Curr Microbiol DOI 10.1007/s00284-014-0608-6
Characterization of Single Spore Isolates of Agaricus bisporus (Lange) Imbach Using Conventional and Molecular Methods Manju Sharma • B. C. Suman • Dharmesh Gupta
Received: 17 February 2014 / Accepted: 26 March 2014 Ó Springer Science+Business Media New York 2014
Abstract Strains A-15, S11, S-140, and U3 of Agaricus bisporus (Lange) Imbach, were used as parent strains for raising single spore homokaryotic isolates. Out of total 1,642 single spore isolates, only 36 single spore isolates were homokaryons and exhibited slow mycelial growth rate (B2.0 mm/day) and appressed colony morphology. All these SSIs failed to produce pinheads in Petri plates even after 65 days of incubation, whereas the strandy slow growing SSIs along with parent strains were able to form the fructification in petriplates after 30 days. Out of 24, six ISSR primers, exhibited scorable bands. In the ISSR fingerprints, single spore isolates, homokaryons, lacked amplification products at multiple loci; they grow slowly and all of them had appressed types of colony morphology. The study revealed losses of ISSR polymorphic patterns in non-fertile homokaryotic single spore isolates compared to the parental control or fertile heterokaryotic single spore isolates.
Introduction Agaricus bisporus (Lange) Imbach, the white button mushroom, is the major edible mushroom species cultivated throughout the world. Major mushroom growing countries of the world are China, USA, Netherlands, M. Sharma (&) B. C. Suman D. Gupta Department of Plant Pathology, Dr Y S Parmar University of Horticulture & Forestry, Nauni, Solan 173230, HP, India e-mail: [email protected] B. C. Suman e-mail: [email protected] D. Gupta e-mail: [email protected]
Poland, etc., and its production is increasing at an annual rate of 6–7 %. In some developed countries of Europe and America, mushroom farming has attained the status of a high-tech industry with very high levels of mechanization and automation. In India, there has been slow, but significant increase in the growth of mushroom research and development. During the past five decades, there has been an increase in the cultivation of all types of temperate, subtropical, and tropical mushrooms due to varied agroclimate, abundance of agricultural wastes and manpower. Mushroom Research Laboratory, Department of Mycology and Plant Pathology, Dr. Yashwant Singh Parmar University of Horticulture and Forestry, Nauni, Solan (H.P.) was the sole supplier and nucleus for catering pure quality spawn requirements of mushroom growers throughout the country but now many organizations are supplying quality spawn. Breeding of A. bisporus is complicated because of its unusual secondary homothallic life-cycle [11, 2]. Instead of forming basidia with four monokaryotic spores, the majority of the basidia produce two spores each containing two nuclei of opposite mating types. These spores thus retain heterozygosity for most of the parental markers and after germination a fertile multinuclear heterokaryotic mycelium is formed. Such heterokaryotic offspring cannot be used directly in our crossing experiments. Approximately 5–7 % of the A. bisporus basidia produce three or four spores [13] and most of these will form homokaryotic mycelia upon germination. Appressed colony morphology, slow mycelial growth and non-fruiting are often used for identification of homokaryons as morphological markers [3]. Inter simple sequence repeat (ISSR), described as stretches of tandemly repeated short motifs, is widely dispersed in most nuclear eukaryotic genomes. They constitute an important source of genetic markers because in
123
M. Sharma et al.: Characterization of Single Spore Isolates
addition to their high polymorphism and wide dispersion in the genome, they are codominant, multi-allelic, and easily scored. Moreover, these markers based on the direct amplification of microsatellite region of DNA have been shown to be more reproducible, because they are longer primers [7]. So, the experiments were carried out to identify the homokaryons and heterokaryons of A. bisporus.
Materials and Methods Strains A-15, S 11, S-140, and U3 of Agaricus bisporus (Lange) Imbach, procured from Spawn Production Laboratory, Chambaghat and Directorate of Mushroom Research, Chambaghat, Solan (H.P.) were used for spore collection in the present study. Young and healthy fruit bodies of selected strains with veil still intact, but tightly stretched were selected for basidiospores collection. Stipe was cut at the soil level using pre-sterilized knife, washed with distilled water and then dipped in 0.1 % mercuric chloride solution for 30 s, washed in distilled sterilized water and dried. The fruit bodies were then mounted on a wire stand placed over the lower part of a Petri plate under a glass beaker previously sterilized in an oven at 180 °C for about 2 h and cooled. The sterilized glass beaker with disinfected fruit bodies was kept for 24–48 h in an incubator at 25 °C. The glass beaker and mushroom along with the wire stand were removed and sterilized lid was placed on the Petri plate to store the discharged spores at (2–4 °C) until used. The homokaryons or single spores were isolated using standard serial dilution technique. A loop full of spores from spore print was transferred aseptically with the help of inoculation needle and serially diluted in 10 ml sterilized distilled water to obtain serial dilution. From the 10-5 dilution, one ml of spore suspension containing about 10–15 spores was transferred with the help of sterilized pipettes over the surface of 2 % wheat extract agar medium in a 90 mm sterilized Petri plates. The Petri plates were incubated for 3 days at 28 °C. After 3 days, the Petri plates were turned upside down and some grains of actively growing mycelium of Agaricus bisporus of respective strains were placed in the upper lids for stimulation of single spore germination and then incubated at 28 °C for 10–15 days. For isolation of spores, the germinated single spores were marked with ink marker under inverted microscope. The marked germinated single spore was lifted with the help of a sterilized fine tip inoculation needle and transferred to malt extract agar medium in test tubes. These test tubes were incubated at 28 °C for 10 days for spore germination. The single spores so isolated were screened in vitro for identification of homokaryons, based on their
123
slow mycelial growth (B2.0 mm/day) and appressed types of colony morphologies on malt extract agar medium. In all, 1642 SSIs were raised from different parent strains SSIs with slow growing ‘‘appressed type’’ of mycelial growth was selected for further studies. A fruiting trial for putative homokaryotic single spore isolates, including parental strains, was carried out in polythene bags of 1 kg capacity, replicated five times. Spawn-run, case-run, primordial initiation, and fruit body maturation were done under controlled conditions. The DNA was extracted using GeneiPure TM Plant Genomic DNA Purification Kit following the protocol of the manufacturer. The quality of the obtained genomic DNA was determined by electrophoresis in 1 % agarose gel stained with ethidium bromide. ISSRs were detected by the use of 24 repeat anchored primers. The micro satellites primers pairs were synthesized from Operon Technologies INC., Alameda (CA, USA). The amplification reactions were carried out in 20 ll volume containing 50 ng DNA template, 35 ng primer, and 0.20 mM dNTPs, 2 mM MgCl2, 1 U Taq DNA polymerase and 19 PCR buffer. The PCR condition was standardized following parameters: initial denaturation at 94 °C for 3 min; denaturation at 94 °C for 45 s; annealing at 50 °C for 1 min; extension 72 °C for 1 min, and final extension at 72 °C for 5 min with 42 cycles of amplification. PCR was performed in triplicates using a thermocycler. For electrophoresis of amplified DNA, 1.0 % agarose gel was used. The molecular weight of bands was estimated using a DNA ruler 100 bp plus. The gel was run at 60 V for 100 min. The amplified DNA was viewed under the UV trans-illuminator, and the image was taken through gel documentation system. The PCR products from ISSR were analyzed by scoring qualitatively for the presence or absence of bands. Genetic similarities between the genotypes were measured by the Jaccard’s similarity coefficient based on the proportion of shared alleles using SIMQUAL sub programme of software NTSYS-pc version 2.02e [12]. The similarity index values found were used to generate a consensus tree using the unweighted pair group method analysis (UPGMA).
