| Dendrobium officinale Kimura et Migo(D.candidum)is a plant of the genus Dendrobium whose stem can be used as a medicine.Its quality stability has been effeated seriously due to the current introduction of D.candidum and provenance confusion.In addition,D.candidum which is cold-resistant likes the warm and humid climate.So it is necessary to evaluate the germplasm resources and assist in the screening of improved varieties.In this paper,33 species of D.candidum cultivars species and 13 wild species were collected from Xiamen,Taining,Shaowu and Yunnan,Zhejiang and other regions.The genetic diversity of D.candidum was analyzed using CDDP,SCo T,TRAP and Co RAP molecular markers.Cold-resistance-related genes was used to screen for cold-resistant D.candidum.The results as follows:1.Analysing genetic diversity of D.candidum by using molecular marker technologyA total of 151 bands were amplified with 16 CDDP primers.The polymorphic band accounted for 95.3% of the total bands,the PIC averaged 0.815,and the RP averaged 1.653;14 SCo T primers amplified a total of 92 bands.The total band was 93.9%.A total of 278 bands were amplified by 21 pairs of TRAP primers.The polymorphic bands accounted for 99.03% of the total bands,the average PIC was 0.899,and the average RP was 2.054;16 pairs of Co RAP primers amplified a total of 208 bands.The polymorphic bands accounted for 93.2% of the total bands,the average PIC was 0.880,and the average RP was 1.806.The amplification results of four molecular markers indicated that the trial provided D.candidum with rich genetic background and high genetic diversity.In addition,the genetic diversity of wild species was higher than that of cultivars in the analysis of TRAP and Co RAP molecular markers developed for cold-resistance genes of D.candidum,indicating that there are greater genetic variations in the genes related to wild-type resistance in complex and changing natural environments.In terms of genetic parameters,the average PPL population of CDDP markers was 69.86%,population H averaged 0.2017,population I averaged 0.4088,interpopulation Gst was 0.1778,population Nm was 2.3118,and SCo T marker,PPL averaged 75.80%.H average 0.2357,I average 0.3360,Gst 0.265,Nm 1.387;TRAP mark,PPL average 76.79%,H average 0.258,I average 0.3892,Gst 0.2017,Nm 1.9793;TRAP mark,PPL average 72.59%,H averaged 0.198,I averaged 0.312,Gst was 0.1808,and Nm was 2.2656.The genetic parameters of the four markers indicated that D.candidum genetic variation occurred mainly within the population,and there was a certain degree of gene exchange among populations.In the cluster analysis,D.candidum was classified into two groups at 0.668 with CDDP markers,but the cultivars in different regions had no obvious boundaries and overlapped each other.The wild species were separated from the cultivar at a similarity coefficient of 0.732.SCo T,TRAP,and Co RAP markers were used to separate D.candidum cultivars and wild species at 0.59,0.536,and 0.61,respectively.Four kinds of molecular markers could not clearly separate cultivars from different regions.The main coordinate analysis showed that there are no obvious correlations between geographical factors and D.candidum in different regions.For wild and cultivars species,four molecular markers can clearly separate them,which TRAP>SCo T>Co RAP>CDDP.2.By using molecular marker technology to assist in screening cold-resistant D.candidumIn the CDDP marker,9 accessions of D.candidum resistant germplasm were screened by using primers for anchored WRKY,MYB,and ERF stress-resistance related transcription factors.In the SCo T marker,SCT-8,SCo T-59,and 11(li)primers of differential bands were generated in the cold-resistant D.candidum,which were used to screen 5 cold-resistance D.candidum species.In TRAP markers,CBF1,DREB1,and CDPK1 cold response-related genes were selected and by using them to screen out 11 excellent accessions of cold-resistance D.candidum.In the Co RAP marker,13 cold-resistance D.candidum were screened by using anchor cold response genes COR15 a,KIN1 and Gs LRPK primers.In the STS markers,DREB1,Gs LRPK,and KIN1 genes were selected to screen out 9 the excellent germplasms of cold-resistance D.candidum.Finally,screening all the CDDP,SCo T,TRAP,Co RAP,and STS markers results,Three cold-resistance D.candidum species were finally selected numbered 10,44,and 13.3.Studying and quality determinating on related genes and physiological changes in D.candidum under low temperature stressThe low-temperature stress screening of D.candidum,the heat shock protein HSP70,protein kinase LRPK,cold-induced KIN1,polysaccharide synthesis Do PMM,transcription factor DREB1 gene expressed,besides the amount of HSP70 gene and cold-induced KIN1 gene expression continued to rise,and the period of treatment had not been reduced;The levels of DREB1 and LRPK e genes increased first and then decreased gradually during treatment,but they were still higher than the control expression;Do PMM,also increased first and then decreased during the low temperature treatment period,it is still higher than the control expression when the stress is released.Under low temperature treatment,the soluble protein,soluble sugar,free proline and SOD activity of D.candidum increased significantly and continued to accumulate;the content of malondialdehyde fluctuates,and the overall change was not significant by the end of treatment;Electrical conductivity increased significantly astime went on;The soluble protein and soluble sugar increased significantly but the content was lower than that of the cold-resistance species,after treatment the free proline and SOD activity decreased,malondialdehyde content and electrical conductivity increased significantly;chlorophyll content showed a significant downward trend in all germplasms,and the rate of decline in uncold-resistanc was greater than that of cold-tolerant species.In the determination of quality,the polysaccharide content of D.candidum 10,44 and 13 was 32.80%,38.60% and 28.30%,respectively,which was higher than the minimum content specified in the Chinese Pharmacopoeia;the content of dantranium was 0.0307%,0.0315%,and 0.0360%,respectively;The contents of naringenin were 0.156 mg/g,0.187 mg/g,and 0.133 mg/g,respectively,and the comprehensive membership function analysis of quality and cold resistance was 13>44>10. |
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