Morchella monospore hybridization breeding method
By using the single-spore hybridization breeding method of morel mushrooms, the problems of germplasm degradation and frequent diseases in the morel mushroom industry have been solved, resulting in improved yield and disease resistance, as well as production stability, and providing an efficient breeding strategy.
Patent Information
- Application Number
- CN202511407482.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-29
- Publication Date
- 2025-12-12
AI Technical Summary
The morel mushroom industry faces problems such as germplasm degradation, unstable fruiting, high deformity rate and frequent disease outbreaks during its rapid development. In particular, the distribution pattern of mating type genes has not been systematically analyzed and hybridization breeding technology is lagging behind, which limits the development of the industry.
The Morchella monosporus hybridization breeding method was adopted. Strains were obtained through tissue isolation and monosporus isolation. The mating type gene was detected, and monosporus isolates carrying the MAT1-1-1 and MAT1-2-1 genes were selected for pairing and hybridization. The hybrid combinations with better yield and disease resistance than the single parent were selected for cultivation experiments.
It improved the yield and disease resistance of morel mushrooms, reduced the risk of disease occurrence, enhanced production stability and economic benefits, and provided an efficient breeding program.
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Figure CN121109374A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of strain selection and breeding technology, and in particular to a method for hybridization and breeding of morel mushrooms. Background Technology
[0002] Morel mushrooms (Morchella spp.), as a rare edible fungus with high economic value, have seen rapid development in artificial cultivation in China, with annual production successfully exceeding 100,000 tons. Among the many cultivated species, *Morchella tessmannii*, *Morchella spp.*, and *Morchella spp.* stand out, contributing over 90% of the yield. Meanwhile, in recent years, successful large-scale field cultivation of morels has been reported in countries such as the United States, Japan, and India, further demonstrating the broad potential of this fungus cultivation. However, while the morel industry is developing rapidly, it also faces the serious challenge of germplasm degradation, with unstable fruiting and a deformity rate exceeding 15%. Furthermore, diseases such as green mold and cobweb disease occur frequently, with incidence rates even exceeding 20%, causing significant economic losses and severely hindering the healthy and sustainable development of the industry.
[0003] Studies have shown that most species in the genus *Morchella* are heterothallic fungi, and mating type genes (MAT1-1-1 and MAT1-2-1) directly regulate their sexual reproduction and fruiting body formation. However, current research has significant gaps; the distribution patterns of mating types among different cultivated *Morchella* species have not been systematically analyzed, and the effects of hybrid combinations on yield and disease resistance lack field evidence, indicating a lag in the development of hybridization breeding technology. Therefore, to overcome breeding bottlenecks and promote the sustainable development of the *Morchella* industry, it is urgent to conduct in-depth research at the level of reproductive and developmental mechanisms to provide strong support for the industry's sustainable development. Summary of the Invention
[0004] To overcome the problems existing in the background technology, the present invention provides a method for breeding morel mushroom single-spore hybridization, which reveals the significant role of mating-type gene hybridization combination in improving morel mushroom yield and enhancing disease resistance, and confirms the positive regulation of yield and stress resistance by mating-type gene complementarity.
[0005] This invention provides a method for hybridization and selection of single spores of morel mushrooms, characterized by the following steps: (1) Morel mushrooms were selected, and tissue isolates were obtained by tissue isolation method, while single-spore isolates were obtained by single-spore isolation method; (2) The mating type gene of the tissue isolates and single-spore isolates was detected respectively; (3) Within the same species, single-spore isolates carrying only the MAT1-1-1 mating type gene and those carrying only the MAT1-2-1 mating type gene were selected for pairing and hybridization. (4) Select successful single-spore hybrid combinations and their corresponding two parent strains for cultivation trials and agronomic trait evaluation, and select hybrid combinations with better yield and disease resistance than single parent strains.
[0006] Preferably, the morel mushroom includes at least one selected from morel mushrooms or morel mushrooms.
[0007] Preferably, the tissue separation method in step (1) specifically includes: taking a 2-4 mm tissue block from the middle of the morel cap under aseptic conditions. 3 The strain was inoculated into PDA medium and cultured at 25°C in the dark. The tissue isolates were obtained by continuous transfer and purification.
