# The genetic linkage map of the medicinal mushroom Agaricus subrufescens reveals highly conserved macrosynteny with the congeneric species Agaricus bisporus

Marie Foulongne-Oriol, Manuela Rocha de Brito, Delphine Cabannes, Aurélien Clement, Cathy Spataro, Magalie Moinard, Eustáquio Souza Dias, Philippe Callac, Jean-Michel Savoie

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Marie Foulongne-Oriol, Manuela Rocha de Brito, Delphine Cabannes, Aurélien Clement, Cathy Spataro, et al.. The genetic linkage map of the medicinal mushroom Agaricus subrufescens reveals highly conserved macrosynteny with the congeneric species Agaricus bisporus. G3, 2016, 6 (5), pp.1217-1226. 10.1534/g3.115.025718. hal-02637089

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# The Genetic Linkage Map of the Medicinal Mushroom *Agaricus subrufescens* Reveals Highly Conserved Macrosynteny with the Congeneric Species *Agaricus bisporus*

Marie Foulongne-Oriol,*,¹ Manuela Rocha de Brito,*,† Delphine Cabannes,* Aurélien Clément,*

Cathy Spataro,* Magalie Moinard,* Eustáquio Souza Dias,† Philippe Callac,* and Jean-Michel Savoie*

*Inra, MycSA, Mycologie et Sécurité des Aliments, 33882 Villenave d'Ornon, France, and †Departamento de Biologia, UFLA, Universidade Federal de Lavras, C.P. 3037, 37200-000, Lavras, MG, Brazil

**ABSTRACT** Comparative linkage mapping can rapidly facilitate the transfer of genetic information from model species to orphan species. This macrosynteny analysis approach has been extensively used in plant species, but few example are available in fungi, and even fewer in mushroom crop species. Among the latter, the *Agaricus* genus comprises the most cultivable or potentially cultivable species. *Agaricus bisporus*, the button mushroom, is the model for edible and cultivable mushrooms. We have developed the first genetic linkage map for the basidiomycete A. *subrufescens*, an emerging mushroom crop known for its therapeutic properties and potential medicinal applications. The map includes 202 markers distributed over 16 linkage groups (LG), and covers a total length of 1701 cM, with an average marker spacing of 8.2 cM. Using 96 homologous loci, we also demonstrated the high level of macrosynteny with the genome of A. bisporus. The 13 main LG of A. *subrufescens* were syntenic to the 13 A. bisporus chromosomes. A disrupted synteny was observed for the three remaining A. *subrufescens* LG. Electronic mapping of a collection of A. *subrufescens* expressed sequence tags on A. bisporus genome showed that the homologous loci were evenly spread, with the exception of a few local hot or cold spots of homology. Our results were discussed in the light of *Agaricus* species evolution process. The map provides a framework for future genetic or genomic studies of the medicinal mushroom A. *subrufescens*.

**KEYWORDS**

comparative
mapping
macrosynteny
almond
mushroom
button
mushroom
chromosome
evolution

The genus *Agaricus* includes several important cultivated mushroom
crop species. Among those, *Agaricus bisporus* (Lange) Imbach, the
button mushroom, is the most widely produced, and is consumed
throughout the world. Given its agronomical importance, *A. bisporus*
has been studied extensively at the genetic, molecular and physiological
levels (Savoie *et al.* 2013). Genome sequence, molecular markers, link-

age maps, QTL for various agronomical traits and breeding methods
are available (Callac et al. 2006; Foulongne-Oriol et al. 2010, 2012a,
2012b; Gao et al. 2013, 2015; Morin et al. 2012), making *A. bisporus* the
model organism for edible and cultivable basidiomycetes.

Lately, another *Agaricus* species, *Agaricus subrufescens* Peck, has received growing attention for its notable therapeutic properties. It is known to produce various bioactive compounds that have potential medicinal applications (Firenzuoli et al. 2008; Wisitrassameewong et al. 2012). Moreover, *A. subrufescens* can convert lignocellulosic residues into highly nutritious food, and in this way contribute to valorization of wastes. This basidiomycete, also known as the almond mushroom due to its particular flavor, has become, within a few years, one of the most important culinary-medicinal cultivable mushrooms, with potentially high added-value products and extended agronomical valorization (Largeteau et al. 2011; Llarena-Hernandez et al. 2013; Okuda et al. 2012). Today, a few commercial cultivars are available, all showing high genetic homogeneity (Colauto et al. 2002; Fukuda et al. 2003; Kerrigan 2005; Tomizawa et al. 2007). Breeding work on this species is in its

<footnote>Copyright © 2016 Foulongne-Oriol et al.
doi: 10.1534/g3.115.025718
Manuscript received December 11, 2015; accepted for publication February 21,
2016; published Early Online February 26, 2016.
This is an open-access article distributed under the terms of the Creative
Commons Attribution 4.0 International License (http://creativecommons.org/
licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction
in any medium, provided the original work is properly cited.
Supplemental material is available online at www.g3journal.org/lookup/suppl/
doi:10.1534/g3.115.025718/-/DC1
¹Corresponding author: INRA, UR1264 Mycologie et Sécurité des Aliments,
bâtiment Qualis, 71 avenue Edouard Bourlaux, CS 20032 F-33882 Villenave
d'Ornon cedex, France. E-mail: mfoulong@bordeaux.inra.fr</footnote>
infancy (Kerrigan and Wach 2008; Llarena-Hernandez et al. 2013) due to several bottlenecks, including a lack of knowledge on its ecology, reproductive biology, biodiversity, and genetics (Largeteau et al. 2011). The recent development of molecular markers and available expressed sequence tag (EST) resources have enriched our toolbox for studying the biology of this mushroom (Foulongne-Oriol et al. 2012c, 2014). The clarification of its amphithallic life cycle, and of the interfertility between field specimens from different continents (Rocha de Brito et al. 2016; Thongklang et al. 2014a), provide a sound basis to exploit the genetic diversity of this species, and to conduct controlled mating of selected genotypes.

