Systematic Identification And Evaluation Of Germplasm Resources Of Salix In Heilongjiang Province

There are many species of Salix,with more than 520 species in the world.There are 257 species,122 varieties and 33 variants in China.Salix is an important tree species for afforestation and timber plantation,which can control water,fix sand and change alkali,and has high ecological and economic value.However,the classification of Salix is a difficult problem in the world,and the research on the innovation and utilization of Salix resources is still to be vigorously developed.Taking the germplasm resources of Salix in Heilongjiang province as the research object,a model combining morphological identification,SEM observation,molecular identification and chemical composition identification was established in this paper,which effectively improved the accuracy of identification and provided scientific evidence for the classification of Salix.In order to exploit and utilize the resources of Salix reasonably,the analytic hierarchy process(AHP)was used to comprehensively evaluate the growth index and medicinal index of Salix,and 6 species of Salix with excellent characteristics were selected,which can be used as the superior varieties in the study of Salix genetics and breeding.The main research contents and results of this paper are as follows:1.The establishment of a systematic identification model of Salix,providing a new idea for the taxonomic study of Salix(1)The traditional morphological identification was used as the preliminary identification:A total of 56 samples of Salix were collected in Harbin,Daqing,Yichun and Tahe County,Heilongjiang Province,described morphologically according to Flora of China and Plant Key List of Heilongjiang Province.Experts were invited to identify the samples,and the results were 14 groups and 29 species of Salix,including Sect Salix(Salix capitata,Salix babylonica,Salix matsudaha,Salix koreensis,Salix eriocarpa,Salix pierotii and Salix fragilis).Sect.Vimen(Salix dasyclados,Salix sachalinensis,Salix viminalis and Salix siuzevii);Sect.Vetrix(Salix taraikensis,Salix raddeana,Salix hsinganica and Salix floderusii);Sect.Daphnella(Salix rorida and Salix skvortzovii);Sect.Helix(Salix linearistipularis and Salix sungkianica);Sect.Amygdalinae(Salix triandra);Sect.Pentandrae(Salix pentandra);Sect.Incubaceae(Salix rosmarinifolia);Sect.Myrtilloides(Salix myrtilloides);Sect.Subviminales(Salix gracilistyla);Sect.Caesiae(Salix integra);Sect.Urbanianae(Salix maximowiczii);Sect.Cheilophilae(Salix cheilophila);Sect.Hastatae(Salix pyrolifolia)and Salix phylicifolia;2 varieties:Salix matsudana var.tortuosa and Salix rosmarinifolia var.Brachypoda;2 variant Salix matsudana var.pseudo-matsudana and Salix matsudan var.matsudanaa f.umbraculifera;and Salix babylonica×S.alba-vitellina(coenospecies).(2)Microscopic morphological characteristics were used as the auxiliary identification:For the species with similar morphology,the leaf epidermis and pollens were observed by SEM,which could distinguish Salix triandra and Salix pentandra,Salix matsudana and Salix matsudana var.pseudo-matsudana.Pollen is characterized by good genetic stability.The morphology and size of pollens,P/E value,number of germination pores,and outer wall decoration were described in detail in this paper.(3)Molecular identification was used as the accurate identification:The ITS2 and psba-tmh sequences of Salix were amplified by PCR and sequenced in two directions by DNA barcoding technology,and phylogenetic trees were constructed.The results showed that the Salix resolution of chloroplast fragment was much lower than that of nuclear genome fragment.The ITS2 sequence can be used to accurately identify the Salix pentandra,Salix rorida,Salix linearistipularis,Salix viminalis and Chosenia arbutifolia,reaching the species level,and some species of Salix can only be identified to the group level.(4)Chemical composition identification was used as the review identification:Components of the extracts of Salix were determined by high efficiency liquid,and HPLC fingerprint was drawn.The similarity of common peaks was compared and analyzed to verify the consistency of the morphological and molecular identification results,and at the same time the uncertain similar species could also be excluded.2.Enrichment of the gene information of Salix and preservation of the excellent gene resources of Salix(1)Flow cytometry was used to determine the chromosome ploidy and genome size of Salix:The chromosome ploidy level and DNA content were calculated according to the fluorescence intensity in the flow cytometry with the diploid tamarisk as the control.The results showed that Salix linearistipularis and Salix cheilophila were diploid,Salix babylonica xSalba-vitellina,Salix matsudana var.Tortuosa,Salix capitata,Salix matsudana,Salix pentandra,Salix triandra,Salix matsudana var.pseudo-matsudana and Salix babylonica were tetraploid,and Salix phylicifolia were hexaploid.Compared with Cucumis sativus(350Mb)and Glycine max(1150Mb),the genome size of Salix was determined as follows:Salix cheilophila(290.69?305.76Mb),Salix capitata(628.22?656.93Mb),Salix pentandra(569.76?612.54Mb),Salix triandra(569.76-612.41Mb),Salix babylonica(565.51?629.09Mb)and Salix integra(285.51?312.63Mb).