The Study Of Genetic Diversity And Physiological Response Under High Temperature And Salt Stress In Narcissus

Daffodil is a fall planting bulb flower from Amaryllidaceae Narcissus L.There are more than 160 species and varieties.The climatic zone of daffodil growth is mainly in the temperate zone.It like cool climate,don't like high temperature and wet.It distribute mainly in the Mediterranean coast and central Europe,distribution center is the Iberian peninsula in northern Morocco(Fernandes,1968).They are widely distributed in many different ecological environments,which has strong disease resistance,cold resistance and salinity tolerance.Narcissus tazetta L.var.chinensis Roem.is a variation of Narcissus tazetta,which is distributed in the southeastern coastal areas of fujian and Zhejiang in subtropical climate zone,and which has strong heat resistance and salinity tolerance(Chen xinqi,1982).Currently,there are more than 20000 horticultural varieties(Worley et al.,2000;Chen JY,2001),and every year there are new varieties,thus the genetic background is complex.But so far,the research on daffodil has been concentrated in the study of the cultivation and kinship relationship,and there is little research on the genetic diversity and resistance physiology.In this paper,the Complete sequence of chloroplasts of daffodil was measured,genome comparative analysis and phylogenetic analysis was carried out,and cpSSR markers in chloroplast genome of daffodil were developed in this study.There are 68 species of daffodil in this study,which genetic diversity and kinship relationship were analyzed by morphological marker,nSSR and cpSSR markers.At the same time,the heat stress and salt stress in a few Narcissus germplasm resources were carried out.The morphological and biochemical reactions of Narcissus were studied under stress.The main results are as follows:1.There are 26 phenotypic traits observed in the experimental materials of daffodil.The results showed that the variation coefficient was obvious and the morphological diversity was significant.The main components of phenotypic traits and the contribution rate of concentration was Obvious.The main characters that cause morphological variation were flower type,flower diameter,flower color,petal width,petal length,deputy crown color,deputy crown shape,deputy crown length and deputy crown width,which is basically the same conclusion as the morphological diversity index.By NTSYS clustering method,the test material were divided into 12 classes,and flower character of daffodil was as its main properties.This is basically consistent with the traditional morphological classification.The agronomic traits of 10 varieties of daffodils were also observed.The results show that they were basically unlikely to flower in the second year after the bulbs of these tested varieties were planted in the nursery with the subtropical climate of Zhangzhou.Narcissus 'Tahiti' and Narcissus 'Mount Hood' planted in Zhangzhou's alpine experimental field possess the following unique characteristics,such as high appreciative value,strong adaptability,strong resistance,high rates of propagation and high yield and the two varieties are very suitable for planting in Zhangzhou alpine areas.2.The Complete sequence of chloroplasts of Narcissus and Lycoris radiata was measured,the results showed that the chloroplast genome in 13 species of Narcissus is 147,865-158,961 bp in length,and exhibits a typical quadripartite structure of the small(SSC,15,806-18,787bp)and the large(LSC,78,201-87,503bp)single-copy regions,separated by a pair of inverted repeats(IRs,26,902-27,414bp).It encodes 165-175 predicted functional genes,including 111-118 protein genes,44-47 tRNA genes and 8 rRNA genes.The gene order,genome structure,codon and GC content are similar to the chloroplast genome of most monocotyledons.Three pairs of reverse repeat sequences,7 tandem repeat sequences and 4 pairs of identical repeat sequences were detected in Narcissus cp genome,of which A/T single base repetition accounts for 77%.The Narcissus plastome contains,The length difference of chloroplast genome in narcissus was mainly determined by the contraction and expansion of IR,which was consistent with previous research results.The chloroplast genome of 13 daffodil materials did not detect structural changes such as the rearrangement or inversion of sequences,but there are a good collinear relationship in gene composition,quantity and arrangement.The sequence variation of LSC and SSC region of chloroplast genome was greater than that