| Pleurotus citrinopileatus,commonly known as Yuhuang Tricholoma,which belongs to Basidiomycetes,Mycoclass,Umbomycetes,Tricholaceae,Tricholaceae fungi genus.The fruiting body of Pleurotus aureus is bright yellow with a strong mushroom fragrance.It was first successfully cultivated in China,which was known as Golden Mushroom.It is a precious edible fungus with promising development prospects.However,most varieties used in production just simply domesticated from wild strains,which were increasingly unable to meet the requirements of high yield and good quality varieties in production.Hybrid breeding is a common breeding method,of which the traditional spore hybrid breeding consumes a long time,the progeny traits are separated seriously,and the workload of screening is large.However,protoplast hybrid breeding can directly isolate mononuclear cells from mycelium,which is not limited by time and season.It can effectively overcome the obstacles of parental traits dispersion and difficult screening which caused by the genetic diversity of spore mononuclear cells in traditional breeding,and the breeding time is short.However,the mating type of protoplast mononucleate and the genetic characteristics of hybrid progeny in protoplast cross-breeding of pleurotus citrinopileatus have not been reported.Therefore,in this study,pleurotus citrinopileatus was used as the test strain,optimizing the preparation conditions of protoplasm system-Preparation of mononuclear protoplasts-The mating type of protoplasts was detected-Protoplast hybridization-Agronomic traits of hybrid offspring(F1)-Esterase isozyme analysis and SRAP Analysis,The aim of this study was to explore the genetic characteristics of the progenies of progeny of protoplast mating type separation hybridization of pleurotus citrinopileatus,creating the fine new germplasm,providing scientific basis and germplasm resources for genetic breeding of pleurotus citrinopileatus.Main results are as follows:(1)Optimizing the preparation conditions of protoplasm system of pleurotus citrinopileatus.The optimal preparation conditions were as follows:2.0%lywallzyme solution,0.6mol/L mannitol as osmotic pressure stabilizer,enzymolysis time of 3h,enzymolysis temperature of30℃,and bacterial age of 5 days.The protoplasm volume was 1.07310~7/m L.(2)The nuclei of the two mating types in the mycelium cells of pleurotus citrinopileatus showed segregation bias during the nucleation process of protoplasts,but the degree of segregation bias was different among strains.Among the 10 strains tested,Y21,Y24,Y26,X18,and X20 could all obtain 2 mating types of mononucleated protoplasts,showing the phenomenon of double polarization,but the number of obtained was different,indicating the existence of the dominant nuclear phenomenon.The other 5 strains only obtained the protoplast of one of the mating types,and the dominant nuclear phenomenon was extremely serious,reaching the degree of single polarization.(3)The hybridization of 15 mononucleated protoplasts showed a rich diversity of esterase isozymes in the F1 strain.The number of bands ranged from 4 to 12,and the RF value ranged from 0.1 to 0.75.Rf values of 0.10 and 0.58 were the common bands of JDL,and the frequency of occurrence reached 100%.When the Rf value was 0.67,the frequency of the enzyme band was low,which was 2.8%.Under the 0.75 level,which is divided into six major categories,the first big groups including eight strains and 60 parents of hybrid strain,the first large groups under the 0.79 level is divided into two groups,the first in the class including the cultivation type X2,parents X20 Y21,Y24,X18,wild type parent and the 31 strains of hybrid strains,including 16 strains of hybrid strains gathered together with one of the parents in this group,there were 10 strains gathered together with the parents in this group.The second subclass consisted of hybrid strains of cultivated parent X3,X13,wild parent Y26,and 29.Eleven of the hybrid strains were clustered together in this group with one of their parents.The second largest group included 4 hybrid strains,the third-largest group included 6 hybrid strains,the fourth largest group included wild-type parent Y13,the fifth largest group included wild-type parent Y22,and the sixth-largest group included hybrid strain 45.(4)A total of 30 DNA bands were amplified by SRAP molecular markers,with a total of 20polymorphic bands(66.6%).At the level of 0.58,the group was divided into 2 groups.The first group included 4 parents and 30 hybrid strains,among which 20 hybrid strains and one of their parents gathered together in this group.The second-largest group was divided into three subgroups at the level of 0.59.The first subgroup included 2 hybrid strains,and the second subgroup included cultivated parent X20 and 39 hybrid strains,among which 11 hybrid strains and cultivated parent X20 were clustered in this group.The third group included 5 wild-type parent strains.(5)Through antagonistic reaction,esterasing isozyme technology,and SRAP molecular marker technology,17 strains of hybrid strains were screened out for cultivation test,and a total of 7 strains with excellent traits were obtained,showing super affinity phenomenon.Six of them with early maturity were screened out and could be used as good breeding materials for early maturity varieties.Z1,Z3,Z4,Z5,Z8,and Z12,respectively.The growth period of hybrid strain Z1 was 53d and 14d shorter than that of its parents.The growth period of hybrid strain Z3was 48d,which was shorter than that of its parents 19d.The growth period of hybrid strain Z4was 58d,which was shorter than that of its parents 9d.The growth period of the hybrid strain Z5 was 52d,which was shorter than that of the parents 23d.The growth period of hybrid strain Z8 was 55 days shorter than that of the parents for 14 days.The growth period of hybrid strain Z12 was 54 days shorter than that of its parents.A hybrid strain Z6 with good comprehensive traits was screened out,which was characterized by the large,and thick bacterial cap,bright color and waterless impregnation stripe. |
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