(2)There are 2047 ITS sequences of Salix in Genbank.In this study,good results were obtained in the amplification and sequencing of nuclear gene fragments.40 ITS2 sequences were submitted to NCBI database with registration numbers:MT177150,MT177151,MT177152,MT177153,MT177154,MT177155,MT177156,MT177157,MT177158,MT177159,MT177160,MT177161,MT177162,MT177163,MT177164,MT177165,MH924219,MT177166,MT177167,MT177168,MT177169,MT177170,MT177171,MT177172,MT177173,MT177174,MT177175,MT177176,MT177177,MT177178,MT177179,MT177180,MT177181,MT177182,MT177183,MT177184,MT177185,MT177186,MT177187,MH924218.Among them,Chosenia arbutifolia(MT177178,MT177179,MT177180);10 ITS2 sequences of Salix pierotii(MT177150),Salix capitata(MH924219,MT177162),Salix cheilophila(MH924218),Salix linearistipularis(MT177181,MT177182)and Salix siuzevii(MT177183,MT177184,MT177185,MT177186)were submitted for the first time,which enriched the DNA information resources of Salix.3.Scientific evaluation of Salix resources and selection of superior speciesThe evaluation model of germplasm resources of Salix was constructed,and two Standard layer indexes,namely growth index and medicinal index were set up.According to the results of the questionnaire,the judgment matrix was established and the weights of 7 factor layer index(tree height 0.1332,DBH 0.1263,crown width 0.0470,number of branches 0.0271,total flavonoids content 0.2079,proanthocyanidin content 0.1317 and luteolin-7-O-?-D-glucosidein content 0.3268)were calculated.The contents of total flavonoids,proanthocyanidins and luteolin-7-O-?-D-glucosidein in Salix leaves were detected by U-V-vis spectrophotometer and high performance liquid chromatography(HPLC).The resource value of Salix was qualitatively and quantitatively judged and the score was calculated.The top six Salix:Salix matsudana var.pseudo-matsudana,Salix pentandra,Salix linearistipularis,Salix babylonica ×S.alba-vitellina,Salix matsudana and Salix babylonica.4.Resource innovation and utilization,and development of the medicinal plant value of Salix(1)Standard deviation method was used to group the contents of total flavone,proanthocyanidin and luteolin-7-O-?-D-glucoside in liulians into three groups:high,medium and low.The differences of the three components among the three groups were very significant(P<0.001).The dynamic changes of three components of 11 species of Salix in Harbin from June to October were investigated.The results showed that the content of total flavonoids was still stable in October,so it could be extracted from deciduous resources,and the content of proanthocyanidins was basically degraded from August to September.Luteolin-7-O-?-D-glucoside was proved to be a good active substance for the treatment of cardiovascular and cerebrovascular diseases,and the content was the highest in August.Among 26 species of Salix,18 species contained luteolin-7-O-?-D-glucopyranoside.The higher contents of luteolin-7-O-?-D-glucopyranoside were Salix pentandra 2.1307mg/g,Salix linearistipularis 1.3557mg/g,Salix rosmarinifolia 1.1947mg/g,Salix integra 1.1210mg/g and Salix capitata 1.0747mg/g.(2)Salicylic acid(salicylic acid)was first discovered from willow tree and then synthesized into aspirin.Eight new salicylic acid schiff base compounds were designed and synthesized with salicylic acid as the substrate to characterize the chemical structure of the products.The antibacterial activity of the product was tested by filter paper method,and the results showed that 3c and 4c had certain inhibitory effect on escherichia coli,staphylococcus aureus and bacillus subtilis.(2)Salicylic acid(salicylic acid)was first found in willow,and then aspirin was synthesized.Using salicylic acid as substrate,eight salicylic acid Schiff base compounds were designed and synthesized and their chemical structures were characterized.Among them,4 kinds of 2-hydroxyl-4-(2-hydroxy-4-diethyl laminophenylidene)benzoic acid(3d),2-hydroxy-5-(2-hydroxy-4-chlorophenylidene)benzoic acid(4b),2-hydroxy-5-(2-hydroxy-4-bromophenylidene)benzoic acid(4c),2-hydroxy-5-(2-hydroxy-5-diethylaminophenylidene)benzoic acid(4d)were new compound synthesized for the first time.The antibacterial activity of the product was tested by filter paper method.The results showed that the product had certain inhibitory effect on Escherichia coli,Staphylococcus aureus and Bacillus subtilis when the concentration was 8mg/mL and 16mg/mL.The bacteriostatic zone diameter of 2-hydroxy-4-(2-hydroxy-5-bromophenylidene)benzoic acid(3c)was 18.5mm,23.5mm,20.2mm,respectively.The bacteriostatic zone diameter of 2-hydroxy-5-(2-hydroxy-4-bromophenylidene)benzoic acid(4c)was 14.1 mm,22.5 mm,12.2 mm,respectively.To sum up,this paper focuses on the identification of Salix plants and the development and utilization of germplasm resources.29 species of Salix and their variants from 14 groups in Heilongjiang.Province were comprehensively studied by traditional morphology,micromorphology,molecular markers,cytobiology and chemical analysis,and a new model for identification of Salix was constructed,which improved the accuracy of identification of Salix.The germplasm resources of Salix were evaluated by Analytic Hierarchy Process(AHP),and 6 excellent germplasms were selected for further genetic breeding of Salix.Through the determination of the contents of total flavonoids,proanthocyanidins and luteolin-7-O-?-D-glucopyranoside and the synthesis of Schiff base compounds of salicylic acid in Salix,the medicinal value of Salix was exploited to provide scientific basis for the development and rational and effective utilization of Salix germplasm resources.