| Leucocalocybe mongolica(S.Imai)X.D.Yu &Y.J.Yao is a prestigious rare wild edible mushroom in Northeast Asia.Its former Latin name is Tricholoma mongolicum S.Imai and its generic name is “Bai–mo” or “Kou–mo”.It is an endemic species of Mongolian Plateau,usually growing in the form of mushroom circles on the prairie.Due to its highly desirable flavour and healthy function,L.mongolica is collected by indigenes and mycophiles unscrupulously;in addition,its habitat is under constant threat from human activities,and its wild resources are becoming rare each passing day.L.mongolica is facing an unprecedented threat and struggling in a critical endangered state.Based on field investigation,the paper aims to explore how to protect the endangered rare “koumo” and how to make rational use of the wild resources from the perspectives of physiology,genetics and molecular ecology.L.mongolica is the unique member of a single-species genus Leucocalocybe,and the study of its phynotype and genetic information is crucial to genus and species classification.The previous studies of it have only involved LSU region while genealogical concordance phylogenetic species recognition(GCPSR),as a vital academic concept,is influencing phylogenetic species recognition in fungi at the present time.We used a multiple gene(loci)phylogeny with three Ribosomal DNA regions(LSU,SSU,ITS)and mitochondrial DNA region(mt SSU)based on maximum likelihood estimation to delimitate species of L.mongolica.Eighty-three mushroom specimens were involved.The results indicated that Lepista have a rather close consanguinity with L.mongolica at molecular level.The genus of Lepista and the genus of Clitocybe are both polyphyletic groups.Physiologic characters of L.mongolica:(1)The mechanism for spore germination was studied.Five media were tested for the germination of spore suspension.Improved rose bengal potato medium was beneficial to spore germination.There was a positive correlation between the diameter of basidiocarp and the number of germination colony.(2)In the study of life cycle,the phenomenon of alternation of nuclear phases was observed using fluorescence microscopy.(3)The biological characteristics of the strains isolated from the wild basidiocarp were studied.For the screening of the single optimal factor,carbon source,nitrogen source,inorganic salt and growth factor were tested for mycelial growth rate and mycelial growth potential.As a result,the fungi grew better with maltose,starch and cellobiose as carbon sources,yeast extract,beef powder and beef extract as nitrogen sources,K2HPO4 and Fe2(SO4)3 as inorganic salt,and decoctions of Pleurotus ostreatus,carrot and soybean sprouts as growth factor.The orthogonal experiments with the above selected conditions for each of the four factors were then performed and the optimal cultivation conditions were determined.A descending order of the impact for the four factors was growth factor > inorganic salt > nitrogen source > carbon source and the F-test showed very significant differences among all the four factors.The optimal culture conditions for L.mongolicum were the combination of starch and beef extract,decoction of 2 % carrot and no added inorganic salt.Based on the optimization of the culture medium by orthogonal experiment,the optimal temperature and initial p H value were studied,with the results of p H=6.5 and at 25 ?.L.mongolica has a restricted distribution globally.Because of its special habitat,the correlative researches of its physiology are essential.The experiment showed that inorganic salt and p H are important factors influencing the mycelial growth.The fact that no transcriptomic or genomic data of L.mongolica are accessible in biological information database is a limitation to further research.Reduced-representation genome sequencing analysis showed that Restriction-site associated DNA sequencing(RAD-seq)was not suitable for identification of SSR markers on L.mongolica.Specific DNA barcoding sequencing about IGS1,ITS,RPB2 and mt SSU did not display ideal polymorphism among 10 specimens.Transcriptome de novo sequencing was carried out.The objective of transcript acquisition was to facilitate gene discovery for understanding of physiological mechanisms about active substance biosynthesis and to identify simple sequence repeat(SSR)markers for developing molecular markers of genetic diversity in L.mongolica.The transcriptome data were obtained by virtue of Illumina paired-end sequencing technology.42,622,958 clean reads were achieved and 37,302 contigs were generated.Based on similarity searches with known proteins,we generated 13,821 unigenes which had a significant BLAST hit.These unigenes were annotated within 7 public databases.Throughout all of the unigenes,6642 were classified as three functional groups;The 3914 unigenes were associated with a COG classification;4019 unigenes were selected from KEGG pathways,3110 had prominent matches in the KEGG database and were classified into five main groups,including 35 KEGG pathways.Fifty-seven genes,potentially involved in terpenoid,steroid,and unsaturated fatty acids biosynthesis,were identified and selected for further research.Secondary metabolites have aroused the concerns of more and more researchers.We gained