| Fraxinus,known as the Oleaceae Fraxinus plants,contain about 70 species,about 30 species in china.Fraxinus is an important species for afforestation in North China,for it's developed root system,wide range of adaptability to salt and strong resistance.It is also widely used in landscaping,timber forest and protection forest construction.Because of it's graceful posture,fast growth,excellent material characteristics,however,the resources consolidation and salt tolerance evaluation of the species of the genus tree is still weak.This study were based on the collection of Fraxinus germplasm resources and through Fraxinus inter and intraspecific hybridization,a new hybrid germplasm were created and which salt tolerance were evaluated,so high-throughput sequencing of Fraxinus velutina transcriptome were conducted and resistant salt DNA molecular marker of which were developed and somatic embryogenesis and plant regeneration of Fraxinus velutina were studied.The main results are as follows:1.In the domestic,Fraxinus mainly distributed in Beijing,Shandong,Xinjiang,Shanxi,Gansu,Heilongjiang,Hunan,Hubei,and so on.We collect germplasm resources of 317 copies,including 16 species(varieties).Based on the analysis and comparison,4 new varieties with genetic stability,salt resistance and comprehensive properties were screened.2.Throughput sequencing technology platform,transcriptome sequence Fraxinus velutina were determined and obtained 51698 Uigene,which average length was 661 bp,N50 value was 980 bp,respectively,the Unigene were annotated to NR,NT,Swiss prot,KEGG,COG,GO library.Through analysis we obtain 3249,which contains SSR sequences and successfully designed 18 pairs of SSR primers,the success rate was 55.4%.3.Cross breeding of Fraxinus velutina×Fraxinus pennsylvanica and 10 crosses of Fraxinus velutina were carried out by use of artificial control pollination.The results showed that hybrid progeny had abundant genetic variation and obvious heterosis,Lu No.3XLu 5 showed that seedling height,ground diameter of hybrid progeny were higher than others,which maximum plant height and ground diameter special combining ability effect value reach 5.21 and 0.83.In order to further identify the hybrids,8 pairs of polymorphic primers were screened by SSR primers,and 8 pairs of primers were used to verify the consistency of parents,hybrids and the stability of SSR,and S81 primers were selected to Identify 240 hybrids,and 196 true hybrids were obtained,which accounted for 81.7%.4.Four crosses were breeded using NaCl salt stress treatment,a strong salt tolerant crosses(Fraxinus velutina Lu 3×Fraxinus pennsylvanica Lu 5)were screened out by phenotypic traits and physiological and biochemical indexes.12 SSR primers were selected,which had the same amplified fragment respectively in salt tolerance and salt sensitive plants and the difference between them was significant.Combined with SSR marker screening results and the rapid-scar markers for screening,SCAR marker association of functional gene were successfully developed,named for S20-592,it's nucleotide sequence length was 592 bp.The marker laid the foundation for the developing salt tolerant Fraxinus velutina molecular marker for shorten the breeding cycle.5.For large-scale production,root tips were taken as explants,somatic embryos were successffully induced and regenerated plants were obtained,induction rate reach to 59.8%,adventitious shoot regeneration rate up to 81.2%,rooting rate up to 97.3%.The seedlings of somatic embryos were transferred to the substrate(sand:soil:matrix volume ratio of 4:1:1),and the survival rate was 97.8%.This study laid the foundation for genetic transformation and large-scale propagation of Fraxinus. |
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