Mating Genotype Analysis of Ganoderma lucidum populations

Mating Genotype Analysis of Ganoderma lucidum populations

Yu-xin CHEN 1,2, Zhi-lan XIA 1,2, Peng LIU 1,2, Xin-cong KANG 1,2, Ya-hui HONG 3, Dong-bo LIU 1,2

1. Hunan Agricultural University, Horticulture and Landscape college, Changsha 410128, China; 2. State Administration of Traditional Chinese Medicine, State Key Laboratory of Subhealth Intervention Technology, Changsha 410128, China; 3. Hunan Agricultural University, College of Bioscience and Biotechnology, Changsha 410128, China

Abstract [Objective]The mating genotype was studied and compared with esterase isozyme of Ganoderma lucidum populations between groups in order to clarify their differences in genetic relationship analysis. [Method] OWE-SOJ technique was applied to identify standard mating types and determinate mating genotype between groups of monokaryons isolates from 24 G. lucidum stains. Genetic relationships were analyzed by combined group mating genotype determination with esterase isozyme assay. [Result] All strains of G. lucidum could be divided into 7 large groups of the mating genotype. Four alleles of A factor, four alleles of B factor and one mixed alleles of A factor were found in this study. Distorted segregation ratio among monokaryon mycelia of G. lucidum had been observed in four kinds of mating types to some extent. Twenty-eight different types of enzyme bands were determined in esterase isozyme test. Twenty-four strains of G. lucidum could be divided into 9 large groups through the cluster analysis when the genetic similarity coefficient was 0.73214. Comparing the results of mating genotype analysis and esterase isozyme analysis, it showed great similarity. [Conclusion] Mating genotype analysis could be used as an important supplementary method for strain identification and genetic diversity research.

Key words Esterase isozyme; OWE-SOJ technique; Mating type; Colony morphology

Ganoderma lucidum, belonging to Ganoderma, Ganodermataceae, Aphyllophorales, Hymenomycetes, Basidiomycotina, Fungi, is a large medicinal fungus. It has the reputation of “immortal grass”, and is the rare medicinal material, with wide application in medicines and foods. G. lucidum is a typical tetrapolar heterothallic higher basidiomycetes, belonging to the double factor incompatibility system, which is composed by incompatibility factors A and B [1].

For tetrapolar heterothallic fungus, the theoretical compatibility rate of various strains of the same species is 25%. However, higher basidiomycetes have wide multiple allele phenomenon, namely a gene locus decided mating type appears multiple alleles. For example, subunit α, β composing A, B factors have a series of multiple alleles, when the allele of α or β subunits changes, new factor A or factor B will form [2]. The existence of multiple alleles increases monokaryons random pairing affinity rate of different strains in natural population [3]. Now, it is found that the Aα and Aβ alleles of Schizophyllum commune are 9 and 32, while alleles of Bα and Bβ are 9. Theoretically, the multiple allele of A factor reaches 288, and the multiple allele of B factor is 81, so they can constitute 23328 different combinations of A, B factor [4]. In addition, it is also found that A, B factors of Pleurotus Ostreatus[5], Flammulina velutipes [6-7] and Lentinula edodes [8] also have multiple alleles.

The existence of multiple alleles results in germplasm diversity of G. lucidum, which is also the basis of crossbreeding. Therefore, accurate identification of different species of G. lucidum mating genotype will provide a reliable theoretical basis for genetic research and parent selection of G. lucidum and other edible fungi. Currently, there are many reports about G. lucidum cultivation and pharmacological effect, but the research on its basic genetics, especially mating genotype has not been reported. With 24 G. lucidum strains as samples, the author studied the number of mating type alleles and compared with esterase isozymes, explored the application of mating type determination in strain difference analysis and variety identification, so as to provide reference for study on germplasm genetic diversity of G. lucidum.

1 Materials and Methods

1.1 Test materials

1.1.1 Test strains

The sources of 24 G. lucidum strains were shown in Table 1, which were preserved by State Key Laboratory of Subhealth Intervention Technology, State Administration of Traditional Chinese Medicine, Hunan Agricultural University.

1.1.2 Media

PDA enrichment medium: potato (peeled) 20%, glucose 2%, KH2PO4 0.3%, MgSO4 0.15%, yeast extract 0.2%, peptone 0.2%, agar 2%, pH 6.5-7.0.

Oak wood extract agar (OWE):5% oak wood extract 20 g, agar 200 mL, adding distilled water to 1000 mL.

Squeezed orange juice agar (SOJ): fresh squeezed orange juice 200 mL, agar 20 g, adding distilled water to 1000 mL.

