Isolation And Cross Breeding Of Single Spore Strain Of Cordyceps Militaris Based On Mating Type Gene

Cordyceps militaris is a kind of edible and medicinal fungus.Strain degeneration leads to the decrease of fruiting body yield and vitality,which is one of the most prominent problems restricting the cultivation of C.militaris.It is a heterozygous fungus with two kinds of mating types of MAT1-1 and MAT1-2.In the present study,fruiting bodies of C.militaris were collected for further strain isolation,mating type identification,monospore isolation and identification,comparison of mycelial culture conditions,degeneration experiment based on mating type genes,single spore cross breeding experiment,mating type gene analysis of hybrid offspring,cloning and expression analysis of cordycepin key enzyme genes,etc.It provides a theoretical basis for the breeding of C.militaris strains with excellent characters and stable heredity.The details are as follows:1.The strains CM1901 and YNCC04 were identified as C.militaris by morphological and ITS identification.CM1901 was a double mating strain containing MAT1-1 and MAT1-2,and YNCC04 was a single mating strain containing MAT1-1.2.102 strains of ascospores were obtained with the strain CM1901 used as material.The mating type genes of monospore strains were determined.The results showed that there were 3 kinds of mating types of ascospore strains,including the single mating type containing only MAT1-1,only MAT1-2,and the parent type containing both MAT1-1 and MAT1-2,with strain number of 57,24,and 21,respectively.The ratio of MAT1-1: MAT1-2 was 78:45.Based on the single factor culture condition experiment,the parent strain and different mating monosporal strains showed different nutritional requirements.3.The results of q PCR based on the degeneration study showed that the expression of MAT1-1 was relatively stable in MAT1-1 and parental monosporal strains and parental strain.Moreover,MAT1-2 was relatively stable only in MAT1-2 monosporal strain,but rapidly decreased in parental monosporal and parental strains.4.Cross breeding experiment of single spore strain of C.militaris was performed including:pairwise crosses of 3 monosporal strains of each mating type with excellent traits(9 groups)and 3monosporal strains each and the parent strain as control(10 groups).The results showed that the parent strain CM1901 produced fruiting bodies with mature perithecia but with low fresh fruiting body weight of 5.77 g/bottle.It suggested that the parent strain degenerated.Among the 9 hybrid combinations,7produced fruiting bodies,with a fruiting rate of 78%,a maturity rate of 100%,and a yield range of 6.37-18.00 g/bottle.The combinations with strain CMSS02 containing MAT1-1 demonstrated highest average yield of 14.79 g/bottle which was 2.5 times higher than that of the parent strain group.The combinations with strain CMSS30 containing MAT1-2(except CMSS102×CMSS30)demonstrated the highest average yield of 15.10 g/bottle which was 2.6 times higher than that of the parent strain group.It suggested that single spore hybridization with different mating types of genes can improve the characters and yield of the fruiting body.5.The results of the mating type gene expression of the fruit body showed that the mating type gene expression was significantly different during mycelial subculture.The expression of MAT1-1 gene was relatively low,while the expression of MAT1-2 gene was higher than that of MAT1-1.6.Adenine phosphoribosyl transferase(AMI),IMP dehydrogenase(Imp),and GMP synthetase(Gmp)were reported as the key enzyme genes for cordycepin synthesis.The q PCR results show that AMI and IMP demonstrated high relative expression levels,while Gmp is low expressed in each strain.