[0008] Preferably, the single-spore isolation method in step (1) specifically includes: scraping morel cap spores to prepare a spore suspension, diluting it with distilled water in a gradient and spreading it on WA medium, culturing it at 25°C in the dark, picking out germinating single spores under micromanipulation, transferring them to PDA medium for expanded culture to obtain single-spore isolated strains.
[0009] Preferably, the mating type gene detection in step (2) specifically includes: using the morel MAT1-1-1 and MAT1-2-1 gene-specific primers and reaction procedures from the public database, performing PCR amplification reactions using the genomic DNA of tissue-isolated strains and single-spore-isolated strains as templates, and detecting the results by 1% agarose gel electrophoresis after the reaction.
[0010] Preferably, the hybridization step (3) is as follows: Select single-spore isolates carrying only the MAT1-1-1 mating type gene and only the MAT1-2-1 mating type gene respectively, cut 5 mm diameter hyphal blocks from the edge of 7-day-old colonies with a sterile punch, and symmetrically inoculate them at both ends of PDA medium. Incubate at 25°C in the dark for 5-7 days. The hybridization is considered successful if there is no isolation zone, no raised line or sclerotium barrier at the junction of hyphae.
[0011] Preferably, the cultivation experiment in step (4) is as follows: Select the single-spore hybrid combination strains that have successfully hybridized and their corresponding two parent strains to prepare first, second and third grade strains and external nutrient bags. When the average daily temperature is 10-15℃, inoculate them into the experimental greenhouse cultivation plots. The uniform inoculation amount is 300g of third grade strains per 1.5m², and 5 external nutrient bags are placed in combination, each weighing 500g.
[0012] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides a scalable and efficient breeding scheme, revealing the significant role of mating-type gene hybridization combinations in increasing morel yield and enhancing disease resistance, and confirming the positive regulation of yield and stress resistance by mating-type gene complementarity. Field trials show that the successfully constructed hybrid strains produce significantly more mushrooms per unit area than the parents, providing a breakthrough germplasm resource for large-scale high-yield morel cultivation. At the same time, the hybrid strains exhibit excellent disease resistance, effectively reducing the risk of disease occurrence during cultivation and improving production stability and economic benefits. Attached Figure Description
[0013] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.
[0014] Figure 1 The electrophoretic detection results of ITS, RPB2, and EF1-α gene amplification products in this embodiment of the invention are as follows: A is the ITS gene; B is the RPB2 gene; C is EF1-α. Figure 2 This is an ML phylogenetic tree constructed based on "ITS+EF1-α+RPB2" in an embodiment of the present invention; Figure 3 Here are the mating type gene amplification patterns of the tissue-isolated strains in this embodiment of the invention: lanes 1-4 represent the MAT1-1-1 gene; lanes 5-8 represent the MAT1-2-1 gene. Figure 4 The following are amplification maps of mating type genes of single-spore isolated strains in the embodiments of the present invention: A is the amplification result of MAT1-1-1 gene of strain YZ01-L1~YZ01-L1, and B is the amplification result of MAT1-2-1 gene of strain YZ01-L1~YZ01-L1. Figure 5 Here are diagrams illustrating single-spore hybridization in this embodiment of the invention: A~B: isolation zone diagram; C~D: raised lines; E~F: sclerotium barrier; G~H: no hyphae antagonism; Figure 6 This is a morphological diagram of a normal fruiting body in a cultivation experiment according to an embodiment of the present invention; Figure 7 These are morphological images of diseased fruiting bodies from cultivation experiments in this embodiment of the invention; Figure 8 This is a morphological diagram of deformed fruiting bodies in the cultivation experiment of this invention. Detailed Implementation
[0015] This invention provides a method for hybridization and selection of single spores of morel mushrooms, comprising the following steps: (1) Morel mushrooms were selected, and tissue isolates were obtained by tissue isolation method, while single-spore isolates were obtained by single-spore isolation method; (2) The mating type gene of the tissue isolates and single-spore isolates was detected respectively; (3) Within the same species, single-spore isolates carrying only the MAT1-1-1 mating type gene and those carrying only the MAT1-2-1 mating type gene were selected for pairing and hybridization. (4) Select successful single-spore hybrid combinations and their corresponding two parent strains for cultivation trials and agronomic trait evaluation, and select hybrid combinations with better yield and disease resistance than single parent strains.
