| Cymbidium has high ornamental,economic and culture value,the key method of new cultivar creation is cross breeding method,whose breeding period is very long and the efficiency is very low.Molecular marker-assisted selection breeding can efficientively shorten the breeding cycle and improve breeding efficiency,however,there are few reports concerning on linkage map construction and molecular marker-assisted selection breeding of Cymbidiums in literature.In this experiment,Cymbidium sinense ‘Huangyehongmo’ and C.‘Yunv’ were employed to produce F1 mapping population,SSR markers were developed by using of transcriptome sequencing of Cymbidium sinense ‘Qijianbaimo’,and used to construct the linkage map of Cymbidium.The main results are as follows:1.Construction of mapping population.Hybrid seeds of C.sinense ‘Huangyehongmo’? Cymbidium ‘Yunv’ were cultured in vitro,and 513 strains were produced.The micropropagation characteristics and charaters of test-tube seedings of these strains were detected.The results showed that there were significant differences in the proliferation and differentiation abilities of intermediate propagule and the growing rate of bud,plant type of the plantlets were diverse,including loose,semi-compact and compact,obvious differences existed on leaf length and leaf width,suggesting that the genetic diversity of hybrid progenies was abundant.94 progenies were randomly sampled to develop the mapping population,the results of analysis indicated the diversity on micropropagation characteristics,palnt type and leaf characters was similar to that of the original population.2.Transcriptome sequencing and develop SSR marker in Cymbidium Transcriprome sequencing of C.sinense ‘Qijuanbaimo’ was conducted by the use of vegetative organs and reproductive organs,51764 unigenes were obtained.After screened with MISA on the unigenes,whose size was more than 1kb,4419 SSR loci were found with the occurrence frequency of 8.54%.Repeat motif of the SSR markers ranged from dinucleotide to six nucleotide,with dinucleotide and three nucleotide being the main types,accounted for 57%and 33.76%,respectively,and the dinucleotide repeat motifs of AG/CT was the predominant repeat types(3.73%).Using Primer Premier 5 design software,318 SSR primers were obtained from Cymbidium sinense,and 185 were sifted from Cymbidium hybridum.Cymbidium sinense ‘Huangyehongmo’ and Cymbidum‘Yunv’ were employed to study the versatility and polymorphism of SSR primers,among them,235 primers from Cymbidum sinense(73.89%)produced clear and reproductive bands,61 primers(19.18%)showed polymorphic;143 primers from Cymbidium hybridum(77.29%)also produced clear and reproductive bands,and 17 pairs of primers(9.19%)were polymorphic.3.Molecular genetic linkage map construction of Cymbidiums 94 offspring of Cymbidium sinense ‘Huangyehongmo’ ? Cymbidium ‘Yunv’ and 78 SSR marker were subjected for construction of a genetic linkage map of Cymbidium by the use of Mapmaker/EXP 3.0 software.The molecular genetic linkage map contained 13 linkage groups and 56 SSR markers,the distance of linkage map was 1608.90 c M,and the average distance were 32.15 c M.Based on estimation,the length of Cymbidium genome was2064.82 c M,therefore,the coverage rate of the linkage map was 77.92%.Through the above research,318 and 185 SSR markers were developed from Cymbidium sinense and Cymbidium hybridum,respectively,and the first molecular linkage map was constructed,these laid a foundation for further developing more perfect genetic linkage map,performing QTL mapping and molecular marker-assisted selection breeding in Cymbidium. |
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