Genetic Linkage and Physical Mapping for an Oyster Mushroom (Pleurotus cornucopiae) and Quantitative Trait Locus Analysis for Cap Color

ABSTRACT

Oyster mushrooms are grown commercially worldwide, especially in many developing countries, for their easy cultivation and high biological efficiency. Pleurotus cornucopiae is one of the main oyster mushroom species because of its gastronomic value and nutraceutical properties. Cap color is an important trait, since consumers prefer dark mushrooms, which are now represented by only a small portion of the commercial varieties. Breeding efforts are required to improve quality-related traits to satisfy various demands of consumers. Here, we present a saturated genetic linkage map of P. cornucopiae constructed by using a segregating population of 122 monokaryons and 3,449 single nucleotide polymorphism (SNP) markers generated by the 2b-RAD approach. The map contains 11 linkage groups covering 961.6 centimorgans (cM), with an average marker spacing of 0.27 cM. The genome of P. cornucopiae was de novo sequenced, resulting in 425 scaffolds (>1,000 bp) with a total genome size of 35.1 Mb. The scaffolds were assembled to the pseudochromosome level with the assistance of the genetic linkage map. A total of 97% SNP markers (3,357) were physically localized on 140 scaffolds that were assigned to 11 pseudochromosomes, with a total of 32.5 Mb, representing 92.5% of the whole genome. Six quantitative trait loci (QTL) controlling cap color of P. cornucopiae were detected, accounting for a total phenotypic variation of 65.6%, with the highest value for the QTL on pseudochromosome 5 (18%). The results of our study provide a solid base for marker-assisted breeding for agronomic traits and especially for studies on biological mechanisms controlling cap color in oyster mushrooms.

IMPORTANCE Oyster mushrooms are produced and consumed all over the world. Pleurotus cornucopiae is one of the main oyster mushroom species. Dark-cap oyster mushrooms are becoming more and more popular with consumers, but dark varieties are rare on the market. Prerequisites for efficient breeding programs are the availability of high-quality whole genomes and genetic linkage maps. Genetic studies to fulfill some of these prerequisites have hardly been done for P. cornucopiae. In this study, we de novo sequenced the genome and constructed a saturated genetic linkage map for P. cornucopiae. The genetic linkage map was effectively used to assist the genome assembly and identify QTL that genetically control the trait cap color. As well, the genome characteristics of P. cornucopiae were compared to the closely related species Pleurotus ostreatus. The results provided a basis for understanding the genetic background and marker-assisted breeding of this economically important mushroom species.

INTRODUCTION

Pleurotus mushrooms, also known as oyster mushrooms, are grown commercially worldwide and are important protein sources for many developing countries, because of their easy cultivation and their high biological efficiency (1, 2). Interest in oyster mushrooms has increased considerably because of their gastronomic value and their nutraceutical properties. The production of oyster mushrooms ranks second in the global mushroom market. Pleurotus cornucopiae and Pleurotus ostreatus are the two main species of oyster mushrooms produced and consumed (3). Although they resemble each other morphologically, P. cornucopiae and P. ostreatus are classified as two distinct species (4). Like other Pleurotus species, P. cornucopiae is a heterothallic basidiomycete.

A shortage of superior cultivars is still a problem for the oyster mushrooms industry. Prerequisites for efficient breeding programs are the availability of high-quality, well-annotated whole genomes and genetic linkage maps, in addition to precise methods to assess agronomic traits. Genetic studies to fulfill some of these prerequisites have hardly been done for P. cornucopiae. Recently, a number of medicinal metabolites were isolated from the fruiting bodies of P. cornucopiae, which increased the attention being paid to this mushroom species among the research community and growers (5, 6). Although there are a number of papers on genetics and breeding of this species (7–9), fundamental knowledge on issues such as genome structure and the genetic basis for agricultural characteristics is still lacking. High-resolution genetic maps are essential for dissecting the genetic control of complex traits and facilitating marker-assisted breeding. The maps are also valuable tools for comparative genomic studies and chromosome-level genome assembly. Genetic linkage maps were generated for several oyster mushroom species, e.g., P. ostreatus (10), Pleurotus pulmonarius (11), Pleurotus eryngii (12), and Pleurotus tuoliensis (13). Genome sequences were available for most of these species. So far, there are no reports about the genetic linkage map and genome sequences of P. cornucopiae. As a closely related species, P. ostreatus has a total of 11 chromosomes and a genome size of 35 Mb (JGI: https://mycocosm.jgi.doe.gov/PleosPC15_2/PleosPC15_2.home.html) (10, 14–16).