Results Isolation and Identification of Homokaryons Out of total 1,642 single spore isolates, 39 single spore isolates from the parent strains namely A-15 (9), S11(9), S-140(11), and U3(10) of Agaricus bisporus were found
0.16 3.28 CD (0.05) 0.14 2.80 CD (0.05) 2.24 CD (0.05)
SSIs single spore isolates
43.17 Check
0.11
Strandy
Strandy 0.09 1.82 CD (0.05)
2.39 50.09 Check Strandy 2.20 46.29 Check Strandy 2.16 45.36 Check Strandy
Appressed
17.77 A15-9
2.06
2.00 42.02 U3-9 Appressed 0.81 17.05 S140-9 Appressed 0.86 18.03 S11-9 Appressed
Strandy
38.44 A15-8
0.85
0.89 18.66 U3-8 Strandy 1.84 38.59 S140-8 Appressed 0.92 19.37 S11-8 Strandy
Appressed
21.21 A15-7
1.83
1.63 34.22 U3-7 Appressed 0.88 18.44 S140-7 Appressed 1.12 23.56 S11-7 Appressed
39.07 A15-6
1.01
Appressed 0.95
1.14 24.00
19.91 U3-5
U3-6 Strandy
Appressed 1.13
1.87 39.32
23.80 S140-5
S140-6 Strandy
Appressed 0.69
1.81 38.03
14.36 S11-5
S11-6 Strandy
Strandy 36.92 A15-5
1.86
17.21
1.76
Appressed
Appressed 0.85 17.88 U3-4 Appressed 0.95 19.91 S140-4 Appressed 0.90 19.00 S11-4 Appressed
Appressed
A15-4
0.82
0.86 18.03 U3-3 Strandy 1.793 37.70 S140-3 Appressed 0.79 16.66 S11-3 Appressed 18.64 A15-3
0.89
Appressed 0.90
0.77 16.21
19.00 U3-1
U3-2 Appressed
Appressed 0.92
0.97 20.25
19.24 S140-1
S140-2 Appressed
Strandy 1.81
1.05 22.05
37.85 S11-1
S11-2 Appressed
Appressed
19.68 A15-2
0.94
18.17 A15-1
0.87
Colony morphology Av. growth rate (mm/ day) Av. colony diameter (mm) in 21 days SSIs Av. colony diameter (mm) in 21 days SSIs
Av. growth rate (mm/ day)
Colony morphology
SSIs
Av. colony diameter (mm) in 21 days
Av. growth rate mm/ day)
Colony morphology
SSIs
Av. colony diameter (mm) in 21 days
Av. growth rate (mm/ day)
Colony morphology
U3 S-140 S11 A-15
Table 1 Linear mycelial growth rate and types of colony morphology of nine putative single spore isolates and parent strains, A-15, S11, S-140, and U3 of Agaricus bisporus on wheat extract agar medium
M. Sharma et al.: Characterization of Single Spore Isolates
slow growing (B2.0 mm/day) and out of those only 36 single spore isolates (nine from each parent strains to maintain uniform pattern) which were supposed to be homokaryons on the basis of slow mycelial growth rate and appressed types colony morphology were selected for future studies. The radial growth of homokaryotic single spore isolates including parent strains was measured after 21 days of incubation at 25 °C temperature, on wheat extract agar medium. It is clear from the data given in Table 1 that among the SSIs isolated from A-15 strains of A. bisporus, A15-4 showed significantly minimum mycelial growth rate (0.82 mm/day) followed by A15-9 (0.85 mm/day), A15-1 (0.87 mm/day), A15-3 (0.89 mm/day), A15-2 (0.94 mm/ day), and A15-7 (1.01 mm/day) on wheat extract agar medium with appressed types of colony morphology. The maximum mycelial growth rate (2.06 mm/day) was observed in parental control i.e., A-15, followed by SSIs A15-6 (1.86 mm/day), A15-8 (1.83 mm/day), and A15-5 (1.76 mm/day) on wheat extract agar medium with strandy types of colony morphology. Similarly, data on average mycelial growth rate (mm/ day) and colony morphology of selected SSIs from S11, S-140, and U3 strains of A. bisporus also recorded significantly minimum mycelial growth by S11-5 (0.69 mm/ day) followed by S11-3 (0.79 mm/day), S11-9 (0.86 mm/ day), S11-4 (0.90 mm/day), S11-8 (0.92 mm/day), and S11-2(1.05 mm/day) with appressed types colony morphology on wheat extract agar medium. Strain S11 supported maximum radial growth (2.16 mm/day) followed by S11-1 and S11-6 (1.81 mm/day) with strandy types colony morphology on wheat extract agar medium. The SSIs selected from parent strain S-140, clearly show that S140-9 had significant minimum mycelial growth rate i.e., 0.81 mm/day followed by S140-7 (0.88 mm/day), S140-1 (0.92 mm/day), S140-4 (0.95 mm/ day), S140-2 (0.97 mm/day), and S140-5 (1.13 mm/day) on wheat extract agar medium with appressed types colony morphology. However, maximum mycelial growth was recorded by parent strain i.e., S-140 (2.20 mm/day) followed by S140-6 (1.87 mm/day), S140-8 (1.84 mm/day), and S140-3 (1.79 mm/day) on wheat extract agar medium with strandy types colony morphology. In case of strain U3 of A. bisporus significantly minimum mycelial growth was recorded by homokaryon U3-2 (0.77 mm/day) followed by U3-4 (0.85 mm/day), U3-3 (0.86 mm/day), U3-8 (0.89 mm/day), U3-1 (0.90 mm/day), U3-5 (0.95 mm/ day), and U3-6 (1.14 mm/day) with appressed types colony morphology on wheat extract agar medium. Significant maximum radial growth was showed by strain U3 (2.39 mm/day) followed by U3-9 (2.00 mm/day) and U3-7 (1.63 mm/day) on wheat extract agar medium with strandy types colony morphology.
123
M. Sharma et al.: Characterization of Single Spore Isolates Table 2 Spawn run and number of fruit bodies of nine putative single spore isolates and parent strains, A-15, S11, S-140 and U3 of Agaricus bisporus A-15
S11
S-140
U3
In 2 kg substrate
In 2 kg substrate
In 2 kg substrate
In 1 kg substrate
SSIs
Spawn run
Average no. of fruit bodies
SSIs
Spawn run
Average no. of fruit bodies
SSIs
Spawn run
Average no. of fruit bodies
SSIs
In 2 kg substrate spawn Run
Average no. of fruit bodies
A15-1
?
0.00
S11-1
??
1.33
S140-1
?
0.00
U3-1
?
0.00
(1.00)a A15-2
?
0.00
(1.52)a S11-2
?
(1.00)
0.00
(1.00)a S140-2
?
(1.00)
0.00
(1.00)a U3-2
?
0.00
(1.00)
(1.00)
A15-3
?
0.00
S11-3
?
0.00
S140-3
??
2.00
U3-3
?
0.00
A15-4
?
(1.00) 0.00
S11-4
?
(1.00) 0.00
S140-4
?
(1.72) 0.00
U3-4
?
(1.00) 0.00
A15-5
??
2.00
S11-5
?
0.00
S140-5
?
0.00
U3-5
?
0.00
(1.00)
(1.00)
(1.72) A15-6
??
1.67
(1.00) S11-6
??
(1.63) A15-7
?
0.00
A15-8
??
2.33
A15-9
?
0.00
Check
???
6.00
S11-7
?
S11-8
?
S140-6
??
0.00 0.00
S11-9
?
0.00
Check
???
5.67
S140-7
?
S140-8
??
4.33
S140-9
?
0.00
Check
???
7.00
0.32 0.15
0.00
0.00
U3-7
??
2.00
U3-8
?
0.00
U3-9
??
6.67
Check
???
8.33
(1.00) (1.72) (1.00)
(1.00)
(2.55) CD (0.05) SE
?
(2.28)
(1.00)
(2.64)
U3-6
(1.00)
(1.00)
(1.00)
4.00
(1.00)
(2.23)
(1.00)
(1.82)
0.23 0.11
1.33
(1.00)
(1.00)
(1.52)
(1.00)
CD (0.05) SE
(1.00)
(2.76)
(2.83) CD (0.05) SE
0.34 0.16
(3.05) CD (0.05) SE
0.20 0.10
Spawn run process in substrate: ?, slow, ??, moderate; ???, fast SSIs single spore isolates a
Figures in parenthesis are square root transformed values
Fruiting Trial Nine putative homokaryotic SSIs selected from each parental strains namely A-15, S11, S-140, and U3 of Agaricus bisporus were tested for fructification in Petri plates containing compost extract agar medium, and the fruiting experiment was also done in polythene bags with 1 kg compost in each bag, in a replicated trial. The spawn run in these SSIs was very slow at incubation temperature of 25 °C and these were not able to colonize the entire Petri plate even after a period of 38 days of incubation in all the replication and were then cased to observe the fructification. All these SSIs with appressed types colony morphology were not able to produce pinheads in Petri plates even after 65 days of incubation, whereas, the strandy slow growing SSIs along with parent strains were able to form the fructification in petriplates after 30 days.
123
The spawn run in these homokaryons during fruiting trial in compost bags was very slow at an incubation temperature of 25 °C and was not completed even after 65 days of incubation, these bags were then cased 65 days after spawning to observe fructification. The number of fruit bodies produced was recorded up to 3 weeks of production. All the slow growing SSIs with appressed types colony morphology did not produce fruit bodies in any replication. However, the strandy slow growing single spore isolates produced fruiting bodies in all the replication; the average number of fruiting bodies produced is shown in Table 2. Slow growing single spore isolates that produced normal fruit bodies similar to those of the parental heterokaryotic control also showed identical banding pattern in ISSR analysis with those of parental heterokaryotic control.
M. Sharma et al.: Characterization of Single Spore Isolates Table 3 Single spore isolates wise variability analysis for strain A-15 of Agaricus bisporus SSIs
Average growth rate (mm/day)a (±SE)
95 % Confidence interval
Colony morphology
In 2 kg substrate Spawn runb
Average no. of fruit bodiesc (±SE)
95 % confidence interval
A15-1
0.87 ± 0.01
0.87 ± 0.02
Appressed
?
±
±
A15-2
0.94 ± 0.02
0.94 ± 0.04
Appressed
?
±
±
A15-3
0.89 ± 0.02
0.89 ± 0.04
Appressed
?
±
±
A15-4
0.82 ± 0.01
0.82 ± 0.02
Appressed
?
±
±
A15-5
1.76 ± 0.01
1.76 ± 0.02
Strandy
??