1.2 Experimental method

Esterase isozyme was analyzed according to reference [9]. Monokaryons isolation adopted conventional dilution separation -- flat coating method, referring to reference [8]. Regular mating type was determined by 3 round of hybrid system test method [10]. After conventional mating type analysis, standard mating type was determined by OWE-SOJ technology [11]. Determination of group mating type genes among strains: a strain was randomly selected, 4 mating type monocaryotic strains were set as standard test strain and mated with 4 mating type monocaryotic strains of other strains; whether there was locking joint was examined under microscope, and analyzed according to mating reaction result, so as to infer strain mating genotype; if this could not infer mating genotype strains, the next round of mating reaction would be conducted, until the mating genotype of all strains was completely inferred.

2 Results and Analysis

2.1 Analysis of esterase isozyme results

The electrophoresis results were shown in Fig. 1, 28 enzyme bands with different migration rate (Rf) were detected from 24 strains, Rf was distributed between 0.0508 and 0.9153. Four groups of bands 0.5631, 0.6911, 0.7552 and 0.51114 were the common bands of most strains; the similarity coefficients of samples were ranged from 0.536 to 0.964, the maximum similarity coefficient appeared between No. 12+2 and No. 40, No. 25 and No. 36, and they might have synonym phenomenon; the minimum similarity coefficient appeared between No. 11 and No. 19, No. 11 and 50+2. The original name of No. 43 and No. 54 was Beihan ganoderma, but they only had similar coefficient of 0.679 in isozyme analysis, which should be the namesake dissimilar phenomenon.

UPGMA clustering method was used for cluster analysis of 24 strains of G. lucidum samples, and clustering tree diagram was shown in Fig. 2. It could be seen that when the similarity coefficient approximately was 0.73214, the samples were divided into 9 categories; 9 strains including 1, 5, 15, 26, 25, 26, 31, 17 and 18 were clustered into one class (marked as I class); 47+2 and 50+2 strains were clustered into one group (marked as II class); 7 strains of 12+2, 3+2, 40, 8, 43 and 51 were clustered into another category (marked as III class); the remaining 6 strains were isolated independently as a class. The name of No. 25 and No. 36, as well as No. 12+2 and No. 40 was different, but clustering analysis showed that their genetic similarity coefficient was very high, which might be the same strain; the original name of No. 43 and 54 was Beihan ganoderma, but they only had little genetic similarity coefficient in clustering analysis, which should be namesake dissimilar phenomenon.

2.2 Standard mating type analysis

Standard mating type analysis results were shown in Table 2. In standard mating type identification, the villous colony without locking joint (A=B ≠, Fig. 4) was detected out by OWE-SOJ technology, but partial combination could be detected to have false locking joint in such mating reaction under microscope. The hook branch formed in false locking joint could not fuse with terminal cell, resulting in the occurrence of false lock joint hyphae (Fig. 5). The palisade colony without locking joint detected by OWE-SOJ technology also had no false lock joint phenomenon (A ≠ B=, Fig. 6).

A total of 437 monokaryons strains were isolated from 24 strains. Viewed from mating type distribution of various strains, the sum of a(AⅠBⅠ) and b(AⅡBⅡ) was quantitatively higher than that of c(AⅠBⅡ) and d(AⅡBⅠ), and its total number ratio was a(AⅠBⅠ):b(AⅡBⅡ):c(AⅠBⅡ):d(AⅡBⅠ)= 131:138:75:93=1: 1.05:0.57:0.71, the separation of 4 mating types was deviated from segregation ratio of 1:1:1:1 to some extent.

2.3 Analysis of determination results of mass mating genotype

After 6 rounds of mating tests, the results were shown in Table 3. The mating gene type of 24 test strains was divided into 7 categories, A factors contained 4 alleles, B factors contained 4 alleles, and a special A mixed gene. A1A2B1B2 and A1A3B1B2 mating type genes have large advantage in test strains, reaching 33.3%, respectively, the remaining 5 mating genotypes occupied small ratio. From the genetic evolution, the mating genotype with dominant proportion was the mainstream of evolution, and the mating genotype with inferior proportion was the variant strain or evolutionary branching, with far genetic relationship.

3 Conclusions and Discussion

The research analyzed esterase isoenzyme of 24 G. lucidum strains, and the results showed that the samples were divided into 9 categories when the similarity coefficient was 0.73214. The population mating type gene determination showed that 24 G. lucidum strains could be divided into 7 categories. Comparing population mating type gene and esterase isozyme analysis, it was found that mating type A1A2B1B2 with large advantage was mainly distributed in the I class (except G.l0050+2 strain), and the other large class of mating type A1A3B1B2 was mainly distributed in the III class (except G.l0047+2 strains), G.l0019, G.l0054 strain and G.l0011 strain with A special mixed mating type gene belonged to their respective clustering. Therefore, mating type analysis and esterase isozyme analysis result had very high degree of similarity and consistency, so mating genotype determination could be used as an important supplementary means for analysis of strain differences and varieties identification.

