Reverse transcriptase-related enzymes are ... - Semantic Scholar

Reverse transcriptase-related enzymes are associated with horizontal chromosome transfer in an asexual pathogen Xiaoqiu Huang, Anindya Das, Binod B Sahu, Subodh K Srivastava, Leonor F Leandro, Kerry O'Donnell, Madan K Bhattacharyya

Supernumerary chromosomes have been shown to transfer horizontally from one isolate to another. However, the mechanism by which horizontal chromosome transfer (HCT) occurs

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is unknown. In this study, we compared the genomes of 11 isolates comprising six Fusarium species that cause soybean sudden death syndrome (SDS) or bean root rot (BRR), and detected numerous instances of HCT in supernumerary chromosomes. We also identified a statistically significant number (21 standard deviations above the mean) of single nucleotide polymorphisms (SNPs) in the supernumerary chromosomes between isolates of the asexual pathogen F. virguliforme. Supernumerary chromosomes carried reverse transcriptase-related genes (RVT); the presence of long RVT open reading frames (ORFs) in the supernumerary chromosome was correlated with the presence of two or more chromosome copies with a significant number of SNPs between them. Our results suggest that supernumerary chromosomes transfer horizontally via an RNA intermediate. Understanding the mechanism by which HCT occurs will have a profound impact on understanding evolution and applying biotechnology as well as accepting HCT as a natural source of genetic variation.

PeerJ PrePrints | https://dx.doi.org/10.7287/peerj.preprints.1324v1 | CC-BY 4.0 Open Access | rec: 25 Aug 2015, publ: 25 Aug 2015

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Reverse transcriptase-related enzymes are

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associated with horizontal chromosome transfer in an asexual pathogen

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Xiaoqiu Huang1,2,3 , Anindya Das1 , Binod B. Sahu4 , Subodh K. Srivastava5 ,

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Leonor F. Leandro6 , Kerry O’Donnell7 , Madan K. Bhattacharyya4

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Department of Computer Science and 2 Plant Sciences Institute, Iowa State University,

Ames, Iowa 50011, USA 4

Department of Agronomy, Iowa State University, Ames, Iowa 50011, USA

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Crop, Soil and Environmental Sciences, University of Arkansas, Fayetteville, Arkansas

72701, USA 6

Department of Plant Pathology, Iowa State University, Ames, Iowa 50011, USA

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National Center for Agricultural Utilization Research, US Department of Agriculture,

Agricultural Research Service, Peoria, Illinois 61604, USA

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Corresponding Author: 3

Xiaoqiu Huang

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Department of Computer Science, Iowa State University, Ames, Iowa 50011, USA

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E-mail: [email protected], Phone: (515) 294-2432, Fax: (515) 294-0258

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Key Words: Asexual reproduction, Horizontal chromosome transfer, Reverse transcriptase-

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related enzymes, Duplication-induced mutation

1 PeerJ PrePrints | https://dx.doi.org/10.7287/peerj.preprints.1324v1 | CC-BY 4.0 Open Access | rec: 25 Aug 2015, publ: 25 Aug 2015

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Abstract

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Supernumerary chromosomes have been shown to transfer horizontally from

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one isolate to another. However, the mechanism by which horizontal chromosome

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transfer (HCT) occurs is unknown. In this study, we compared the genomes of 11

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isolates comprising six Fusarium species that cause soybean sudden death syndrome

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(SDS) or bean root rot (BRR), and detected numerous instances of HCT in super-

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numerary chromosomes. We also identified a statistically significant number (21

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standard deviations above the mean) of single nucleotide polymorphisms (SNPs) in

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the supernumerary chromosomes between isolates of the asexual pathogen F. vir-

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guliforme. Supernumerary chromosomes carried reverse transcriptase-related genes

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(RVT); the presence of long RVT open reading frames (ORFs) in the supernu-

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merary chromosome was correlated with the presence of two or more chromosome

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copies with a significant number of SNPs between them. Our results suggest that

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supernumerary chromosomes transfer horizontally via an RNA intermediate. Un-

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derstanding the mechanism by which HCT occurs will have a profound impact on

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understanding evolution and applying biotechnology as well as accepting HCT as

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a natural source of genetic variation.