The availability of a high-quality whole-genome sequence (WGS) allows the development of genome-wide markers and construction of a saturated genetic linkage map covering all chromosomes. This facilitates quantitative trait locus (QTL) mapping and thus marker-assisted breeding (17). With the development of the next-generation sequencing techniques, single-nucleotide polymorphisms (SNPs) have been used widely in genetic linkage studies. Genotyping by sequencing (GBS) is a high-throughput method for simultaneously discovering and genotyping thousands of SNP markers over the whole genome. It is rapid and cost-effective compared to other SNP detection and genotyping methods (18–20). Restriction site-associated DNA tag sequencing (RADseq) further cuts the cost by reducing the complexity of the genome and has been used widely in marker detection and genetic mapping (13, 19).

Cap color is one of the most important commercial traits of mushrooms. Oyster mushrooms on the fresh market present a wide range of cap colors, varying from gray, brown, light brown, and cream to pure white. Dark-cap oyster mushrooms are more and more popular, based on consumers’ preferences, but these dark varieties are rare on the market. Breeding superior cultivars of oyster mushrooms with darker caps is, therefore, needed. The genetic control of the trait cap color must be revealed in order to perform marker-assisted breeding in P. cornucopiae. The cap color of the button mushroom Agaricus bisporus (brown versus white) is determined by a major locus (PPC1) on chromosome 8 and modified by several smaller QTL. The locus PPC1 explained more than 80% phenotypic variation (17, 21, 22). Cap color of mushrooms is determined by a wide range of components that can also be involved in discoloration after mechanical damage (23). The discoloration of bruised or sliced caps was considered also to be due to the biosynthesis of melanin (24). By using white-brown and white-white cap color segregating populations, Gao et al. obtained indications that the pigmentation of cap color and postharvest discoloration of A. bisporus are caused by different mechanisms (21). Distinct from A. bisporus, the cap color of oyster mushrooms is probably under the genetic control of multiple loci with minor effects rather than a major locus. The genetic determinants for cap color of P. ostreatus were previously reported on chromosome IV and X with explained variance of 9.93% and 7.26%, respectively (14). Next, Sivolapova et al. reported 6 QTL, but only the one on LG1 was significant at a P value of 0.01 (25). A previous inheritance study showed that the cap color of P. cornucopiae was a quantitative trait and controlled by two pairs of major alleles (9). Nevertheless, the genetic controls of cap color in oyster mushrooms are still unclear.

P. cornucopiae strain CCMSSC00406 is one of the most popular oyster mushroom cultivars, having dark gray caps. In this study, we de novo sequenced the genome and constructed a saturated genetic linkage map of this cultivar. The genome characteristics of P. cornucopiae were compared to those of the closely related species P. ostreatus. The genetic linkage map was effectively used to assist the genome assembly and identify QTL that genetically control the trait cap color. The genetic linkage map can be used to map other economically important traits of oyster mushrooms, since QTL for agronomic traits of oyster mushrooms are still scarce (12). The newly found markers, which are closely linked to the loci controlling cap color, will facilitate marker-assisted breeding of oyster mushrooms.

RESULTS

Genotyping by the 2b-RAD approach.

The 2b-RAD libraries of the parental lines and the progeny were constructed using adaptors with 5′-NNC-3′ overhangs to target a subset of BsaXI fragments in the genome. Sequencing the libraries produced an average of 2.2 million high-quality reads per individual. The average sequencing depth was 70×, which controlled the genome coverage and the error rate of sequencing well. The high-quality reads were aligned to the reference genome of 406P1 (NCBI accession number WQMT00000000). A total of 4,443 SNP markers were identified. Repetitive and doubtful markers (showing errors and/or more than 20% missing values) were omitted in subsequent analyses.

High-resolution genetic linkage mapping.

A total of 3,449 SNP markers were assigned to 11 linkage groups (LGs) (Fig. 1). The map covers a total of 961.6 centimorgans (cM), with an average map distance between adjacent markers of 0.28 cM (Table 1).

FIG 1.

Genetic linkage map of P. cornucopiae. Black bars indicate SNP markers; the x axis represents the number of linkage groups, and the y axis indicates the marker locations (in centimorgans) on genetic linkage maps.

TABLE 1.