The development of an attractive panel of cultivars of *A. subrufescens* is a challenging and long-term process. As a cultivable species, the traits of interest for selection are related to yield, quality, or disease control. Considering its medicinal properties, enrichment in bioactive compounds could also represent a target for selection. Most of those traits are under complex inheritance, making breeding much more arduous. Unraveling genetic control, in terms of gene numbers, effect, and genome position, is essential. Thus, as a prerequisite to develop such quantitative genetic approaches, the primary aim of this work was to develop a genetic linkage map of *A. subrufescens*. Markers with known homologs in *A. bisporus* (Foulongne-Oriol et al. 2014) used for the construction of the *A. subrufescens* map provide ground to explore the synteny between the two species. The conservation of chromosome-scale gene linkage relationships (macrosynteny), including, or not, the preserved order of the loci (collinearity) was assessed by a comparative mapping approach. Identifying commonalities and differences between the two *Agaricus* genome species will provide a basis to investigate evolutionary mechanisms and possible genomic divergence that have contributed to speciation process (Duran et al. 2009; Guyot et al. 2012). Besides phylogenetic considerations, comparative mapping enables the transfer of information between related species (Dirlewanger et al. 2004; Khazaei et al. 2014) and knowledge from the model species *A. bisporus* can benefit the orphan species *A. subrufescens*.

In the present study, we constructed the first genetic linkage map of *A. subrufescens*. By comparing the mapping position of *A. subrufescens* sequence tagged site markers with that of their homologs on the *A. bisporus* genome, we describe the macrosyntenic relationship between the two congeneric species. We interpret our results in the light of the species evolution process. Further possible applied outcomes for *A. subrufescens* breeding purposes are also discussed.

# MATERIALS AND METHODS

## Mapping population

The parental hybrid strain CA487-100 × CA454-3 was previously obtained by crossing the two homokaryons CA487-100 and CA454-3, which are single spore isolates (SSIs) of the French and Brazilian strains CA487 and CA454 (a subculture of WC837), respectively (Llarena-Hernandez et al. 2013; Thongklang et al. 2014a). Single spores isolates were obtained from a spore print of the hybrid strain following the methodology described in Thongklang et al. (2014a). For each SSI, the level of ploidy (n + *n* vs. *n*) was determined with a multilocus genotype test based on 21 genomic microsatellite markers (hereinafter referred to as SubSSR for *A. Subrufescens* simple sequence repeats) (Foulongne-Oriol et al. 2012c) that showed polymorphism between the two parental homokaryons: SSIs that did not exhibit heteromorphic profile at any loci were considered homokaryotic (Thongklang et al. 2014a). Among the 380 SSIs tested, 79 were found homokaryotic and were thus selected for further mapping purposes. The three parental strains (CA487-100, CA454-3, and the hybrid CA487-100 × CA454-3) and the haploid

progeny of 79 homokaryons (referred hereafter as Eᵢ), are maintained in the "Collection du Germplasm des Agarics à Bordeaux" (CGAB), INRA-Bordeaux (http://www6.bordeaux-aquitaine.inra.fr/mycsa_eng/Biological-resources-of-value/The-Agaricus-culture-collection-CGAB).

## Genotyping

Total DNAs were extracted from freeze-dried mycelium following the protocol described in Zhao et al. (2011). DNA concentration was adjusted to 25 ng/µl, and the samples were stored at -20°.

The genotyping of genomic SubSSR markers (Foulongne-Oriol et al. 2012c) followed from the homokaryon selection test described above. In addition to this first set of 21 microsatellite markers, EST-SSRs (hereinafter referred to as ES-SSR) were also used for mapping (Foulongne-Oriol et al. 2014). Amplification conditions, electrophoretic separation, and visualization were performed according to Foulongne-Oriol et al. (2012c). All microsatellite primers used for mapping are listed in Supplementary Material, Table A in File S1.

Sequence-tagged site (STS) markers were developed on the basis of sequences available from the literature (Foulongne-Oriol et al. 2012c, 2014; Matsumoto-Akanuma et al. 2006; Thongklang et al. 2014a). The cleaved amplified polymorphic sequence (CAPS) approach was used to reveal polymorphism in STS. PCR primer pairs were designed to amplify products between 300 and 500 bp using Primer 3 software with default parameters (Rozen and Skaletsky 2000). Amplifications, digestion, electrophoretic separation, and visualization were performed according to Foulongne-Oriol et al. (2010). For each STS marker (hereinafter referred to as PRS), primers and corresponding restriction enzymes are described in Table A in File S1.