2.00 ± 0.22
2.00 ± 0.47
A15-6
1.86 ± 0.01
1.86 ± 0.02
Strandy
??
1.67 ± 0.13
1.67 ± 0.28
A15-7
1.01 ± 0.01
1.01 ± 0.02
Appressed
?
±
±
A15-8
1.83 ± 0.01
1.83 ± 0.02
Strandy
??
2.33 ± 0.13
2.33 ± 0.28
A15-9
0.85 ± 0.02
0.85 ± 0.04
Appressed
?
±
±
C
2.06 ± 0.01
2.06 ± 0.02
Strandy
???
6.00 ± 0.22
6.00 ± 0.47
a
Average growth rate calculated from the results of three separate experiments with five replications each on wheat extract agar medium incubation at 25 °C
b
Spawn run process in substrate: ?, slow; ??, moderate; ???, fast; ±, no primordial like structure; C, parental control
c
Average numbers of fruit bodies calculated from the results of one set of experiment with three replications
Table 4 Single spore isolates wise variability analysis for strain S11 of Agaricus bisporus SSIs
Average growth rate (mm/day)a (±SE)
95 % confidence interval
Colony morphology
In 2 kg substrate Spawn runb
Average no. of fruit bodiesc (± SE)
95 % confidence interval
S11-1
1.81 ± 0.03
1.81 ± 0.06
Strandy
??
1.33 ± 0.13
1.33 ± 0.28
S11-2
1.05 ± 0.03
1.05 ± 0.06
Appressed
?
±
±
S11-3
0.79 ± 0.02
0.79 ± 0.04
Appressed
?
±
±
S11-4
0.90 ± 0.01
0.90 ± 0.02
Appressed
?
±
±
S11-5
0.69 ± 0.01
0.69 ± 0.02
Appressed
?
±
±
S11-6
1.81 ± 0.01
1.81 ± 0.02
Strandy
??
1.33 ± 0.13
1.33 ± 0.28
S11-7
1.12 ± 0.01
1.12 ± 0.02
Appressed
?
±
±
S11-8
0.92 ± 0.01
0.92 ± 0.02
Appressed
?
±
±
S11-9
0.86 ± 0.02
0.86 ± 0.04
Appressed
?
±
±
C
2.16 ± 0.03
2.16 ± 0.06
Strandy
???
5.67 ± 0.63
5.67 ± 1.34
a
Average growth rate calculated from the results of three separate experiments with five replications each on wheat extract agar medium incubation at 25 °C
b
Spawn run process in substrate: ?, slow; ??, moderate; ???, fast; ±, no primordial like structure; C, parental control
c
Average numbers of fruit bodies calculated from the results of one set of experiment with three replications
Slow spawn run and no fruit bodies production were recorded in all SSIs whose growth rate was (\1.2 mm/day), and these were statistically at par with each other. The maximum number of fruit bodies was formed by heterokaryotic parental control i.e., A-15, S11, S-140, and U3 followed by the slow growing single spore isolates, from each parents with strandy types of colony morphology.
Perusal of the data given in Table 3 indicated that SSIs with appressed types of colony morphology of strain A-15 with growth rate B1.1 mm/day exhibited slow spawn run and failed to complete the spawn running even after 65 days of incubation. Data recorded in Table 4 showed the SSIs wise variability analysis for strain S11 of Agaricus bisporus. It is
123
M. Sharma et al.: Characterization of Single Spore Isolates Table 5 Single spore isolates wise variability analysis for strain S-140 of Agaricus bisporus SSIs
Average growth rate (mm/day)a (±SE)
95 % confidence interval
Colony morphology
In 2 kg substrate Spawn runb
Average no. of fruit bodiesc (±SE)
95 % confidence interval
S140-1
0.92 ± 0.01
0.92 ± 0.02
Appressed
?
±
±
S140-2
0.97 ± 0.03
0.97 ± 0.06
Appressed
?
±
±
S140-3
1.79 ± 0.02
1.79 ± 0.04
Strandy
??
2.00 ± 0.22
2.00 ± 0.47
S140-4
0.95 ± 0.01
0.95 ± 0.02
Appressed
?
±
±
S140-5
1.13 ± 0.03
1.13 ± 0.06
Appressed
?
±
±
S140-6
1.87 ± 0.03
1.87 ± 0.06
Strandy
??
4.00 ± 0.22
4.00 ± 0.47
S140-7
0.88 ± 0.01
0.88 ± 0.02
Appressed
?
±
±
S140-8
1.84 ± 0.02
1.84 ± 0.04
Strandy
??
4.33 ± 0.45
4.33 ± 0.96
S140-9
0.81 ± 0.03
0.81 ± 0.06
Appressed
?
±
±
C
2.20 ± 0.02
2.20 ± 0.04
Strandy
???
7.00 ± 0.22
7.00 ± 0.22
a
Average growth rate calculated from the results of three separate experiments with five replications each on wheat extract agar medium incubation at 25 °C
b
Spawn run process in substrate: ?, slow; ??, moderate; ???, fast; ±, no primordial like structure; C, parental control
c
Average numbers of fruit bodies calculated from the results of one set of experiment with three replications
Table 6 Single spore isolates wise variability analysis for strain U3 of Agaricus bisporus SSIs
Average growth rate (mm/day)a (±SE)
95 % confidence interval
Colony morphology
In 2 kg substrate Spawn runb
Average no. of fruit bodiesc (±SE)
95 % confidence interval
U3-1
0.90 ± 0.01
0.90 ± 0.02
Appressed
?
±
±
U3-2
0.77 ± 0.01
0.77 ± 0.02
Appressed
?
±
±
U3-3
0.86 ± 0.01
0.86 ± 0.02
Appressed
?
±
±
U3-4
0.85 ± 0.02
0.85 ± 0.04
Appressed
?
±
±
U3-5
0.95 ± 0.01
0.95 ± 0.02
Appressed
?
±
±
U3-6
1.14 ± 0.01
1.14 ± 0.02
Appressed
?
±
±
U3-7
1.63 ± 0.01
1.63 ± 0.02
Strandy
??
2.00 ± 0.22
2.00 ± 0.47
U3-8
0.89 ± 0.02
0.89 ± 0.04
Appressed
?
±
±
U3-9
2.00 ± 0.01
2.00 ± 0.02
Strandy
??
6.67 ± 0.25
6.67 ± 0.53
C
2.39 ± 0.01
2.39 ± 0.02
Strandy
???
8.33 ± 0.13
8.33 ± 0.28
a
Average growth rate calculated from the results of three separate experiments with five replications each on wheat extract agar medium incubation at 25 °C
b
Spawn run process in substrate: ?, slow; ??, moderate; ???, fast; ±, no primordial like structure; C, parental control
c
Average numbers of fruit bodies calculated from the results of one set of experiment with three replications
clear from the data that the homokaryons having growth rate B1.12 mm/day were not able to form primordia. It is obvious from the data recorded in Table 5 that the single spore isolates of strain S-140 which showed B1.13 growth mm/day were infertile or the monokaryons. Similarly from the data recorded in Table 6, it is clear that the appressed type of colony morphology of strain U3
123
having growth rate B1.14 mm/day was not able to complete the spawn run even after 65 days of incubation and did not produce fruit bodies. All single spore isolates with appressed types of colony morphologies did not produce fruit bodies in any of the replication. However, the strandy slow growing single spore isolates, produced fruiting bodies in each replication.