Features of the genetic linkage map of oyster mushroom P. cornucopiae

LG	Size of assembled genome (Mb)	No. of scaffolds	Homologous chromosome of PC15	Size of PC15 (Mb)	Map length (cM)	Size ratio (kb/cM)	No. of markers	No. of cosegregating markers	Largest marker interval (cM)	No. of distorted markers	No. of crossovers/chromosome						Mean crossover frequency
											aa	bb	1	2	3	≥4
LG1	2.7	8	7	3.3	65.4	40.5	314	273	8.3	0	25	26	64	7	0	0	0.64
LG2	6.0	26	1 + partial 11c	4.8	175.8	33.9	630	519	9.8	76	10	6	54	37	13	2	1.43
LG3	2.6	9	8	2.7	113.1	22.8	312	248	7.9	31	26	28	54	11	1	1	0.69
LG4	2.6	13	6	2.7	78.2	33.0	275	222	4.9	2	19	20	74	9	0	0	0.75
LG5	4.4	14	3	4.6	107.9	40.8	561	486	7.8	5	17	25	59	16	4	1	0.88
LG6	2.0	12	2	2.4	63.1	31.5	170	145	17.5	137	4	54	63	0	0	0	0.52
LG7	3.6	14	5	3.6	73.6	49.2	373	320	4.1	5	22	27	62	10	1	0	0.7
LG8	1.6	16	10	1.6	62.0	26.1	153	121	11.7	150	7	51	61	2	0	1	0.57
LG9	3.4	8	4	3.6	89.3	38.5	393	335	9.9	149	25	12	68	16	0	1	0.85
LG10	1.5	11	11 - partial 11c	2.9	77.7	18.7	110	81	10.8	28	21	44	53	2	0	1	0.5
LG11	1.6	9	9	1.9	55.6	29.3	158	126	17.0	1	31	30	52	8	0	1	0.59
Total	31.9	140	11	34.1	961.6		3,449	2,876		584
Avg	2.9	12.7		3.1	87.4	33.2	313.5	261.5	10.0	53.1							0.74

^aNumber of individuals that inherited the entire LG from 406P1.

^bNumber of individuals that inherited the entire LG from 406P2.

^cScaffold 11 of PC15 splits onto two pseudochromosomes of 406P1 (LG2 and LG10).

The cosegregating markers represented 83% of all the mapped markers. LG2 has the largest number of markers (630), and LG10 the smallest (110). The LGs range in size from 55.6 cM (LG11) to 175.8 cM (LG2). There is a significant correlation between marker numbers and map size per linkage group (R² = 0.89, P = 0.01). The largest marker interval per group ranges from 4.1 cM (LG7) to 17.5 cM (LG6). A total of 584 markers (16.9%) showed distorted segregation (P = 0.05). These markers were mostly assigned to LG2, LG3, LG6, LG8, LG9, and LG10. For LG8 the distortion of markers is 98% (see Table S1 in the supplemental material).

The maximum percentages of the parental genotypes among individuals of the segregating population are 78.6% for 406P1 and 75.9% for 406P2. Most of the individuals inherited half of the genetic information from each monokaryotic parental line. The mean crossover frequency (the number of crossovers per LG per individual) is 0.74. The number of crossovers per individual ranges from 3 to 14, with an average of 10 showing a normal frequency distribution (Fig. S1).

De novo genome sequencing of P. cornucopiae.

The genome was consisted of 425 scaffolds (>1,000 bp), representing a total size of 35.1 Mb. The ratio of the genome size to map length is 36.5 kb/cM. The longest scaffold is 1.49 Mb (N₅₀, 0.08 Mb; N₉₀, 0.01 Mb). The G+C content of the assembled sequence is 50.0%, and the size of the total Ns in the genome is 0.63 Mb. A total of 11,615 gene models were predicted, of which 96.7% (11,271) were confirmed by aligning to NCBI nonredundant database (NR) using the E value threshold 1e⁻⁵. The predicted gene models have an average length of 508 amino acids (aa). The coding sequences fully account for 50.4% (17.7 Mb) of the whole genome of P. cornucopiae.

Genome assembly using the genetic linkage map.

In order to assemble the scaffolds to the chromosome level, 3,357 mapped markers (97%) were positioned on the genome of 406P1 through sequence blasting. According to the genetic linkage map, 140 scaffolds were assigned to 11 pseudochromosomes (CHR) with a total of 32.5 Mb, representing 92.5% of the whole genome (Fig. 2). Most scaffolds were mapped to single linkage groups, except scaffolds 8 and 31. Scaffold 8 was divided between LG2 and LG8; scaffold 31 was divided between LG4 and LG6. This may suggest misassemblies of these scaffolds. The SNP markers were found evenly distributed on the pseudochromosomes (Fig. S2). A total of 58.4% (2014) of the SNP markers were located within coding regions (Table S2). CHR 2 was the longest, consisting of 26 scaffolds with a total length of 6.0 Mb; CHR 10 was the shortest, consisting of 11 scaffolds with a total length of 1.5 Mb (Table 1).

