Title : Molecular characterization of Termitomyces protoclones through PCR and RFLP based DNA typing

A total of five protoclones were successfully cultured on PDA medium out of regenerated twenty two colonies of Termitomyces protoplast and further studied. Liquid MYG grown mycelial tissue is used for protoplast isolation by enzymatic digestionin a mixture containing Lysing enzyme 2% and Cellulase R10 2% in 0.6 M mannitol. The incubation conditions like temperature, shaking and time were standardized at 24oC, 60 rpm and 10 hours, respectively for healthy protoplasts liberation. The purified protoplasts showed an average yield of 1.2×107 cells/gm tissue with 31.60±9.31% regeneration efficiency on specific medium and 77.12±2.72% viability by FDA test. Four ISSR primers were used in this study resulting a total of 27 reproducible bands with mean value of 6.75. They showed similar banding pattern in all the lines with zero percent polymorphism ranged from 280bp 2700 bp. The amplified rRNA-ITS gene showed ~ 600 bp size in gel and found a single restriction site for enzyme HaeIII in all the protoclones and parent with similar fragment size in all.


Introduction
Edible mushrooms are rich in protein content and thus used as food supplement worldwide.They can be commercially cultivated but still the wild collection is very popular to the poor tribal family in Asian and African countries.Termitomyces sp. is such popular wild edible mushroom grown in termite gut and found in Birbhum, Purulia and Medinipur districts of West Bengal, India.Apart from protein, Termitomyces is an affluent source of sugar, fibre, lipid, vitamin, mineral in addition to medicinal value which is used in lower blood pressure, rheumatism, kwashiorkor, obesity, diarrhea and purgative [1][2].So, it is now an important issue to improve the Termitomyces strains in terms of substrate specificity, yield and nutritional attributes.The mushroom crop improvement includes the two major aspects i.e., genome shuffling via protoplast fusion technology and development of transgenic via genetic engineering.Due to the lack of available genetic resources and many other disadvantages, researchers prefer the protoplast fusion technology for mushroom breeding where even mating can be possible between the sexually incompatible strains [3][4].However, it has been proved that the protoplasts are good source material for mushroom crop improvement where researchers had successfully developed somatic hybrids between the different genera followed by fruit body production [3][4][5].Development of Termitomyces protoplast and study about their genome stability is a crucial factor for somatic breeding with other genera.Sometimes, the fruit body derived mushroom strains show genome instability with their parent analyzed by molecular DNA markers [6].
In the past, few Chinese mushroom researchers developed protoclones of Agaricus bisporus and characterized them using morphological and molecular parameters [7].They showed the genome instability in the protoclones analyzed by ISSR markers.ISSR is a reproducible molecular DNA marker for genetic analysis [8] and hence have been used to study the genetic diversity of mushrooms [3-4, 7, 9, 10].The RFLP of nuclear rRNA-ITS genes are also productive molecular markers for mushroom genome analysis [4,16].
However, in this study we wanted to study about the genome stability of Termitomyces heimii protoclones developed from mycelial tissues and characterization by productive molecular DNA markers.

Protoplast isolation, purification and culture
Fruit body of Termitomyces heimii was collected from Bankura district of West Bengal, India and routinely maintained the vegetative culture on PDA medium (Potato Dextrose Agar, pH 6.2) at 28°C.The 7 days old MYG (10 g/l malt extract, 4 g/l yeast extract and 10 g/l glucose, pH 6.2) grown mycelial tissue used for enzymatic digestion.Protoplast isolation and purification was carried out according to the protocol of Mallick & Sikdar [3].
Protoplast yield was measured using a hemocytometer and viability was calculated by FDA staining [11].Culture media containing MYG supplemented with 0.7M NaCl as an osmotic stabilizer and 1.5% agar with purified protoplast was kept at 28°C for regeneration.

Extraction of genomic DNA and ISSR
The genomic DNA was extracted from MYG grown mycelial tissues using modified CTAB method [12].Four ISSR primers were used (Table1) in each single reaction according to the protocol of Bornet & Branchard [8] with slight modifications.The PCR was performed in 25µl of reaction volume in a DNA thermal cycler (Applied Bio-systems 2027) containing 25ng template DNA, 10X Taq buffer (+KCl), 25mM MgCl2, 2mM dNTPs mix, primer and Taq DNA polymerase (5U/µl).The initial denaturation at 94ºC for 5 min, followed by 40 cycles consisting of denaturation at 94ºC for 1 min, primer annealing (37-61ºC) for 1 min and extension at 72ºC for 3 min and a final extension for 10 min at 72ºC.

Amplification of rRNA-ITS gene and RFLP
According to the protocol of White et al. [13], the rRNA-ITS gene was amplified by PCR in a 25µl of reaction volume containing 15ng template DNA, 10X Taq buffer (+KCl), 25mM MgCl2, 2mM dNTPs, primer ITS1-F(5′ TCCGTAGGTGAACCTGCGG 3′) and ITS4-R(5′ TCCTCCGCTTATTGATATGC 3′) (Table1), Taq DNA polymerase (5U/µl).The amplification was conducted in a DNA thermal cycler (Applied Bio-systems 2027) by preliminary denaturation at 95°C for 4 min followed by 40 cycles; denaturation at 94°C for 1 min, primer annealing at 55°C for 1 min, initial extension at 72°C for 1 min 30 sec, followed by a final extension at 72°C for 8 min.For restriction analysis, PCR products were purified by sodium acetate precipitation.10μl of purified PCR product was used for restriction digestion according to the supplier's specification with enzyme HaeIII (Fermentas).

