Phynotype And Gene Clone Of Mutants With Embryo And Endosperm Defects In Arabidopsis Thaliana

The reproductive development of higher plants is very complicated and exquisite,and it is of great significance to explore its characteristics of growth and molecular regulatory mechanisms.As a typical model plant,Arabidopsis has the advantages of short life cycle,small genome,clear genetic background,high homogeneity of genes and easy access to defective mutants,making it an ideal material for the research on development process and internal molecular regulatory mechanisms in plants.Therefore,the acquisition of mutants with the abnormal phenotype we are interested in and the isolation of the target genes are essential.In this study,four mutants with abnormal embryo and endosperm(275-3,350,535-1and 578-1)as well as one male sterile mutant(241-2)were used as experimental materials.The near-isogenic lines of the mutants were obtained by the heterotypic hybridization,then screened to explore the target genes,loss of function of which contributed to certain phenotypes,through map-based cloning technique.After preliminary positioning and fine mapping,the target genes were identified within a certain segment of a chromosome.Whereafter,combined with whole-genome resequencing analysis,the target genes were likely to be found out.At the same time,a series of methods,including ovule overall transparency,endosperm autofluorescence experiment,I₂-KI staining,FDA staining,Alexander staining and anther semi-thin section,were applied for their phenotype investigation.The main results obtained in the studies are described as follows:1.The ovule transparency and endosperm autofluorescence techniques were applied in investigation of the 275-3,350,535-1 and 578-1 mutants.The results showed that the embryo development of mutant 275-3 delayed when the embryo of wild type reached the globular stage,but it would continue to grow until the globular stage.Meanwhile,the free nuclei of the endosperm of 275-3 decreased compared with wild type,and no endosperm cellularization was observed.When the wild-type embryo reached globular stage,the embryo of mutant 350 was at the same stage,but the division pattern was disordered.And when the wild-type embryo developed to torpedo stage,the embryos of mutant 350 stagnated at the mid-globular or late-globular stage,and its division pattern was disordered as well.In addition,the endosperm failed to cellularize and its free nucleus were swollen and scarce in mutant 350.The mutant 535-1 began to show abnormal embryo,from the eight-cell to sixteen-cell stage,and eventually stagnated at the early globular phase,showing that indistinct boundary was present between the embryo body and the suspensor and the embryonic division was obviously irregular.When the wild-type embryo came to the globular stage,the embryos of mutant 578-1were still at two-cell stage,and the embryonic development stagnated before the globular phase,with disordered division pattern,and the development of suspensor was abnormal as well.Both mutants,535-1 and 578-1,showed sparse and enlarged endosperm free nucleus existed as syncytia.2.Preliminary observation of mutant 241-2 revealed that the siliques were short and can not produce seeds.Staining of pollen and anther showed that the pollens were aborted due to the abnormal development of anther and male gametophyte.Further observations of semi-thin section indicated that the pollens of mutant 241-2 were normal during the phase of pollen mother cell and tetrad,but thet began to show highly vacuolated and abnormally enlarged microspore during the polarized microspore period,and the shape of bicellular pollen was still abnormal and the number was reduced obviously.The pollens of mutant 241-2 shrinked and dented eventually,degrading into minor remnants of cell debris.Therefore,the pollens were likely to abort between the stage of tetrad and polarized microspore in Arabidopsis mutant 241-2.3.Genetics and molecular biology techniques were used to analyse the phenotype and genetic stability of the mutant 275-3.The results showed that the phenotype of mutant 275-3 was stable during the process of hybridization and backcrossing,its ovule abortion rate was about 25%,and the separation ratio was close to 2:1.Thus it is suggested that the variation in mutant 275-3 should be a recessive mutation controlled by a single gene,and the homozygous variation of the locus was lethal.To map the target genes of mutants 275-3,350,535-1 and 578-1,we used the BC₂F₁generation population obtained by hybridization and backcrossing for preliminary positioning analysis,and the BC₃F₁generation population for fine mapping and whole-genome resequencing.Whereas in mapping the mutated gene of mutant 241-2,the F₂generation was collected for whole-genome resequencing analysis.The positional cloning results show as follows:(1)The mutation site of mutant 275-3 is located between the molecular markers T22F11 and T9J22 in chromosome 2,that is,from Chr02:10779026 to Chr02:11328569,and the physical distance is 549543bp.After the whole-genome resequencing,it was found that an insertion of a single base C occurs after Chr02:11104201.And sequencing of the DNA fragment containing the mutated site was performed,confirming that the mutation does occur at this site.Therefore,At2g26060 corresponding to this site was listed as the candidate gene.Information of the gene was searched on NCBI,At2g26060 was also called At CIA1,and there had been reports on its functional studies.The phenotype of mutant 275-3 was consistent with the mutants used in the published papers.(2)The mutation site of mutant 350 is located between the molecular markers MGL6 and MKP6 of chromosome 3,namely from Chr03:5634662 to Chr03:6014083,and the physical distance is 379421bp.The whole-genome resequencing analysis revealed that a deletion of 37 bases occurs between Chr03:5727293 and Chr03:5727329,and the corresponding gene was At3g16810,which was listed as the candidate gene.Its information was searched on NCBI,and the gene was named APUM24.Functional studies on APUM24 had been reported,and the phenotype of mutant 350 was consistent with the mutants reported in the papers.(3)The mutation site of mutant 535-1 was located between the molecular markers In Del 2 and In Del 3 of chromosome 2,namely between Chr02:17446704 and Chr02:17574439,and the physical distance is 127735bp.However,we can't find possible mutation sites even after resequencing analysis within this interval.(4)The mutation site of mutant 578-1 was located between the molecular markers Indel 1 and Indel 2 of chromosome 2,namely from Chr02:17240191 to Chr02:17446704,and the physical distance was 206513bp.Nevertheless,we failed to find possible mutation sites after whole-genome resequencing as well.(5)Through whole-genome resequencing of mutant 241-2,we found a deletion of single base G at Chr04:11384008,a replacement of base A by base G at Chr04:11384015,and an insertion of single base T after Chr04:11384018.The three sites are very close to one another,and their corresponding gene is At4g21370.The DNA fragment amplification containing the candidate sites was conducted,but we just failed to amplify the target band,therefore the results of whole-genome resequencing analysis could not be confirmed.