[0016] In this invention, morel mushrooms include at least one selected from morel mushrooms or morel mushrooms.
[0017] In this invention, the tissue separation method specifically includes: taking a 2-4 mm tissue block from the middle of the cap of a morel mushroom under aseptic conditions. 3 The strain was inoculated into PDA medium and cultured at 25°C in the dark. The tissue isolates were obtained by continuous transfer and purification.
[0018] In this invention, the single-spore isolation method specifically includes: scraping morel cap spores to prepare a spore suspension, diluting it with distilled water in a gradient and spreading it on WA medium, culturing it at 25°C in the dark, picking out germinating single spores under micromanipulation, and transferring it to PDA medium for further culture to obtain a single-spore isolated strain.
[0019] In this invention, the mating type gene detection specifically includes: using morel MAT1-1-1 and MAT1-2-1 gene-specific primers and reaction procedures from publicly available databases, performing PCR amplification reactions using genomic DNA from tissue-isolated strains and single-spore-isolated strains as templates, and detecting the results by 1% agarose gel electrophoresis after the reaction.
[0020] In this invention, the mating type gene of the tissue-isolated strains is first detected to determine that the selected morel fruiting bodies are all heterokaryotic, and their hyphal cells carry nuclei containing two mating type genes simultaneously; based on this, the mating type gene of the single-spore isolated strains is detected and genotyped.
[0021] In this invention, the hybridization steps are as follows: single-spore isolates carrying only the MAT1-1-1 mating type gene and only the MAT1-2-1 mating type gene are selected respectively. Mycelial blocks with a diameter of 5 mm are cut from the edge of 7-day-old colonies using a sterile punch and symmetrically inoculated at both ends of PDA medium. The culture is carried out at 25°C in the dark for 5-7 days. The absence of an isolation zone, raised line, or sclerotium barrier at the mycelial junction indicates successful hybridization. The absence of antagonistic reaction at the mycelial junction is used as the criterion for successful hybridization of the strain, providing a feasible solution for high-throughput screening of rare hybridization events.
[0022] In this invention, the cultivation experiment is as follows: Select successful hybrid single-spore hybrid strains and their corresponding two parent strains to prepare first, second, and third-level inoculum and external nutrient bags. Inoculate them into the experimental greenhouse cultivation plots when the average daily temperature is 10-15℃. The uniform inoculation amount is 300g of third-level inoculum per 1.5m², and five external nutrient bags are placed, each weighing about 500g. After the fruiting bodies mature, the agronomic traits of the hybrid combinations and parent strains are evaluated, and hybrid combinations with agronomic traits such as yield and disease resistance that are superior to single parent strains are selected.
[0023] To further illustrate the present invention, a method for breeding morel mushroom single-spore hybridization provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.
[0024] Example 1 1. Biomaterials Two morel fruiting bodies collected from a morel cultivation base in Yunnan Province, China, were preliminarily identified as belonging to the black group based on external morphological characteristics such as cap morphology, vein structure, and color. Sample information is shown in Table 1. Table 1 Materials and related information 2. Isolation and culture of bacterial strains Two morel fruiting bodies (YZ01; YZ02) were isolated under aseptic conditions using a tissue isolation method: 2-4 mm sections were cut from the center of the morel cap. 3 Tissue blocks were inoculated into PDA medium and cultured at 25°C in the dark until mycelial germination. After continuous transfer and purification, two tissue isolates were obtained (a total of four isolates, numbered: YZ01-L1, YZ01-L2; YZ02-Q1, YZ02-Q2).
[0025] Meanwhile, spore suspensions were prepared by scraping spores from the caps of morel mushrooms (YZ01; YZ02) using the single-spore isolation method. After being serially diluted with distilled water, the suspensions were spread on WA medium and cultured at 25°C in the dark. Germinating single spores were picked by micromanipulation and transferred to PDA medium for further culture to obtain 68 and 63 single-spore isolates respectively (numbered as: YZ01: M1~M68; YZ02: N1~N63).
