| Rhododendron fortunei,a species of the genus Rhododendron,is unique to China.It is mainly distributed in Anhui,Hunan,Hubei,Zhejiang and other southern provinces.Due to its beautiful flowers,bright colors,easy artificial cultivation,it has high horticultural value.In this study,the transcriptome of Rhododendron fortunei was sequenced by using the clone ZL-01.Functional annotation of the assembled Unigenes was performed based on the similarity comparison against the public databases.The MISA software was used for SSR analysis of Rhododendron fortunei transcriptome,and the SSR motif and frequency was determined.Based on the Unigenes containing SSR,primers were designed for polymorphic and transferability analysis.On the other hand,the seeds of R.fortunei were used for polyploid induction by treatment with colchicine and oryzalin.The polyploidy level and leaf-related traits were further analyzed.The main results were listed as follows:(1)Through sequence assembly,84633 unigenes were obtained with mean length of691.4 bp.And,35526 unigenes were successfully annotated by public databases.GO(Gene Ontology)analysis showed that 23215 unigenes were potentially involved in 55 functional category of biological process,cellular component and molecular function.KOG(Eukaryotic orthologous groups)analysis showed that 11 085 unigenes were classified into26 functional groups.For KEGG(Kyoto Encyclopedia of Genes and Genomes)analysis,9887 unigenes were assigned to 272 known pathways.21900 SSR loci were found using the Misa software.The frequency was 20.58%,and the average distribution distance was2.67 kb.Mono-,di-,and tri-nucleotide repeats were the main SSR locus types.This study will provide the foundation and reference for genetic diversity analysis,marker development,hybrids identification,molecular assistant breeding and functional genes isolation in R.fortunei.(2)8439 pairs of SSR primers were designed according to SSR sequences of R.fortunei,and 45 pairs of SSR primers were randomly selected for polymorphism analysis.As the result,32 pairs of primers showed polymorphism in 32 different germplasms of R.fortunei,with the number of alleles varied from 2 to 8,and the mean number of alleles was 5.25.The average of the effective number of alleles,the observed heterozygosity,the expected heterozygosity,the Shannon’s diversity index and the polymorphism information content(PIC)values were 3.1755,0.7006,0.6404,1.3264 and 0.7017,respectively.Furthermore,25 pairs of SSR primers could amplify polymorphic bands in other 31 species of Rhododendron,with a transferability rate of 78.125%.This indicated the high transferability of SSR primers from R.fortunei in species of Rhododendron.All these results showed that the SSR primers of R.fortunei could provide abundant marker resources for genetic diversity analysis,molecular marker assisted breeding of R.fortunei as well as the analysis of genetic relationships between Rhododendron species.(3)In order to induce polyploidy of R.fortunei,the seeds were treated with different concentrations of colchicine and oryzalin.The results showed that there was no significant change in the germination rate of colchicine-treated seeds,and low-concentration colchicine had the effect of promoting seed germination.Compared with the control,oryzalin treatment resulted in a decrease in seed germination rate.The germination rate of10mg/L oryzalin treatment was 66.80%,and the germination rate of control was 70.41%.However,the seed germination rate of oryzalin treatment was not significantly different from the control.After 15 days after germination,it was found that the radicles of the control group were the longest,and the radicle length of the seedlings became shorter with the increase of colchicine concentration,and when the concentration reached to 0.2%,the root tip became spherical,and the embryo axis was thicker and shorter than control.On the other hand,the seedlings after treatment with oryzalin have no obvious radicle outgrowth,and the embryonic axis of the seedling was thicker than that of the control.And it can be clearly seen that there were tiny trichomes on the cotyledons.The 5-month-old treated seedlings were examined by flow cytometry.The proportion of plants with ploidy change was about 47.5%.The average number size of the control plants was 27.87.The average size of diploid was 27.73,the average value of tetraploid was 56.81,the hexaploid was75.43,and the octoploid was 106.61.Among them,the proportion of tetraploids was the highest,reaching to 23%;the proportion of chimera was 17.5%,and the proportion of octoploid plants was 2.5%.Compared with diploids,the tetraploids and octoploid became smaller,height and leaf size were significantly smaller,the leaves became rounder,the color became darker,the leaves became harder,and the thickness of the leaves was also different. |
PDF Full Text Request