Mitochondrial DNA restriction fragment length polymorphisms and phenetic relationships in natural populations of the oyster mushroom, Pleurotus ostreatus

Mycol. Res. 99 (5): 562-566 (1995)

562

Printed in Great Britain

Mitochondrial DNA restriction fragment length polymorphisms and phenetic relationships in natural populations of the oyster mushroom, Pleurotus ostreatus*

TERUYUKI MATSUMOTO AND YUKIT AKA FUKUMASA-NAKAI The Tottor; Mycological Institute, Kokoge

211,

Tottori

689-11,

Japan

Restriction fragment length polymorphisms (RFLPs) of mitochondrial DNA (mtDNA) in 34 isolates of the oyster mushroom, Pleurotus ostreatus, from throughout the northern hemisphere, were employed to evaluate genetic relatedness among natural populations. Eighteen isolates were from Japan, ten from Europe, five from U.S.A., and one from Korea. BamHL EeaR! and EcoRV digests of mtDNAs produced 18, 19 and 19 distinct RFLP patterns, respectively. By combining all RFLP patterns obtained with the three endonucleases, mtDNAs could be assigned to 22 different classes that were clustered phenetically into three major similarity groups, each of which represents a geographically distinct population. The results suggest that geographical distance between natural populations of P. ostreatus is correlated with genetic divergence.

The oyster mushroom, Pleurotus ostreatus (Jacq.: Fr.) P. Kumm., and related species in the P. ostreatus complex, are commercially important edible mushrooms in many countries of the world. Annual production of P. ostreatus complex (Chang & Miles, 1991) is second only to that of the cultivated mushroom, Agaricus bispoyus (J. E. Lange) Imbach. Development of superior new cultivars of P. ostreatus is important for promoting commercial production. Egger, Eden & Wissig (1976) have suggested that the fruiting process in P. ostreatus involves many genes. Eugenio & Anderson (1968) have reported that dikaryons of P. ostreatus derived from interstock matings produce more fruiting bodies than those from intrastock matings. These studies suggest that wild isolates of P. ostreatus may provide valuable genetic resources for breeding purposes. Although the taxonomy of species belonging in P. ostreatus complex was recently studied in detail (Vilgalys, Smith & Sun, 1992), there has been little research aimed at understanding genetic relatedness among wild isolates of P. ostreatus. Here we employ restriction fragment length polymorphisms (RFLPs) in mtDNA to evaluate genetic similarity among geographically distinct P. ostreatus populations. RFLPs of mtDNA have been shown to be useful genetic markers for estimating genetic divergence and phylogenetic relationships between natural populations in various taxa (Kozlowski, Bartnik & Stepien, 1982; Taylor, Smolich & May, 1986; Bruns et al., 1988; Smith & Anderson, 1989; Forster et al., 1989; Jeng et aI., 1991). By using RFLP analysis in mtDNA, Fukuda et al. (1994) revealed that in the edible mushroom, Lentinula edodes (Berk.) Pegler, mtDNA divergence is significantly correlated with geographical distribution. In this study,

• Contribution No. 298 from The ToUori Mycological Institute.

we examined mtDNA similarity among wild isolates of P. ostreatus from geographically different populations in the northern hemisphere by mtDNA RFLP analysis.

MA TERIALS AND METHODS Strains and culture conditions Thirty-four P. ostreatus wild dikaryotic isolates were used: eighteen from different prefectures in Japan (Shizuoka, Mie, Hyogo, Tottori, Yamaguchi, Oita), ten from Europe (Germany, France, Hungary, Czechoslovakia), five from the U.S.A., and one from Korea (Table 1). Vilgalys et al. (1992) showed that a mating compatibility test could distinguish P. ostreatus from other species of the P. ostreatus complex, such as P. pulmonarius (Fr.) Que!' and P. populinus O. Hilber & O. K. Mil!. To avoid confusion with other species of the P. ostreatus complex, mating compatibility tests were carried out. All isolates used in this study were interfertile with the two P. ostreatus tester strains from the culture collections of Duke University (D337 and D261). Cultures were grown in MYG liquid medium (2 % malt extract, 0'2 % yeast extract, 2 % glucose) at 25°C for 14 d, and then fragmented with a Waring blender. Ten ml of the fragmented MYG culture were used to inoculate a 500 ml Erlenmeyer flask containing 100 ml of MYG. The flask cultures were incubated in a stationary state at 25° for 10-14 d, harvested onto nylon cloth (300 Ilm pore size), washed with distilled water, and lyophilized.