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Introduction

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Conventional wisdom holds that asexual organisms lack a mechanism for generating ge-

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netic variation. However, evidence of sexual reproduction has not been detected in many

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important plant pathogens (Masel et al., 1996; Michielse & Rep, 2009). Asexual fungal

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pathogens are known to have variable electrophoretic karyotypes (Kistler & Miao, 1992).

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It was shown through pulsed-field gel eletrophoresis that extra nonessential chromosomes

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(called supernumerary chromosomes) are present only in some individuals of a species

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(Masel, Irwin & Manners, 1993). It was demonstrated under laboratory conditions that a

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2-Mb supernumerary chromosome was transferred between two vegetatively incompatible

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isolates of an asexual fungus (He et al., 1998). A whole-genome comparative study also

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suggests that supernumerary chromosomes were horizontally acquired (Ma et al., 2010).

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In addition, the genomes of some asexual fungal pathogens contain lineage-specific (LS)

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regions that are highly variable among isolates (Klosterman et al., 2011). An analysis of

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several nonhomologous recombination forms and polymorphic sequence types of each form

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in LS regions from different isolates suggests that LS sequences were horizontally acquired

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(Huang, 2014). These results indicate that horizontal transfer generates genetic variation

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in asexual fungal pathogens, yet the mechanism and extent of transfer are unknown. It is

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also unclear whether transfer creates not only presence/absence polymorphisms but also

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single nucleotide polymorphisms (SNPs).

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Fusarium virguliforme is an economically important fungal pathogen that causes sud-

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den death syndrome (SDS) in soybean in North and South America (Aoki et al., 2003).

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Although different F. virguliforme isolates show variation in aggressiveness on soybean

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plants, studies with various molecular markers detected an extremely low level of genetic

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variation within F. virguliforme isolates from North and South America (O’Donnell et

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al., 2010; Mbofung et al., 2012). Moreover, mating experiments with 17 US isolates of

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F. virguliforme indicated that they all belonged to a single mating type (Covert et al.,

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2007). A genome assembly of a F. virguliforme isolate was produced recently (Srivastava

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et al., 2014), and the mating type locus in F. virguliforme and its six close relatives were

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characterized. A PCR assay based on both mating type sequences revealed that all 129


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isolates of F. virguliforme in North and South America possessed the MAT1-1 mating type

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(Hughes et al., 2014). These data suggest that the reproduction mode of F. virguliforme

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on soybean is asexual. It is unknown how genetic variation in this asexual pathogen is

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generated for the disease to have reached all major soybean-growing areas in the USA

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since its first detection in the early 1970s.

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F. virguliforme is related to the sexual fungal pathogen Nectria haematococca MPVI,

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which is known to contain supernumerary chromosomes (Miao, Covert & VanEtten, 1991).

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These supernumerary chromosomes contain genes involved in resistance to plant antimi-

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crobial compounds and in host-specific pathogenicity (Covert, 1998). Sequences of the

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N. haematococca MPVI supernumerary chromosomes (Coleman et al., 2009) can be used

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to determine if their homologs are present in F. virguliforme.

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F. virguliforme is closely related to five morphologically distinct Fusarium species that

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cause SDS or bean root rot (BRR): F. azukicola, F. brasiliense, F. cuneirostrum, F.

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phaseoli and F. tucumaniae (Aoki et al., 2012). F. tucumaniae is the only known sexually

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reproducing fungus among these species (Covert et al., 2007). In this study, we selected

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ten isolates of these closely related species — three (F. virguliforme), three (F. tucuma-

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niae), and one (each of the other four species) — for next-generation genome sequencing

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and analysis in comparison with the F. virguliforme genome assembly (Srivastava et al.,

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2014) as a reference (Table 1). Note that including the reference isolate leads to a total

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of eleven isolates, four of which are F. virguliforme isolates.