[0026] 3. DNA extraction and phylogenetic identification Genomic DNA was extracted from tissue isolates and single-spore isolates of mycelium. Ribosome ITS region, elongation factor (EF1-α), and RNA polymerase II second large subunit (RPB2) gene fragments were amplified by PCR (primer sequences are shown in Table 2). The reaction volume was 25 μL, containing 1.0 μL DNA template (50 ng / μL), 12.5 μL 2×Easy Taq PCR Master Mix, 0.5 μL each of forward and reverse primers (10 μmol / L), and ddH2O to bring the volume to 25 μL. The reaction parameters were: 94℃ pre-denaturation for 4 min, 94℃ denaturation for 60 s, 50℃ annealing for 30 s, 72℃ extension for 60 s, 35 cycles, followed by a final extension at 72℃ for 10 min. The amplified products were detected by 1.5% agarose gel electrophoresis. The results are shown below. Figure 1 As shown, the amplification products of the ITS, RPB2, and EF1-α genes are 750bp, 800bp, and 1100bp in length, respectively, which are consistent with the expected fragments of the target genes.
[0027] Table 2 Primer sequences for ITS, RPB2, and EF1-α gene amplification The amplified products were verified by bidirectional sequencing. The obtained sequences were submitted to the NCBI database for BLASTn homology comparison analysis, and the GenBank accession numbers of the corresponding gene sequences were obtained through the GenBank platform (see Table 1). After splicing the three gene fragments into a 1710 bp combined sequence, phylogenetic analysis showed that the two morel fruiting bodies, YZ01 and YZ02, exhibited high clustering support rates in the phylogenetic tree. Morel fruiting body YZ01 clustered with *Morchella importuna* in the same branch with a 98% expansion value, while morel fruiting body YZ02 clustered with *Morchella esxtelata* in the same branch with an 85% expansion value. Based on the above molecular phylogenetic tree analysis results, it can be clearly determined that the two tested morel fruiting bodies belong to the species *Morchella importuna* and *Morchella esxtelata*, respectively.
[0028] High support rates (98% and 85%, respectively) confirm the reliability of the identification results, providing a crucial and accurate taxonomic basis for subsequent mating type studies. Numerous studies have shown that relying solely on single fragments such as ITS is insufficient to accurately distinguish more than 60 phylogenetic species within the genus *Morchella*, and the NCBI database contains numerous mis-annotated sequences for this genus. Therefore, this application carefully selected pattern sequences validated by authoritative research for alignment. Specifically, ClustalW was used for multiple sequence alignment, and after removing ambiguous regions, a phylogenetic tree was constructed using the maximum likelihood (ML) method based on the "ITS+EF1-α+RPB2" combined sequence. A 1000-fold expansion test was set to evaluate node support rates, and *Morchella rubiginosa* was selected as the outgroup.
[0029] This invention constructs a phylogenetic tree using multiple genes, which not only accurately identifies the species attribution of two samples, but also further highlights the significant advantages of multi-gene joint analysis in the identification of morel species.
[0030] 4. Mating type gene testing First, mating type gene detection was performed on two morel mushroom tissue isolates (YZ01-L1, YZ01-L2; YZ02-Q1, YZ02-Q2). Morel MAT1-1-1 and MAT1-2-1 gene-specific primers published in the NCBI database (primer sequences are shown in Table 3) were used to perform PCR amplification using the genomic DNA of the tissue isolates as templates. The reaction parameters were: 94℃ pre-denaturation for 4 min, 94℃ denaturation for 60 s, 50℃ annealing for 30 s, 72℃ extension for 60 s, and 35 cycles followed by a 72℃ extension for 10 min. The amplified products were detected by 1% agarose gel electrophoresis, and a bright band was selected for sequencing.
[0031] Table 3 Primer sequences for MAT1-1-1 and MAT1-2-1 gene amplification PCR amplification and sequencing revealed that specific bands of MAT1-1-1 and MAT1-2-1 were stably and simultaneously amplified in the DNA of all tissue isolates. The specific fragment of the MAT1-1-1 gene was approximately 700 bp in size, while the specific fragment of the MAT1-2-1 gene was approximately 500 bp in size. These sequences were highly homologous to known MAT mating type genes, with a sequence identity of 99%. These results demonstrate that the *Morchella tessmannii* (YZ01) and *Morchella septemlobus* (YZ02) collected in the examples are genetically heterokaryotes, with their mycelial cells containing both mating type nuclei.