49.02 25 26 44.90 22
Fig. 1 ISSR fingerprints observed with primers P3 in A-15 strains of Agaricus bisporus
49 43.40
Molecular Characterization of Single Spore Isolates Using ISSR (Inter Simple Sequence Repeat) Marker Systems Extraction of Genomic DNA
Total
MB monomorphic bands, PB polymorphic bands, P polymorphism
52
30
22
42.30
53
30
23
27
62.50 33.33 8 9 55.56 50.00 56 54 (CT)8RG (CAAGG)3 P31 P39
9 10
5 6
4 4
44.44 40.00
9 10
4 5
5 5
3 6
5 3
51
250–2,500
200–1,800 250–2,500 50.00 44.44 4 4 4 5
100–1,500
8 44.44 55 (GA)8C P30
8
4
4
50.00
9
5
4
5
37.50
8 9
57.14 4 3
125–2,500
3
7
50.00
50.00 5
4 4
5
28.57
8
50.00
2 7 25.00 2
5
8 33.33 3
6 8
9 44.44
28.57 2
4 5 9
5 53 (AG)8YC P22
55 (CT)8AGA P8
7
6
9 50.00 52 (GA)8T P3
9
5
4
44.44
8
4
4
4
4
10
250–2,500 44.44 4 5 9 55.56 5 4
Total bands Total bands MB
PB
P Total bands
MB
PB
P
Total bands
P PB MB
U3 S-140 S11 A-15 Tm (°C) Sequence (50 –30 ) Primers
Table 7 ISSR analysis for selected homokaryotic single spore isolates from four parent strains, A-15, S11, S-140 and U3 of Agaricus bisporus
MB
PB
P
Size range (bp)
M. Sharma et al.: Characterization of Single Spore Isolates
Out of 24, six primers were selected, generated excellent results and were used in this investigation (Table 7). Results clearly indicate that each primer gave rich and clear bands ranging from 100 to 2,500 bp, and their combination was sufficient to differentiate all of the tested homokaryons. The six chosen primers generated a total of 52 bands (A-15), 53 bands (S11), 49 bands (S-140) and 51 bands (U3) of high intensity and good separation. These were selected as markers, of which 42.30 % (A-15), 43.40 % (S11), 44.90 % (S-140), and 49.02 % (U3) were polymorphic. The results obtained for the same single spore isolates gave identical ISSR fingerprints after electrophoresis. It could be noted that the genome of A. bisporus has more microsatellite sequences related to these six primers than to the other primers. Primers P3 (A-15), P39(S11), P30(S140), and P8(U3) strains of Agaricus bisporus were proved to be the best due to the sharpness of banding patterns. Representative ISSR fingerprints obtained with primers P3 in A-15 and P39 in S11 strains of A. bisporus are shown in Figs. 1 and 2. Similarly, ISSR fingerprints observed with primers P30 in S-140 and P8 in U3 strains of A. bisporus are shown in Figs. 3 and 4. In the ISSR fingerprints of nine putative single spore isolates from A-15 strains with P3 primer, six homokaryons (A15-1, A15-2, A15-3, A15-4, A15-7, and A15-8) lacked amplification products at multiple loci; they grow
123
M. Sharma et al.: Characterization of Single Spore Isolates
Fig. 2 ISSR fingerprints observed with primers P39 in S-11 strains of Agaricus bisporus
Fig. 4 ISSR fingerprints observed with primers P8 in U3 and strains of Agaricus bisporus
(U3-7, U3-9) displaying amplification products at all loci are likely heterokaryons that received non-sister postmeiotic nuclei. The study revealed losses of ISSR polymorphic patterns in non-fertile homokaryotic single spore isolates compared to the parental control or fertile heterokaryotic single spore isolates. ISSR Data Analysis
Fig. 3 ISSR fingerprints observed with primers P30 in S-140 strains of Agaricus bisporus
slowly and all of them had appressed types of colony morphology. These may either be homokaryons derived from uninucleate basidiospores or heterokaryons composed of post-meiotic sister nuclei. The other single spore isolates (A15-5, A15-6, and A15-8) displaying amplification products at all loci are likely heterokaryons that received nonsister post-meiotic nuclei. Similar results were recorded in other three parental strains i.e., S11, S-140 and U3 of Agaricus bisporus in which homokaryons; (S11-2, S11-3, S11-4, S11-5 S11-7, S11-8, S11-9), (S140-1, S140-2, S140-4, S140-5, S140-7, S140-9), and (U3-1, U3-2, U3-3, U3-4, U3-5, U3-6, U3-8) did not produce amplification products at multiple loci. However, other single spore isolates; (S11-1, S11-6), (S-140-3, S-140-6, S-140-8) and
123
ISSR data was analyzed as described in Materials and Methods considering all the bands amplified with six primers.The first major bifurcation of the dendrogram (Fig. 5) divided the single spore isolates into two major clusters consisted of the homokaryons of A-15 and S140 strains of A. bisporus. However, the second cluster was further divided into six sub-clusters, included homokaryons, heterokaryons, and control of all the parental strains. Therefore, it is apparent from the data recorded by fruiting trial or by ISSR characterization of the single spore isolates that from A-15 strain of Agaricus bisporus, six single spore isolates were monokaryons. Similarly from strains S11, S-140, and U3, seven, six, and seven single spore isolates were homokaryons, respectively. Selected homokaryons were used in further studies. In particular, ISSR markers can be highly variable with in a species and reveal many more polymorphic fragments by utilizing longer primers that allow more stringent annealing temperature. It was for this reason that we adopted the technology in order to generate more specific and easily recognizable bands for identifying homokaryons from A-15, S11, S-140, and U3 strains of A. bisporus. In the ISSR fingerprints of putative single spore isolates, homokaryons lacked amplification products at multiple loci; these grows slowly and all of them had appressed types of
M. Sharma et al.: Characterization of Single Spore Isolates Fig. 5 Consensus cluster analysis of homokaryotic single spores of parental strains of Agaricus bisporus using ISSR fingerprints data from a Jaccard’s similarity matrix and the UPGMA method; the numbers assigned at the right margin of the tree branches representing homokaryotic single spore isolates and the letter C for parental heterokaryotic control
colony morphology. The study revealed losses of ISSR polymorphic patterns in nonfertile homokaryotic single spore isolates, compared to the parent strains or fertile heterokaryotic single spore isolates. ISSR data indicated that each primer gave rich and clear bands ranging from 100 to 2,500 bp. Primers P3 in A-15, P39 in S11, P30 in S-140, and P8 in U3 strains of Agaricus bisporus were proved to be the best due to the sharpness of banding patterns. These results also confirmed the identity of these single spore isolates to be homokaryons.
Discussion Button mushroom (Agaricus bisporus) is an economically important crop. However, most of the cultivated strains in the world were derived from or similar to the ‘‘hybrid’’ strains HU1 and HU3, thereby leaving the mushroom industry with a crop that was genetically very limited and had a high risk of sensitivity to disease. Most of the spores of A. bisporus produced heterokaryotic progenies. Genetic analysis and selective breeding of Agaricus bisporus, however, require the isolation of homokaryons from the heterokaryotic stocks. However, homokaryons are difficult to obtain by conventional basidiospore isolation from A. bisporus strains because of secondary homothallic life cycle. In the present study, from parent strains A-15, S 11, S-140 and U3 of Agaricus bisporus (Lange) Imbach, 1,642 single spore isolates were raised and characterized on wheat extract agar medium. These findings are in conformity with Jianping et al. [4]. All single spore isolates with
appressed types of colony morphologies did not produce fruit bodies in any of the replication. However, the strandy slow growing single spore isolates produced fruiting bodies in each replication. These results are in the agreement with Horgen and Anderson [3], Yadav [14]. In particular, ISSR markers can be highly variable with in a species and reveal many more polymorphic fragments by utilizing longer primers that allow more stringent annealing temperature. It was for this reason that we adopted the technology in order to generate more specific and easily recognizable bands for identifying homokaryons from A-15, S11, S-140, and U3 strains of A. bisporus. In the ISSR fingerprints of putative single spore isolates, homokaryons lacked amplification products at multiple loci; they grow slowly and all of them had appressed types of colony morphology. The study revealed losses of ISSR polymorphic patterns in nonfertile homokaryotic single spore isolates, compared to the parent strains or fertile heterokaryotic single spore isolates. These results also confirmed the identity of these single spore isolates to be homokaryons. ISSR PCR has also been used previously to detect the homokaryons using the same primers derived from stretches of tandemly repeated short motifs [8–10]. However, Khush et al. [6], Challen et al. [1], Yan and Jiang [15], Kavousi et al. [4, 5] used RAPD markers for the molecular characterization of homokaryons. Khush et al. [6] found that homokaryons carried a subset of the RAPD markers found in the parental heterokaryons. Challen et al. [1] observed that homokaryotic progeny from the secondarily homothallic A. bisporus was less variable (12 %) with fewer segregating loci than the known heterothallic
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M. Sharma et al.: Characterization of Single Spore Isolates
species, A. bitorquis (50 %) and A. nivescens (32 %). Yan and Jiang [15] reported that RAPD-based molecular genetic distance of the monokaryotic parents might be a suitable criterion for selecting monokaryotic parents of Stropharia rugoso-annulata and predicting the performance of hybrids in mushroom cross breeding. Kavousi et al. [5], reported that RAPD markers could discriminate homokaryons from heterokaryons, based on number of bands generated. The number of bands in homokaryons was significantly less than the heterokaryons.
6.
7.
8.
9.
References 1. Challen MP, Kerrigan RW, Callac P (2003) A phylogenetic reconstruction and emendation of Agaricus section Duploannulatae. Mycologia 95:61–73 2. Elliott TJ (1985) The genetics and breeding of species of Agaricus. Bio tech culti mush. John Wiley and Sons, Chichestar, pp 111–139 3. Horgen PA, Anderson JB (1989) The germination of basidiospores from commercial and wild collected isolates of Agaricus bisporus. Can J Microbiol 35:492–498 4. Jianping X, Kerrigan W, Paul A, Horgen PA, Anderson JB (1993) Localization of the mating type gene in Agaricus bisporus. Appl Environ Microbiol 59(9):3044–3049 5. Kavousi HR, Farsi M, Shahriari F (2008) Comparison of RAPD markers and morphological characters in identification of
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10.
11. 12. 13.
14.
