Genetic Mapping Of Fertility Restoration Gene Of Petaloid Cytoplasmic Male Sterility In Carrot

Carrot?Daucus carota L.?,with high nutritional value and economic benefits,is one of the top ten vegetable crops worldwide.Currently,carrot hybrids have been widely used in the large-scale bases.Cytoplasmic male sterility including petaloid type and brown anther type is the main method to breed hybrids in carrot,of which the petaloid type is most widely used due to stable genetic and easy identification.Petaloid cytoplasmic male sterility?CMS?has also been found in other crops but it is rarely used because its genetic is instabe and it is difficulty to find the good restorer lines.Carrot petaloid type CMS has been widely used in hybrid breeding production,but the research focusing on fertility restoration is limited.In this study,two F₂ populations were constructed using sterile lines P2S,P25A,and inbred line 17166,respectively.Combination of QTL-seq and traditional genetic mapping was used to locate fertility restoration gene?Rf?.Bioinformatic analyses were subsequently performed to facilitate the candidate genes.The main results are as followed:1.Based on the previous investigation of 65 carrot hybrids and 11 combinations from different seed companies,the fertilities were ranged from 0 to 100%.The fertilities of two combination H1601and H1604 were completely recovered.The segregation ratio of H1604 F₂ population was suitable for the expected value of 3:1 by the Chi-square test,but it is not for H1601 F₂ population.Some part restored plants were also found.This result suggested that fertility restore might be regulated by single main or multiple minor genes.2.In the H1604 F₂ population,the fertile and sterile pools were constructed with twenty three individual plants of typical fertility and petaloid CMS,respectively.Two parental accessions and two pools were re-sequenced with the NGS-based high-throughput whole-genome.A 0–5.1 Mb interval candidate region on chromosome 9 was found and associated with fertility recovery by QTL-seq analysis.3.Based on the genome-wide re-sequencing data,66 pairs of InDel markers and 68 pairs of CAPS markers were developed in the 0–5.1 Mb interval on chromosome 9.Only 16 pairs of InDel markers and 9 pairs of CAPS markers with polymorphism were obtained through screened their parents and the two pools,which were also polymorphic in H1601 F₂ population.In the H1604 F₂ population,Rf1 was located in a major genomic region[InDel2328988–InDel3352891]which was corresponding to the physical position of 2.33 Mb–3.35 Mb on chromosome 9 with 88 annotated genes.In the H1601 F₂population,Rf2 was located between the flanking markers[CAP9?1857204–CAP9?2177501]which was corresponding to the physical position of 1.86 Mb–2.18 Mb on chromosome 9 with 36 annotated genes.Rf1 and Rf2 were neighboured accoreding to the flanking markers.4.Based on the carrot genome annotation,six SNPs causing non-synonymous mutations in exons were obtained in the Rf1 region,five of which were encoded pentatricopeptide repeat?PPR?proteins.Phylogenetic analysis indicated that these five genes were clustered with 11 Rfs PPR cloned from other6 species.The identity of the four genes LOC108201828,LOC108201825,LOC108201831,LOC108202465 was 81.5%,which was 72.8%with petunia Rf-PPR592 and 72.31%with Arabidopsis RPF1.