Isolation

0/ mtDNA and restriction

analyses

The mtDNA was isolated according to the procedure of Fukumasa-Nakai, Matsumoto & Fukuda (1992). MtDNA

1. Matsumoto and Y. Fukumasa-Nakai

563

Table 1. Geographical origin and source of Pleurotus ostreatus wild isolates employed Stock No.

Origin

Source

161 164 165 318 517 552 739 1052 1122 1595 1598 1600 2883 2934 3I 95 3476 3484 M336

Tottori, Japan Hyogo, Japan Yamaguchi, Japan Mie, Japan Tottori, Japan Tottori, Japan Tottori, Japan Tottori, Japan Hyogo, Japan Hyogo, Japan Shizuoka, Japan Tottori, Japan Tottori, Japan Oita, Japan Tottori, Japan Tottori, Japan Tottori, Japan Tottori, Japan

TMI-30052 a TMI-30054a TMI-30055' TMI-32214" TMI-32215' TMI-30682" TMI-30696" TMI-30050a TMI-30011' TMI-31810' TMI-30250a TMI-30251" TMI-30855 a TMI-30882' TMI-30567a TMI-31054a TMI-31057a TMI-32216'

a b C

Stock No.

Origin

Source

281 284 286 287 761 1182 1183 1184 1185 1187 1189 1196 1204 1205 4073 4074

Czechoslovakia Bayern, Germany Bayern, Germany Bayern, Germany Hungary France France France France France u.s.A. Korea California, U.S.A. California, U.s.A. Mississippi, U.S.A. Tennessee, U.S.A.

O. Hilber PI 5F O. Hilber PI lqu O. Hilber PI 4Y O. Hilber PI 2Z J. Lelley H-7 F. Zadrazil P4 F. Zadrazil P6 F. Zadrazil P8 F. Zadrazil Pll F. Zadrazil P13 F. Zadrazil PI J. H. Ree 2-1 R. A. Kerrigan R. A. Kerrigan R. Vilgalys D337b R. Vilgalys 0261" (ATCC 66376')

Tottori Mycological Institute Culture Collection number. Duke University Department of Botany Mycology Culture Collection ref. number. American Type Culture Collection number.

derived from each isolate was digested separately with three restriction endonucleases, BamHI, feoRI and feoRV, following the supplier's specification (Nippon Gene Co. Ltd). Electrophoresis of all digests was carried out on 0'7-1'0% agarose (Nippon Gene Co. Ltd, Type HS) slab gel in TAE (40 roM Tris/acetate, 10 mM EDTA, pH 8'0) at 5 V cm- 1 for 3 h. Gels were stained with ethidium bromide (0'5 IJg ml- 1) for observation on a uv transilluminator. Lambda phage DNA digested with HindIII was used as a molecular size standard.

ABCDEFGHI

JKLMNOPOR

Phenetic analysis Presence or absence of individual restriction fragments derived from BamHI, feoRI and feoRV digests were scored, and a distance value based on the scoring data was calculated between pairs of different mtDNA phenotypes using the dice coefficient as ~(Nxy)/(Nx+Ny), in which N XY is the number of restriction fragments shared between a pair of the phenotypes, and N x and Ny are the total numbers of restriction fragments in all digests in respective phenotype x and y. Dendrograms based on the distance values were constructed by group-average cluster analysis and by neighbor-joining analysis using the computer package FACOM OS IV/ESP III by Fujitsu corporation (version 1'0, June 1985).

