Mitochondrial Genomic Analysis Of Carrot Petaloid Cytoplasmic Male Sterile And Maintainer Line

Carrot?Daucus carota L.?is one of the ten toppest vegetables and rich with ?-and ?-carotene.Carrot hybrids bred with cytoplasmic male sterile?CMS?method have been widely used in the bases.Two morphologically different CMS systems were found: brown anther type and petaloid type.Petaloid type is a maternally inherited loss of male sterility based on flower male organ dysfunctions due to a complete conversion of stamens into petal-like organs and has been widely utilized.Previous studies showed that mitochondrial reorganization in the petaloid CMS line caused atp9 13 amino acids longer than that in the fertile line.In carrot,the generation of homeotic floral organ resulted from an impaired expression of homologs of Antirrhinum GLOBOSA and DEFICIENS caused by a disturbed interaction between mitochondrial and nuclear genes in an unknown way.However,the mitochondrial phenotype and genome research has not been reported.In this study,the petaloid CMS line?P2S?and its maintainer line?P2M?were used to observe the phenotype of mitochondrion and re-sequenced to compared the mitochondrial genome differences.This study will provide an improved understanding of the regulation mechanism about carrot CMS.1.The length of mitochondria was about 0.5-1.5 ?m and most mitochondrial cristae was vesicular in P2 S and P2 M with transmission electron microscope.There were 8.4 mitochondria per leaf cell in P2 M,but only 4.8 in P2 S.The P2 S in inflorescence cells contains 5.2 mitochondria per cell,and P2 M contains 9.0.Whatever,inflorescence mitochondrial cristae and super size of mitochondrion?>5 ?m?was found in P2 S.There was a certain degree of cavitation in mitochondria in P2 S and P2 M inflorescence cells.P2 S contains 1.3 cavitation mitochondria per inflorescence cell,while P2 M contains 0.7 per cell.Through genomic resequencing,the mitochondrial genome sizes of P2 S and P2 M were 274,155 bp and 281,120 bp,respectively.Compared with P2 M,seven structural variations and three ORFs?orf27,orf32,orf34?deletions were found in P2 S,which might be the main reason to cause the change of mitochondrial cristae structure and mitochondria reduce.2.Compared with P2 M,there were 46 SNP variants and 6 InDel variants in the P2 S gene region.The transmembrane structure of these genes was predicted with TMHMM software and six male sterility related ORFs were primitively screened: orf27,orf30,orf32,orf34,orf40 a and orf26.RT-PCR results showed that the expression of orf27,orf30,orf32,orf34 were significantly different between P2 S and P2 M in three periods of flower development,and their functions need to be further studied.3.Four specific markers of MtD₁-4 were designed according to the SV in P2 S.Different genotype of sterile lines and maintainer lines,land races and hybrids were used to detected the polymorphism of these markers.The results showed that only single sterile fragment occurred in the landraces and four sterile fragments exist in the sterile lines.The coincidence rates of MtD₁-4 markers were 96.2%,100%,96.2%,and 100%,respectively in the sterile lines and maintainer lines,which can be used to detect petaloid sterility in carrot.