Amplified fragment length polymorphism (AFLP) markers were genotyped using the settings described by Foulongne-Oriol et al. (2010). Total DNA was digested with the EcoRI and MseI endonucleases. EcoRI and MseI primers with, for both, two selective nucleotides, were used for amplification through seven pair combinations (Table B in File S1). The amplified fragments were separated and visualized on an ABI3130 sequencer (Applied Biosystems). Electropherograms were analyzed with GENEMAPPER V4.0 software. For each primer pair, the reference panel used for allele calling in the progeny was based on the AFLP patterns of the three parental strains (presence/absence in one of the two homokaryons, presence in the hybrid).

The allelic segregation at the mating-type locus (MAT) was determined by pairing each homokaryon Eᵢ with the two homokaryons CA454-3 (MAT-2), and CA487-100 (MAT-4). The MAT allele of each Eᵢ was deduced from unambiguous compatible reaction with one tester or the other. Mating tests were performed as described in Thongklang et al. (2014a) with two replicates per confrontation. Thus, the allele at the MAT locus could be determined for 74 Eᵢ (93.6%).

## Linkage map construction

Genotypic data were independently scored by two experimenters to minimize scoring errors. Locus segregation was tested for deviation from expected Mendelian ratios 1:1 with a $\chi^2$ test. Linkage and mapping analysis was performed using MAPMAKER/EXP V3.0b software (Lander et al. 1987). The recombination frequency was converted into Kosambi centimorgan (cM) units. In a first round of mapping, markers having more than 30% of missing data, and/or markers with a skewed segregation ratio (*P* < 0.05), were omitted to limit spurious linkage. AFLP markers were kept out of this first step, and those with unbalanced allelic segregation were definitely removed from the genotyping data set. A minimum LOD score of 4.0 and maximum distance of 30 cM were set as thresholds for linkage groups (LG) determination with the 'GROUP' command. For each group, the most likely marker order was
<table><caption>Table 1 Characteristics of the A. subrufescens genetic linkage groups</caption><thead><tr><th>Linkage Group</th><th>Length (cM)</th><th>No. of Markers</th><th>No. of Distorted Markers<sup>a</sup></th><th>Average Marker Spacing (cM)</th><th>Largest Interval (cM)</th></tr></thead><tbody><tr><td>1</td><td>202</td><td>26</td><td>2</td><td>7.8</td><td>23</td></tr><tr><td>2</td><td>196</td><td>22</td><td>0</td><td>8.9</td><td>27</td></tr><tr><td>3</td><td>161</td><td>22</td><td>2</td><td>7.3</td><td>19</td></tr><tr><td>4</td><td>141</td><td>15</td><td>3</td><td>9.4</td><td>27</td></tr><tr><td>5</td><td>138</td><td>13</td><td>1</td><td>10.6</td><td>26</td></tr><tr><td>6</td><td>130</td><td>21</td><td>1</td><td>6.2</td><td>19</td></tr><tr><td>7</td><td>129</td><td>13</td><td>1</td><td>9.9</td><td>24</td></tr><tr><td>8</td><td>113</td><td>10</td><td>1</td><td>11.3</td><td>24</td></tr><tr><td>9</td><td>113</td><td>12</td><td>0</td><td>9.4</td><td>24</td></tr><tr><td>10</td><td>102</td><td>10</td><td>1</td><td>10.2</td><td>26</td></tr><tr><td>11</td><td>76</td><td>8</td><td>6</td><td>9.5</td><td>30</td></tr><tr><td>12</td><td>70</td><td>6</td><td>0</td><td>11.7</td><td>26</td></tr><tr><td>13</td><td>65</td><td>9</td><td>2</td><td>7.2</td><td>17</td></tr><tr><td>14</td><td>42</td><td>9</td><td>0</td><td>4.7</td><td>6</td></tr><tr><td>15</td><td>13</td><td>3</td><td>0</td><td>4.3</td><td>9</td></tr><tr><td>16</td><td>10</td><td>3</td><td>1</td><td>3.3</td><td>5</td></tr><tr><td>Total</td><td>16</td><td>1701</td><td>202</td><td>—</td><td>—</td></tr><tr><td>Mean</td><td>—</td><td>106.3</td><td>12.6</td><td>8.2</td><td>21</td></tr></tbody></table>

established using the 'ORDER' command. Marker orders were confirmed with the 'RIPPLE' command. A framework map was thus established. In a second step, markers excluded from the first round of mapping were tested for linkage using the 'GROUP' and 'LINKS ANY' commands. The 'ASSIGN' command (LOD > 6) allowed the assignation of these markers to the LG defined in the first step. For each LG, additional markers were sequentially placed using the 'TRY' commands. Markers that were difficult to place with several likely positions were dropped. Subsequent orders were again tested with the 'RIPPLE' command.

MAPCHART software (Voorrips 2002) was used for graphical representation of the linkage map. Linkage groups were numbered in descending order based on their length in centimorgans. The occurrence of crossovers for each LG and each individual was analyzed using Graphical Genotyping (GGT 2.0) (van Berloo 2008). Estimated genome length $L_e$ was determined from the linkage data according to Hulbert et al. (1988), as modified by Chakravarti (Chakravarti et al. 1991, Method 3). Map coverage was calculated using the formula $P = 1 - (1 - 2c/L_e)^m$, where P is the proportion of the genome within 2c cM of a marker, m is the number of informative markers on the map, and $L_e$ is the estimated genome length (Beckmann and Soller 1983).