Fig. 1. Eighteen representative rntDNA restriction fragment patterns produced from 34 Pleurotus ostreatus wild isolates with BamH! digestion electrophoresed on 1 % agarose. The isolates associated with respective RFLP patterns of A to R are presented in Table 2. Far left lane: HindIII-digested lambda phage DNA.

ABCDEFGH

I

JKLMNOPORS

RESUL TS AND DISCUSSION By digestion with BamHI, fcoRI and fcoRV, P. ostreatus mtDNA was found to be highly polymorphic, as has been described previously for other fungi, including Neurospora crassa Shear & Dodge (Collins & Lamboeitz, 1983), Schizophyllum commune Fr. (Specht, Novotny & Ullrich, 1983), Agaricus bitorquis (Quel.) Sacco (Hintz et ai., 1985), and L. edodes (Fukuda et ai., 1994). Endonuclease digests of mtDNAs from these isolates produced 18 different BamHI RFLP patterns (A

Fig. 2. Nineteen representative mtDNA restriction fragment patterns produced from 34 Pleurotus ostreatus wild isolates with feoR! digestion electrophoresed on 1 % agarose. The isolates associated with respective RFLP patterns of A to 5 are presented in Table 2. Far left lane: HindIII-digested lambda phage DNA.

MtDNA RFLPs in Pleurotus ostreatus ABCDEFGH

I

564

JKLMNOPQRS

summation of restriction fragment sizes from BamHI, EcoRI, and EcoRV digests, the sizes of mtDNAs were estimated to range from 72'9 kb for X phenotype to 80'4 kb for XIII phenotype (Table 2), The average size of the mtDNAs was 77'2 kb, The genome sizes of mtDNA obtained in the present study generally agree with those described previously in P, ostrearns by Katayose, Yui & Shishido (1989), Molecular size variation of mtDNAs, due to length mutations, is a common feature generally recognized in fungi (Turner, Earl & Greaves, 1982)_

Fig. 3. Nineteen representative mtDNA restriction fragment patterns produced from 34 Pleurotus ostreatus wild isolates with EcoRV digestion electrophoresed on 1 % agarose, The isolates associated with respective RFLP patterns of A to S are presented in Table 2, Far left lane: HindIII-digested lambda phage DNA.

to R), 19 EcoRI patterns (A to S), and 19 EcoRV patterns (A to S) (Figs, 1, 2 and 3), When all these RFLP patterns were combined, the mtDNAs from 34 wild isolates could be divided into 22 different phenotypes (Table 2), Based on the

Distance values calculated between all pairs of the 22 mtDNA,phenotypes of P_ ostreatus are shown in Table 3. In general. lower distance values were obtained among the mtDNA phenotypes of wild isolates from the same geographic region than among those from different regions, Based on the distance matrix, a dendrogram was constructed by the groupaverage analysis. The dendrogram groups the 22 mtDNA RFLP phenotypes into three major clusters: (1) ten Far Eastern Asian types of I to X; (2) seven European types of XI to XVII; (3) five U,S.A. types of XVIII to XXII. Results of the neighbourjoining analysis were very similar to that of the group-average analysis, although some different relationships within the

Table 2. MtDNA phenotypes and size among Pleurolus ostrealus wild isolates from different geographical origins Size (kb)

RFLP pattern MtDNA phenotype

II

III N V VI

VII VIII IX

X XI XII XIII

XIV XV XVI XVII

XVIII XIX

XX XXI XXII

Stock No, 161 552 1122 1196 1595 2883 2934 739 3195 3484 1600 1052 318 517 3476 M336 164 165 IS98 284 287 1183 1184 286 1185 1182 281 761 4073 4074 1187 1189 1205 1204