FIG 2.

Graphical representation of syntenic relationship between the genetic linkage map and the physical map of P. cornucopiae. Linkage groups (LGs) and the corresponding scaffolds are depicted in colors in the circle. Lines of the same colors connect the markers on LGs and physical positions on scaffolds. The diagram was plotted using Circos (39).

Comparative genomic analysis between of P. cornucopiae and P. ostreatus.

We compared the genetic linkage map to the genome of P. ostreatus. In total, 2,863 (83%) and 2,756 markers (80%) were anchored on the genome sequence of PC9 and PC15, respectively (Table S2). The percentage difference might indicate the dissimilarity between the genome sequences of PC9 and PC15. According to the marker positions on the chromosomes of PC15, we observed that scaffold 11 of PC15 splits onto two pseudochromosomes of 406P1 (CHR 2 and 10), and this might indicate that a large translocation occurred between the two chromosomes (Fig. S3). As a result, the largest CHR of 406P1 (6.0 Mb) was larger than its homologous chromosome of PC15 (4.8 Mb) (Table 1). In addition, colinearity analysis indicated the existence of inversions and translocations among the genome sequences of 406P1, PC9, and PC15. More inversions and translocations were observed between 406P1 and PC9 than between 406P1 and PC15 (Fig. 3).

FIG 3.

Colinear comparison of the genomes of P. cornucopiae (406P1) and P. ostreatus (PC9 and PC15) using MUMmer plots. The red dots represent colinear sequences, and blue dots represent sequence inversion. Scattered dots represent repetitive sequences aligned on different genomic positions. (A) Comparison of the genomes of 406P1 and PC9 (32); (B) comparison of the genomes of 406P1 and PC15 (16). The scaffolds are represented by gray dotted lines and named in order from top to bottom (y axis) and left to right (x axis). The genome of 406P1 (GenBank accession number WQMT01000000) were used for the comparison, and the short scaffolds (<30 kb) were removed in order to make the symbols readable.

Statistical analysis of trait performance.

The heterokaryotic population displayed a large continuous variation in cap color, with a minimum whiteness index (WI) of 17.2 and a maximum of 55.9, which suggested quantitative and polygenic control (Fig. 4). The parental line CCMSSC00406 and the tester line CCMSSC00358 showed dark caps (34.4) and pure white caps (60.58), respectively. Transgressive segregation was detected for both sides by using multiple comparison (Student-Newman-Keuls [SNK] test, P < 0.05), i.e., significantly darker (23 strains) and lighter (25 strains) mushrooms compared to the hybrid strain of 406P1 × 358P1. The cap color of the population was tested as a normal distribution by using the Kolmogorov-Smirnov test (P < 0.05).

FIG 4.

Frequency distribution of the value of cap color. The horizontal axis indicates the value range of cap color, and the vertical axis indicates the frequency of individuals.

Analysis of variance was conducted for the trait cap color, and results showed that genotype (G) was a significant factor to generate the differences in cap color among individuals of the population (α = 0.001) (Table S3). Measurements (M) and G × M effects did not significantly influence the phenotype of cap color (α = 0.05). Broad-sense heritability (H²) was calculated as 0.99, which indicated that the cap color of P. cornucopiae is a highly heritable trait.

QTL for cap color of P. cornucopiae.

An average threshold of a −log₁₀(P) value of 3.65 (26, 27) was used to identify QTL significant for cap color. Results of composite interval mapping (CIM) were presented (Fig. 5). Six QTL were detected for cap color, with explained variances (EV) varying from 5.8% (LG9) to 18.0% (LG5). Three major QTL explained the high phenotypic variance, i.e., 10.8% (LG4), 16.85% (LG7), and 18.0% (LG5). The total variation in cap color explained by all the QTL accounted for 65.6%. The high-value alleles of the QTL on LG1, −4, −5, and −7 came from 406P1, and that of QTL on LG3 and LG9 were from 406P2 (Table 2). This confirmed the heterozygosity of the parental line 406 for the trait of cap color.

FIG 5.

