Assembly Of Satsuma Mandarin Mitochondrial Genome And Identification Of Candidate Cytoplasmic Male Sterility Associated Genes In A Somatic Cybrid Of HB Pummelo

Seedlessness is highly desirable traits for citrus.Male sterility is mainly responsible for seedlessness in widely-cultivated citrus varieties.Male sterility is caused by mitochondrial genes with coupled nuclear genes in Satsuma mandarin.Sexual crosses that utilized germplasm with Satsuma mandarin as the female parent yielded new seedless varieties of citrus.However,the mitochondrial genome infromation and mechanisms of cytoplasmic male sterility(CMS)remain to be elucidated in citrus.In our previous breeding program,male sterile and seedless cybrid pummelo(G1+HBP)was regenerated by fusing protoplasts from seedless ?Guoqing No.1‘ Satsuma mandarin(G1,the callus parent)and seedy ?Hirado Buntan‘ pummelo(HBP,C.grandis(L.)Osbeck,the leaf parent),which is an ideal material for cytoplasmic male sterility analysis.The high-quality mitochondrial genomes of G1+HBP and its fusion parents were sequenced and assembled,and determined the origins of the nuclear and cytoplasmic genomes of the cybrid.Compared with the fertile HBP mitochondrial genome and transcriptome,57 ORFs that co-transcribed with the same mitochondrial genes in the cybrid and G1 were identified as the CMS-specific ORFs.The orf88 was then selected as a candidate gene,and its potential mechanism of regulating CMS was studied.The main results are as follows: 1.Characterization of the G1 and HBP mitochondrial genomePurified mt DNA of G1 was extracted from a fresh subculture of embryoniccallus while mt DNA of HBP was purified from 45-d-old etiolated seedlings.HBPtotal DNA was isolated from frozen leaves using a simplified CTAB protocol,andG1 total DNA was isolated from embryonic callus.The mitochondrial genomes ofG1 and HBP were assembled into circular molecules of 521,599 bp and 518,274 bpwith GC contents of 45.06% and 45.14% by using the total DNA Pac Bio Sequelsequencing reads and mt DNA sequencing reads,respectively.The mitochondrialgenome of citrus encodes 35 to 36 proteins,three ribosomal RNAs and 21 t RNAs.Atotal of 460,067 bp(88.8%)of HBP is homologous to the mitochondrial genomefrom G1,and at least 28 apparent rearrangements in both the syntenic order and therelative direction of the ORFs.In addition,species specificity of RNA sites andefficiency existed between G1 and HBP.2.Origins of the cytoplasmic and nuclear genomes of the cybrid pummelo G1+HBPThe mitochondrial genome of the cybrid(G1+HBP)was assembled into a circle of 538,434 bp.The cybrid inherited the entire mitochondrial genome of the G1 with an extra recombinant fragment of 16.8 kb,and 471.9 kb(87.6%)of the cybrid sequence is homologous to the HBP sequences.The synteny analysis showed that up to 96.9% the mitochondrial genome sequence from the cybrid is continuously co-linear with the mitochondrial genome sequence from G1;the remaining 3.1% of the mitochondrial genome sequence from the cybrid is an extra homologous recombinant fragment;and at least 29 rearrangements in the cybrid relative to HBP.A total of 28 of these rearrangements were identical to the rearrangements we observed in G1 relative to HBP.The chloroplast genomes of the cybrid,G1 and HBP were assembled into circular molecules of 160,423,157,088 and 160,590 bp,respectively.Each of these genomes has a GC content of 38.47%.The chloroplast genome sequence similarity was >99% among the cybrid and its parents,and the cybrid had 11 SNPs and 42 In Dels relative to the chloroplast genome sequence of HBP,which is much less than the 384 SNPs and 120 In Dels relative to the chloroplast genome sequence of G1.Total DNA of the cybrid and its parents were sequenced using the Illumina platform to identify SNPs and In Dels relative to the sweet orange genome.A total of 946,383 SNPs and 200,661 In Dels relative to the sweet orange genome are shared by the parents and the cybrid,which are unidentical in parents.Among these polymorphisms,99.8% of the SNPs and 96.9% of the In Dels are shared by the genomic sequences of HBP and the cybrid.These data indicate that the chloroplast and nuclear genomes of the cybrid was inherited from HBP.3.Screening and function verification of candidate CMS gene of the cybrid pummelo G1+HBP.Compared with the fertile HBP mitochondrial genome and transcriptome,21 and 20 CMS-specific mitotype-specific sequences(MSSs),and 89 and 70 CMS-specific ORFs were identified in the cybrid and G1 mitochondrial genomes,respectively.We identified two reverse CMS-specific ORFs(orf59 and orf88)and one forward CMS-specific ORF(orf55)co-transcribed with the 138 bp reverse-complementary sequence from the 3‘ end of rps1(?rps1)on one 1705 bp transcript DN16978 in the cybrid and G1.The transcript DN16978 was ectopically expressed in Arabidopsis and transgenic lines shown typical male stertile.Transmembrane helices and subcellular location prediction shown the deduced amino acid sequence of orf88 has mitochondrial localization signal peptide and a membrane-spanning helice,indicating that the orf88 was a mitochondrial-anchored protein and more likely to the causal gene for CMS induction in citrus.The candidate protein Cs NADH,subunit 12 of the NADH dehydrogenase 1 alpha sub complex,was identified to interact with DN16978 by yeast two hybrid screening and interact with orf88 validated by point to point and luciferase complementation imaging assays verification.The results of subcellular localization showed that orf88 and Cs NADH were localized to the mitochondria.In summary,the mitochondrial genomes of seedless cybrid pummelo(G1+HBP)and its parents were sequenced and assembled.A chimeric mitochondrial protein orf88 which interacts with the Cs NADH in the G1+HBP was then verified;moreover,mechanism of orf88 for regulating CMS in the somatic cybrid pummelo was also discussed.