Morphology And Molecular Mechanism Study Of Pollen Abortion In Oncidium Honey Angel

Oncidium is a plant of Epidendroideae in Orchidaceae.Its inflorescence has good branching,and it is like a dancing girl when it is in full bloom,so it is also known as a dancing orchid.As the largest export flower cut flower in Taiwan,Oncidium mainly focuses on One.Gower Ramsey and One.Honey Angel,and the color is single yellow.However,the acceptance of yellow flowers in European countries is not high,because the yellow flowers in these countries represent separation or sadness.Therefore,in order to expand the export market of Oncidium,it is necessary to breed varieties of different colors to improve the market acceptance of cut Oncidium.However,One.HA and One.GR have self-incompatibility and hybrid incompatibility.Under natural conditions,natural pollination cannot be carried out to obtain fruit pods,and many efforts have been made to carry out hybridization tests,and new varieties cannot be bred.At present,some researchers have made a preliminary study on the cross incompatibility of One.GR,and found that its pollen abortion is the main reason for the difficulty of cross-pollination.Therefore,this study intends to explore the pollen abortion of One.HA from two aspects of cell morphology and molecular biology,so as to find out the reasons of pollen abortion and provide a theoretical basis for the study of hybridization of Oncidium.1 Morphological observation of the pollen abortion in OncidiumBy observing the pollen cluster morphology of sterile cultivar One.Honey Angel,One.Gower Ramsey and fertile cultivar Onc.Sharry Baby,it was found that the anther chamber of sterile cultivar One.HA and One.HA was obviously flattened,the outer wall was sunken and shrunk,and the pollen grains of pollen cluster were arranged very closely and irregularly.There was no obvious pollen grains attachment in the mature pollen clusters.The pollen grains morphology of the sterile cultivar One.HA and the fertile cultivar One.SB were observed.Compared with the fertile cultivar,the pollen grains of sterile cultivar were different in size and shape,the development of pollen cell wall was discontinuous and there were many holes(not germination holes).The pollen cells of the sterile varieties are irregular in size and shape,and many damaged cells are found under the microscope field,and the cell contents are less and the vacuolization is serious.DAPI staining of pollen mother cells showed that the genetic material in the daughter cells of One.HA pollen mother cells at the telophase of meiosis I and meiosis II was not evenly distributed,resulting in the microspore containing different numbers of chromosomes.After meiosis is completed,trispore or polysporophyte may be formed.In conclusion,the pollen abortion of One.HA may be related to the following problems:1.Abnormal development of pollen wall.This problem will result in the outflow of pollen cell contents,which will not provide the necessary nutrients for the normal development of microspore,thus affecting the normal development of microspore.2.The meiosis of pollen mother cells is abnormal.Onc.HA pollen mother cells may have abnormal homologous chromosome pairing,synapse and recombination during meiosis,which may lead to different number of chromosomes in microspore and affect its normal development.2 Comparative analysis of pollen transcriptome sequencing between Oncidium and MiltoniaRNA-Seq was applied to analysis the pollen transcriptome differences between Oncidium and Miltonia.In total,we obtained 132,789 Unigenes.Furthermore,48,066 DEGs were screened out according to the condition that the difference multiple was not less than 4(|log2Ratio|≥4)and the false discovery rate was not higher than 0.001(FDR≤0.001),among which 16,527 genes were up-regulated and 31,539 genes were down-regulated.The number of differentially expressed genes associated with pollen development was 1,200 of which 475 were up-regulated and 725 were down-regulated,involving genes involved in pollen development,pollen germination,pollen tube growth,pollen wall formation,meiosis,etc.From the above 1200 gene differences in 145 selected by hierarchical cluster analysis,a total of 31 has significantly differentially expressed genes(7 genes were up-regulated,and 24 genes were up-regulated).To verify the quality of RNA-Seq sequencing,9 genes were selected from 31 significantly differentially expressed genes for qRT-PCR analysis.The results showed that the qRT-PCR results of the nine differentially expressed genes were in good agreement with the sequencing