QTL of cap color located on the genetic linkage groups (pseudochromosomes) of P. cornucopiae. The red line indicates the threshold of the QTL analysis [–log₁₀(P) = 3.65]. The curves above the red lines are significant QTL, and the peaks of the curves indicate their significance. The two-color scales of the bars indicate the two parents contributing high-value alleles of QTL. The blue color (varying from dark to light) represents alleles from 406P1: the darker the color, the higher the effect of the QTL. Similarly, brown (varying from light to dark) represents the high-value alleles from 406P2.

TABLE 2.

QTL of cap color detected in the oyster mushroom P. cornucopiae

QTL	LG	Position	−log10(P)	EV (%)	Additive effects	High-value allele	Marker interval	Position interval (cM)	Range of −log10(P)
c3554	1	29.2	4.4	6.3	1.9	406_P1	c1657–c87	14.3–29.2	3.7–4.8
c3577	3	33.9	5.5	7.9	2.1	406_P2	c296–c959	22.4–52.4	4.2–6.1
c215	4	76.6	7.1	10.8	2.5	406_P1	c1253–c722	52.7–78.2	4.3–8.0
c2972	5	68.4	10.8	18.0	3.2	406_P1	c1545–c3050	44.8–93.1	4.9–13.2
c558	7	18.4	10.2	16.8	3.1	406_P1	c2755–c4296	0–37.3	4.3–12.3
c1229	9	42.1	4.1	5.8	1.8	406_P2	c1384–c2226	39.6–52.3	3.7–4.4

The three major QTL were physically located on CHR 4 (bp 1813213 to 2581190), CHR 5 (bp 737001 to 3028103), and CHR 7 (bp 276338 to 1557471), with region sizes of 0.77 Mb, 2.29 Mb, and 1.28 Mb, respectively. Totals of 257, 886, and 434 genes were identified in the QTL regions of LG4, −5, and −7, respectively. The GO analysis showed that the genes in three major QTL regions were mainly involved in the pathways of cellular and metabolic process (biological process category), cell and cell part (cellular component category), and catalytic activity and binding (molecular function category). In addition, the three major QTL regions incorporated a total of 7 genes involved in the melanin synthesis pathway (Table S4) (24). These genes might be candidate genes controlling the cap color of oyster mushrooms.

DISCUSSION

A whole-genome sequence was generated for P. cornucopiae using a combination of deep sequencing (70×) and a dense genetic linkage map. This provides a sound base for future analysis of the genetic base of agronomic traits in this species. Based on this map, six QTL were detected for the trait cap color, explaining a total of 65% of the phenotypic variation, which contributes considerably to knowledge needed to improve this economically important trait.

The map and genome characteristics of P. cornucopiae were mainly compared to those of the closely related species P. ostreatus (Table S5). The size ratio of the linkage map to the genome of P. cornucopiae (36.5 kb/cM) is similar to that of P. ostreatus (35.1 kb/cM) (10). Although the 2b-RAD method obviously generates an excess of SNP markers, it explains the high number of cosegregating markers and thus the low marker interval. The similarity in map length and crossover frequency observed in P. cornucopiae and P. ostreatus indicates that mapping resolution mainly depends on crossover frequency rather than marker density. Crossovers are generally evenly distributed over most of the pseudochromosomes of P. cornucopiae (Fig. S4). Higher crossover frequency was observed at the ends of CHR 5 and 8 (Fig. S5). The percentage of skewed segregation of markers in P. cornucopiae is a bit higher than that found in P. ostreatus (14%). The skewed segregation was mainly found on three linkage groups (LG6, LG8, and LG9), and directional distortion was observed toward one of the two parental lines (406P2) for markers on LG6 and LG8. Skewed segregation in homokaryotic offspring of mushrooms is not an uncommon phenomenon (10, 13, 28, 29). The disruption of allele combinations on the three chromosomes might be sublethal or lead to slow germination of basidiospores, thereby causing a bias in isolation of single-spore offspring (13).

