Development of a Segregating Population with Biological Efficiency in Agaricus bisporus

Introduction

Agaricus bisporus is one of the most widely consumed mushrooms in the world [1]. In Korea, A. bisporus has been one of the most popular edible mushrooms and the total production of A. bisporus was 9,732 MT (63 billion won) in 2015 [2].

There are two varieties of A. bisporus, i.e., bisporus and burnettii. A. bisporus var. bisporus mostly generates two spores with two non-sister nuceli. Around 10~15% of basidia actually produce four spores with haploid nuclei that are compatible with the other haploid nuclei (homokaryons) [3]. A. bisporus var. burnettii typically has four spores with a single nucleus after meiosis. Among single spore isolates of this variety, approximately 90% were homokayons and 10% were heterokaryons [4, 5]. However, A. bisporus var. burnetti shows poor features under commercial conditions with respect to pinning, scaling, cap shaped mushroom distribution and yield, compared with those of A. bisporus var.bisporus [6, 7].

Yield is the most important trait for commercial cultivars in breeding. The genus Agaricus is known for its potential to degrade lignocellulosic materials [8, 9]. The bioconversion of the substrate and the biodegradation of lignocelluloses in the substrate, i.e., biological efficiency (BE) are related to yield. The BE is expressed as the yield of fresh fruiting bodies per 100 g of dry substrate according to Chang et al. [10]. Current studies define it as the percent conversion of dry substrate to dry matter in fruiting bodies [11].

A segregating population is required to perform linkage mapping. In segregating populations, parental lines should be selected based on strains that are significantly and tightly linked to the goal of the mapping study. Additionally, population size is important. The smaller the size of population, the less robust of the linkage map is. Population size has an effect on the detection of linkage, fragmented linkage groups, and locus order [12, 13].

The purpose of this study was to develop a segregating population to perform linkage mapping and quantitative trait locus mapping for traits related to BE.

Materials and Methods

Mushroom strains

The commercial strain A. bisporus A15 was obtained from the strain collection of Wageningen UR Plant Breeding. Eight wild strains of A. bisporus were obtained from the Agaricus Resource Program (ARP) (Table 1) [14].

Preparation of hybrids

Two parental strains (p1 and p2) were isolated as protoclones by established protocols [15] from 9 strains, and strains were intercrossed using a diallel crossing scheme (Fig. 1). Hybrid compatibility was confirmed based on mycelia morphology or molecule markers.

Spawn preparation and mushroom cultivation

Independent cultivation experiments were performed for each set of 153 hybrids at the mushroom farm of Unifarm in Wageningen UR under a controlled climate. Mushrooms were grown on commercial compost (CNC substrates) and spawned in 0.1 m² boxes (40 × 30 × 21 cm) filled with 8 kg of compost. Each individual was grown once in one box [7].

Biological efficiency (BE)

Total weights of all harvested fruiting bodies were measured as the total yield of mushrooms. The BE (mushroom yield per kg of substrate on a dry wt basis) was calculated according to the following formula [10].