15.
homokaryon isolates in white button mushroom, Agaricus bisporus. Pak J Biol Sci 11(4):1771–1778 Khush RS, Becker E, Wach M (1992) DNA amplification polymorphisim of the cultivated mushroom Agaricus bisporus. Appl Environ Microbiol 57:1735–1739 Malekzadeh K, Shahri BJM, Mohsenifard E (2011) Use of ISSR markers for strain identification in the button mushroom, Agaricus bisporus. In: Proceedings of the 7th international conference on mushroom biology and mushroom products (ICMBMP7), pp 30–34 Nazrul MI, Yinbing B (2009) ISSR as new markers for identification of homokaryotic protoclones of Agaricus bisporus. Curr Microbiol 60:92–98 Nazrul MI, Yinbing B (2011) Differentiation of homokaryons and heterokaryons of Agaricus bisporus with inter-simple sequence repeat markers. Microbiol Res 166(3):226–236 Nazrul MI, Lin FX, Yinbing B (2010) Screening of homokaryotic protoclones of Agaricus bisporus (J. Lge) Imbach by colony characters and ISSR markers. Bangladesh J Bot 39(1):119–122 Raper CA, Raper JR, Miller RE (1972) Genetic analysis of the life cycle of Agaricus bisporus. Mycologia 64:1088–1117 Rohlf FJ (1993) NTSYS-P-numerical taxonomy and multivariate analysis system, version 2.0. Exeter Software, New York Summerbell RC, Castle AJ, Horgen PA, Anderson JB (1989) Inheritance of restriction fragment length polymorphism in Agaricus brunnescens. Genetics 123:293–300 Yadav MC (2003) Molecular breeding for development of genetically improved strain and hybrids of Agaricus bisporus. Current vistas in mushroom biology and production. Nirmal Vijay Press, New Delhi, pp 261–274 Yan PS, Jiang JH (2005) Preliminary research of the RAPD molecular marker-assisted breeding of the edible Basidiomycete Stropharia rugoso-annulata. J Microbiol Biotechnol 21:559–563
Characterization of Single Spore Isolates of Agaricus bisporus (Lange) Imbach Using Conventional and Molecular Methods Manju Sharma • B. C. Suman • Dharmesh Gupta
Received: 17 February 2014 / Accepted: 26 March 2014 Ó Springer Science+Business Media New York 2014
Abstract Strains A-15, S11, S-140, and U3 of Agaricus bisporus (Lange) Imbach, were used as parent strains for raising single spore homokaryotic isolates. Out of total 1,642 single spore isolates, only 36 single spore isolates were homokaryons and exhibited slow mycelial growth rate (B2.0 mm/day) and appressed colony morphology. All these SSIs failed to produce pinheads in Petri plates even after 65 days of incubation, whereas the strandy slow growing SSIs along with parent strains were able to form the fructification in petriplates after 30 days. Out of 24, six ISSR primers, exhibited scorable bands. In the ISSR fingerprints, single spore isolates, homokaryons, lacked amplification products at multiple loci; they grow slowly and all of them had appressed types of colony morphology. The study revealed losses of ISSR polymorphic patterns in non-fertile homokaryotic single spore isolates compared to the parental control or fertile heterokaryotic single spore isolates.
Introduction Agaricus bisporus (Lange) Imbach, the white button mushroom, is the major edible mushroom species cultivated throughout the world. Major mushroom growing countries of the world are China, USA, Netherlands, M. Sharma (&) B. C. Suman D. Gupta Department of Plant Pathology, Dr Y S Parmar University of Horticulture & Forestry, Nauni, Solan 173230, HP, India e-mail: [email protected] B. C. Suman e-mail: [email protected] D. Gupta e-mail: [email protected]
Poland, etc., and its production is increasing at an annual rate of 6–7 %. In some developed countries of Europe and America, mushroom farming has attained the status of a high-tech industry with very high levels of mechanization and automation. In India, there has been slow, but significant increase in the growth of mushroom research and development. During the past five decades, there has been an increase in the cultivation of all types of temperate, subtropical, and tropical mushrooms due to varied agroclimate, abundance of agricultural wastes and manpower. Mushroom Research Laboratory, Department of Mycology and Plant Pathology, Dr. Yashwant Singh Parmar University of Horticulture and Forestry, Nauni, Solan (H.P.) was the sole supplier and nucleus for catering pure quality spawn requirements of mushroom growers throughout the country but now many organizations are supplying quality spawn. Breeding of A. bisporus is complicated because of its unusual secondary homothallic life-cycle [11, 2]. Instead of forming basidia with four monokaryotic spores, the majority of the basidia produce two spores each containing two nuclei of opposite mating types. These spores thus retain heterozygosity for most of the parental markers and after germination a fertile multinuclear heterokaryotic mycelium is formed. Such heterokaryotic offspring cannot be used directly in our crossing experiments. Approximately 5–7 % of the A. bisporus basidia produce three or four spores [13] and most of these will form homokaryotic mycelia upon germination. Appressed colony morphology, slow mycelial growth and non-fruiting are often used for identification of homokaryons as morphological markers [3]. Inter simple sequence repeat (ISSR), described as stretches of tandemly repeated short motifs, is widely dispersed in most nuclear eukaryotic genomes. They constitute an important source of genetic markers because in
123
M. Sharma et al.: Characterization of Single Spore Isolates
addition to their high polymorphism and wide dispersion in the genome, they are codominant, multi-allelic, and easily scored. Moreover, these markers based on the direct amplification of microsatellite region of DNA have been shown to be more reproducible, because they are longer primers [7]. So, the experiments were carried out to identify the homokaryons and heterokaryons of A. bisporus.
Materials and Methods Strains A-15, S 11, S-140, and U3 of Agaricus bisporus (Lange) Imbach, procured from Spawn Production Laboratory, Chambaghat and Directorate of Mushroom Research, Chambaghat, Solan (H.P.) were used for spore collection in the present study. Young and healthy fruit bodies of selected strains with veil still intact, but tightly stretched were selected for basidiospores collection. Stipe was cut at the soil level using pre-sterilized knife, washed with distilled water and then dipped in 0.1 % mercuric chloride solution for 30 s, washed in distilled sterilized water and dried. The fruit bodies were then mounted on a wire stand placed over the lower part of a Petri plate under a glass beaker previously sterilized in an oven at 180 °C for about 2 h and cooled. The sterilized glass beaker with disinfected fruit bodies was kept for 24–48 h in an incubator at 25 °C. The glass beaker and mushroom along with the wire stand were removed and sterilized lid was placed on the Petri plate to store the discharged spores at (2–4 °C) until used. The homokaryons or single spores were isolated using standard serial dilution technique. A loop full of spores from spore print was transferred aseptically with the help of inoculation needle and serially diluted in 10 ml sterilized distilled water to obtain serial dilution. From the 10-5 dilution, one ml of spore suspension containing about 10–15 spores was transferred with the help of sterilized pipettes over the surface of 2 % wheat extract agar medium in a 90 mm sterilized Petri plates. The Petri plates were incubated for 3 days at 28 °C. After 3 days, the Petri plates were turned upside down and some grains of actively growing mycelium of Agaricus bisporus of respective strains were placed in the upper lids for stimulation of single spore germination and then incubated at 28 °C for 10–15 days. For isolation of spores, the germinated single spores were marked with ink marker under inverted microscope. The marked germinated single spore was lifted with the help of a sterilized fine tip inoculation needle and transferred to malt extract agar medium in test tubes. These test tubes were incubated at 28 °C for 10 days for spore germination. The single spores so isolated were screened in vitro for identification of homokaryons, based on their
123
slow mycelial growth (B2.0 mm/day) and appressed types of colony morphologies on malt extract agar medium. In all, 1642 SSIs were raised from different parent strains SSIs with slow growing ‘‘appressed type’’ of mycelial growth was selected for further studies. A fruiting trial for putative homokaryotic single spore isolates, including parental strains, was carried out in polythene bags of 1 kg capacity, replicated five times. Spawn-run, case-run, primordial initiation, and fruit body maturation were done under controlled conditions. The DNA was extracted using GeneiPure TM Plant Genomic DNA Purification Kit following the protocol of the manufacturer. The quality of the obtained genomic DNA was determined by electrophoresis in 1 % agarose gel stained with ethidium bromide. ISSRs were detected by the use of 24 repeat anchored primers. The micro satellites primers pairs were synthesized from Operon Technologies INC., Alameda (CA, USA). The amplification reactions were carried out in 20 ll volume containing 50 ng DNA template, 35 ng primer, and 0.20 mM dNTPs, 2 mM MgCl2, 1 U Taq DNA polymerase and 19 PCR buffer. The PCR condition was standardized following parameters: initial denaturation at 94 °C for 3 min; denaturation at 94 °C for 45 s; annealing at 50 °C for 1 min; extension 72 °C for 1 min, and final extension at 72 °C for 5 min with 42 cycles of amplification. PCR was performed in triplicates using a thermocycler. For electrophoresis of amplified DNA, 1.0 % agarose gel was used. The molecular weight of bands was estimated using a DNA ruler 100 bp plus. The gel was run at 60 V for 100 min. The amplified DNA was viewed under the UV trans-illuminator, and the image was taken through gel documentation system. The PCR products from ISSR were analyzed by scoring qualitatively for the presence or absence of bands. Genetic similarities between the genotypes were measured by the Jaccard’s similarity coefficient based on the proportion of shared alleles using SIMQUAL sub programme of software NTSYS-pc version 2.02e [12]. The similarity index values found were used to generate a consensus tree using the unweighted pair group method analysis (UPGMA).