The colinear analyses were conducted among the genomes of five Pleurotus species, i.e., P. cornucopiae, P. ostreatus, P. pulmonarius, P. eryngii, and P. tuoliensis. (Fig. S6). Inversions and translocations exist among genomes of different species. The genomes of P. cornucopiae and P. ostreatus showed higher degree of colinearity than those of P. cornucopiae and the other three Pleurotus species. This demonstrated that P. cornucopiae and P. ostreatus are genetically closely related. Consistent results were observed in the phylogenetic tree analysis, which revealed close evolutionary affinity between P. cornucopiae and P. ostreatus, with divergence approximately 21.64 million years ago (Fig. S7). The genomes of P. eryngii and P. tuoliensis showed a high degree of colinearity, as detected previously (13). During the evolution of species, chromosome rearrangement events occur constantly (30). A major chromosomal translocation occurred between homologous chromosomes, with the result that CHR 2 of P. cornucopiae was homologous to CHR 1 and part of CHR 11 of P. ostreatus (Fig. S3). CHR 11 of P. ostreatus was homologous to LG 10 and part of LG 2 of P. cornucopiae. In addition, a high degree of synteny was observed between the gene models of P. cornucopiae and P. ostreatus, which is highly consistent with that of the genomes (Fig. S8). The number of predicted gene models (11,615 proteins) in P. cornucopiae was less than that reported in P. ostreatus PC9 (11,875 proteins) (31) and PC15 (12,330 proteins) (16). Nevertheless, a total of 1,984 (17.1%) and 2,242 (19.3%) unique gene models were detected for 406P1 compared to PC9 and PC15, respectively (Table S6). A transcriptome analysis might be helpful to confirm and increase the number of the gene models.

The mapped markers are distributed homogeneously on the anchored scaffolds and cover the majority of the genome, although the marker order on the genetic linkage map was not always coherent with their physical position on scaffolds (Table S2 and Fig. S2). The lack of coherence was often seen in mapping regions with very low or no recombination (13, 21). The even spreading of markers on the assigned chromosomes indicated that the genetic linkage map generated here is generally saturated and encompasses most of the genome of P. cornucopiae. The ratio of SNP markers located in coding regions is comparable with that of the coding sequences accounting for the whole genome. The SNP markers in the coding regions may facilitate the prediction of candidate genes and functional analysis in the QTL studies. The sequence divergence of the genomes between the parental strains 406P1 and 406P2 might explain the fact that 3% of markers could not be positioned on the genome of 406P1. Thus, even between haplotypes within the same species, not all markers will map. Additionally, it might indicate that transposable elements or other nonessential sequences play a role in the sequence divergence between haplotypes. Approximately 80% of the P. cornucopiae markers could be mapped on the genome of P. ostreatus, which might partly reflect the genetic dissimilarity between the two species.

The monokaryotic population was mated with a compatible tester strain to generate dikaryons for obtaining the trait cap color. Although darker than the other present-day commercial lines, strain 406 has gray caps, not dark enough for the current fresh market. Previous research has shown that CCMSSC00406 is heterozygous for the dark alleles controlling cap color, and dark is dominant over white (9). The purpose of this study was, therefore, to map alleles linked to the darkness of cap color and use this information for future marker-assisted selection to generate varieties homozygous for dark alleles. The tester line represents one of the constituent nucleus isolates via protoclones of a pure white strain. Since the dark color is dominant, it allows the expression of the trait. Due to limited space, small bottles were used to cultivate the population, and only one fruiting body was allowed to grow in each bottle. Four replications of each individual were used to obtain reliable phenotypes for QTL analyses. Since there is sometimes variation of color on a single cap, three positions on each cap were selected for measurement, to minimize the influence of variation on trait screening.

QTL analyses for oyster mushrooms have been conducted so far mainly in P. ostreatus on traits related to production, quality, and mycelium growth rate (14). The power of QTL mapping can be strengthened by using high-resolution genetic mapping and a composite interval mapping procedure. The 0.28-cM marker interval contributed to better precision of the QTL position in this study. A total of 6 QTL were detected for cap color, with a total explained variance of 65.6%, which was higher than that reported in other oyster mushroom species, e.g., total explained variance of 17.31% in P. ostreatus (14) and 44.87% in P. eryngii (12). The cap color of mushroom species can also be influenced by environmental factors. For instance, the caps of oyster mushrooms are darker when mushrooms are cultivated at lower temperature and with higher light intensity (32). An appropriate experimental design might thus contribute to understanding the interaction between genotype and phenotype. The continuous distribution indicates a polygenic control of cap color in P. cornucopiae, as was also reported for other Pleurotus species. A homologous QTL was detected for P. cornucopiae and P. ostreatus, which was located on pseudochromosome 9 of P. cornucopiae and chromosome IV of P. ostreatus (14). This QTL might indicate homologous genes controlling cap color of the two species of oyster mushrooms. Nevertheless, it was a minor QTL for the cap color of P. cornucopiae. It might be worthy of further exploration for the 7 genes involved in the melanin synthesis pathway incorporated on CHR 5 and 7 (homologous chromosomes III and V for P. ostreatus) with higher explained variance (18.0% and 16.8%) (Fig. S8) (24). The small population size of previous studies might result in low mapping accuracy in terms of low confidence in the estimation of recombination frequency and low power to detect linkage. The high-value alleles did not originate from a single parent, indicating the heterozygosity of the parental lines for the beneficial (dark cap) alleles. With the assistance of closely linked markers, individuals with stacked beneficial alleles for the trait cap color can be selected as breeding stocks. Further studies are still required to fine map the major QTL and thus find candidate genes involved in the control of cap color of oyster mushrooms.

