| Gastrodia elata is a precious is a traditional and precious Chinese medicinal material in my country.Armillaria mellea is a symbiotic fungus necessary for the growth of Gastrodia elata and has a great impact on the quality and yield of Gastrodia elata.The confusion of Gastrodia elata and Armillaria mellea germplasm restricts the development of Gastrodia elata industry.To screen elite germplasm of Gastrodia elata and Armillaria mellea.In this experiment,three variants of Gastrodia elata(Gastrodia.elata Bl f.elata,Gastrodia.elata Bl f.glauca S.Chow and Gastrodia.elata Bl f.viridis Makino)and 11 Armillaria mellea stains were identified at the molecular level,and the growth and yield characteristics were studied.,with the main conclusions are as follows:1.The primers P18S/P26S amplify the r DNA-ITS of the three variants of Gastrodia.elata,allowing the differentiation of asparagus from other species and the identification of the Gastrodia.elata Bl f.elata variant.The genetic stability within the three Gastrodia.elata variants is strong,with the Gastrodia.elata Bl f.elata being more distantly related to the Gastrodia.elata Bl f.glauca S.Chow and Gastrodia.elata Bl f.viridis Makino,and the latter two being more closely related.The amplified r DNA-ITS sequences of all three variants of Gastrodia.elata were able to segregate from outgroups,and the Gastrodia.elata Bl f.elata could be segregated from the Gastrodia.elata Bl f.glauca S.Chow and Gastrodia.elata Bl f.viridis Makino.The genetic distance within all three Gastrodia.elata variants is 0.000,between the Gastrodia.elata Bl f.elata and the Gastrodia.elata Bl f.glauca S.Chow,Gastrodia.elata Bl f.viridis Makino variants is 0.010,and between the latter two variants is 0.000.2.The three variants of Gastrodia elata have good HPLC specific chromatogram,which can be used as specific chromatogram of Gastrodia elata,but it is not possible to qualitatively distinguish the three variants of Gastrodia elata.The degree of variation is greater in Gastrodia.elata Bl f.glauca S.Chow,followed by Gastrodia.elata Bl f.viridis Makino and to a lesser extent Gastrodia.elata Bl f.elata.The similarity between the three variant of Gastrodia.elata control profiles ranged from 0.935～0.997,meeting the requirements for a characteristic profile.The similarity within the Gastrodia.elata Bl f.glauca S.Chow variant ranges from 0.859～0.997,within the Gastrodia.elata Bl f.viridis Makino variant ranges from 0.978～1.000,and within the Gastrodia.elata Bl f.elata variant ranges from 0.992～0.996.3.HA,W3A and L2A were better in yield,and HA,W3A3 and L4 were better in gastrodin and p-hydroxybenzyl alcohol content.HA,W3A and L2A had higher total yields of 2.41,0.91 and 2.73 kg/m~2respectively,There is no significant difference between HA and HA1,but it is significantly different from other Gastrodia.elata Bl f.elata stains,and the latter two were significantly higher than other stains in the variant.The total amount of gastrodin and p-hydroxybenzyl alcohol in HA,W3A3 and L4 was0.78%,0.74%and 0.56%,respectively,and all three were significantly higher than other stains in the variant.4.Screen out three varieties of Gastrodia elata with good comprehensive performance,which are HA1,W3A1 and L2.The combined evaluation results show that HA1 has high values of the affiliation function for total quantity,arrow hemp quantity,and leachate,with 0.542 for the former two and 0.653 for the latter,in Gastrodia.elata Bl f.elata stains.W3A1 has a high affiliation function value of 0.663and 0.434 for total yield and single weight of arrow hemp,respectively,in Gastrodia.elata Bl f.glauca S.Chow stains.L2 had high affiliation function values of 0.706,0.596 and 0.653 for arrow hemp yield,leachate and p-hydroxybenzyl alcohol content,respectively,in Gastrodia.elata Bl f.viridis Makino stains,and three stains had high average affiliation function values of 0.479,0.496 and 0.535,respectively,with better overall performance.5.The r DNA-ITS amplified by primers ITS1/ITS4 allowed the identification of11 Armillaria mellea stains to species and were suitable for the identification of partial Armillaria mellea genes.The 11 Armillaria mellea stains could also be classified by r DNA-IGS amplified by primer 5SA/CNL12 and observed by transmission electron microscope.The primers ITS1/ITS4 amplify r DNA-ITS of Armillaria mellea,which can divide the 11 Armillaria mellea stains into two major groups,and all of them can be separated from the outgroup.Group I includes M-01～M-05 and M-08～M-10,and Group II includes M-06,they were comparatively identified in the NCBI database,M-07 and M-11.M-01～M-02,M-04～M-05 and M-08～M-10 maybe Armillaria.gallica,M-03 and M-11 maybe Armillaria.sinapina,M-06 and M-07 maybe Armillaria.mellea.The r DNA-IGS amplified by primers5SA/CNL12,and the classification results are consistent with the above,but it is isolated from the known sequences in the NCBI database,and 11 Armillaria mellea stains cannot be identified.In transmission electron microscopic observations,Group I Armillaria mellea stains had more bacilli than Group II at low magnification,and the overall structure(nucleus,cell wall,cell membrane,etc.)of Group I Armillaria mellea stains was thicker than that of class II at high magnification.6.M-05 and M-08～M-10 grow well in the bottle.M-10 had a faster bacteria speed of 1.18 cm/d,which was not significantly different from M-09.The laccase and cellulase activities of M-09 were higher,at 25.93 and 10660.39 U/L,respectively,during the breeder seed activation.M-05,M-01 and M-08 had a faster bacteria speed of 0.22 cm/d,and M-05 had a higher laccase activity of 37.53 U/L and M-10 had a higher cellulase activity of 9067.53 U/L,during the mother species cultivated.M-05had a larger mycelium length of 31.30 mm,which was significantly different from M-02,M-04 and M-07.M-09 had a faster bacteria speed of of 0.34 cm/d and a higher laccase activity of 37.30 U/L,and M-10 had a higher cellulase activity of 10047.916U/L,during the cultivated species expanding propagation.7.In the bottle of good growth of Armillaria mellea and Gastrodia.elata Bl f.elata mixed planting although in the yield,quality are different,but will not appear"empty nest"phenomenon,while the poor growth of Armillaria mellea not only very low yield,and appear"empty nest"phenomenon.M-10 had a higher total yield of2.98 kg/m~2,which was significantly different from the other Armillaria mellea stains,followed by M-08 at 2.12 kg/m~2,and the total content of gastrodin and p-benzyl alcohol in Gastrodia.elata Bl f.elata were higher thanthe pharmacopoeia standard,while M-06,M-07 and M-11 had low total yields of 0.01,0.08 and 0.04 kg/hole,respectively,with M-06 and M-07 appear"empty nest"phenomenon.8.M-05 was screened for good overall performance of the Armillaria mellea stains.The results of the comprehensive evaluation showed that Gastrodia.elata Bl f.elata mixed with M-05 Armillaria mellea stain had the highest affiliation function values for total yield,arrow hemp single weigh,moisture,ash,gastrodin,p-hydroxybenzyl alcohol and balisonoside B content of 0.639,0.350,0.649,0.629,0.622,0.647 and 0.580,respectively,and a higher mean affiliation function value(0.496).To sum up,In this experiment,one copy of each of the three varieties of Gastrodia elata and Armillaria mellea elite germplasm was screened out,which were HA1,W3A1,L2 and M-05 respectively,which could provide a reference for the subsequent screening of elite germplasm of Gastrodia elata and Armillaria mellea. |
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