Gel electrophoresis
ISSR fragments, rRNA-ITS products and restriction digested products were separated on 1.8%, 1.5% and also 1.5% agarose gel with pre-stained ethidium bromide solution using 1X TAE buffer, respectively.The gels were run at 80 volts for 3 hrs (ISSR), 2 h (rRNA-ITS)

Protoplast culture and regeneration
The enzyme mixture containing lysing enzyme 2% and cellulase R10 2% in 0.6 M mannitol as osmoticum showed the best result with gentle shaking at 60 rpm for 10 hat 24ºC.
The average yield of purified protoplast was 1.2×10 7 cells/gm of 7 days old tissues (Fig. 1a) as optimum.They showed the maximum 31.60±9.31%regeneration and 77.12±2.72%viability (Fig. 1c).The purified protoplasts (Fig. 1b) were cultured on MYG medium supplemented with 0.7M NaCl and 1.5% agar, pH 6.2 and kept at 28°C for regeneration.Protoplast started germ tube formation in 24 h followed by micro-colonies (Fig. 1d) formation in 72 hrs onwards.A total of twenty two macro-colonies were observed in four replicate regeneration plates after 5, 7 (from two replicate) and 8 days, respectively.Immediately, the colonies were transferred in PDA mediumfor further growth and kept at 28°C.Among them only five putative protoclones were selected based on their growth performance and named as TPC1, TPC2, TPC3, TPC4 and TPC5 (Fig. 1e&f).

ISSR profile
Four reproducible ISSR primers amplified a total of 27 bands in all the TPC lines including parent with mean value of 6.75.ISSR-01 produced the lowest number of band i.e., 5 and ranged from 520 bp -1850 bp where ISSR-12 produced the highest number of band i.e., 9 and ranged from 280 bp -2700 bp (Table1).All the TPC lines showed similar banding patterns in size and number with the parentusing ISSR primers (Fig. 2).However, the ISSR profiles resulted that there is no dissimilarities or polymorphism inall the protoclones with respect to their single parent.

Amplification of rRNA-ITS region and RFLP analysis
The primer set ITS1(F) and ITS4(R) amplified the rRNA-ITS gene in all the TPC lines and parentand the size was analyzed in agarose gel.The PCR product showed a single band in all the lines with approx.600 bp in size (Fig. 3a).The primer set ITS1(F) and ITS4(R) can amplify only the ITS1-5.8S-ITS2region which is basically a conserved sequence.We also found that the TPC lines are genetically similar with the parent bythe restriction enzyme digestion.Restriction enzyme HaeIII showed only single restriction site in the entire ITS1-5.8S-ITS2region in all the lines including parent (Fig. 3b).It was also noted that the sizes of restriction fragments were same in all.

Discussion
Development of protoclones of agricultural crop usually helps in breeding for development of superior quality lines.In plant system several reports have been made where protoclones are developed from the protoplast and interestingly they showed phenotypic diversity where genotypic identity was maintained and vice-versa [14][15].Theoretically, protoclones are identical lines of the existing parent whether it may be plant, animal, fungi or other living organisms.For rapid multiplication of crop plant and mammalian cell lines this technique is used in tissue culture industry.
Reports on characterization of mushroom protoclones are limited and it depends on the regeneration percentage of protoplast in the suitable culture medium.Isolation and purification of Termitomyces protoplast was carried out following the standardized protocol with slight modifications and the protoplasts were cultured in such suitable medium where the regeneration percentage was highly achieved.However, the resulted genetic structures were identical in all the TPC lines with parent based on ISSR profile and RFLP of rRNA-ITS gene.The ISSR, RFLP of rRNA-ITS gene has already been proved as reproducible and suitable genetic markers for analysis of mushroom somatic hybrids and fruit body derived lines [3][4]16].Even the mushroom fruit body derived lines showed genetic diversity which might be occurred due to the gene environmental factor followed by elimination or rearrangement of genome structure during subsequent culture (Mallick & Sikdar, 2015).In this case, all the TPC lines are genetically identical with little morphological dissimilarity like heterogeneous colony nature, growth rate on PDA medium (not details in this manuscript).
This might be happen due to the presence of inactive compounds in the medium or the protoplast cultures were not exposed to the environment where direct gene environmental factor works.The morphological variation maybe due to the effects of vegetative culture or the growth responsive gene might be in the dominant allele or induced during sub-culturing.
Protoplast technology is very useful technique for mushroom crop improvement specially when it is essential to develop somatic hybrid between two sexually incompatible strains.In mushroom crop breeding there are still lacks of genetic resources and hence transformation is restricted.However, principally it is essential to be identical the protoclones so that they can be used as stable genetic material for transformation.Mushroom protoplast can be used for gene transformation having stable genetic makeup in future research.In this manuscript we have successfully standardized the Termitomyces's protoplast isolation, purification and culture.We have also showed the homokaryotic nature of each protoclones with single parent.However, in this study we have identified the similar banding profiles through ISSR and RFLP of rRNA-ITS gene in all the protoclones with parent.As they are derived from the protoplasts, the genetic makeup remains identical in this case and we could claim that these lines are identical clone of a single parent i.e., Termitomyces.The further study regarding their yield at field level, detail genetic attributes by FISH, nutritional profiles of the existing cloud give a good conclusion.

Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 25 April 2018 doi:10.20944/preprints201804.0326.v1 and
3 h (RFLP) and banding profiles were visualized under UV light using a transilluinator system.Data were recorded in a Molecular Analyst Gel Documentation System.Gene Ruler 100 bp plus DNA ladder, (MBI, Fermentas) is used as a standard molecular weight marker in each case.[Table1 near here]