In conclusion, our study presents a de novo-sequenced genome and a saturated genetic linkage map of an oyster mushroom, P. cornucopiae. The map facilitated the assembly of the de novo-sequenced and annotated genome, which provides a solid foundation for further genetic analysis of agronomic traits of this essential oyster mushroom species. The QTL detected in this study are valuable tools for marker-assisted breeding for oyster mushrooms of desirable colors.

MATERIALS AND METHODS

P. cornucopiae strain and segregating population.

All strains used in this study were obtained from China Center for Mushroom Spawn Standards and Control (CCMSSC). The segregating population consists of 122 monokaryons derived from a traditional hybrid strain, CCMSSC00406 (406), by single-spore isolation as described previously (13). The monokaryotic parental lines (406P1 and 406P2) of 406 were recovered by protoplasting (33). The mycelium was confirmed as a monokaryon by the absence of clamp connections. In order to measure the cap color of the fruiting body, a heterokaryotic population was generated by crossing the 122 monokaryons with a monokaryotic tester line, 358P1, which was obtained by protoplasting a pure white dikaryotic strain CCMSSC00358 (33). Successful pairing was confirmed by the presence of clamp connections on the hyphae of the hybrids.

Genotyping the population by the 2b-RAD approach.

Genomic DNA of the individuals of the population was extracted from freeze-dried mycelium with the Wizard Magnetic 96 DNA plant system (Promega) according to the manufacturer’s instructions, and the concentration was adjusted to 25 ng/μl. 2b-RAD libraries were prepared and analyzed at Oebiotech (Shanghai) for the two parents and 122 progeny by following the protocol developed by Wang et al. (34). Briefly, the libraries were constructed using adaptors with NNC overhangs to target the subset of BsaXI fragments. All the libraries were pooled for paired-end sequencing on the HiSeq Xten platform. The trimmed, high-quality reads of the parents and progeny were mapped to the reference genome of 406P1 by SOAP2 (35). Sequence tags mapping to the repetitive region and reads with less than 3× sequence depth were discarded. According to the results of sequence alignments, SNP loci of the populations were genotyped.

Linkage analysis and map construction.

Expected segregation (1:1) was tested for each SNP marker by a χ² test. Linkage mapping was performed using the Highmap program (36). Markers showing more than 20% missing data were discarded. The recombination frequencies and the modified logarithm of odds (MLOD) between markers were calculated by two-point analysis. Markers were assigned to linkage groups according to the values of MLOD (>3.0) and ordered within groups with the maximum-likelihood (ML) algorithm. Kosambi’s mapping function was used to determine the map distances. The crossover frequency of each chromosome and each individual was analyzed using GGT 2.0 (37).

De novo genome sequence and scaffold reassembly.

The whole genome of 406P1 was de novo sequenced on the HiSeq4000 platform with a read length of 150 bp (BGI, Wuhan). The filtered reads were assembled by SOAPdenovo to generate scaffolds. Gene prediction was performed by using BRAKER1 (version 1.9). The protein-coding genes were confirmed by using BLAST+ (version 2.2.31) against the NR database with an E value cutoff of 1e⁻⁵. The core sequence tags of SNP marker were subjected to a BLAST search against the genome by using BLAST+ (version 2.2.31) with an E value cutoff of 1e⁻³ and a word size of 20 to anchor markers on scaffolds of P. cornucopiae and P. ostreatus. Scaffolds were assembled onto chromosomes according to genetic positions of the anchored markers. Graphical representations of the assembled genome and genetic linkage map were plotted with Circos 0.69 (38).

Comparative genomic analysis between of P. cornucopiae and P. ostreatus.

