PAV3, PAV4 and PAV5 (Fig 1).
Fig 2.
The mt segment, (KX394364: 33962..34226) encompasses a unique sequence of T. indica (PAV2).
This is used for the design of primers (Table 3) in the LAMP assay. The primer sequences of the two outer primers (F3 and B3) and the two inner primers (FIP, BIP) are highlighted in yellow and the two loop primers are highlighted in blue. The orientations of the primer sequences in the assay are as indicated.
Table 3.
Sequences of primers for the LAMP assay of T. indica.
Table 4.
Distribution of SNPs in mt genomes between a T. indica (Ps2) and a T. walkeri (TJ23) isolate, and between two T. indica isolates, F11 and Ps2.
Fig 3.
Distribution of PAVs in T. indica isolates.
Amplification of PAV elements, PAV1 to PAV5 (Fig 1) using primers (Table 2) designed from analysis of the mt genomes alignment. The lengths of the amplicons with the PAV elements; PAV1, PAV2, PAV3, PAV4 and PAV5 are 1289, 370, 1521, 1715 and 1232 nt respectively (Table 2). NTC refers to no template control. T. walkeri isolate, Tw6, is a replicate of Tw4 (Table 1).
Table 5.
Profiles of the five PAVs in T. indica isolates.
Fig 4.
An unrooted phylogenetic tree generated by the maximum likelihood algorithm on a data file of amino acid/protein sequences of intron-encoded homing endonuclease genes (HEGs; LAGLIDAG and GIY-YIG endonucleases).
Analysis used the Jones Taylor Thornton model with uniform substitution rates in the program MEGA6 [25]. Each of the four clusters of the intron sequences containing these HEGs are inserted in the same position in the mt gene indicated. The analysis demonstrated that the HEGs in PAV1, PAV4 and PAV5 of T. indica are more closely related with intron-encoded HEGs inserted at the same site in the same gene from diverse fungal species than the phylogeny of the fungal species, and is evidence of the horizontal transfer of these mobile introns, across taxonomic boundaries in fungi.
Fig 5.
A positive LAMP reaction can be visualized as a ladder of DNA loop amplicons on an agarose gel using the nucleic acid fluorescent stain, GelRed. Sensitivity of the LAMP assay was determined at approximately 10 pg (lane 5) using a 1 in 10 DNA dilution series from 10 ng (lane 8) to 0.01 pg (lane 2). Lane 1 is no template control.
Fig 6.
False negative results from LAMP assay by Gao et al. [37].
A positive reaction is indicated as a ladder of DNA fragments on an agarose gel. Eight of thirteen T. indica isolates were tested ‘negative’ as predicted by the absence of PAV5 (Fig 3). The positive results obtained for isolates Ps6, Ps21 and MKTi9 with assay by [37] suggested the presence of PAV5 in a few copies of mt DNA in these isolates.