Genetic Features Of Self-Compatible Mutant,'ZiZhenZhu'(Crataegus Pinnatifida Bge.)

Hawthorn?Crataegus pinnatifida Bge.?is a fruit tree of the genus Crataegus of Rosaceae.As one of the important fruit tree germplasm resources in China,the research on self-incompatibility mechanism has not been reported yet.As the only germplasm resource of Hawthorn self-compatibility found at present,‘Zizhenzhu'provides important research materials for exploring the molecular mechanism of Hawthorn self-incompatibility and the self-incompatibility mechanism of hawthorn plants.To explore the underlying mechanism of self-compatibility of‘Zizhenzhu',two full-length S-RNase genes were cloned in this study.Bioinformatics analysis of the nucleotide sequences and their deduced amino acid sequences,also genetic analysis of self-pollinated progeny were processed in order to facilitate mechanism research of self-incompatibility in Crataegus,which would provide scientific basis for process of pollination in agricultural production.On the one hand,Primers were designed towards 5'UTR and 3'UTR of S-RNase based on the bioinformatics analysis of data available through Genome Database for Rosaceae?https://www.rosaceae.org?.S-RNases were cloned from leaf and style of hawthorn‘Zizhenzhu'by PCR.Homology analysis was carried out by Basic Local Alignment Search Tool?BLAST?.Multiple sequence alignment of the deduced amino acid sequences of hawthorn‘Zizhenzhu'S-RNases with homologues S-RNases from some genera in Rosaceae was analyzed by Lasergene.Phylogenetic analysis between hawthorn‘Zizhenzhu'S-RNases and S-RNases from other species?Prunus webbii,Prunus avium,Prunus dulcis,Prunus mume,Prunus salicina,Malus,Pyrus and Petuniahy brida?was performed by MEGA7.0.To further analyze the nature of the self-compatible mutation in‘Zizhenzhu',genetic analysis of populations of selfed progeny of‘Zizhenzhu'were performed.And the segregation of S-RNases in selfed progeny of‘Zizhenzhu'was investigated by PCR using S-haplotype-specific S-RNase primers.On the other hand,self-pollination and cross-pollination experiments of‘Zizhenzhu'were designed to discuss the causes of self-compatibility mutation through genetic analysis of offspring.The results are as follows:?1?Two full-length S-RNase genes,with 960 bp and 843 bp in length respectively,were successfully cloned from‘Zizhenzhu'.The two novel genes were named ZZZS₁-RNase and ZZZS₂-RNase respectively?GenBank accession number:ZZZS₁-RNase MG385836 and ZZZS₂-RNase MG385837?.In phylogenetic trees,ZZZS₁-RNase and ZZZS₂-RNase were clustered with Malus and Pyrus,suggesting that the self-incompatibility type of Hawthorn might be the same as that of Malus and Pyrus.?2?‘Zizhenzhu'and its inbred progeny‘No.6'are all S₁S₂ genotypes.Self-pollination affinity index of‘Zizhenzhu'was 1.27,showing self-compatibility;self-pollination affinity index of‘No.6'was 0.57,showing self-incompatibility,and mutual pollination found that‘Zizhenzhu'pollinated‘No.6'with an affinity index of 0,showing incompatibility;while‘No.6'pollinated with‘Zizhenzhu'as a male parent,the affinity index was 0.965,showing self-compatibility.It was preliminarily judged that the mutation of self-compatibility of‘Zizhenzhu'occurred at the stylar part.?3?Two pairs of specific primers were designed to identify S genotypes of self-crossing and cross-breeding progenies by using the sequence polymorphism of two alleles of‘zizhenzhu'.Three genotypes of S₁S₁,S₁S₂ and S₂S₂?3:26:5?appeared in34 self-crossing progenies,resulting in partial segregation;The ratio of three genotypes S₁S₁,S₁S₂ and S₂S₂ of 34 Hybrid Progenies of‘Zizhenzhu×No.6'was7:16:11??1:2:1?,which accorded with Mendel's inheritance rule.It was inferred that the self-compatibility of‘Zizhenzhu'was caused by the mutation of non-S factor on the stylar part.?4?By designing universal primers F-5-3/R-5-3,S-RNase genes of Hawthorn‘Dahuang'and‘Xuzhoudahuo'were cloned,named DHS₃-RNase and XZS₄-RNase,and their S genotypes were identified as S₃S₃ and S₄S₄,respectively.It was predicted theoretically that the genotypes of the progeny populations of‘Zizhenzhu'and‘Dahuang'should be S₁S₃ and S₂S₃?1:1?.The genotypes of the progeny populations of‘Zizhenzhu'and‘Xuzhoudahuo'should be S₁S₄ and S₂S₄?1:1?.However,four pairs of specific primers,S1F/S1R,S2F/S2R,DHS3F/DHS3R and XZS4F/XZS4R,were designed based on the sequence polymorphisms of four S-RNase genes of Hawthorn‘Zizhenzhu',‘Dahuang'and‘Xuzhoudahuo'to identify the genotypes of the progenies of‘Zizhenzhu×Dahuang'and‘Zizhenzhu×Xuzhoudahuo'.It was found that there were S₁S₂ genotypes in both progenies.It was consistent with the genotype of the female parent‘Zizhenzhu'.Therefore,we speculated that there may be apomixis in the seed of the offspring when‘Zizhenzhu'was the female parent.