| Stropharia rugosoannulata(Daqiugaigu)is a nutritious and crispy grass-rot edible fungus that is being widely promoted for cultivation in China.The production of S.rugosoannulata can convert a large amount of crop straw,improve the utilization efficiency of crop straw,especially when it’s rotated with rice,it can fully utilize idle winter fields and rice straw resources,improve the planting benefit of farmland,and increase soil organic matter.Currently,there are issues such as long mycelium growth cycle,overlapping planting period with the second crop of rice,and rapid decrease of commodity characteristics of fruiting bodies due to the fast rise of spring temperature,so it is urgent to breed strains that are suitable for converting rice straw,have short growth periods,and good commodity characteristics.This study uses 5 cultivated strains of S.rugosoannulata with different growth rates on rice straw as parents for hybrid breeding.The whole genome of the parent strains was re-sequenced,and the main research results are as follows.Spores isolated by dilution in liquid.five cultivated strains of S.rugosoannulata,SA06,SA09,SA29,SA30,and SA47,and a total of 137 monokaryon were obtained,including 10,9,35,63,and 20 from each strain,respectively.Based on the ability to produce clamp connections,three different mating types of monokaryon were initially identified from SA09.Dikaryon-monokaryon mating was carried out using 32 strains and three monokaryon,resulting in four hybrids,while Mon-mom crossing was conducted using 128 monokaryon from four other parents and three monokaryon,resulting in 87 hybrids out of 300 combinations.The affinity rate of S.rugosoannulata monokaryon was29.0%,and there was no significant difference in affinity rate between different cultivated strains.Using the second-generation high-throughput sequencing technology,the whole genome of five tested strains of S.rugosoannulata was re-sequenced.A total of 612,120 variant sites were obtained from the five strains,including 549,316 single nucleotide polymorphisms(SNPs)and 62,804 insertions and deletions(In Dels).PCR amplification product sequencing verification showed that mating type A locus has two homologous homeodomain genes(HD1 and HD2).Among the five tested strains,there are only two genotypes for the mating type A locus and no multiple alleles exist.Similar phenomenon was observed in the B locus as well.These results indicate that parental strains of S.rugosoannulata have a relatively uniform gene type at mating type loci with small differences,possibly derived from the same ancestral strain,which is the main reason for the low affinity between monokaryon during hybridization.Focusing on the growth of S.rugosoannulata mycelium on rice straw,the differences in main characteristics of hybrids during the mycelial stage were analyzed.The study found that the coefficient of variation for mycelial growth rate in test tubes was 15.97%,while the coefficient of variation for mycelial density was 23.64%.Correlation analysis showed that the time to full mother spawn could better reflect the mycelial growth rate on the straw culture dish,while mycelial growth in test tubes did not match the growth on the culture dish,indicating that mycelial growth rate is easily influenced by the culture vessel.However,regardless of whether it was in a culture dish or a test tube,mycelial density was significantly correlated with mycelial growth rate.Based mainly on mycelial density and time to full mother spawn,combined with activity determination of carboxymethyl cellulase and lacquer enzyme,50 hybrids were selected and produced spawns.After eliminating hybrids with contamination rates exceeding 70%,cultivation experiments were conducted on the 30 hybrids and 5 parents.Focusing on the main characteristics of S.rugosoannulata fruiting bodies,the main traits of hybrids during the fruiting stage was examined and analyzed.Among the 30 hybrids,17 produced fruit bodies while 13 did not.The earliest fruiting hybrids were81-23 and SA15,with a fruiting period of 90 days.Combined with analysis of changes in temperature and humidity,it was found that S.rugosoannulata requires a temperature above 10 ℃ and air humidity above 70% for fruiting.Even if the mycelium has fully grown into the substrate,fruiting may still be difficult without these conditions.Correlation analysis showed that there is a significant positive correlation between the cap height and single fruit body weight.Using entropy values for analysis,the results showed that fruiting period,proportion of high-quality fruit,and stem diameter are the most important characteristics for S.rugosoannulata fruiting bodies.Compared with commercially available high-quality fruit phenotypes,stem diameter and length are more suitable as screening indicators than cap height and diameter.Using grey relational analysis,entropy method,and technique for order preference by similarity to an ideal solution,a comprehensive evaluation of 13 main traits was conducted on 22 strains.The results showed that 81-30,81-23,and 32-20 were the three optimal hybrids based on comprehensive characteristics.Among all hybrids,81-23 had fast mycelial growth rate and the earliest fruiting time on rice straw.Its fruiting period was only 90 days,35 days earlier than the average fruiting period.The mycelial growth rate of 81-23 on rice straw culture dish at 10 ℃ and in rice straw test tube at 25 ℃ was0.97 mm/d and 2.09 mm/d,respectively,and the growth rate in cultivation reached 0.26mm/d,which were all higher than the average values.In this study,we performed whole-genome resequencing analysis of 5 cultivated strains of S.rugosoannulata,identified different mating-type spores of strain SA09,and conducted both Mon-mom crossing and dikaryon-monokaryon hybridization.We analyzed the weight of different traits and correlations among them,and preliminarily screened three excellent hybrid strains(81-30,81-23,and 32-20).Among them,32-20 had the fastest mycelial growth rate on rice straw,81-23 had the shortest fruiting period. |
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