&rnHl

£CaRl

£CoRV

BamHI

£CaRl

EeoRV

Mean± standard deviation

A

A

A

81'0

78'1

77-1

78-7± 2'0

B

B

B

80'0

79'3

78'1

79'1 ±0-9

B

E

E

C D

D C

D C

80'0 78'6 74'5

79'8 78'9 73'9

77'S 78'8 74'S

79'1 ± 1'3 79'2±O'6 74'3±0-3

77-2 76'6 74'2 77'4 72'8 77'4 76'7 82'6

76'5 78'1 74'0 75'5 74'0 75'9 76'3 81'1

79'8 76-5 73'6 '76'8 72'1 74'9 76'6 77'7

77-8± 1'7 77'O±O'8 73'9±O'3 76-5±O'9 72'9 ± 0-9 76'O±I'2 76'S±O'7 80'4±2'S

73'7 76'S 76'S 76'S 78'4 80'2

75'7 76'6 75-3 75'7 80'S 77'6

74'8± 1'0 76'5±0-1 76-1±O'7 76-2±O'4 79'O± 1'2 78'6± 1'3

80-1

77'7

78'7 ± 1'2

79-3 79'7 S 80'5 79'3 S Average size mtDNA 77'2±2'O

76'6 70'0

7B-B± 1'1

F

F

G H I H M N L

G H I

M N N N

J

P

K K K

0 P

0 P

S P

M S

75'2 76'6 76'6 76'6 78'3 78'2

Q

R

R

78'3

R R

0

E F G H I

J

K L

See Fig, I (BamHI), 2 (EeoR!) and 3 (EcoRV),

Q

J N

K Q

L

79'1

±1'4

565

T. Matsumoto and Y. Fukumasa-Nakai Table 3. Distance matrix based on mtDNA phenotypes of Pleuratus aslrealus wild isolates MtDNA phenotype II I II III IV V VI VII VIII IX X XI XII XIII XIV XV XVI XVII XVIII XIX XX XXI XXII

III

IV

V

VI

0-000 0-211 0-000 0-351 0-288 0-000 0-440 0-378 0-221 0-000 0-324 0-284 0-429 0-465 0-000 0-417 0-380 0-216 0-200 0-412 0-000 0-479 0-361 0-307 0-184 0-420 0-178 0-472 0-352 0-351 0-227 0-529 0-306 0-4790-4170-413 0-421 0-507 0-370 0-514 0-420 0-389 0-288 0-545 0-314 0-697 0-692 0-647 0-623 0-742 0-606 0-701 0-667 0-681 0-6860-7460-701 0-606 0-631 0-618 0-652 0-677 0-606 0-742 0-738 0-750 0-723 0-655 0-710 0-662 0-719 0-672 0-706 0-705 0-662 0-656 0-714 0-667 0-701 0-700 0-656 0-631 0-688 0-672 0-706 0-672 0-662 0-881 0-909 0-884 0-886 0-841 0-881 0-824 0-851 0-8570'831 0-813 0-853 0-882 0-881 0-8290-803 0-813 0-824 0'821 0'848 0'855 0'829 0'746 0'851 0'818 0'846 0'853 0'826 0'742 0'848

VII

VIII

0-000 0-260 0-000 0-324 0-288 0-239 0-171 0-612 0-606 0-676 0-672 0-672 0-667 0-746 0-806 0-697 0-692 0-692 0-688 0-697 0-692 0-882 0_881 0-8260-824 0-797 0-794 0'853 0'821 0'851 0'818

IX

X

XI

XII

0-000 0-380 0-672 0-765 0-731 0-810 0-727 0-723 0-727 0-853 0-797 0'797 0'824 0'821

0-000 0-625 0-692 0-656 0-800 0-683 0-677 0-683 0-877 0-848 0'818 0'846 0'844

0-000 0-279 0-500 0-571 0-458 0-448 0-492 0-738 0-774 0'774 0'803

0-000 0-574 0-509 0-500 0-525 0-500 0-677 0-746 0'746 0'774 0'800 0'770

XIII

XIV

XV

XVI

XVII XVIII XIX

0-000 0-643 0-492 0-483 0-492 0-738 0-806 0'871 0'836 0'833

0-000 0-382 0-370 0-273 0-684 0-724 0'759 0'684 0'679

0-000 0-053 0-000 0-103 0'088 0-000 0-600 0-627 0-667 0-000 0-738 0-733 0'705 0-587 0'738 0'733 0'738 0'651 0'733 0'729 0'700 0'581 0'729 0'724 0'695 0'639