Results Isolation and Identification of Homokaryons Out of total 1,642 single spore isolates, 39 single spore isolates from the parent strains namely A-15 (9), S11(9), S-140(11), and U3(10) of Agaricus bisporus were found
0.16 3.28 CD (0.05) 0.14 2.80 CD (0.05) 2.24 CD (0.05)
SSIs single spore isolates
43.17 Check
0.11
Strandy
Strandy 0.09 1.82 CD (0.05)
2.39 50.09 Check Strandy 2.20 46.29 Check Strandy 2.16 45.36 Check Strandy
Appressed
17.77 A15-9
2.06
2.00 42.02 U3-9 Appressed 0.81 17.05 S140-9 Appressed 0.86 18.03 S11-9 Appressed
Strandy
38.44 A15-8
0.85
0.89 18.66 U3-8 Strandy 1.84 38.59 S140-8 Appressed 0.92 19.37 S11-8 Strandy
Appressed
21.21 A15-7
1.83
1.63 34.22 U3-7 Appressed 0.88 18.44 S140-7 Appressed 1.12 23.56 S11-7 Appressed
39.07 A15-6
1.01
Appressed 0.95
1.14 24.00
19.91 U3-5
U3-6 Strandy
Appressed 1.13
1.87 39.32
23.80 S140-5
S140-6 Strandy
Appressed 0.69
1.81 38.03
14.36 S11-5
S11-6 Strandy
Strandy 36.92 A15-5
1.86
17.21
1.76
Appressed
Appressed 0.85 17.88 U3-4 Appressed 0.95 19.91 S140-4 Appressed 0.90 19.00 S11-4 Appressed
Appressed
A15-4
0.82
0.86 18.03 U3-3 Strandy 1.793 37.70 S140-3 Appressed 0.79 16.66 S11-3 Appressed 18.64 A15-3
0.89
Appressed 0.90
0.77 16.21
19.00 U3-1
U3-2 Appressed
Appressed 0.92
0.97 20.25
19.24 S140-1
S140-2 Appressed
Strandy 1.81
1.05 22.05
37.85 S11-1
S11-2 Appressed
Appressed
19.68 A15-2
0.94
18.17 A15-1
0.87
Colony morphology Av. growth rate (mm/ day) Av. colony diameter (mm) in 21 days SSIs Av. colony diameter (mm) in 21 days SSIs
Av. growth rate (mm/ day)
Colony morphology
SSIs
Av. colony diameter (mm) in 21 days
Av. growth rate mm/ day)
Colony morphology
SSIs
Av. colony diameter (mm) in 21 days
Av. growth rate (mm/ day)
Colony morphology
U3 S-140 S11 A-15
Table 1 Linear mycelial growth rate and types of colony morphology of nine putative single spore isolates and parent strains, A-15, S11, S-140, and U3 of Agaricus bisporus on wheat extract agar medium
M. Sharma et al.: Characterization of Single Spore Isolates
slow growing (B2.0 mm/day) and out of those only 36 single spore isolates (nine from each parent strains to maintain uniform pattern) which were supposed to be homokaryons on the basis of slow mycelial growth rate and appressed types colony morphology were selected for future studies. The radial growth of homokaryotic single spore isolates including parent strains was measured after 21 days of incubation at 25 °C temperature, on wheat extract agar medium. It is clear from the data given in Table 1 that among the SSIs isolated from A-15 strains of A. bisporus, A15-4 showed significantly minimum mycelial growth rate (0.82 mm/day) followed by A15-9 (0.85 mm/day), A15-1 (0.87 mm/day), A15-3 (0.89 mm/day), A15-2 (0.94 mm/ day), and A15-7 (1.01 mm/day) on wheat extract agar medium with appressed types of colony morphology. The maximum mycelial growth rate (2.06 mm/day) was observed in parental control i.e., A-15, followed by SSIs A15-6 (1.86 mm/day), A15-8 (1.83 mm/day), and A15-5 (1.76 mm/day) on wheat extract agar medium with strandy types of colony morphology. Similarly, data on average mycelial growth rate (mm/ day) and colony morphology of selected SSIs from S11, S-140, and U3 strains of A. bisporus also recorded significantly minimum mycelial growth by S11-5 (0.69 mm/ day) followed by S11-3 (0.79 mm/day), S11-9 (0.86 mm/ day), S11-4 (0.90 mm/day), S11-8 (0.92 mm/day), and S11-2(1.05 mm/day) with appressed types colony morphology on wheat extract agar medium. Strain S11 supported maximum radial growth (2.16 mm/day) followed by S11-1 and S11-6 (1.81 mm/day) with strandy types colony morphology on wheat extract agar medium. The SSIs selected from parent strain S-140, clearly show that S140-9 had significant minimum mycelial growth rate i.e., 0.81 mm/day followed by S140-7 (0.88 mm/day), S140-1 (0.92 mm/day), S140-4 (0.95 mm/ day), S140-2 (0.97 mm/day), and S140-5 (1.13 mm/day) on wheat extract agar medium with appressed types colony morphology. However, maximum mycelial growth was recorded by parent strain i.e., S-140 (2.20 mm/day) followed by S140-6 (1.87 mm/day), S140-8 (1.84 mm/day), and S140-3 (1.79 mm/day) on wheat extract agar medium with strandy types colony morphology. In case of strain U3 of A. bisporus significantly minimum mycelial growth was recorded by homokaryon U3-2 (0.77 mm/day) followed by U3-4 (0.85 mm/day), U3-3 (0.86 mm/day), U3-8 (0.89 mm/day), U3-1 (0.90 mm/day), U3-5 (0.95 mm/ day), and U3-6 (1.14 mm/day) with appressed types colony morphology on wheat extract agar medium. Significant maximum radial growth was showed by strain U3 (2.39 mm/day) followed by U3-9 (2.00 mm/day) and U3-7 (1.63 mm/day) on wheat extract agar medium with strandy types colony morphology.
123
M. Sharma et al.: Characterization of Single Spore Isolates Table 2 Spawn run and number of fruit bodies of nine putative single spore isolates and parent strains, A-15, S11, S-140 and U3 of Agaricus bisporus A-15
S11
S-140
U3
In 2 kg substrate
In 2 kg substrate
In 2 kg substrate
In 1 kg substrate
SSIs
Spawn run
Average no. of fruit bodies
SSIs
Spawn run
Average no. of fruit bodies
SSIs
Spawn run
Average no. of fruit bodies
SSIs
In 2 kg substrate spawn Run
Average no. of fruit bodies
A15-1
?
0.00
S11-1
??
1.33
S140-1
?
0.00
U3-1
?
0.00
(1.00)a A15-2
?
0.00
(1.52)a S11-2
?
(1.00)
0.00
(1.00)a S140-2
?
(1.00)
0.00
(1.00)a U3-2
?
0.00
(1.00)
(1.00)
A15-3
?
0.00
S11-3
?
0.00
S140-3
??
2.00
U3-3
?
0.00
A15-4
?
(1.00) 0.00
S11-4
?
(1.00) 0.00
S140-4
?
(1.72) 0.00
U3-4
?
(1.00) 0.00
A15-5
??
2.00
S11-5
?
0.00
S140-5
?
0.00
U3-5
?
0.00
(1.00)
(1.00)
(1.72) A15-6
??
1.67
(1.00) S11-6
??
(1.63) A15-7
?
0.00
A15-8
??
2.33
A15-9
?
0.00
Check
???
6.00
S11-7
?
S11-8
?
S140-6
??
0.00 0.00
S11-9
?
0.00
Check
???
5.67
S140-7
?
S140-8
??
4.33
S140-9
?
0.00
Check
???
7.00
0.32 0.15
0.00
0.00
U3-7
??
2.00
U3-8
?
0.00
U3-9
??
6.67
Check
???
8.33
(1.00) (1.72) (1.00)
(1.00)
(2.55) CD (0.05) SE
?
(2.28)
(1.00)
(2.64)
U3-6
(1.00)
(1.00)
(1.00)
4.00
(1.00)
(2.23)
(1.00)
(1.82)
0.23 0.11
1.33
(1.00)
(1.00)
(1.52)
(1.00)
CD (0.05) SE
(1.00)
(2.76)
(2.83) CD (0.05) SE
0.34 0.16
(3.05) CD (0.05) SE
0.20 0.10
Spawn run process in substrate: ?, slow, ??, moderate; ???, fast SSIs single spore isolates a
Figures in parenthesis are square root transformed values
Fruiting Trial Nine putative homokaryotic SSIs selected from each parental strains namely A-15, S11, S-140, and U3 of Agaricus bisporus were tested for fructification in Petri plates containing compost extract agar medium, and the fruiting experiment was also done in polythene bags with 1 kg compost in each bag, in a replicated trial. The spawn run in these SSIs was very slow at incubation temperature of 25 °C and these were not able to colonize the entire Petri plate even after a period of 38 days of incubation in all the replication and were then cased to observe the fructification. All these SSIs with appressed types colony morphology were not able to produce pinheads in Petri plates even after 65 days of incubation, whereas, the strandy slow growing SSIs along with parent strains were able to form the fructification in petriplates after 30 days.
