Research And Application Of Male Sterility Of Tagetes Patula And Tagetes Erecta

Tagetes patula and Tagetes erecta belonging to the Asteraceae family.They are grown in world-wide for medicinal,industrial and ornamental purpose.Because of the capitulum structure which combined by multiple individual florets,the artificial emasculation of flowers is a highly labor intensive process.Thus,the development of a pollination control system would greatly facilitate the production of F₁ hybrids.The objectives of this research were to study on the characterization and application of male sterile mutant of Tagetes patula and the molecular mechanism of male sterility of Tagetes erecta.Our study will play an important role in the breeding and hybrid seed production of Tagetes patula and Tagetes erecta.The main findings are as follows:1.In this study,we reported the characterization of a novel pollen abortion mutant of Tagetes patula which was isolated from 1796 heat shock induced cutting seedlings.Histological and cytological observation demonstrated that the abnormal development of the microspores during the binucleate pollen stage led to pollen abortion,and this was correlated to premature tapetum degeneration.Cross hybridization experiments indicated that recessive nuclear gene was responsible for the trait.2.Two male sterile lines（Ms-1,Ms-2）and ten inbred lines（K3,K4,K5,K8,K13,K15,K17,K20,K30,K33）were crossed according to Comstock and Robinson’s（1952）North Carolina Design II to estimate heterosis,combining ability,heritability and the phenotypic correlation coefficient for eleven horticultural characteristics of Tagetes patula.The results showed that the hybrid combinations displayed higher ornamental values and showed obvious heterosis over the male-parent for most of the ornamental traits evaluated.Both GCA effects and SCA effects were found to be important for the performance of individual hybrid combinations,and the relative importance of GCA and SCA to progeny quality varied for different traits.Estimates of heritability showed that many of the quantitative ornamental traits of Tagetes patula were highly heritable.By contrast,environmental factors influenced markedly on the traits of plant height,crown size,number of days between opening and wilting of inflorescences and the maximum number of flowers present each day.Phenotypic correlation coefficient analysis showed that the various vegetative growth characteristics were closely related to one another and,more interestingly,significant phenotypic correlations were found between the growth of the vegetative and reproductive structures of Tagetes patula.3.We generated a reference transcriptome and eleven digital gene expression（DGE）of flower buds sized 1 mm and 4 mm from the male sterile and male fertile plants by using Illumine sequencing.A total of 90,547,072 raw tags were sequenced in the transcriptome library of Tagetes erecta.After filtering out low quality reads,87,473,431 clean tags remained in the library.There were 128,937 transcripts were assembled of clean data by using Trinity software,and produced 65,857 unigenes.34176 unigenes（51.89%）were annotated in Nr,Nt,Pfam,Swiss-Prot,KOG,KEGG and/or GO.We found that there were557 differential expressed genes（DEGs）detected in the 1 mm flower bud libraries between the male sterile and male fertile（S1 vs F1）,while there were totally 785 DEGs detected in the 4 mm flower bud libraries between the male sterile and male fertile（S2 vs F2）.From the DEGs results,there were 12 unigenes belong to MADS-box family with significantly different expression in the S2 vs F2,and only one MADS-box unigene with significant different expression in the S1 vs F1.26 unigenes were randomly selected for quantitative real-time PCR analysis,and their expression levels agreed well with the DGE analysis,confirming the accuracy and reproducibility of the Illumina expression profile.4.Five B class genes（Te PI,Te AP3-1,Te AP3-2,Te TM6-1 and Te TM6-2）were isolated from the male sterile plant and male fertile plant of Tagetes erecta.Sequence comparisons showed that the sequence of Te PI gene was different in the male-fertile plant and male-sterile plant,named as Te PI-1 and Te PI-2,respectively.Compared to the sequence of Te PI-1,the Te PI-2 gene has an extra 1108 nucleotides before the fourth exons in the g DNA sequence,which lead to an extra 12 nucleotides of c DNA sequence and 4 amino acids in the K domain of amino acid sequence.A co-dominant candidate gene marker based on the different sequences of Te PI genes was used to segregating populations,and the results showed that all the PCR products amplified by the candidate gene marker were consistent to the phenotypic of the two segregating population with 988 individuals.Expression analysis showed that all the B class genes of Tagetes erecta were strongly expression in stamen of male fertile plant,and the expression level was significantly higher than the expression in stamen of male sterile plant.The same result occurred in the expression level of flower buds.Protein interactions of the B class proteins showed that both Te PI-1and Te PI-2 protein can form a homodimers and heterodimer with all the other four DEF/AP3 proteins,Te AP3-1,Te AP3-2,Te TM6-1 and Te TM6-2.But the interaction strength of Te PI-2/AP3 dimerization was significantly weaker than the interaction strength of Te PI-1/AP3 dimerization.Constitutive expression of Te PI genes of Tagetes eracta in tobacco showed that the transformed 35S::Te PI-1 tobacco plants had smaller flower organs and obvious shorter pistil,but none of transgenic tobacco plants of35S::Te PI-2,35S::Te PI-2,35S::Te AP3-1,35S::Te AP3-2,35S::Te TM6-1 and35S::Te TM6-2 showed any obvious change from wild-type morphology.In summary,we concluded that the mutation of Te PI led to the male sterility trait of Tagetes eracta.