The rapid development of next-generation sequencing technologies over the past decade has led to a flood of both de-novo sequencing and re-sequencing projects in almost every branch of the tree of life. Within the fungal kingdom, comparative genome studies have led to the unexpected finding that large genomic regions may be variable among isolates of a given species. One category of these variable regions are unique chromosomes referred to as supernumerary or conditionally dispensable because they are not typically required for saprophytic growth [1–3]. These chromosomes have been identified in many fungi including Magnaporthe oryzae [4–6], Fusarium oxysporum , Nectria haematococca [8, 9], Mycosphaerella graminicola , Cochliobolus heterostrophus , Leptosphaeria maculans , and Alternaria alternata [13, 14].
Plant pathogenic fungi in the genus Alternaria infect a remarkable range of host plants and are major causes of agricultural yield losses . Conditionally dispensable chromosomes (CDCs) are carried by several of the small-spored, plant-pathogenic Alternaria species [13, 14, 16]. These chromosomes are generally less than 2.0MB in size, and may be transmitted horizontally between isolates in a population, potentially conferring new pathogenic attributes to the receiving isolate [17–20]. Loss of the CDC can also occur during repeated sub-culturing, resulting in the transition from a pathogenic to saprophytic form of the fungus . Several genes coding host specific toxins (HSTs) have been located to gene clusters on CDCs, including those producing AF-toxin from the strawberry pathotype , AK-toxin from the Japanese pear pathotype , and ACT-toxin from the tangerine pathotype . These toxins share a common 9,10-epoxy-8-hydroxy-9-methyl-decatrienoic acid structural moiety, with the genes encoding each toxin sharing a high degree of homology [21–25]. In addition, the AMT gene from the apple pathotype, a gene involved in host-specific AM-toxin cyclic peptide biosynthesis, is located on a small chromosome of 1.1 to 1.7 Mb [13, 26], with at least four copies involved in AM-toxin biosynthesis . The only other gene sequences identified to date on CDCs are extended families of transposon-like sequences (TLSs) .
Horizontal gene transfer (HGT) is the movement, without recombination, of stable genetic material between two individuals . HGT may not only occur between different individuals of the same species, but also between species or even between bacteria and fungi or between fungi and oomycetes [29, 30]. In fungi, the movement of plasmids, mycoviruses, transposable elements, gene clusters, and whole chromosomes have been demonstrated from one individual to another . The first theory to explain gain and loss of HSTs was proposed in 1983 . It has then been hypothesized that the genome content of CDCs in Alternaria species were acquired through HGT events . The most well studied example of HGT in fungi is the movement of the ToxA gene from the wheat blotch pathogen Stagonospora nodorum to Pyrenophora tritici-repentis, the causal agent of tan spot of wheat [33, 34]. This horizontal transfer event was identified by nucleotide sequence similarity and structural comparisons between genes from both species. The direction of transfer was inferred by the fact that the ToxA gene consisted of a single haplotype in P. tritici-repentis but 11 haplotypes in S. nodorum isolates.
Alternaria arborescens (synonym A. alternata f. sp. lycopersici), the fungus that produces host-specific AAL toxin, is the causal agent of stem canker of tomato [35, 36]. It has been observed in pulsed field gel electrophoresis (PFGE) studies that A. arborescens carries one CDC of 1.0-Mb [16, 37]. To date, only two genes have been reported to be carried on this CDC including ALT1, which is a PKS gene involved in AAL toxin biosynthesis [38, 39], and AaMSAS, also a PKS gene [40, 41]. A CDC deletion mutant of A. arborescens generated through restriction enzyme mediated integration (REMI) showed a toxin and pathogenicity minus phenotype . In addition, in protoplast fusion experiments, a CDC from A. arborescens was observed to transfer into the strawberry pathotype, and subsequently introduced new tomato pathogenicity to the fusant .
In this study, we used a next generation sequencing approach to produce a draft sequence of the A. arborescens genome and used a novel bioinformatics approach to separate CDC contigs from the essential chromosome (EC) contigs. The gene content of the CDC was analyzed to answer the following questions: (1) What is the difference between the CDC and EC genome content at the nucleotide level? (2) Are CDC genes under positive selection and could they represent additional virulence factors in addition to the known toxin encoding genes? (3) Is the evolutionary history of the CDC the same as that of the ECs, and is there any evidence of a HGT event? In answering these questions, we confirmed a different genome content pattern of the A. arborescens CDC and found evidence for HGT.