[0032] Based on this, mating type gene detection was performed on 131 single-spore isolates from the fruiting bodies of *Morchella tessmannii* (YZ01) and *Morchella spp.* (YZ02). The results showed that six single-spore isolates (M1, M16, M30, M31, M40, and N19) could simultaneously amplify both MAT1-1-1 and MAT1-2-1 fragments, indicating that these single-spore isolates were heterokaryotic strains. Further analysis of the heterokaryotic ratio in the two morel species (statistics shown in Table 4) revealed 7% for YZ01 and 2% for YZ02. Based on existing research and detection results, it is speculated that during sporulation in morels, immature nuclear migration or somatic cell fusion events occur, resulting in a small number of spores containing both parental nuclei. The remaining single-spore isolates could only amplify one MAT gene, either MAT1-1-1 or MAT1-2-1. Among the single-spore isolates of species YZ01, 40 strains (59%) contained the MAT1-1-1 gene, and 23 strains (34%) contained the MAT1-2-1 gene. Among the single-spore isolates of species YZ02, 36 strains (57%) contained the MAT1-1-1 gene, and 26 strains (41%) contained the MAT1-2-1 gene.
[0033] Based on the detection results of mating type genes, it can be clearly seen that 95.4% of the single-celled strains carry only one mating type gene, MAT1-1-1 or MAT1-2-1, which is consistent with the typical characteristics of heterothallic fungi.
[0034] Table 4. Statistics on mating type genes of single-spore isolated strains 5. Single-spore hybridization From the single-spore isolates obtained from the fruiting bodies of two morel mushrooms, 25 strains of each strain with good mycelial growth and containing only MAT1-1-1 and MAT1-2-1 were screened out, and a total of 1250 pairs of intraspecific single-spore hybrid combinations were constructed. 625 pairs of fruiting bodies from each of the two morel mushrooms were used. Mycelial blocks with a diameter of 5 mm were cut from the edge of 7-day-old colonies using a sterile punch and symmetrically inoculated at both ends of PDA medium. After incubating the PDA medium at 25℃ in the dark for 5-7 days, the mycelial growth and interaction were observed. The absence of an isolation zone, raised line or sclerotium barrier at the mycelial junction was used as the morphological affinity standard.
[0035] The results of the single-cell hybridization experiment showed that even some single-cell strains from the same fruiting body exhibited significant differences in phenotypic characteristics such as hyphal color depth and sclerotium production capacity. The vast majority of hybrid combinations showed nutritional incompatibility, specifically manifested in three typical characteristics: (1) a clear isolation zone was formed at the hyphal contact area; (2) clear raised lines appeared in the hyphal contact area; and (3) a row of regular sclerotium barriers were formed in the contact area.
[0036] Of all the hybrid combinations, only four pairs showed nutritional compatibility during the mycelial growth stage, exhibiting interpenetration of mycelia with no obvious antagonism or nutritional incompatibility observed between the two parental mycelia. These combinations included: M11×M37 from morel fruiting bodies YZ01, and N7×N18, N07×N32, and N16×N35 from morel fruiting bodies YZ02. The success rate of single-cell hybridization between the two morel species was extremely low, with a success rate of 0.16% for YZ01 and 0.48% for YZ02.
[0037] 6. Cultivation Experiment 6.1 Preparation of microbial strains and external nutrient bags The successfully hybridized single-spore hybrid strains (M11×M37; N07×N32) and their corresponding two parental strains were selected for strain preparation: (1) Preparation of primary seed The culture medium is PDA medium; Preparation materials: 200g / L potato, 20g / L glucose, 20g / L agar, natural pH; Preparation process: After inoculation, culture at 25℃ in the dark until the mycelium has fully grown on the slant or plate for later use.