data,indicating that the transcriptome sequencing results were highly reliable.Based on the functional annotation of differentially expressed genes,5 genes were selected from the above 9 differentially expressed genes for further quantitative analysis.The results showed that the five differential genes expression levels were in the fertile plants Miltonia and One.SB were significantly higher than One.HS,and the difference multiples were all greater than 3.The results showed that the selected genes were indeed differentially expressed in the pollen development of One.SB and Miltonia.3 Cloning,bioinformatics and expression analysis of OnMAD2 gene in OncidiumBased on the transcriptional database data of One.HA,the OnMAD2 gene was cloned,bioinformatics analysis and quantitative expression analysis were carried out.The RT-PCR results showed that the gene coding region of OnMAD2 was 624 bp,encoding 207 amino acids.The length of gDNA is 961bp,with 2 introns and 3 exons.Bioinformatics analysis showes that OnMAD2 belongs to the HORMA superfamily with a HORMA domain.The relationship between Oncidium OnMAD2 and Dendrobium MAD2(PKU87023.1)is the closest,followed by Apostasia shenzhenica(PKA55503.1).The OnMAD2 protein sequence has high similarity to the other species MAD2,both above 70%,indicating that the MAD2 protein is highly conserved in plants.The qRT-PCR results of different tissues and organs showed that OnMAD2 had a very high expression level in the root and the lowest relative expression in the pseudosperm,and had obvious tissue specificity.It is speculated that this gene may be expressed normally in roots.The results of qRT-PCR at different flowering stages showed that the relative expression of OnMAD2 was the highest in the semi-open period,followed by the early blooming stage and the lowest relative expression level in the blooming stage.It is speculated that this gene may play a role in controlling chromosome segregation during meiosis of pollen mother cells.4 Cloning,bioinformatics and expression analysis of OnMYB106 gene in OncidiumThe RT-PCR showed that the coding region of OnMYB106 was 819 bp,encoding 272 amino acids.The length of gDNA is 1005 bp,with 2 introns and 3 exons.Bioinformatics analysis showes that OnMYB106 belongs to the SANT superfamily and has two consecutive MYB domains.It is a typical R2R3-MYB.The evolutionary distance of OnMYB 106 and rice OsMYB 106(OC_Os08g33660.1)is the closest.It is speculated that these two genes may have similar functions and belong to a class of transcription factors regulating pollen development.The qRT-PCR results of different tissues and organs showed that OnMYB106 has a high expression level in the floral organs of Oncidium,but the expression level is low in other tissues.Therefore,it is speculated that the gene may play an important role in the growth and development of floral organs.The results of qRT-PCR at different flowering stages showed that the relative expression of OnMYB106 was the highest in bud stage,and only a small amount in blooming stage.It was speculated that this gene belongs to the "early" expression gene of pollen development and mainly regulates the synthesis of pollen wall.5 Construction and transient transformation of overexpression vectors of OnMAD2 and OnMYB106 genes in OncidiumIn this experiment,two overexpression vectors p1301-MAD2-GUS and p1301-MYB106-GUS is successfully constructed by the traditional restriction enzyme ligation method.The overexpression vector was transferred into Oncidium PLBs by Agrobacterium-mediated transformation for instantaneous conversion.RT-PCR analysis showed that both GUS and hygromycin genes were found in both positive control and transgenic PLBs.The detection of qRT-PCR and RT-PCR was consistent with the expression of Gus gene only in positive control and transgenic PLBs.Objective qRT-PCR analysis showed that OnMAD2 and OnMYB106 were significantly increased in transgenic PLBs compared with negative control and positive control.These results indicated that the transient transformation of p1301-MAD2-GUS and p1301-MYB106-GUS overexpression vectors was successful.qRT-PCR is consistent with the detection of RT-PCR,and the GUS gene is only expressed in positive controls and transgenic PLBs.qRT-PCR analysis of the target gene showed that OnMAD2 and OnMYB106 were significantly increased in the transgenic PLBs compared with the negative control and the positive control.Taken together,the above results indicated that the two overexpression vectors p1301-MAD2-GUS and p1301-MYB106-GUS were instantaneous conversion successfully.