The genome sequence and the genetic linkage map of P. cornucopiae were compared to those of P. ostreatus (10), since these species are closely related. The assembled genomes of the P. ostreatus strains PC9 (31) and PC15 (16) were used for the comparative analyses in this study. The sequence tags of the SNP markers of P. cornucopiae were subjected to a BLAST search against the genomes of PC9 and PC15 by using BLAST+ (version 2.2.31) with an E value cutoff of 1e⁻³ and a word size of 20 to anchor markers on the genomes of P. ostreatus. Homology and synteny between the genetic linkage groups (LGs) of P. cornucopiae and chromosomes of P. ostreatus (PC15) were presented by Circos (39). The synteny analysis of gene models between P. cornucopiae and P. ostreatus was analyzed by MCScanx (40). Colinearity among genomes of the five Pleurotus species, i.e., P. cornucopiae, P. ostreatus (16, 31), P. pulmonarius (NCBI accession number ASM1298053v1), P. eryngii (41), and P. tuoliensis (13), were analyzed by using MUMmer version 3.1 (42) with the setting “mincluster = 2000.”

Cultivation tests and phenotypic evaluation.

Mushrooms were grown in 280-ml (cylindrical; depth = 70 mm; height = 90 mm) polypropylene bottles. Each bottle was filled with 140 g premixed substrate using the ingredients (dry weight) cotton seed hull (94%), wheat bran (5%), and gypsum (1%) and moistened to 65% using tap water. The substrate bottles were sterilized at 121°C for 2 h. The heterokaryotic population, consisting of 122 strains, was cultured on potato dextrose agar (PDA) medium at 28°C for 7 days. Afterward, a piece of mycelium (3 cm²) was inoculated into each bottle. Four replicate bottles were inoculated for an individual strain. Mycelia achieved full colonization in the bottle within 2 weeks of incubation at 25°C in the dark. All the bottles were opened, and the surface layer of the substrate was ruffled to stimulate the mycelia and cultured at 25°C in the dark for another 3 days. The fully colonized bottles were subsequently transferred to the fruiting room, with a controlled climate at 16 to 18°C and 80 to 90% relative humidity and a 12-h photoperiod (300 to 350 lx). After the formation of primordia, caps were removed from the bottles. The concentration of CO₂ was controlled below 1,000 ppm by ventilation.

All mushrooms were picked at the developmental stage 3 (43). Cap color was measured by using a Minolta Chroma Meter (CM-700d) with the tristimulus coordinate system CIELAB scale (Commission Internationale de L’Eclairage 1976, L*a*b color system) immediately after picking. The two types of color value are generated with the specular component included (SCI) or excluded (SCE). Positions on mushroom caps were selected to measure each color type either surrounding the cap (ST) or vertically from the outside border to the center of the cap (VT) (Fig. S9). The four measurements for each genotype were designated SCI-ST, SCI-VT, SCE-ST, and SCI-VT. Cap color was quantified as the whiteness index (WI), i.e., the value of L-3b (44). The mean WI of the four measurements was defined as the value of cap color.

Statistical analysis of phenotypic traits.

Statistical analyses were performed in GenStat (version 18) and SPSS. ANOVA was performed for cap color according to the following model: Y = μ + G + M + G × M + ε, where μ is the mean value, G is the genotypic effect, M is the measurement effect, G × M is the genotype and measurement interaction effect, and ε is the residual effect. A normal distribution was tested for the phenotypic data. Multiple comparison was performed to detect transgressed segregation of the offspring by using the Student-Newman-Keuls (SNK) test. Broad-sense heritability (H²) was calculated for data of each measurement with the model${\text{σ}}_G^2/\left[{\text{σ}}_G^2+\left({\text{σ}}_e^2/r\right)\right]$ and ${\text{σ}}_G^2/[{\text{σ}}_G^2+({\text{σ}}_{G\times M}^2/\mathit{nr})+({\text{σ}}_e^2/r)]$ for the combined data, where ${\text{σ}}_G^2$ represents the genetic variance, ${\text{σ}}_e^2$ is the error variance (mean square of residual), and${\text{σ}}_{G\times M}^2$ is the variance of genotype and measurement interaction. In this study, n was the number of measurements (n = 4) and r was the number of replicates of each measurement (r = 4).

QTL mapping for cap color.

QTL detection for cap color was performed in GenStat version 18 with the model of single-trait (single-environment) QTL using simple interval mapping (SIM) and composite interval mapping (CIM) as described previously (21). After SIM, the detected loci having a test statistic [−log₁₀(P)] larger than the threshold were selected as cofactors for CIM. CIM were conducted for several rounds until no new QTL were detected. The total explained variation of QTL of each trait was calculated by a multiple-regression analysis using the most significant markers for each putative QTL (45).

Data availability.

The assembled and annotated genome of CCMSSC00406 (406P1) was deposited at GenBank under accession number WQMT00000000. The version of the originally assembled genome of 406P1 has accession number WQMT01000000, and that assembled onto pseudochromosomes has accession number WQMT02000000.