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We compared the genomes of 11 isolates comprising six Fusarium species that cause

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SDS or BRR, and detected numerous instances of HCT (horizontal chromosome transfer)

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in supernumerary chromosomes. The genome of the asexual pathogen F. virguliforme

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was composed of a large core genome and a small supernumerary portion; there was little

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variation in the core between isolates, but there are a statistically significant number (21

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standard deviations above the mean) of SNPs in the supernumerary chromosomes between

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isolates. Supernumerary chromosomes carry reverse transcriptase-related genes (RVT);

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they were highly variable in length, and the presence of long RVT open reading frames

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(ORFs) in the supernumerary chromosome was correlated with the presence of two or more

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chromosome copies with a significant number of SNPs between them. Our results suggest 4


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that supernumerary chromosomes transfer horizontally via an RNA intermediate; their high

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SNP and length variation rates were attributed to the high error rates of the RVT enzymes.

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We report an extensive body of evidence to suggest that the F. virguliforme genome

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evolved by two mechanisms: duplication-induced mutation for the core and replication via

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an RNA intermediate for the supernumerary.

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Materials & Methods

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Sequence data

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We selected 10 isolates of six Fusarium species and produced Illumina paired-end reads of

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102 bp for each of them. We previously produced a genome assembly (NCBI BioProject

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Accession: PRJNA63281) of isolate F. virguliforme Mont-1 (Srivastava et al., 2014),

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which was used as a reference genome assembly in this study. The origin, year of collection,

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and name abbreviation of each of these 11 isolates are in Table 1.

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Read mapping and SNP detection

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A SNP between the reference isolate and another isolate (query) has two or more alleles

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called REF and ALT. The REF allele refers to the allele in the reference and ALT alleles

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refer to alternate non-reference alleles. A SNP is of type 2 if both the REF allele and the

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ALT allele are present in the query isolate, and of type 1 if only the ALT allele is present

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in the query isolate.

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The sets of Illumina paired-end reads for each query isolate were mapped onto the

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reference genome assembly with Bowtie2 (Langmead & Salzberg, 2012). The output from

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Bowtie2 in SAM format was redirected to Samtools (Li et al., 2009) with the view com-

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mand to produce output in BAM format, which was sorted with the sort command. The

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sorted output in BAM format was piled up on the reference with the mpileup command.

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For command options and parameter values, see Huang (2014). The sorted BAM output

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files for all the isolates along with the reference genome assembly were uploaded into Inte-


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grative Genomics Viewer (Robinson et al., 2011) for viewing SNPs and presence/absence

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polymorphisms in each isolate.

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Assembly of short reads

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An assembly of paired-end reads for each isolate was performed with an Illumina version

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of PCAP (Huang et al., 2003) with the following data and options: a pair of mate files

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in fastq format; a minimum insert length of 100 bp and a maximum insert length of 700

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bp; an average insert length of 400 bp with a standard deviation of 100. The minimum

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length of overlaps with no base mismatch match was set to 84 bp, and that of overlaps

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with up to three base mismatches was set to 90 bp. No overlap with more than three

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base mismatches was accepted. Each data set was of size up to 49 Gb, and each assembly

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could be produced in a day on a processor with 100 Gb of main memory. One feature of

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the program is that it is conservative in joining reads into contigs by avoiding reads in the

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overlap between two potential contigs that can not be merged into one.

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Assembly mapping

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Each assembly of Illumina reads was mapped to the reference genome assembly by using

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BWA-MEM (Li, 2013) with the default options. The output from BWA-MEM in SAM

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format was redirected to Samtools (Li et al., 2009) with the view command and -bS

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options to produce output in BAM format, which was sorted with the sort command. An

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output file of SNPs and indels in VCF format was produced in the same way as in the

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read mapping. The assembly mapping was useful in finding long indels between contigs

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in the reference assembly and query assembly, respectively. The coordinates of an indel

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between two contigs were found by computing an alignment of the contigs with GAP3

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(Huang & Chao, 2003).