Development of the BC₁F₁ population with CAPS markers

For one hybrid with high BE, over one thousand single spore isolates (SSIs) were obtained to develop a segregating population by microscopy. The mycelia of SSIs were incubated for 7~8 days in malt mops pepton (MMP) media. Then, 15 µL of PCR mix (10× PCR buffer; Qiagen, Germantown, MD, USA), 25 mM MgCl₂ (Qiagen), 5 mM dNTP mix (Qiagen), and 5unit of HotStarTaq (Qiagen) was added to each well of a 96 wells PCR plate on ice, and 1 mm² of mycelium from each culture were transferred to 50 µL of DNA extraction buffer (T₁₀E_0.1buffer) in each well of a 96 well plate. The 96 well plates with mycelia were placed in a microwave for 90 sec. The plates were removed from the microwave and directly placed on ice. The mycelia were mixed with the DNA extraction buffer by pipetting up and down and 10 µL of solvent was transferred to the plate containing the PCR mix with three markers followed by PCR. The cleaved amplified polymorphic sequence (CAPS) markers MatA_HD2.1 and MatA_HD1 were in the A-mating type homeodomain region of different A. bisporus strains (H97, H37 and Bisp119/9-p4). The PIN50 marker was made following previous protocols [16, 17]. Amplifications was performed using a GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA, USA) using an initial denaturation step at 95°C for 15 min, 35 cycles consisting of 30 sec at 94°C, 30 sec at the appropriate annealing temperature (Table 2), and 90 sec at 72°C and a final extension step at 72°C for 5 min. The 10 µL of restriction digest master mix (10× restriction buffer, 10 U/µL restriction enzyme) was added to each well of a 96-wells PCR plate on ice. 5 µL of PCR product was added to each well, and the plate was sealed and spun to collect the liquid at the bottom of the wells. After restriction digest, the sample was incubated at 37°C for 2 hr. After incubation, the sample was resolved on 1% (w/v) agarose gels. One hundred selected homokaryons were crossed with the other parental line (bisp15-p1) to develop the BC1F1 population.

Results and Discussion

BE of hybrids

One hundred fifty-three hybrids were obtained with eighteen protoclones from nine strains. Five strains were A. bisporus var. bisporus and bisp 141/03, bisp015 and bisp119/9 were A. bisporus var. burnettii. After intercrossing, 64 lines were used to investigate mycelial morphology and examine markers, and 60 hybrids were cultivated for mushroom production to measure BE (Fig. 1). The average BE of hybrids ranged from 0.096 in MB001 (crossed between bisp 015-p1 and bisp 034-p1) to 0.394 in MB-310 (crossing between 141/3p1 and Z8) (Fig. 2). The average BE in each homokaryon was calculated as the mean of the BE of all hybrids with the same homokaryons. The values for homokaryons ranged from 0.223 in bisp 119/9p1 to 0.304 in bisp 119/9 p4. The homokaryons were successfully crossed from 6 to 11 times. The crossing frequency of bisp 141/3p1, A15-p1, and Z8 was the highest, and that of bisp 103-p1 and bisp 119/9p1 was the lowest among homokaryons (Table 3). A. bisporus var. bisporus was not different from A. bisporus var. burnettii with respect to the frequency of crossing. Some hybrids were not cultivated because the parental lines were not crossed [6]. MB-013 is an intervarietal hybrid resulting from the crossing between with bisp 034-p2 and bisp 015-p2. The hybrid had a BE of 0.152 and was selected to construct an F₁ population.

BC₁F₁ population

Three hundred SSIs were collected in the MB-013 line from the crossing between bisp 034-p2 and bisp 015-p2 to select over 100 of homokaryons, because one parental strain, bisp 015, was tetrasporic, A. bisprorus var. burnettii. After intercrossing A. bisporus var. bisporus with A. bisporrus var. brunettii, the first-generation hybrids were homokaryotic tetrasporic strains, dominant trait as observed by Kerrigan et al. [5]. Mycelia of SSIs of MB-013 were subjected to direct PCR with PIN 150 and the PCR product was digested PCR with HaeIII (Fig. 3) as a CAPS marker [18]. One hundred and seventy homokaryons were selected in the F₁ population. The PIN 150 marker was designed based on the gene sequences located on chromosome I and near the mating (MAT) gene were tested to screen putative homokaryotic protoclones [16, 17]. Therefore, the marker has the potential to discriminate between heterokaryons and homokaryons. The 100 selected homokaryons were crossed with bisp 015-p1 to construct the BC₁F₁ population (Fig. 4). The selected F₁ population will be  re-sequenced including bisp 034-p2 and bisp 015-p2, to perform linkage mapping. Furthermore, mushrooms of the BC₁F₁ populations will be used to calculate BE and for quantitative trait locus (QTL) mapping.