## Assessment of A. subrufescens–A. bisporus macrosynteny

All the sequence-based markers used for the presented linkage map construction were used to infer syntenic relationships between A. subrufescens and A. bisporus chromosomes.

In a previous study, A. subrufescens EST sequences were aligned on the genome sequence of A. bisporus (Foulongne-Oriol et al. 2014). As a brief reminder, the 10,114 A. subrufescens sequences were mapped to A. bisporus scaffolds (H97 sequence V2.0, http://genome.jgi-psf.org/Agabi_varbisH97_2/Agabi_varbisH97_2.home.html; Morin et al. 2012) using the BLAT algorithm (Kent 2002). A score of 60%, which corresponds to the product of the first quartile value for alignments coverage (75%) and identity (80%), was used as threshold to filter out unreliable results. Putative A. bisporus genes homologous to A. subrufescens ESTs were searched for by comparing best hit mapping coordinates to A. bisporus gene model (GM) coordinates. Thus, 6752 A. subrufescens sequences were mapped on the A. bisporus genome, among which 6570 overlapped with 3620 A. bisporus GM (34.93% of

all GM) (Foulongne-Oriol et al. 2014). To go further in the present study, A. bisporus homolog distribution along the genome was examined per scaffold, and, on each scaffold, per window of 50 kb. For the others markers, significant homology with A. bisporus gene was inferred by blastn search similarity (e-value < 1 × 10e-10).

The syntenic relationships established between A. subrufescens linkage map and A. bisporus chromosome sequences were visualized using CIR-COS (Krzywinski et al. 2009). The level of collinearity between genetic and physical orders was assessed by comparing, between map and genome, the coordinate and orientation of each homologous pairs' intervals.

## Data availability

Strains are available upon request. Information about markers used or developed specifically for this project can be found in Table A and Table B of File S1. The genotype dataset used to build the genetic map is available in Table S1. The A. subrufescens EST sequences are accessed in GenBank with the reference GBEJ00000000.1. Coordinate data resulting from the alignment of these A. subrufescens EST sequences on the A. bisporus genome are available in Foulongne-Oriol et al. 2014 or upon request.

## RESULTS

### Genotyping, segregation and linkage analysis, and map construction

In addition to the 21 SubSSR markers used for homokaryon selection, 18 ES-SSR found polymorphic between the two parental strains were used for mapping. Ninety-four polymorphic CAPS markers were developed from available A. subrufescens sequences. The AFLP technique was used in order to increase mapping coverage. A total of 90 polymorphic and reliable AFLP markers were identified using seven primer pairs.

A set of 224 markers (90 AFLPs, 39 SSRs, 94 CAPSs, and the MAT locus) was analyzed for mapping purposes. Twenty-eight markers (12.5%) showed a segregation ratio that deviated from the expected 1:1 ratio (P < 0.05). The total number of genotyped individuals per locus varied from 45 to 79, with an average of 72. Nearly 97% of the scored markers showed less than 20% of missing data.

The first round of mapping included 106 markers representing the most confident and informative set of genotyped loci (only unbiased

<footnote>a Markers showing unbalanced segregation ratios (P < 0.05).</footnote>
<figure><img src="image_2.png" /><figcaption>**Figure 1** Genetic linkage map of *A. subrufescens*. Markers with homologs found in *A. bisporus* genome are in bold. Italicized markers show inconsistent mapping location according to the physical position of their homologs. Markers exhibiting significant segregation distortion are indicated with asterisk (* P < 0.05, ** P < 0.01, *** P < 0.001).</figcaption></figure>
<table><caption>■ Table 2 Oxford grid showing conservation of synteny between *A. subrufescens* linkage groups (rows) and *A. bisporus* chromosomes (columns), sorted by the As-LG number</caption><thead><tr><th rowspan="2"></th><th rowspan="2">LG\chrom.</th><th colspan="10">A. bisporus</th><th rowspan="2">Total</th></tr><tr><th>VI</th><th>II</th><th>III</th><th>VII</th><th>VIII</th><th>I</th><th>IV</th><th>V</th><th>XIII</th><th>XI</th><th>IX</th><th>X</th><th>XII</th></tr></thead><tbody><tr><td rowspan="10">A. subrufescens</td><td>1</td><td>8</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>8</td></tr><tr><td>2</td><td>0</td><td>10</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>10</td></tr><tr><td>3</td><td>0</td><td>0</td><td>8</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>1</td><td>0</td><td>0</td><td>0</td><td>9</td></tr><tr><td>4</td><td>0</td><td>0</td><td>0</td><td>8</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>8</td></tr><tr><td>5</td><td>0</td><td>0</td><td>0</td><td>0</td><td>8</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>1</td><td>0</td><td>0</td><td>9</td></tr><tr><td>6</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>11</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>11</td></tr><tr><td>7</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>10</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>10</td></tr><tr><td>8</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>6</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>6</td></tr><tr><td>9</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>3</td><td>0</td><td>0</td><td>0</td><td>0</td><td>3</td></tr><tr><td>10</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>3</td><td>0</td><td>0</td><td>0</td><td>3</td></tr><tr><td>11</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>7</td><td>0</td><td>0</td><td>7</td></tr><tr><td>12</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>3</td><td>0</td><td>3</td></tr><tr><td>13</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>4</td><td>4</td></tr><tr><td>14</td><td>0</td><td>0</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>1</td></tr><tr><td>15</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>2</td><td>0</td><td>0</td><td>2</td></tr><tr><td>16</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>2</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>2</td></tr><tr><td>Total</td><td></td><td>8</td><td>10</td><td>9</td><td>8</td><td>10</td><td>11</td><td>10</td><td>6</td><td>3</td><td>5</td><td>9</td><td>3</td><td>4</td><td>96</td></tr></tbody></table>