123
The spawn run in these homokaryons during fruiting trial in compost bags was very slow at an incubation temperature of 25 °C and was not completed even after 65 days of incubation, these bags were then cased 65 days after spawning to observe fructification. The number of fruit bodies produced was recorded up to 3 weeks of production. All the slow growing SSIs with appressed types colony morphology did not produce fruit bodies in any replication. However, the strandy slow growing single spore isolates produced fruiting bodies in all the replication; the average number of fruiting bodies produced is shown in Table 2. Slow growing single spore isolates that produced normal fruit bodies similar to those of the parental heterokaryotic control also showed identical banding pattern in ISSR analysis with those of parental heterokaryotic control.
M. Sharma et al.: Characterization of Single Spore Isolates Table 3 Single spore isolates wise variability analysis for strain A-15 of Agaricus bisporus SSIs
Average growth rate (mm/day)a (±SE)
95 % Confidence interval
Colony morphology
In 2 kg substrate Spawn runb
Average no. of fruit bodiesc (±SE)
95 % confidence interval
A15-1
0.87 ± 0.01
0.87 ± 0.02
Appressed
?
±
±
A15-2
0.94 ± 0.02
0.94 ± 0.04
Appressed
?
±
±
A15-3
0.89 ± 0.02
0.89 ± 0.04
Appressed
?
±
±
A15-4
0.82 ± 0.01
0.82 ± 0.02
Appressed
?
±
±
A15-5
1.76 ± 0.01
1.76 ± 0.02
Strandy
??
2.00 ± 0.22
2.00 ± 0.47
A15-6
1.86 ± 0.01
1.86 ± 0.02
Strandy
??
1.67 ± 0.13
1.67 ± 0.28
A15-7
1.01 ± 0.01
1.01 ± 0.02
Appressed
?
±
±
A15-8
1.83 ± 0.01
1.83 ± 0.02
Strandy
??
2.33 ± 0.13
2.33 ± 0.28
A15-9
0.85 ± 0.02
0.85 ± 0.04
Appressed
?
±
±
C
2.06 ± 0.01
2.06 ± 0.02
Strandy
???
6.00 ± 0.22
6.00 ± 0.47
a
Average growth rate calculated from the results of three separate experiments with five replications each on wheat extract agar medium incubation at 25 °C
b
Spawn run process in substrate: ?, slow; ??, moderate; ???, fast; ±, no primordial like structure; C, parental control
c
Average numbers of fruit bodies calculated from the results of one set of experiment with three replications
Table 4 Single spore isolates wise variability analysis for strain S11 of Agaricus bisporus SSIs
Average growth rate (mm/day)a (±SE)
95 % confidence interval
Colony morphology
In 2 kg substrate Spawn runb
Average no. of fruit bodiesc (± SE)
95 % confidence interval
S11-1
1.81 ± 0.03
1.81 ± 0.06
Strandy
??
1.33 ± 0.13
1.33 ± 0.28
S11-2
1.05 ± 0.03
1.05 ± 0.06
Appressed
?
±
±
S11-3
0.79 ± 0.02
0.79 ± 0.04
Appressed
?
±
±
S11-4
0.90 ± 0.01
0.90 ± 0.02
Appressed
?
±
±
S11-5
0.69 ± 0.01
0.69 ± 0.02
Appressed
?
±
±
S11-6
1.81 ± 0.01
1.81 ± 0.02
Strandy
??
1.33 ± 0.13
1.33 ± 0.28
S11-7
1.12 ± 0.01
1.12 ± 0.02
Appressed
?
±
±
S11-8
0.92 ± 0.01
0.92 ± 0.02
Appressed
?
±
±
S11-9
0.86 ± 0.02
0.86 ± 0.04
Appressed
?
±
±
C
2.16 ± 0.03
2.16 ± 0.06
Strandy
???
5.67 ± 0.63
5.67 ± 1.34
a
Average growth rate calculated from the results of three separate experiments with five replications each on wheat extract agar medium incubation at 25 °C
b
Spawn run process in substrate: ?, slow; ??, moderate; ???, fast; ±, no primordial like structure; C, parental control
c
Average numbers of fruit bodies calculated from the results of one set of experiment with three replications
Slow spawn run and no fruit bodies production were recorded in all SSIs whose growth rate was (\1.2 mm/day), and these were statistically at par with each other. The maximum number of fruit bodies was formed by heterokaryotic parental control i.e., A-15, S11, S-140, and U3 followed by the slow growing single spore isolates, from each parents with strandy types of colony morphology.
Perusal of the data given in Table 3 indicated that SSIs with appressed types of colony morphology of strain A-15 with growth rate B1.1 mm/day exhibited slow spawn run and failed to complete the spawn running even after 65 days of incubation. Data recorded in Table 4 showed the SSIs wise variability analysis for strain S11 of Agaricus bisporus. It is
123
M. Sharma et al.: Characterization of Single Spore Isolates Table 5 Single spore isolates wise variability analysis for strain S-140 of Agaricus bisporus SSIs
Average growth rate (mm/day)a (±SE)
95 % confidence interval
Colony morphology
In 2 kg substrate Spawn runb
Average no. of fruit bodiesc (±SE)
95 % confidence interval
S140-1
0.92 ± 0.01
0.92 ± 0.02
Appressed
?
±
±
S140-2
0.97 ± 0.03
0.97 ± 0.06
Appressed
?
±
±
S140-3
1.79 ± 0.02
1.79 ± 0.04
Strandy
??
2.00 ± 0.22
2.00 ± 0.47
S140-4
0.95 ± 0.01
0.95 ± 0.02
Appressed
?
±
±
S140-5
1.13 ± 0.03
1.13 ± 0.06
Appressed
?
±
±
S140-6
1.87 ± 0.03
1.87 ± 0.06
Strandy
??
4.00 ± 0.22
4.00 ± 0.47
S140-7
0.88 ± 0.01
0.88 ± 0.02
Appressed
?
±
±
S140-8
1.84 ± 0.02
1.84 ± 0.04
Strandy
??
4.33 ± 0.45
4.33 ± 0.96
S140-9
0.81 ± 0.03
0.81 ± 0.06
Appressed
?
±
±
C
2.20 ± 0.02
2.20 ± 0.04
Strandy
???
7.00 ± 0.22
7.00 ± 0.22
a
Average growth rate calculated from the results of three separate experiments with five replications each on wheat extract agar medium incubation at 25 °C
b
Spawn run process in substrate: ?, slow; ??, moderate; ???, fast; ±, no primordial like structure; C, parental control
c
Average numbers of fruit bodies calculated from the results of one set of experiment with three replications
Table 6 Single spore isolates wise variability analysis for strain U3 of Agaricus bisporus SSIs
Average growth rate (mm/day)a (±SE)
95 % confidence interval
Colony morphology
In 2 kg substrate Spawn runb
Average no. of fruit bodiesc (±SE)
95 % confidence interval
U3-1
0.90 ± 0.01
0.90 ± 0.02
Appressed
?
±
±
U3-2
0.77 ± 0.01
0.77 ± 0.02
Appressed
?
±
±
U3-3
0.86 ± 0.01
0.86 ± 0.02
Appressed
?
±
±
U3-4
0.85 ± 0.02
0.85 ± 0.04
Appressed
?
±
±
U3-5
0.95 ± 0.01
0.95 ± 0.02
Appressed
?
±
±
U3-6
1.14 ± 0.01
1.14 ± 0.02
Appressed
?
±
±
U3-7
1.63 ± 0.01
1.63 ± 0.02
Strandy
??
2.00 ± 0.22
2.00 ± 0.47
U3-8
0.89 ± 0.02
0.89 ± 0.04
Appressed
?
±
±
U3-9
2.00 ± 0.01
2.00 ± 0.02
Strandy
??
6.67 ± 0.25
6.67 ± 0.53
C
2.39 ± 0.01
2.39 ± 0.02
Strandy
???
8.33 ± 0.13
8.33 ± 0.28
a
Average growth rate calculated from the results of three separate experiments with five replications each on wheat extract agar medium incubation at 25 °C
b
Spawn run process in substrate: ?, slow; ??, moderate; ???, fast; ±, no primordial like structure; C, parental control
c
Average numbers of fruit bodies calculated from the results of one set of experiment with three replications
clear from the data that the homokaryons having growth rate B1.12 mm/day were not able to form primordia. It is obvious from the data recorded in Table 5 that the single spore isolates of strain S-140 which showed B1.13 growth mm/day were infertile or the monokaryons. Similarly from the data recorded in Table 6, it is clear that the appressed type of colony morphology of strain U3
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having growth rate B1.14 mm/day was not able to complete the spawn run even after 65 days of incubation and did not produce fruit bodies. All single spore isolates with appressed types of colony morphologies did not produce fruit bodies in any of the replication. However, the strandy slow growing single spore isolates, produced fruiting bodies in each replication.
