| The Auricularia is a wood-rotting fungus widely distributed in the world,with very important edible and medicinal values,it mainly includes Auricularia heimuer,Auricularia cornea,Auricularia delicata,Auricularia fibrillifera and other species.With the increasing demand for fungus in recent years and President Xi Jinping’s praise for the "small fungus,big industry",the fungus has become a major industry,the fungus industry continues to usher in new developments,but at the same time the degradation of the original fungus strains shows up year by year,the development of new germplasm of fungus is becoming more and more urgent.Most of the current fungus industry is still using the previous varieties or mainly exploring the improvement of cultivation methods and the development of different substrates to replace them,there are few reports on biological breeding through protoplast mononucleation,and even fewer reports on interspecific distant hybridization,distant crosses can not only pool the superior traits of both parents into the hybrid offspring,but also have the potential to produce a superparental hybrid strain,we provide more material for genetic breeding of fungus and new varieties for production,and at the same time enrich fungus germplasm resources and provide more possibilities for producers to choose.Our team collected strains of the main species of the Auricularia,including strains of Auricularia cornea,Auricularia delicata,Auricularia heimuer and Auricularia fibrillifera,the experiments were carried out in three aspects: agronomic trait evaluation,molecular marker evaluation,protoplast mononuclearization and distant hybridization research,to provide a reference for better evaluation of germplasm resources of Mucor spp.And to screen for superior strains with exploitation value.The specific research results are as follows:1.Agronomic trait evaluation.The test strain was cultivated in bags,and the most scientific,practical and efficient way of ear emergence was used to cultivate the strain substrate,and the data collected in this test after averaging several measurements showed that:In terms of strain cotyledon length,Auricularia fibrillifera strain >Auricularia delicata strain> Auricularia cornea strain > Auricularia heimuer strain,and cotyledon width Auricularia delicata strain > Auricularia cornea strain > Auricularia fibrillifera strain > Auricularia heimuer strain,substrates of thick Auricularia delicata strains >Auricularia cornea strains >Auricularia heimuer strains > Auricularia fibrillifera strain.In terms of the fertility of the strains,the strains were in the order of shortest to longest in terms of Auricularia fibrillifera strain < Auricularia delicata strain <Auricularia cornea strains < Auricularia heimuer strains.the degree of soaking was from highest to lowest in the strains of Auricularia fibrillifera strain> Auricularia cornea strain > Auricularia heimuer strain > Auricularia delicata strain,the strains were ranked from highest to lowest in terms of substrate yield as Auricularia heimuer strain >Auricularia cornea strain > Auricularia delicata strain > Auricularia fibrillifera strain.The strains with greater exploitation value were selected by combining all the agronomic trait indexes of this experiment,The Auricularia delicata Z1,which has a short reproductive period and a brittle texture,Auricularia delicata Y1,which has a nice uniform pattern and a thicker cotyledon,and the Auricularia heimuer A14.It also provides a reference for future germplasm evaluation of Auricularia.2.Molecular marker evaluation.The SSR primer design of this experiment was based on the whole genome of Auricularia heimuer,the whole genome of Auricularia cornea and the transcriptome of Auricularia fibrillifera crispate sequencing data.The main part of this experiment was mainly evaluated from two aspects,SSR molecular marker evaluation and ITS molecular marker evaluation,18 pairs of primers with good specificity and high stability were selected from 48 pairs of primers for the clustering analysis of strains in this experiment,the results of NTSYS-PC cluster analysis showed that the test strains could be classified into four major groups at a genetic similarity coefficient of 0.36.The first group includes 7 strains,mainly Auricularia cornea strain,specifically including W1,W6,W9,W10,AP66,P4,Z and other 7 strains,Auricularia delicata Z containing in this taxon;Cluster II contains four strains,mainly of Auricularia delicata,specifically Z1,Z2,Y1 and Y2;Taxon III is Auricularia heimuer strain A14;Taxon IV is Auricularia fibrillifera 5513.In the SSR clustering analysis of this experiment,the test strains were accurately classified into four groups,namely Auricularia cornea strain,Auricularia delicata strains,Auricularia heimuer strain and Auricularia fibrillifera,ITS molecular markers were used to construct a phylogenetic tree based on PCR amplification of ITS fragments,in which the test strains were divided into 2major groups,the first taxon is a total of 10 dark strain taxa,mainly containing Z,Z1,Z2,Y1,Y2,A14,5513,AP66,W6,P4,five plants of Auricularia delicata,two plants of Auricularia cornea,one plant of Auricularia heimuer and one plant of Auricularia fibrillifera,taxon II is a light-colored strain taxon of three strains,all of which are Auricularia cornea strain,W1,W9 and W10.Among the two molecular evaluation methods,the SSR molecular marker method based on the design of primers for the genome of Mucor has higher accuracy by classifying each strain in more detail.3.This experiment optimized the best protoplasmic preparation conditions suitable for the strains in this experiment at the same time: The enzyme concentration was 1.95%,the enzymatic digestion time was 2.5h,the enzymatic digestion temperature was 28.5°C,and 0.63mol/L mannitol was the stable osmosis solution,and the optimal regeneration concentration of protoplasts was 150 pcs/μL and the regeneration effect was better with sucrose medium as the regeneration medium carbon source.The 53 mononuclear strains obtained in the experiment can be continued in interspecific hybridization tests,it provides test materials for further obtaining more excellent breeding strains,providing more varieties for production,also found:In interspecific hybridization tests,the closer the genetic similarity,the easier it is to cross between species,but the ear rate of hybrid strains is very low and the probability of having a good strain is low.4.The test strains were subjected to protoplast mononucleation,after mycelial culture,staining,microscopic examination and other experimental treatments,a total of 10 strains and53 mononuclear strains were collected,then the mononuclear strains were subjected to interspecific hybridization tests,and a total of 32 hybrid combinations of interspecific crosses were made,and finally 19 hybrid strains were obtained,Finally,only A14×Z1 and A14×Y1collected substrates,and the ear emergence rate of interspecific hybrid strains was 10.53%.The results showed that the transverse diameter,longitudinal diameter,thickness,and color of the seeds of the hybrid strain were between those of the parental strain,and strain A14 × Z1 showed superparental advantage in yield,which has greater value for exploitation.It was also found that the closer the relative genetic distance between SSR and ITS molecular markers in the interspecific hybridization test,the easier it was to cross,but the ear rate of the hybrid strains was low and the probability of having good strains was low. |
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