Each number in cell denotes the number of homologous pair of loci.

codominant markers with less than one-third of missing data). With our mapping condition (LOD > 4, $d_{max} < 30$ cM), a preliminary framework map was established with 97 markers distributed over 18 LG. The next rounds of mapping consisted in placing the remaining markers. The addition of markers allowed the merging of four groups into two larger ones. For the other groups, the sequential mapping procedure led to an increase of marker density and length. Twenty-two markers (14 AFLPs, two SSRs, and six CAPSs) were discarded from the current mapping dataset due to inconsistent localization or substantial increases of map length.

## Map features

The final *A. subrufescens* linkage map includes 202 markers (76 AFLPs, 37 SSRs, 88 CAPS, and the *MAT* locus; segregating data are available in Table S1) distributed over 16 LG, and covers a total length of 1701 cM, with an average distance between adjacent markers of 8.2 cM (Table 1 and Figure 1). According to their number of constituent markers and their relative length, 14 major LGs (from 1 to 14), built with more than six markers (length > 40 cM), and two minors ones (15, 16), based on only three linked markers (length < 15 cM) can be distinguished (Table 1). Most of the intervals between two adjacent markers (89.7%) are less than 20 cM (60.3% in less than 10 cM), and the largest interval is of 30 cM (between ES47 and PRS234 on LG11). Considering the major LGs, the mean crossover frequency per LG per individual varies from 0.39 (LG 14) to 2.06 (LG 1), with an average of 1.13, and was highly correlated with the number of markers ($r = 0.90, P = 8.6 \times 10^{-6}$). Of the 202 markers used for map construction, 21 (10.4%) show significant segregation deviation from the expected 1:1 ratio ($P < 0.05$) (Table S1). One-third concerns isolated markers, suggesting that the observed bias could be due to possible genotyping scoring errors. The other markers showing skewed segregation are not found randomly distributed, but tend to cluster on distal position of linkage groups (Figure 1). The genetic segments formed by successive biased markers represent less than 4.3% of the linkage map. Linkage group 11 alone exhibits a large proportion of markers with unbalanced segregation (6/8). Of the 21 mapped markers with skewed segregation, 19 have an excess of CA487-100 parental strain alleles. The only two markers showing a bias toward an excess of CA454-3 allele are PRS217 (LG10) and ES31 (LG7).

The estimated genome length $L_e$ was 2458 cM, expanding the observed map length of 44.5%. According to this estimate, the proportion of the genome within 20 cM of a marker is 79%. To reach 95% of saturation with the same mapping precision, the number of markers should be increased 2.3-fold.

## Synteny with the A. bisporus genome

Among the 125 *A. subrufescens* (As) sequence-based markers, 96 showed similarities to *A. bisporus* genes (Ab) (Table 2). The map location of these markers, and the physical position of their corresponding homologs in the genome of *A. bisporus* were compared. In most cases (98%), the markers genetically linked in *A. subrufescens* are physically related in *A. bisporus* (Table 2, Figure 2, and Figure A in File S1) and bound syntenic blocks. Two exceptions are observed for PRS267 and PRS011. These two markers, mapped on As-LG3 and As-LG5, respectively, have their homologs (Ab genes ID 1982365 and 195996) physically assigned to the Ab-chromXI instead of Ab-chromIII, and Ab-chromVIII as expected regarding the other adjacent mapped loci. The marker PRS011 derives from an As-EST sequence that shows strong homology with a gene encoding a cytochrome P450 monooxygenase. Therefore, the development of a locus-specific marker from sequence belonging to such a multigenic family could be quite hazardous. This suspicion was confirmed by the results of electronic mapping, since several hits on various scaffolds were returned. Among them, an equally likely hit (e-value < $1 \times 10^{-17}$) was found on scaffold 8 (Ab-chromVIII), consistent with the genetic location. For PRS267, a unique homolog (gene ID 212249) was found on Ab-chromIX. However, this gene is annotated as encoding for a hypothetical protein containing an abhydrolase_3 domain. In the genome of *A. bisporus* (Morin et al. 2012), six other genes with the same functional domain encode putatively proteins of the alpha/beta hydrolase superfamily, but none of them is located on Ab-chromIII. A local chromosomal rearrangement could not be excluded to explain the position of PRS267. The other As-markers without Ab-homologs are spread evenly over the As LG.