of IR,and the variation of intergene region was the largest,and the sequence variation of coding region was much lower than that of non-coding region.Sequence analysis discovered the 12 most divergent genes,in which 6 genes including ndhF,accD,c1pP,matK,Ycf1 and ycf2 can be used as candidate genes for the phylogeny of daffodil.This also supports that gene sequence evolution not only has the location effect but also has the function effect.Further,phylogenetic analysis which based on 80 chloroplast protein coding gene sequences by ML and MP method demonstrated that there are clearly identified between the Chinese daffodil and the European daffodil,that there is a sister relationship between Narcissus'Jinzhanyintai'and Narcissus'Yulinglong',and the European daffodil is divided into two branches.3.The DNA polymorphism from 68 species of daffodil was studied using cpSSR marker and nSSR marker in the experiment,the results showed that a total of 106 clear and reproducible bands were generated from 6 cpSSR primers,of which 94 were polymorphic,with an average of 15.6 polymorphic bands and an average of 86.13%polymorphism rate,which indicated that the cpSSR primers had hign level of genetic diversity.The results showed that a total of 255 clear and reproducible bands were generated from 6 nSSR primers,of which 243 were polymorphic,with an average of 42,5 polymorphic bands and an average of 94.89%polymorphism rate,which indicated that the nSSR primers had hign level of genetic diversity.The results of both molecular markers indicated that the daffodil germplasm resources had high level of genetic diversity.The main characteristics band of all the primers are obvious,which showed that the daffodil internal genome has the same identity.The dendrogram based on cpSSR and nSSR data were constructed by using UPGMA analysis.The clustering results showed that the similarity coefficient between the 68 daffodil germplasm resources varied greatly,that they clustered into two groups with this two molecular markers.The first group included 21 Narcissus tazetta,which can be divided into two subgroups.The second group included 47 single flower European daffodils,which can be divided into 10 subgroups.The result showed that there were distant genetic relationship between the Narcissus tazetta and single flower European daffodils,and the genetic relationship was close in Chinese daffodils.4.To study the response mechanism of Daffodils to high temperature,and select some Physiological indexes which can reflect the different system of heat-resistance for the Daffodils and higher heat-resistance germplasm,leaf anatomical structure,membrane permeability,Pro content and MDA content were measured of 10 varieties of narcissus in 37? high temperature stress.The result showed that:The changes of heat resistant physiological indexes leaf was large in the different Daffodils;There were higher heat resistance of Narcissus 'Minnow'?Narcissus'Graford' and Narcissus'Scarlet Gem';Leaf Stomata density,the ratio of palisade tissue/spongy tissue thickness,leaf relative membrane permeability,Pro content and MDA content show some variation rule,so they can be used as heat resistant study of narcissus indicator.In order to compare the mechanism of salt damage on halophyte Narcissus tazetta L.seedlings,the seedlings were treated in greenhouse through substrate salinity and salt spray,respectively.The growth rate,the allocation of mineral elements and ash in the different ages and parts of the leaf were measured and compared.The results showed that the growth rate of N.tazetta seedling treated with substrate salinity and salt spray was slower than that of control group.Na+ and Cl-in the different parts of the leaf mostly distributed in the leaf tip,which coincided with the symptom of lesions distributed mostly on the leaf tip of N.tazetta seedling.Under the two ways of salt stress,the mass fraction of ash in the different ages and parts of the leaf was significantly higher than that of control group(p<0.05).Therefore the accumulation of ash in the plant increased with increasing salinity.Na+ and Cl-in the different ages of the leaf mostly distributed in the young leaf under salt spray,and that mostly distributed in the old leaf under substrate salinity.Moreover,the ratio of K+/Na+ under salt spray was lower than that under substrate salinity.The ion toxicity on N.tazetta seedling were more serious under salt spray in leaf than that in root.There was significant positive correlation between contents of Na+ and Cl-after both salt stress pathways.