49.02 25 26 44.90 22
Fig. 1 ISSR fingerprints observed with primers P3 in A-15 strains of Agaricus bisporus
49 43.40
Molecular Characterization of Single Spore Isolates Using ISSR (Inter Simple Sequence Repeat) Marker Systems Extraction of Genomic DNA
Total
MB monomorphic bands, PB polymorphic bands, P polymorphism
52
30
22
42.30
53
30
23
27
62.50 33.33 8 9 55.56 50.00 56 54 (CT)8RG (CAAGG)3 P31 P39
9 10
5 6
4 4
44.44 40.00
9 10
4 5
5 5
3 6
5 3
51
250–2,500
200–1,800 250–2,500 50.00 44.44 4 4 4 5
100–1,500
8 44.44 55 (GA)8C P30
8
4
4
50.00
9
5
4
5
37.50
8 9
57.14 4 3
125–2,500
3
7
50.00
50.00 5
4 4
5
28.57
8
50.00
2 7 25.00 2
5
8 33.33 3
6 8
9 44.44
28.57 2
4 5 9
5 53 (AG)8YC P22
55 (CT)8AGA P8
7
6
9 50.00 52 (GA)8T P3
9
5
4
44.44
8
4
4
4
4
10
250–2,500 44.44 4 5 9 55.56 5 4
Total bands Total bands MB
PB
P Total bands
MB
PB
P
Total bands
P PB MB
U3 S-140 S11 A-15 Tm (°C) Sequence (50 –30 ) Primers
Table 7 ISSR analysis for selected homokaryotic single spore isolates from four parent strains, A-15, S11, S-140 and U3 of Agaricus bisporus
MB
PB
P
Size range (bp)
M. Sharma et al.: Characterization of Single Spore Isolates
Out of 24, six primers were selected, generated excellent results and were used in this investigation (Table 7). Results clearly indicate that each primer gave rich and clear bands ranging from 100 to 2,500 bp, and their combination was sufficient to differentiate all of the tested homokaryons. The six chosen primers generated a total of 52 bands (A-15), 53 bands (S11), 49 bands (S-140) and 51 bands (U3) of high intensity and good separation. These were selected as markers, of which 42.30 % (A-15), 43.40 % (S11), 44.90 % (S-140), and 49.02 % (U3) were polymorphic. The results obtained for the same single spore isolates gave identical ISSR fingerprints after electrophoresis. It could be noted that the genome of A. bisporus has more microsatellite sequences related to these six primers than to the other primers. Primers P3 (A-15), P39(S11), P30(S140), and P8(U3) strains of Agaricus bisporus were proved to be the best due to the sharpness of banding patterns. Representative ISSR fingerprints obtained with primers P3 in A-15 and P39 in S11 strains of A. bisporus are shown in Figs. 1 and 2. Similarly, ISSR fingerprints observed with primers P30 in S-140 and P8 in U3 strains of A. bisporus are shown in Figs. 3 and 4. In the ISSR fingerprints of nine putative single spore isolates from A-15 strains with P3 primer, six homokaryons (A15-1, A15-2, A15-3, A15-4, A15-7, and A15-8) lacked amplification products at multiple loci; they grow
123
M. Sharma et al.: Characterization of Single Spore Isolates
Fig. 2 ISSR fingerprints observed with primers P39 in S-11 strains of Agaricus bisporus
Fig. 4 ISSR fingerprints observed with primers P8 in U3 and strains of Agaricus bisporus
(U3-7, U3-9) displaying amplification products at all loci are likely heterokaryons that received non-sister postmeiotic nuclei. The study revealed losses of ISSR polymorphic patterns in non-fertile homokaryotic single spore isolates compared to the parental control or fertile heterokaryotic single spore isolates. ISSR Data Analysis
Fig. 3 ISSR fingerprints observed with primers P30 in S-140 strains of Agaricus bisporus
slowly and all of them had appressed types of colony morphology. These may either be homokaryons derived from uninucleate basidiospores or heterokaryons composed of post-meiotic sister nuclei. The other single spore isolates (A15-5, A15-6, and A15-8) displaying amplification products at all loci are likely heterokaryons that received nonsister post-meiotic nuclei. Similar results were recorded in other three parental strains i.e., S11, S-140 and U3 of Agaricus bisporus in which homokaryons; (S11-2, S11-3, S11-4, S11-5 S11-7, S11-8, S11-9), (S140-1, S140-2, S140-4, S140-5, S140-7, S140-9), and (U3-1, U3-2, U3-3, U3-4, U3-5, U3-6, U3-8) did not produce amplification products at multiple loci. However, other single spore isolates; (S11-1, S11-6), (S-140-3, S-140-6, S-140-8) and
123
ISSR data was analyzed as described in Materials and Methods considering all the bands amplified with six primers.The first major bifurcation of the dendrogram (Fig. 5) divided the single spore isolates into two major clusters consisted of the homokaryons of A-15 and S140 strains of A. bisporus. However, the second cluster was further divided into six sub-clusters, included homokaryons, heterokaryons, and control of all the parental strains. Therefore, it is apparent from the data recorded by fruiting trial or by ISSR characterization of the single spore isolates that from A-15 strain of Agaricus bisporus, six single spore isolates were monokaryons. Similarly from strains S11, S-140, and U3, seven, six, and seven single spore isolates were homokaryons, respectively. Selected homokaryons were used in further studies. In particular, ISSR markers can be highly variable with in a species and reveal many more polymorphic fragments by utilizing longer primers that allow more stringent annealing temperature. It was for this reason that we adopted the technology in order to generate more specific and easily recognizable bands for identifying homokaryons from A-15, S11, S-140, and U3 strains of A. bisporus. In the ISSR fingerprints of putative single spore isolates, homokaryons lacked amplification products at multiple loci; these grows slowly and all of them had appressed types of
M. Sharma et al.: Characterization of Single Spore Isolates Fig. 5 Consensus cluster analysis of homokaryotic single spores of parental strains of Agaricus bisporus using ISSR fingerprints data from a Jaccard’s similarity matrix and the UPGMA method; the numbers assigned at the right margin of the tree branches representing homokaryotic single spore isolates and the letter C for parental heterokaryotic control
colony morphology. The study revealed losses of ISSR polymorphic patterns in nonfertile homokaryotic single spore isolates, compared to the parent strains or fertile heterokaryotic single spore isolates. ISSR data indicated that each primer gave rich and clear bands ranging from 100 to 2,500 bp. Primers P3 in A-15, P39 in S11, P30 in S-140, and P8 in U3 strains of Agaricus bisporus were proved to be the best due to the sharpness of banding patterns. These results also confirmed the identity of these single spore isolates to be homokaryons.
Discussion Button mushroom (Agaricus bisporus) is an economically important crop. However, most of the cultivated strains in the world were derived from or similar to the ‘‘hybrid’’ strains HU1 and HU3, thereby leaving the mushroom industry with a crop that was genetically very limited and had a high risk of sensitivity to disease. Most of the spores of A. bisporus produced heterokaryotic progenies. Genetic analysis and selective breeding of Agaricus bisporus, however, require the isolation of homokaryons from the heterokaryotic stocks. However, homokaryons are difficult to obtain by conventional basidiospore isolation from A. bisporus strains because of secondary homothallic life cycle. In the present study, from parent strains A-15, S 11, S-140 and U3 of Agaricus bisporus (Lange) Imbach, 1,642 single spore isolates were raised and characterized on wheat extract agar medium. These findings are in conformity with Jianping et al. [4]. All single spore isolates with
appressed types of colony morphologies did not produce fruit bodies in any of the replication. However, the strandy slow growing single spore isolates produced fruiting bodies in each replication. These results are in the agreement with Horgen and Anderson [3], Yadav [14]. In particular, ISSR markers can be highly variable with in a species and reveal many more polymorphic fragments by utilizing longer primers that allow more stringent annealing temperature. It was for this reason that we adopted the technology in order to generate more specific and easily recognizable bands for identifying homokaryons from A-15, S11, S-140, and U3 strains of A. bisporus. In the ISSR fingerprints of putative single spore isolates, homokaryons lacked amplification products at multiple loci; they grow slowly and all of them had appressed types of colony morphology. The study revealed losses of ISSR polymorphic patterns in nonfertile homokaryotic single spore isolates, compared to the parent strains or fertile heterokaryotic single spore isolates. These results also confirmed the identity of these single spore isolates to be homokaryons. ISSR PCR has also been used previously to detect the homokaryons using the same primers derived from stretches of tandemly repeated short motifs [8–10]. However, Khush et al. [6], Challen et al. [1], Yan and Jiang [15], Kavousi et al. [4, 5] used RAPD markers for the molecular characterization of homokaryons. Khush et al. [6] found that homokaryons carried a subset of the RAPD markers found in the parental heterokaryons. Challen et al. [1] observed that homokaryotic progeny from the secondarily homothallic A. bisporus was less variable (12 %) with fewer segregating loci than the known heterothallic
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M. Sharma et al.: Characterization of Single Spore Isolates
species, A. bitorquis (50 %) and A. nivescens (32 %). Yan and Jiang [15] reported that RAPD-based molecular genetic distance of the monokaryotic parents might be a suitable criterion for selecting monokaryotic parents of Stropharia rugoso-annulata and predicting the performance of hybrids in mushroom cross breeding. Kavousi et al. [5], reported that RAPD markers could discriminate homokaryons from heterokaryons, based on number of bands generated. The number of bands in homokaryons was significantly less than the heterokaryons.
6.
7.
8.
9.
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