The alignment of As marker-Ab homolog pairs along LG and scaffolds reveals an extensive macrosyntenic relationship (Table 2, Figure 2, and Figure A in File S1), together with a strong collinearity. Between one (As-LG14/Ab-chromVI) and 11 (As-LG6/Ab-chromI)
<figure><img src="image_3.png" /><figcaption>**Figure 2** Graphical representation of syntenic relationship between *A. subrufescens* linkage map and *A. bisporus* genome. Linkage groups (LG) of *A. subrufescens* are depicted in colors. The chromosomes and the corresponding scaffolds (chr/scaff) of *A. bisporus* (Morin et al. 2012) are in gray, and have been reordered to minimize lines overlapping. Lines of the same color as the *A. subrufescens* LG connect genetic molecular markers with the physical position of their homologs on *A. bisporus* genome. The units of the LG are given in centimorgan for *A. subrufescens* map and 10⁴ bases for *A. bisporus* genome.</figcaption></figure>

pairs of As-marker/Ab-homologs per LG, with an average of six, allowed the analysis of synteny. Indeed, nearly 80% of the *A. subrufescens* linkage map can be related to the genome of *A. bisporus*. In most cases, synteny blocks span whole As-LG and enables the inference of homologous chromosomes between *A. subrufescens* and *A. bisporus*.

The main As LG are found syntenic to the 13 Ab chromosomes. The As-LG14 and the As-LG3 are both found syntenic with the Ab-chromIII. The As-LG14 is anchored on the distal part of the chromosome (Figure 2 and Figure A in File S1). The same pattern of synteny is also observed between As-LG5/As-LG16 and Ab-chromVIII, the corresponding Ab-homologs
<table><caption>Table 3 Results of A. subrufescens EST sequence similarity searches against A. bisporus gene models</caption><thead><tr><th>A. bisporus<br>Chromosome</th><th>Scaffold on A. bisporus<br>Genome V2.0</th><th>No. of A.<br>subrufescens Mapped<br>Sequences</th><th>No. of A. subrufescens<br>Sequences Overlapping A.<br>bisporus GM</th><th>No. of A.<br>bisporus GM<br>Overlapped</th><th>% of A.<br>bisporus<br>GM<br>Overlapped</th></tr></thead><tbody><tr><td>I</td><td>scaffold 01</td><td>1065</td><td>1038</td><td>540</td><td>43.34</td></tr><tr><td>III</td><td>scaffold 02</td><td>842</td><td>828</td><td>449</td><td>40.20</td></tr><tr><td>V</td><td>scaffold 03</td><td>495</td><td>477</td><td>280</td><td>31.96</td></tr><tr><td>II, IV</td><td>scaffold 04</td><td>638</td><td>621</td><td>340</td><td>37.74</td></tr><tr><td>II</td><td>scaffold 05</td><td>659</td><td>638</td><td>364</td><td>41.65</td></tr><tr><td>VI</td><td>scaffold 06</td><td>558</td><td>540</td><td>285</td><td>36.26</td></tr><tr><td>VII</td><td>scaffold 07</td><td>485</td><td>476</td><td>259</td><td>36.79</td></tr><tr><td>VIII</td><td>scaffold 08</td><td>258</td><td>251</td><td>145</td><td>23.81</td></tr><tr><td>X</td><td>scaffold 09</td><td>289</td><td>270</td><td>153</td><td>26.33</td></tr><tr><td>IX</td><td>scaffold 10</td><td>313</td><td>308</td><td>172</td><td>32.09</td></tr><tr><td>XIII</td><td>scaffold 11</td><td>229</td><td>228</td><td>127</td><td>30.98</td></tr><tr><td>XII</td><td>scaffold 12</td><td>191</td><td>184</td><td>106</td><td>30.11</td></tr><tr><td>XI</td><td>scaffold 13</td><td>170</td><td>162</td><td>93</td><td>27.43</td></tr><tr><td>IV</td><td>scaffold 14</td><td>220</td><td>217</td><td>121</td><td>38.91</td></tr><tr><td>XI</td><td>scaffold 15</td><td>105</td><td>104</td><td>57</td><td>27.27</td></tr><tr><td>II</td><td>scaffold 16</td><td>97</td><td>95</td><td>48</td><td>25.40</td></tr><tr><td>XII</td><td>scaffold 17</td><td>67</td><td>63</td><td>44</td><td>25.43</td></tr><tr><td>IV</td><td>scaffold 18</td><td>33</td><td>33</td><td>22</td><td>28.21</td></tr><tr><td>VII</td><td>scaffold 19</td><td>34</td><td>33</td><td>13</td><td>21.31</td></tr><tr><td>ND</td><td>scaffolds 22, 29</td><td>4</td><td>4</td><td>2</td><td>17.15</td></tr><tr><td>Total</td><td></td><td>6752</td><td>6570</td><td>3620</td><td>—</td></tr><tr><td>Mean</td><td></td><td>337.60</td><td>328.50</td><td>181.00</td><td>31.12</td></tr></tbody></table>

of As-LG16 markers being located on one end of the chromosome. For these two cases of synteny discrepancies, the LG under consideration are found linked at mapping thresholds less stringent (LOD > 3, $d_{max} < 35$ cM). Regarding the synteny between As-LG11 and As LG15 with Ab-chromIX, the Ab-homologs of the markers mapped to LG15 were found flanked on each side by Ab-homologs of As-LG11 markers (Figure 2). A most permissive mapping procedure did not allow a genetic linkage to be surmised between these LG. The collinearity is mostly respected, with the genetic order of the mapped markers being consistent with the physical order of their homologs (Figure 2 and Figure A in File S1). Indeed, 77.3% of the marker pairs' intervals were found collinear with their homologous ones. Some local inversions are observed on LG1, LG2, LG3, LG4 or LG12. The pattern observed on LG8 suggests an inversion of the genetic interval comprising between SubSSR76 and PRS075. Several breakdown of collinearity were observed on LG11.