[0038] (2) Preparation of secondary seeds The culture medium is wheat culture medium; Raw materials for preparation: 88% wheat grains, 10% sawdust, 1% gypsum, 1% calcium carbonate; Preparation process: Soak wheat grains for 12 hours, then boil until cooked but not mushy. Drain the water and mix evenly with sawdust, gypsum, calcium carbonate, and other auxiliary materials. Dispense into 500mL inoculum bottles and autoclave at 121℃ for 2 hours. After cooling, inoculate and incubate in the dark at 20-22℃. Use when the mycelium has fully colonized the bottle.
[0039] (3) Preparation of tertiary seeds The culture medium is a solid matrix culture medium.
[0040] Raw materials for preparation: 70% sawdust, 20% wheat bran, 8% corn flour, 1% gypsum, 1% lime; Preparation process: Mix all raw materials evenly, and adjust the moisture content to 60-65% (the material should leave water marks between the fingers when squeezed tightly, but not dripping). Dispense into 17cm×33cm polypropylene plastic bags, and autoclave at 121℃ for 3 hours or at normal pressure for 8-10 hours; after cooling, inoculate with secondary inoculum and cultivate in the dark at 20-22℃ for 25-30 days. Once the mycelium has fully colonized the bag, it can be used for field sowing.
[0041] (4) Preparation of external aid nutrition bags Raw materials for preparation: 50% wheat grains, 30% sawdust, 18% wheat bran, 1% gypsum, 1% lime; Preparation process: Mix the raw materials evenly and adjust the moisture content to 60-65%. Dispense into special polypropylene nutrient bags with a specification of 12cm×24cm. One end of the nutrient bag is pre-opened and equipped with a special cotton-free cover. Sterilize at 121℃ for more than 3 hours. Use after thorough cooling. The weight of each bag is 500g.
[0042] 6.2 Field Cultivation In early November, when the average daily temperature is 10-15℃, the tertiary spawn, fully colonized with mycelium, is removed from the bag and broken into pieces approximately 1-2 cm³ in size. Using a layer-sowing method, the spawn pieces are evenly sown onto pre-prepared cultivation beds (1-1.2m wide, 10-15cm deep), with a uniform inoculation rate of 300g of tertiary spawn per 1.5m². This is then covered with a 2-3cm layer of sterilized loam or peat moss.
[0043] 15-20 days after sowing, when the mycelium on the surface of the seedbed has germinated in large quantities and formed a mycelial bloom, the external nutrient bags can be placed. Place the sterilized external nutrient bags with the opening facing down directly and tightly on the seedbed after it has been covered with soil, with 5 external nutrient bags evenly placed per 1.5m².
[0044] Four biological replicates were set up (1.5 m² / replica), with an interval of 1 m between each replicate. 300 g of tertiary seed was inoculated and 5 nutrient bags (500 g / bag) were provided.
[0045] Temperature control: During primordia differentiation, maintain diurnal temperature variation stimulation, with daytime temperatures of 12-18℃ and nighttime temperatures of 6-10℃; during the fruiting body growth period, maintain the temperature at 10-20℃.
[0046] Humidity control: Maintain the relative humidity inside the cultivation greenhouse at 85%-95%. Use a micro-spraying system to increase humidity, avoiding direct rinsing of the seedbeds and external nutrient bags.
[0047] Ventilation and lighting: Keep the air inside the greenhouse fresh, with CO2 concentration below 800 ppm. Ventilate as needed daily and provide adequate diffused light (200-800 Lux).
[0048] Harvest when the fruiting bodies reach commercial maturity (with a plant height ≥7 cm as the main morphological indicator and the cap morphology being basically complete). Statistical analysis is performed on the following within each replicate unit area: 1) total number of fruiting bodies; 2) number of diseased fruiting bodies (with typical disease symptoms); 3) number of deformed fruiting bodies (with abnormal cap or stipe morphology).
[0049] All data are expressed as mean ± standard error (Mean ± SE). Statistical analysis was performed using IBM SPSS Statistics 26.0 software. One-way ANOVA was used to test the significance of differences between different treatment groups, and Duncan's multiple range test was used for multiple comparisons within groups.