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Gene identification and functional annotation

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Ab initio gene identification in a Fusarium genomic sequence was performed using Augus-

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tus (Stanke & Waack, 2003) with training data from F. graminearum. A non-redundant 6


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protein sequence database at National Center for Biotechnology Information was searched

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using Blastx (Gish & States, 1993) with a genomic coding region as a query to find a set

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of protein database sequences that are most similar to the coding region. The gene struc-

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ture from Augustus was refined by using AAT (Huang et al., 1997) on the set of protein

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database sequences. Functional annotation of genes was performed using the HMMER

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web server (Finn, Clements & Eddy, 2011).

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Phylogenetic analysis

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A maximum-likelihood tree of the 11 SDS/BRR Fusarium isolates was inferred from

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genome-wide SNP data. The data were produced by mapping reads from each of 10

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of the 11 isolates onto a genome assembly of the reference Fv Mont-1. A covered SNP

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position is a position of the reference that was sufficiently covered by reads from each

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isolate and had an alternative allele (a SNP) in the read coverage of this position from

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one of the 10 isolates. A total of 297,076 covered SNP positions were aligned in the

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11 isolates. The multiple sequence alignment was analyzed to infer the tree with 200

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bootstrap samples.

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Results

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Rapid evolution in a small portion of the F. virguliforme genome

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We mapped short reads from each of the ten isolates onto a 50.5-Mb genome assembly

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of isolate Fv Mont-1 (Srivastava et al., 2014) as a reference. The length of the reference

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covered by reads from the isolate and the distribution of SNPs between the reference

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and the isolate are given in Table 2. Table 2 reveals significant variation in evolutionary

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rate among the isolates. First, the four F. virguliforme isolates possessed a low genome-

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wide SNP rate of less than 1 in 10,000 bp, which is consistent with an asexual mode of

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reproduction. Isolate Fv 34551 collected in South America in 2002 was closer to Fv Mont-

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1 collected in the USA in 1991 than the other two F. virguliforme isolates collected in

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the USA. Second, the genome-wide SNP rate of about 1 in 200 bp between the reference 7


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and each non-F. virguliforme isolate was at least 58 times higher than that between

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the reference and each F. virguliforme isolate, indicating a significantly higher level of

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polymorphism and suggesting a much longer divergence time between F. virguliforme and

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the other species. Third, the genome-wide SNP rate of 1 in 200 bp between the reference

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and each non-F. virguliforme isolate was not high enough to explain why at least 10 Mb

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of the reference genome was covered by reads from every F. virguliforme isolate, but not

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by reads from any non-F. virguliforme isolate.

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To shed light on the last observation, we selected all of the contigs that were at least

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1 kb in the reference assembly and calculated the total number of contig bases covered by

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reads from Fc 31157 as well as that not covered by reads from this isolate. The size of the

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covered portion was 39.5 Mb; that of the uncovered portion was 10.9 Mb. The uncovered

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portion was A+T rich (68%), whereas the covered portion was A+T poor (45%). The

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duplicated content of the uncovered portion was 70%, with 48% made up of sequences

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with copy numbers above 20. In sharp contrast, the duplicated content of the covered

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portion was 3.8%, with 0.56% made up of 20-plus-copy sequences. These results indicate

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that since the divergence between F. virguliforme and F. cuneirostrum, the uncovered

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portion of the F. virguliforme genome evolved much faster in association with duplication

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and C-to-T/G-to-A mutation. For example, a maximum likelihood tree of 13 duplicated

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sequences 3,772 bp in length from the genome assembly of Fv Mont-1 showed that the

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more recently duplicated sequences have a higher A+T content (Fig. 1).

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Although the genome-wide SNP rate between the reference F. virguliforme isolate and

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each of the other three F. virguliforme isolates was at most 0.00007, we found high levels

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of variation among the four F. virguliforme isolates in a small portion (