Alignment of the A. subrufescens EST sequences on the A. bisporus genome showed that hits to homologous loci were found on all the main scaffolds (Table 3). The average distance between any two electronically mapped sequences based on their A. bisporus homologs locations was 7.2 kb. The number of GM overlapped by A. subrufescens sequences per chromosome was found to be highly correlated with the total number of GM per chromosome ($r = 0.97, P = 5.35 \times 10^{-13}$). Taken as a whole, the distribution per 50 kb of overlapped GM along the chromosomes is consistent with the GM distribution within the same interval. For illustration, the distribution of overlapped GM on chromosome I (scaffold 1) follows the distribution of the GM (Figure 3A), with the lowest density of overlapped GM observed between 2.9 Mb and 3.2 Mb, which corresponded to the lowest density in GM due to the presence of repeated elements. However, an unexpected pattern of homolog distribution was observed for some chromosomes (V, VI, VIII, IX, and XI) ($\chi^2$ test, $P < 0.01$), with hot spots (higher number of homologs than expected), and/or cold spots (lower number

than expected), of homology. These distinct regions were found distributed randomly on the linkage group, as illustrated for chromosome VI on Figure 3B. Beyond cold spots of homology related to repeats region (around 0.22 Mb and around 1.52 Mb), other genomic segments exhibit either fewer (1075–1125 kb) or more (1175–1225 kb) homologs.

## DISCUSSION

The construction of genetic linkage maps in cultivable mushrooms has been restricted mostly to economically important agaricomycete species such as A. bisporus (Foulongne-Oriol et al. 2010, 2011), Pleurotus spp. (Larraya et al. 2000; Okuda et al. 2012), and Lentinula edodes (Gong et al. 2014; Kwan and Xu 2002). In this study, we present the first genetic linkage map for the medicinal mushroom A. subrufescens using sequence tag site and AFLP markers. The availability of such a molecular tool constitutes a milestone for further genetic studies in this species. The near-complete map presented is a solid backbone of the A. subrufescens genome, and makes possible the initiation of QTL analyses. The dissection of complex inherited traits of economic importance, such as yield components, can be done (Foulongne-Oriol 2012). In the particular case of this medicinal mushroom, the identification of QTL controlling the production of bioactive compounds can contribute to unraveling their biosynthetic pathways, as well as their biological functions in the mushroom. This approach was undertaken successfully to identify the genetic basis of biotechnological added-value metabolites variations in yeast (Breunig et al. 2014), and in P. ostreatus (Santoyo et al. 2008). Further applications, such as fine mapping or map-based cloning, can also be considered. In this way, the use of nonanonymous markers would facilitate subsequent resolution of mapped genomic regions to candidate genes. The availability of A. subrufescens EST catalogs as a source of molecular markers will greatly enhance the development of targeted loci related to specific pathway or traits of interest. Our results confirm also that the recombination events occur normally in A. subrufescens, as already suggested (Thongklang et al.
<figure><img src="image_4.png" /><figcaption>**Figure 3** Distribution of the percentage of overlapped *A. bisporus* gene models (GM) (dark gray) by *A. subrufescens* sequence per 50 kb-window on chromosome I (scaffold 1) (A) and chromosome VI (scaffold 6) (B), piled at 100% of GM. The dotted line is the average percentage of overlapped *A. bisporus* GM for each chromosome.</figcaption></figure>

2014a; Rocha da Brito *et al.* 2016). Such recombination ability is of particular interest for mushroom breeding purposes since it will facilitate the creation of favorable allelic combinations in breeding schemes.

Syntenic relationships have been studied widely in plant and animal species, but until recently such comparisons have been performed only rarely in fungi. The availability of complete fungal genome sequences has made the analysis of synteny between genomes possible through comparative genomics studies (Thon *et al.* 2006; Hane *et al.* 2011; Ohm *et al.* 2012). In our case, the lack of a reference genome sequence for *A. subrufescens* did not allow such an approach. Therefore, the construction of the *A. subrufescens* linkage map affords an opportunity to explore the syntenic relationship with the species *A. bisporus* thanks to a set of 96 homologous loci. In this way, we demonstrate the extensive conservation of synteny and gene order between *A. bisporus* and *A. subrufescens*, suggesting that the genome structures of these two species have not diverged drastically. Such a comparative mapping strategy has been applied only rarely in fungi between congeneric species. For example, De Vos *et al.* (2014) demonstrated genome-wide macrosynteny, with a few chromosome rearrangements, among the *Gibberella fujikoroi* species complex. The present study is the first devoted to synteny analysis between mushroom species. For each As LG, homology with