[0050] 6.3 Fruiting performance of mating-type gene-complementary hybrid strains Morel mushroom (YZ01) entered the fruiting period 65 days after sowing, and morel mushroom (YZ02) entered the fruiting period 75 days after sowing. Compared with single-mating parent strains, the successfully bred hybrid combinations M11×M37 and N07×N32 maintained a 100% fruiting rate while significantly or extremely significantly increasing the number of fruits per unit area (see Table 5). Specifically, the average number of fruits produced by the morel mushroom hybrid combination M11×M37 was significantly higher than that of the MAT1-1-1 type parent M11 (P≤0.05), and extremely significantly higher than that of the MAT1-2-1 type parent M37 (P<0.01). The Six Sisters morel hybrid combination N07×N32 showed a more significant yield increase, with an average fruiting number of 33.00±4.55, which was significantly different from the MAT1-1-1 parent N07 (P≤0.05) and extremely significantly different from the MAT1-2-1 parent N32 (P<0.01). At the same time, this combination also showed excellent yield stability, with the fruiting number concentrated between 27 and 38.
[0051] In this embodiment, single-spore strains of *Morchella tessmannii* and *Morchella spp.* (containing only one mating type gene, MAT1-1-1 or MAT1-2-1) could produce fruiting bodies normally. Some single mating type strains, such as M37 and N07, could also achieve 100% fruiting, but the yield was significantly lower than that of hybrid strains. The fruiting rate of strain N32 was only 75%. The mating type gene detection results confirmed that the above strains indeed contained only one mating type gene. Subsequently, mating type gene detection was performed on the cap tissue of some fruiting bodies produced by the above single-cell strains, and both MAT1-1-1 and MAT1-2-1 mating type genes were detected. Based on the above phenomena, combined with existing research and detection results, it is speculated that the above strains may have a small number of heterokaryotic nuclei when mating type gene detection is performed. However, due to the extremely small number of such nuclei, the copy number of the mating type gene they carry is too low to be detected by PCR amplification. Therefore, single-cell strains can produce fruiting bodies, but because the extremely small number of heterokaryotic nuclei cannot fully activate the complete regulatory pathway of sexual reproduction, it is difficult to support high-efficiency fruiting.
[0052] Experimental data show that the coexistence and synergistic expression of MAT1-1-1 and MAT1-2-1 genes are the key mechanisms driving high yield. By forming binucleates, they activate the complete transcriptional regulatory network required for sexual reproduction, thereby more efficiently initiating the development of fruiting body primordia. Creating binucleated strains through artificial hybridization is an effective breeding strategy to break through the yield bottleneck of morel mushrooms.
[0053] Table 5. Statistics on fruiting numbers of mating-type gene-complementary hybrid strains Note: Data are expressed as mean ± standard error. Within-group statistical comparisons were performed independently (M. importuna and M. sextelata groups were tested separately): within the same species, "-" indicates no statistical significance; "*" indicates a significant difference (p≤0.05); "**" indicates an extremely significant difference (p<0.01).
[0054] 6.4 Disease resistance of mating-type gene-complementary hybrid strains As shown in Table 6, in *Morchella tessmannii*, the number of diseased fruiting bodies in the hybrid strain M11×M37 was significantly lower than that in its single-cross parent (P≤0.05). In *Morchella spp.*, the improvement in disease resistance was even more significant: the number of diseased fruiting bodies in the hybrid combination N07×N32 decreased to 0, significantly lower than that in its parent (P≤0.05). In both species, there were no statistically significant differences in the number of deformed fruiting bodies between the hybrid strains and the parental strains (P>0.05), and no significant differences in the number of deformed fruiting bodies among the individual strains.
[0055] Morel mating type genes regulate only sexual reproduction, while fruiting body morphogenesis is dominated by developmental regulatory networks and environmental signals. Related studies have verified the regulatory function of mating type genes using RNA interference technology. However, they found no significant difference in the appearance of morel fruiting bodies cultivated from single mating type strains (MAT1-1 or MAT1-2), but the asci and ascospores of strain MAT1-1 showed slight malformations. This indicates that both mating type genes are involved in reproductive development regulation, and their regulatory effects differ at the microstructural level.
