Identification Of Gene Related To The Formation Of 2n Male Gamete In Cymbidium And Creation Of Polyploid Germplasm Resources

Polyploid breeding is an important breeding method of Cymbidium.The 2n gamete pathway is an important way to create plant polyploid germplasm resources and develop polyploid varieties.However,there are few reports on the creation of Cymbidium polyploids byusing of 2n gamete,andthe molecular mechanism of 2n male gamete formationin orchids has not been reported.In this study,the occurrenceof 2n male gametes was observed in the hybrid offspringof Cymbidium sinens?Cymbidium lancifolium,the comparative analysis of the transcriptome of the hybrid offspring with significant difference in the incidence rate of 2n male gamete,SDS geneassociated with the occurrence of 2n male gamete was identified and cloned,and polyploids were created through 2n gamete pathway.The main results are as follows:1.2n male gametes were evaluated in mature pollen of the hybrids of C.sinense‘Xiaoxiang'?C.lancifolium and C.sinense‘Jinzui'? C.lancifolium.The average incidence rate of 2n male gametes in hybrid offspring was significantly higher than that in the parents.Tere were 4 and 8 hybrids with the incidence rate of 2n male gamete being more than 3% in the above combinations,respectively.2.By the use of germplasm resourceswith high occurrence rate of 2n male gamete as parents,41 cross or self-pollination combinations were made up,and 592 seedlings were produced by seed culture in vitro.A tetraploid was identified from the self-pollination combination of 45-17,and a triploid was obtained in the cross combination of 45-32 ? 45-17.3.The proliferation and differentiation characteristics of different polyploids were different.The proliferation and bud differentiation rates of tetraploid HX-2-21 rhizome were significantly higher than those of diploid,the same as thetriploid HX-3-3,however,the multiplication and bud differentiation rates of triploid HX-4-35 rhizome were not significantly different from those of diploid.4.Comparative analysis of the transcriptome swas carried out on hybrid progenies 67-80 and 67-109 of C.sinense‘Jinzui'? C.lancifolium.A total of 13 candidate genes associated with 2n male gametogenesis were screened.Real-time fluorescence quantitative PCR results showed thatthere were significant differences in expressions of SDS?NACK11?NACK12?MAP2K2?MAP2K91?MAP3K1?MAP3K3 and MAP3K7 genesbetween67-80 with the high incidence rate of 2n male gamete and67-109 ith thelow incidence rate of 2n malegameteduring meiosis.The expressions of NACK11?NACK12?MAP2K2?MAP2K91?MAP3K1and MAP3K3 genes were up-regulated,while those of SDS and MAP3K7 wasdown-regulatedin 67-80,suggesting that these genes could be the related genes resulting inthe inprovement of 2n male gamete formation in 67-80.5.The SDS homologous gene of Cymbidium was obtained by RACE technique,named Csl SDS(accession number: KY873304.The c DNA was 2203 bp in length,encoding 582 amino acids with a molecular weight of 65.66 KD and a theoretical isoelectric point of 5.02.It had a transmembrane domain at the position of the 500 th amino acid and probably encoded a kind of membrane protein.The homology comparison results showed the similaritywas 56%with Amborella trichopodagene,and phylogenetic tree analysis indicated that the gene was closely related to Asparagus officinalis.According to the above research,the occurrence rate of 2n male gamete in hybrid progeny of C.sinense?C.lancifolium was higher and stable,andtwo new polyploids were obtained by the use of these hybrids,13 genes associated with a high incidence of 2n male gamete were identified and the c DN A full length of SDS gene in the hrbrid was obtained.These laid a soli foundation for further developingsexual polyploidsin Cymbidiumand studying molecular mechanism of polyploidization evolutionary in orchid.