0-8

0-6

0-4 Distance

0·2

0.0

XXI

XXII

0-000 0'250 0'000 0'175 0'302 0'000 0'226 0'290 0'049 0'000

I II V III IV VI VII VIII X IX XI XII XIV XV XVI XVII XIII XVIII XIX XXI XXII XX

I II V III IV VI VII VIII X IX XI XII XIV XV XVI XVII XIII XVIII XIX XXI XXII XX 1-0

XX

0·8

0·6

0-4 Distance

0·2

0.0

Fig. 4. Dendrogram based on mtDNA phenotypes of Pleuraius as/rea/us analysed by group-average analysis.

Fig. 5. Dendrogram based on mtDNA phenotypes of Pleura/us as/realus analyzed by neighbour-joining analysis.

major clusters were observed (Fig. 5). The correlation between mtDNA divergence and geographical distribution in basidiomycetes and some other fungi has been recognized in worldwide samples of Cochliobolus heterostrophus Drechsler (Garber & Yoder, 1984), N. crassa (Taylor et al., 1986) and Aspergillus flavus Link: Fr. (Moody & Tyler, 1990). In recent work (Fukuda et al., 1994) on mtDNA polymorphisms of L. edades, a similar correlation was obtained. The present results similarly show that P. ostreatus mtDNA phenotypes can be separated into three distinct groups that are correlated with geographical

origin, except for the case of phenotype XX. This phenotype consisted of an American isolate and a European isolate. However, because the two isolates are shown to have the same mating factors (unpublished data) and the cultivation of P. astreatus is worldwide (Chang & Miles, 1991), it may be that either of the isolates in the phenotype XX is an escaped nonnative cultivar. In summary, from the present results, it may be pOSSible to accept that isolates of P_ astreatus from geographically different origins are genetically divergent, supporting the idea that there is little or no mitochondrial and

MtDNA RFLPs in Pieurotus ostreatus nuclear DNA gene flow between these geographically distant population groups in P. ostreatus. This suggests that geographically separated isolates are genetic resources for use inbreeding. Within the Japanese populations of L. edodes it has been reported (Fukuda et ai., 1994) that there was no apparent correlation between geographical distribution and mtDNA phenotypes, and that the dissimilarity values among these mtDNA phenotypes ranged from 0'026 to 0'3. P. ostreatus mtDNA phenotypes among Japanese isolates also show no significant correlation between geographical distribution and mtDNA types. This result suggests that sexual matings may often occur within the P. ostreatus Japanese population and such mating behavior may accelerate a random geographical distribution of the mtDNAs. Nevertheless, it is noteworthy that the degree of dissimilarity among the mtDNA phenotypes in Japanese populations of P. ostreatus showed larger values, ranging from 0'171 to 0'545 (Table 3), than that among the mtDNA phenotypes in Japanese populations of L. edodes (Fukuda et ai., 1994). Generally, mtDNA polymorphisms might be generated by loss or gain of a restrictionendonuclease recognition site in point mutation, or by arrangement of DNA sequences in insertions or deletions (Turner et ai., 1982). On the other hand, it has been considered that the recombination in mtDNA increases the degree of differences among restriction fragment patterns in plant and some fungi (e.g. Small, Suffolk & Leaver, 1989; Takano et ai., 1990). In our ongoing study on mtDNA transmission during sexual crosses of P. ostreatus, it has also been recognized that mtDNA recombination occurs in dikaryons only from the junction zone, although the mitochondrial genome was inherited uniparentally in every dikaryon from the colony periphery at both sides (Matsumoto & Fukumasa-Nakai, unpublished data). In conclusion, it may be considered that the occurrence of mtDNA recombination might be associated with the very large variations found among the RFLPs of mtDNA in P. ostreatus local populations. We are grateful to Dr N. Hiratsuka for his support and encouragement of this work. We also thank Dr D. S. Hibbett for his critical reading of the manuscript.

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(Accepted 5 December 1994)

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