[0056] In summary, the complementation of the double mating type genes in *Morchella esculenta* may globally enhance the physiological state of the mycelium or activate a wider range of disease resistance-related pathways. However, the morphological stability of the fruiting body is mainly dominated by other polygenic genetic backgrounds (such as transcription factors regulating development) or environmental factors (such as temperature, humidity, and light). The role of the mating type system is more focused on initiating sexual reproduction and maintaining the overall vigor and stress resistance of the strain. This discovery decouples the genetic regulation of agronomic traits such as yield and disease resistance from morphogenesis to some extent, providing a theoretical basis for separately breeding specialized strains with "high yield and disease resistance" and "beautiful morphology".
[0057] Table 6. Statistics on disease resistance and morphological normality rate of mating type genes to morel mushrooms. Note: Data are expressed as mean ± standard error. Within-group statistical comparisons were performed independently (M. importuna and M. sextelata groups were tested separately): within the same species, "-" indicates no statistical significance; "*" indicates a significant difference (p≤0.05); "**" indicates an extremely significant difference (p<0.01).
[0058] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made to it in form and detail without departing from the scope defined by the claims of the present invention.
Claims
1. A method for hybridization and selection of morel mushrooms, characterized in that: Includes the following steps: (1) Morel mushrooms were selected, and tissue isolates were obtained by tissue isolation method, while single-spore isolates were obtained by single-spore isolation method; (2) The mating type gene of the tissue isolates and single-spore isolates was detected respectively; (3) Within the same species, single-spore isolates carrying only the MAT1-1-1 mating type gene and those carrying only the MAT1-2-1 mating type gene were selected for pairing and hybridization. (4) Select successful single-spore hybrid combinations and their corresponding two parent strains for cultivation trials and agronomic trait evaluation, and select hybrid combinations with better yield and disease resistance than single parent strains.
2. The method for breeding morel mushroom single-spore hybridization according to claim 1, characterized in that: The morel mushrooms include at least one selected from morel mushrooms or morel mushrooms.
3. A method for breeding morel mushroom single-spore hybridization according to any one of claims 1 or 2, characterized in that: The tissue separation method described in step (1) specifically includes: taking a 2-4 mm tissue block from the middle of the cap of morel mushrooms under aseptic conditions. 3 The strain was inoculated into PDA medium and cultured at 25°C in the dark. The tissue isolates were obtained by continuous transfer and purification.
4. A method for hybridization and selection of morel mushrooms according to any one of claims 1 or 2, characterized in that: The single-spore isolation method described in step (1) specifically includes: scraping morel cap spores to prepare a spore suspension, diluting it with distilled water in a gradient and spreading it on WA medium, culturing it at 25°C in the dark, picking out germinating single spores under a micromanipulation, and transferring it to PDA medium for further culture to obtain a single-spore isolated strain.
5. The method for breeding morel mushroom single-spore hybridization according to claim 1, characterized in that: Step (2) specifically includes the following: using the MAT1-1-1 and MAT1-2-1 gene-specific primers and reaction procedures from the publicly available database, PCR amplification reaction is performed using the genomic DNA of tissue isolates and single-spore isolates as templates, respectively. After the reaction, 1% agarose gel electrophoresis is used for detection.
6. The method for single-spore hybridization and selection of morel mushrooms according to claim 1, characterized in that: The hybridization step (3) is as follows: Select single-spore isolates carrying only the MAT1-1-1 mating type gene and only the MAT1-2-1 mating type gene respectively. Use a sterile punch to cut 5 mm diameter hyphal blocks from the edge of 7-day-old colonies and inoculate them symmetrically at both ends of PDA medium. Incubate at 25°C in the dark for 5-7 days. The hybridization is considered successful if there is no isolation zone, no raised line or sclerotium barrier at the junction of hyphae.
7. The method for hybridization and selection of morel mushrooms according to claim 1, characterized in that: The cultivation experiment described in step (4) is as follows: Select the single-spore hybrid combination strains that have successfully hybridized and their corresponding two parent strains to prepare first, second and third grade strains and external nutrient bags. When the average daily temperature is 10-15℃, inoculate them into the experimental greenhouse cultivation plots. The uniform inoculation amount is 300g of third grade strains per 1.5m², and 5 external nutrient bags are placed with each bag weighing 500g.