the Ab chromosome could be inferred, but one-to-one correspondence was not fully established. The 13 main As linkage groups were found syntenic to the 13 Ab chromosomes, but the situation was not clear for the three remaining As linkage groups with an observed disrupted synteny relationship. In this case, increasing marker density from an additional mapping effort could answer the question of whether there is a linkage gap between two parts of a unique chromosome, or if there are several segregating chromosomes. The number of haploid chromosomes in *A. subrufescens* remains unknown to date, but our results suggest that this number is greater than, or equal to, 13. The two *Agaricus* sections *Arvenses* and *Bivelares* to which *A. subrufescens* and *A. bisporus*, respectively, belong, have diverged from their common ancestor approximately 30 million years ago (MYA) according to Lebel and Syme (2012). Chromosome rearrangements are likely to occur during speciation processes, and represent an important factor in fungal genome evolution (Giraud *et al.* 2008). In *A. bisporus*, the location of a unique putative centromere per chromosome (Foulongne-Oriol *et al.* 2010), and the absence of interstitial trace of remnant of telomere (Foulongne-Oriol *et al.* 2013), suggest that the 13 chromosomes of the species did not resulted from fusion. Thus, if the ancestral chromosome number is 13, a possible fission of chromosomes could
explain the macrosyntenic pattern observed between *A. subrufescens* and *A. bisporus*. Cytological chromosome counts and electrophoretic karyotyping would ascertain the haploid number of chromosomes in *A. subrufescens*. In the light of our results, the overall gene collinearity between the two species was evidenced, but local structural changes could not be completely excluded. Possible rearrangement has been already suggested in a previous mapping study based on another progeny analysis to explain the loss of synteny for the rDNA (ITS) locus chromosome assignment between the two species (Rocha de Brito *et al.* 2016).

To go further, the high level of synteny between Ab-chromosome I and As-LG6 deserves particular attention. The *A. bisporus* chromosome I carries the *MAT* locus, located in the pericentromeric region and governing sexual compatibility (Xu *et al.* 1993). Thongklang *et al.* (2014a) showed that *A. subrufescens*, like *A. bisporus*, has a unifactorial system of sexual incompatibility with a *MAT* locus. In the present study, we localized the As-*MAT* locus on As-LG6, homologous to Ab-chromosome I. This result suggests a possible functional conservation of the locus. A deeper analysis of the microsynteny at the sequence level could confirm such an observation. The tight genetic linkage between the As-*MAT* locus and the marker PRS091, which is derived from the putative *A. subrufescens* mip gene (Thongklang *et al.* 2014a) known to flank the *MAT* locus (James *et al.* 2004), represents the first evidence in this direction.

Beyond evolutive considerations, consequences of the observed conserved macrosynteny between these two agronomic mushroom species can be discussed in the light of applied issues. An analogy between *A. bisporus* and *A. subrufescens* can be drawn. The cultivation process of *A. subrufescens* has been modeled mainly on the methods used to grow *A. bisporus*. Both are secondary decomposers, and can be cultivated on similar substrates, but *A. subrusfescens* requires higher temperature conditions for fruiting. Others agronomic parallels between the two cultivated species can be pointed out: similar yield components, common biotic disorders, comparable postharvest issues (Largeteau *et al.* 2011; Llarena-Hernandez *et al.* 2013, 2014). The high level of synteny demonstrated between the two species renders the possible transfer of knowledge gained on *A. bisporus* to *A. subrufescens* conceivable. We may therefore expect that QTL controlling traits such as yield would be found conserved between the two species. Such comparative QTL mapping studies have been done successfully in various plants (Chagné *et al.* 2003; Li *et al.* 2013), and our results provide the first evidence that this approach could also be applied to fungal species. Since we have demonstrated that *A. subrufescens* EST with known *A. bisporus* homologs are well distributed on the *A. bisporus* genome, these sequences provides a sound basis from which to perform further synteny-based genomic studies. Besides applications in genetics and breeding, comparative QTL mapping could be seen as a tool to predict regions of homology, and could provide information on functional synteny.

To conclude, we have constructed the first genetic linkage map of *A. subrufescens*, providing a framework for the future genetic studies of this medicinal species. This map, based on sequence-tagged site markers, will be very useful for prospective *A. subrufescens* genome sequencing projects. We have proved, through a comparative mapping approach with the *A. bisporus* genome, a high degree of synteny conservation between the two species. Since *Agaricus* is potentially the most cultivable mushroom genus, our results are also promising for other species such as *A. bitorquis*, *A. arvensis*, or *A. flocculosipes* (Thawthong *et al.* 2014; Thongklang *et al.* 2014b). In addition, we have also demonstrated that the *A. subrufescens* EST sequences provide a potential source of syntenic markers that could be custom-made to target specific genomic regions. Beyond macrosynteny, we may imagine a potential transferability of the genetic information between the two species. We have found the first

evidence of possible functional conservation between the two species with the location of the *MAT* locus on homolog chromosomes. The present work provides landmarks to guide further exploration of the evolutionary story of these two species, and their process of speciation.

## ACKNOWLEDGMENTS

The authors acknowledge the technical assistance provided by Ombeline Hueber. Manuela Brito gratefully acknowledges the Brazilian agencies "Coordenação de Aperfeiçoamento de Pessoal de Nível Superior" (CAPES) and "Programa de Doutorado Sanduiche no Exterior" (PDSE-CAPES) for financial support. This work was supported by a research project funded by a bilateral cooperation between Mexico (project 115790 CONACYT) and France (ANR-09-BLAN-0391-01).

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Communicating editor: B. J. Andrews