In the sexual reproduction process of edible fungus, mating type genes play a decisive role in monocaryon mycelium sex control and dikaryon hyphal formation. G. lucidum has tetrapolar mating system, and its mating is controlled by two independent distribution factors (commonly known as A, B factor). A factor controls the development process of cytoplasmic fusion, karyon pairing, nuclear fission and lock cell formation through HD1-HD2 hetero dimmer; B factor codes pheromone and receptor, and controls nuclear migration [12]. In the generation of heterokaryon, only A, B factors are in the heteroallelic gene state, stable heterokaryon can be formed, completing sexual life history. If only the B factor is in heteroallelic gene state, and A factor is in allele status (A=B ≠), the nuclear migration can not be stabilized, hook branch can not fuse with terminal cell, resulting in production of false locking joint mycelium[11,13], and this is consistent with the results in the test. However, in A=B ≠ combination, only partial not all false locking joints are produced, this is similar to the mating type of L. edodes by LIN Fang-can[11], and the specific reasons are worthy of further study.

In the test, G. lucidum strain also appears segregation phenomenon of sporidium mating type factors, which also appears in other fungi, such as L. edodes [14] and F. velutiper[15]. Kawa Bata et al. believed that mating type proportional deviation had relationship with cytoplasm selection ability to nuclear, which also had relation with some growth inhibition factor with lethal effects concatenated with A, B mating type factors[16]. Raper pointed out that the partial deviation had relationship with B factor, B mating type factor affected the viability of cell nuclear from heterokaryon cell separation, and this was the genetic basis of asymmetry nuclear ratio phenomenon [17]. Kay et al. believed that after protoplasted monokaryon of binuclear mycelium of P. ostreatus, the deviation of 2 nuclear proportions might be associated with some form of nuclear selection[18]. Whether the reason for this phenomenon in P. ostreatus is the same as other fungi still deserves further studying.

OWE-SOJ technology is a new technology developed in the early 1990's, which is first used in mating reaction identification of Armillaria mellea[19]. The technical advantage is intuitive experimental results with simple operation, and the shortage is long experimental period, but single use can not distinguish the reaction of same A. So we must combine with conventional mating type analysis, so as to accurately distinguish 4 mating types.

Table 1 Sources of test strains

Table 2 Standard mating type results of 24 G. lucidum strains

Table 3 Group mating genotype determination results of 24 G. lucidum strains

Fig. 1 Esterase isozyme electrophoresis of 24 G. lucidum strains

Fig. 2 Esterase isozyme genetic clustering map of 24 G. lucidum strains

Fig. 3 Lock joint (indicates with arrow) (400×)

Fig. 4 Villous colony without locking joint (A=B≠)

Fig. 5 False locking joint (indicates with arrow) (400×)

Fig. 6 Fence type colony without locking joint(A≠B=)

灵芝群体交配基因型分析

陈裕新1,2，夏志兰1,2，刘   鹏1,2，康信聪1,2，洪亚辉3，刘东波1,2

（1湖南农业大学园艺园林学院，长沙 410128；2国家中医药管理局亚健康干预技术实验室，长沙 410128；

3湖南农业大学生物技术学院，长沙 410128）

摘   要：【目的】研究灵芝群体交配基因型，并与酯酶同工酶试验相比较，探讨它们在群体亲缘关系分析上的异同点。【方法】采用OWE-SOJ技术对24个灵芝菌株的单孢分离菌株进行标准交配型鉴定，及群体间交配基因型的测定，并结合酯酶同工酶试验对群体间的亲缘关系进行分析。【结果】鉴定出7大类交配基因型，并发现A因子含有4个等位基因，B因子含有4个等位基因，及一个特殊的A混合基因，四类交配型出现了一定程度的偏分离。酯酶同工酶试验检测出了28条不同酯酶酶谱带，在相似系数为0.73214时，样品被分为9大类。比较交配基因型分析与酯酶同工酶试验结果，发现两者具有很高的相似性。【结论】交配基因型测定可以作为分析菌株间差异和鉴定品种的一种重要补充手段。

关键词：酯酶同工酶；OWE-SOJ技术；交配型；菌落形态

Received: June 08, 2012 Accepted:

Supported by National Science and Technology Support Program "Regional Development of Chinese Traditional Medicine Industry and Research of Characteristic Product" (2006BIA06A20) & Personnel Services Business Action Plan of Ministry of Science and Technology "Development of Series Products of Rare Edible Fungus" (2009GJD20012).

ØCorresponding author. E-mail: chinasaga@163.com

Responsible editor: YIN Jian-li

Responsible proofreader: WU Xiao-yan

Agricultural Science&Technology,2012,13(8):1651-1654