Characterization of SNP and Structural Variations in the Mitochondrial Genomes of <i>Tilletia indica</i> and Its Closely Related Species Formed Basis for a Simple Diagnostic Assay

<div><p><i>Tilletia indica</i> causes the disease Karnal bunt in wheat. The disease is under international quarantine regulations. Comparative mitochondrial (mt) genome analysis of <i>T</i>. <i>indica</i> (KX394364 and DQ993184) and <i>T</i>. <i>walkeri</i> (EF536375) has found 325 to 328 SNPs, 57 to 60 short InDels (from 1 to 13 nt), two InDels (30 and 61 nt) and five (>200 nt) presence/absence variations (PAVs) between the two species. The mt genomes of both species have identical gene order. The numbers of SNPs and InDels between the mt genomes of the two species are approximately nine times of the corresponding numbers between the two <i>T</i>. <i>indica</i> isolates. There are eight SNPs between <i>T</i>. <i>indica</i> and <i>T</i>. <i>walkeri</i> that resulted in amino acid substitutions in the mt genes of <i>cob</i>, <i>nad2</i> and <i>nad5</i>. In contrast, there is no amino acid substitution in the mt genes of the <i>T</i>. <i>indica</i> isolates from the SNPs found. The five PAVs present in <i>T</i>. <i>indica</i> (DQ993184) are absent in <i>T</i>. <i>walkeri</i>. Four PAVs are more than 1 kb and are not present in every <i>T</i>. <i>indica</i> isolate. Analysis of their presence and absence separates a collection of <i>T</i>. <i>indica</i> isolates into 11 subgroups. Two PAVs have ORFs for the LAGLIDAG endonuclease and two have ORFs for the GIY-YIG endonuclease family, which are representatives of homing endonuclease genes (HEGs). These intron- encoded HEGs confer intron mobility and account for their fluid distribution in <i>T</i>. <i>indica</i> isolates. The small PAV of 221 bp, present in every <i>T</i>. <i>indica</i> isolate and unique to the species, was used as the genetic fingerprint for the successful development of a rapid, highly sensitive and specific loop mediated isothermal amplification (LAMP) assay. The simple procedure of the LAMP assay and the easy detection formats will enable the assay to be automated for high throughput diagnosis.</p></div

False negative results from LAMP assay by Gao et al. [37].

<p>A positive reaction is indicated as a ladder of DNA fragments on an agarose gel. Eight of thirteen <i>T</i>. <i>indica</i> isolates were tested ‘negative’ as predicted by the absence of PAV5 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166086#pone.0166086.g003" target="_blank">Fig 3</a>). The positive results obtained for isolates Ps6, Ps21 and MKTi9 with assay by [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166086#pone.0166086.ref037" target="_blank">37</a>] suggested the presence of PAV5 in a few copies of mt DNA in these isolates.</p

Sequences of primer pairs for the amplification of the five PAV elements; PAV1, PAV2, PAV3, PAV4 and PAV5 (Fig 1).

<p>Sequences of primer pairs for the amplification of the five PAV elements; PAV1, PAV2, PAV3, PAV4 and PAV5 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166086#pone.0166086.g001" target="_blank">Fig 1</a>).</p

The mt segment, (KX394364: 33962..34226) encompasses a unique sequence of <i>T</i>. <i>indica</i> (PAV2).

<p>This is used for the design of primers (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166086#pone.0166086.t003" target="_blank">Table 3</a>) 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.</p

Distribution of SNPs in mt genomes between a <i>T</i>. <i>indica</i> (Ps2) and a <i>T</i>. <i>walkeri</i> (TJ23) isolate, and between two <i>T</i>. <i>indica</i> isolates, F11 and Ps2.

<p>Distribution of SNPs in mt genomes between a <i>T</i>. <i>indica</i> (Ps2) and a <i>T</i>. <i>walkeri</i> (TJ23) isolate, and between two <i>T</i>. <i>indica</i> isolates, F11 and Ps2.</p

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).

<p>Analysis used the Jones Taylor Thornton model with uniform substitution rates in the program MEGA6 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166086#pone.0166086.ref025" target="_blank">25</a>]. 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 <i>T</i>. <i>indica</i> 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.</p

Profiles of the five PAVs in <i>T</i>. <i>indica</i> isolates.

<p>Profiles of the five PAVs in <i>T</i>. <i>indica</i> isolates.</p

Distribution of PAVs in <i>T</i>. <i>indica</i> isolates.

<p>Amplification of PAV elements, PAV1 to PAV5 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166086#pone.0166086.g001" target="_blank">Fig 1</a>) using primers (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166086#pone.0166086.t002" target="_blank">Table 2</a>) 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 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166086#pone.0166086.t002" target="_blank">Table 2</a>). NTC refers to no template control. <i>T</i>. <i>walkeri</i> isolate, Tw6, is a replicate of Tw4 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166086#pone.0166086.t001" target="_blank">Table 1</a>).</p