| As a tall dioecious tree of salicaceae,Idesia polycarpa is an important woody oil plant.In fact,it has a long juvenile period of 4-5 years.At present,the economic demand of female plants is obviously greater than that of male plants.It is difficult to distinguish the sex of Idesia polycarpa in seedling stage,which makes it difficult to carry out the research on breeding and genetic cultivation and utilization of Idesia polycarpa.Therefore,the development of the technology for early sex identification of Idesia polycarpa seedlings can effectively shorten the breeding cycle and reduce the breeding cost,which is of great significance in production and breeding.In this study,PCR amplification was used to verify the universality of sex identification molecular markers previously reported in Idesia polycarpa.A large number of SSR markers were developed by using simplified genome sequencing.The reaction system of SRAP-PCR was optimized and a large number of SRAP molecular markers were screened out.The differentially expressed genes were screened according to the public database of flower expression profiles,and the differentially expressed genes were screened by bioinformatics analysis.Primers were designed according to the differentially expressed gene sequences and verified by PCR amplification.The results are as follows:(1)The universality of one ISSR marker and one SCAR marker was verified by using Idesia polycarpa with known gender.Results The two groups of markers used in this study did not show good universality,so it is necessary to develop molecular markers based on early sex identification of Idesia polycarpa chinensis.(2)Simplified genome sequencing was performed using poplar genome as reference genome,SSR loci were identified using MISA software for polymorphic SLAF tags,and primers were designed for verification.Based on the analysis of SSR number,SSR loci,SSR repeat distribution and sequence length on SLAF tag of Idesia polycarpa,it was concluded that the largest number of SSR in Idesia polycarpa genome were mononucleotide,dinucleotide and trinucleotide.The proportion of A/T type in mononucleotide was the highest.Among dinucleotides and trinucleotides,TA/AT and AA?(A,G,C and T were all available)had the highest repeat proportion,and 78.52%SSR length was mainly 10-15 bp.The proportion of 100 pairs of randomly synthesized primers amplified polymorphic primers was 35%,among which,STZ-SSR1 had a verification rate of 68.33% for specific fragments of male and female plants of Idesia polycarpa,and 47 samples were used to detect different bands when they were transformed into SCAR markers.The second and third repeated tests showed that there was no specificity between male and female.Finally,the marker was not a marker of gender difference;Six pairs of polymorphic primers were randomly selected for genetic analysis of 30 samples from Idesia polycarpa,and the clustering results reflected the source,geographical distribution and resource characteristics of samples well.(3)The SRAP reaction system of Idesia polycarpa chinensis was optimized by orthogonal experimental design,and 846 pairs of primer combinations were amplified by this system.Results The ranking of the influence of different factors on SRAP-PCR was obtained,and the optimal combination was determined.Among 846 pairs of primers,792 pairs of primers could amplify bands in Idesia polycarpa seeds.Using 6pairs of polymorphic primers,24 Idesia polycarpa germplasm were amplified by PCR,and the cluster results of genetic relationship were highly consistent with the results of SSR molecular marker analysis.(4)Based on the transcriptome data library,a large amount of basic data were obtained,and a total of 104,732 Unigene genes with an average length of 845 bp were assembled.The number of annotated Unigene in each database was 51,531,21,025,26,669,34,860 and 32,302,respectively.The three species with the largest number were Populus trichocarpa,Populus deltoides and Populus alba sinicum,which indicated that therelationship of Idesia polycarpa was close to these three species.A total of 14,785 SSR loci were retrieved from the transcriptome of Idesia polycarpa,among which mononucleotide repeats,dinucleotide repeats and trinucleotide repeats were the most,and the dominant repeating motifs were A/T,TC/CT and AT?(A,G,C and T were all available).The number of SSR repeats in the transcriptome of Idesia polycarpa was mainly between 8-24 bp,and the length of SSR in Idesia polycarpa was between 10-15 bp.By comparing transcriptome data of Idesia polycarpa in different periods,267 female flower-specific genes and 170 male flower-specific genes were detected.Specific primer were design and synthesize based on these flower-specific genes,and PCR verificationwere conducted by using DNA samples from male and female plants of Idesia polycarpa.The results showed that 115 pairs of male specific gene primers and117 pairs of female specific gene primers could amplify clear bands.Howerever,no oneshowed sex specificity. |
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