insight into the complex transcriptome of the biosynthesis about active substance and established a biotechnological platform for further research.The total number of carbohydrate-active enzymes of L.mongolica is 446 and the number of carbohydrate binding module(CBM)is relatively low.CE11,GT19,GT51,GT56,GH131,GH133 and GH135 constitute the characteristic carbohydrate-active enzymes subfamily compared to other edible mushrooms.The characteristic carbohydrate enzymes relative to other mushrooms can play a vital role in the metabolism of nutrients.In these generated sequences,1860 SSRs were identified and characterized as molecular candidate markers existing L.mongolica.Exploration of genetic diversity and population structure of L.mongolica is an important work for conservation biology.We investigated the main distribution areas and took field survey from July 2015 to September 2017.The sampling scope mainly covered Xilingol grassland,Hulunbuir grassland,Gonggeer grassland and Bashang grassland of Zhangjiakou.Ninety expressed sequence tag-sample sequence repeat(EST-SSR)primers were developed by software eprimer3 v6.6.0 based on transcriptome data.Eight loci of polymorphism for genotyping were screened from 90 EST-SSR by polyacrylamide gel electrophoresis.In total,402 alleles of 223 individuals from 17 wild geographical populations were obtained based on 8 simple sequence repeat loci of polymorphism using Capillary electrophoresis typing.Total number of alleles from each population ranged from 11 to 31 with an average of 24 per population.The average observed heterozygosity(HO)and expected heterozygosity(HE)ranged from 0.300 to 0.875 and from 0.188 to 0.621,respectively.Analysis of molecular variance(AMOVA)indicated that 14.19% of the total molecular variance could be attributed to differences between populations(p < 0.001),whereas 85.81% could be affiliated to differences within populations.The mean value of FST(0.171)suggested low genetic differentiation among populations.The mantel test also showed lack of significant relationship between genetic distance(FST)and geographical distance(r=0.049,p=0.28).The UPGMA dendrogram generated from SSR data suggested that 17 populations separated into 3 main sister clusters on the Nei's genetic identity coefficient 0.8.Two hundred and twenty-three individuals of L.mongolica were divided into three groups by STRUCTURE analysis.The population structure was characterized,and the integral distributional situation about the three groups was mapped to the 17 geographic populations through the visual analysis of map.Spatial genetic structure of the 17 populations was revealed by “Interpolate genetic landscape shapes(IGLS)” procedures of the software “Alleles in space(AIS)”.Comprehensive analysis indicated that habitat characteristics had greater correlation with population genetic diversity and population structure.The barrier analysis indicated that the southern distribution area of the whole distribution region(Latitude and longitude range 43.2-45 N,116.2E-118E)were destroyed into several spatially distant patchy habitats.It is obvious that the barriers existed.Habitat fragmentation contributed to the destruction of continuous habitat into several spatially distant patchy habitats.Maintaining and enriching population diversity is a crucial issue for the conservation of L.mongolica.The data of population structure and genetic diversity were obtained in this study,which will provide basis for the establishment of conservation measures.For in situ conservation,areas with high genetic diversity identified in this study should be listed as priority protected areas.Nowadays,there is still a lack of an actual breakthrough on cultivation techniques of L.mongolica,and as another approach as centralized and economical utilization of resources,fermentation technology shows a bright future.Liquid fermentation and solid fermentation were separately studied.The optimal formula for liquid fermentation was successively tested by single factor test,Plackett-Burman experiment,steepest ascent experiment and response surface methodology.The formula is sucrose 35.13 g/L,soybean meal 16.55 g/L and corn steep powder 33 g/L.On this basis,the optimal fermentation conditions were 25 ?,initial p H 6.5,120 rpm,liquid volume in flask 75 m L/250 m L and inoculum size 12.5 %.For screening the optimal substrates for solid fermentation,5 substrates,including corn kernel,wheat,etc.were tested.The results showed that corn kernel is a better substrate for solid fermentation.Technological process of solid fermentation on L.mongolica whose main raw material is corn kernel was established.Nutritional ingredients and volatile components of the products by the two fermentation techniques were evaluated.The results of GC-MS showed that 21 volatile components are likely to be the flavor compounds of basidiocarp.The active components before and after solid fermentation on L.mongolica were evaluated.The test showed that the content of flavonoid,polysaccharides,triterpenoids,sterols and phenols after solid fermentation was enhanced compared with unleavened substrate.Solid fermentation technology of L.mongolica is worth further research. |
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