Identification Of Loquat Distant Hybrids And Expression Analysis Of Genes Related To Anthocyanin Synthesis

Common loquat(Eriobotrya japonica Lindl.)(2n=2x=34)is the only cultivated species in the loquat genus of Rosaceae.Therefore,the genetic background of cultivated loquat is narrow,and the evaluation and utilization of loquat genus wild resources are seriously insufficient,and the traits of many loquats can‘t be improved for a long time.Loquat genus wild resources have many excellent traits,among them,bronze loquat(Eriobotrya deflexa),the flowering period is from late spring to early summer,and the maturity period is from late summer to early autumn.The fruit is yellow-red,hairless,seeds 1-2 and highly resistant to leaf tip scorch.These characteristics are loquat maturity,fruit color,number of seeds,and resistance improved traits provide ideal materials.So far,the breeding methods of loquat still use seed selection and cross breeding as the main methods,and there are few related researches on the hybridization between distantly related species.In this study,the progeny of the cross between the loquat variety ‘Ninghaibai’ and the bronze loquat were used as materials to conduct ploidy analysis and hybrid identification of the offspring,and to conduct a preliminary study on the expression of its genes.The main findings are as follows:1.Identification of distant hybrids of loquat and acquisition of true hybridsPloidy identification of the progeny obtained from ‘Ninghaibai’ × bronze loquat showed that the hybrid progeny had diploid and triploid.Morphological observation of the 1-year-old plants showed that the leaf morphology of the hybrid offspring was generally between the parents,and the hybrid offspring all had the stipule morphological characteristics of the male parent.Using bronze loquat g DNA as a probe,GISH analysis found that the number of chromosomes with hybridization signals in diploid and triploid hybrids is the same.In order to further clarify the origin of triploids,five pairs of SSR primers were screened to identify the hybrids of triploids.Among them,the primer CH04c06 can amplify 2 specific bands of the parent,and only one of the triploids was amplified specific bands of male parent(bronze loquat)and 2 female parent(‘Ninghaibai’).It can be seen that the offspring of different ploidy hybrids are all true hybrids,and the triploids are formed due to the unreduced number of female gametes(‘Ninghaibai’)(2n gametes).2.Parental transcriptome analysisTranscriptome sequencing analysis of mixed samples of leaves and stem tips of‘Ninghaibai’ and bronze loquat at different developmental stages found that there were a total of 8566 differentially expressed genes in the stem tip comparison group,of which 3147 genes were up-regulated and 5419 genes were down-regulated in bronze loquat.Differentially expressed genes are mainly enriched in monoterpene biosynthesis,phenylpropane biosynthesis,cyanoamino acid metabolism,α-linolenic acid metabolism,flavonoid biosynthesis and carotenoid biosynthesis.There are 9554 differentially expressed genes in the leaf comparison group.Among them,bronze loquat has 3842up-regulated genes and 5712 down-regulated genes.Differentially expressed genes are mainly enriched in carbon metabolism,glycolysis/gluconeogenesis,carbon fixation in photosynthetic organisms,cysteine and methionine metabolism and peroxidation.A total of 31 differentially expressed genes in the anthocyanin synthesis pathway were screened in the two comparison groups,mainly structural genes involved in anthocyanin synthesis,indicating that structural genes that regulate anthocyanin synthesis play an important role in the coloring of loquat leaf.3.Expression analysis and cloning of key genes of anthocyanin pathway in different ploidy materialsAnalyzing the anthocyanin content and structural gene expression in the parents and the hybrid offspring,the anthocyanin content in the leaves of bronze loquat is high,while the anthocyanin content in the leaves of ‘Ninghaibai’ is very low.This may be the main reason why the young leaves of bronze loquat are purple-red while the young leaves of ‘Ninghaibai’ are white.In the hybrid offspring,the anthocyanin content in the leaves of triploids at different developmental stages was higher than that of diploids.It can be seen that the synthetic activity of anthocyanin in the offspring of triploid is higher than that of diploid.The expression levels of key genes in the anthocyanin pathway were tested,and the results showed that in the early and late stages of leaf development(S1,S2,and S4),the expression of most key genomes in triploids was higher than that in diploids,and the number of genomes with higher expressions in diploids increased in the middle and late stages(S3).It can be seen that the expression of anthocyanin synthesis genes is affected by the number of genomes,but the effects are different in different periods.Generally speaking,anthocyanin synthesis in triploids is positively regulated.In addition,comprehensive analysis of the expression of key genes in the anthocyanin synthesis pathway in parents and offspring showed that ANS,CHS,F3 H and C4 H may be the main differential genes in anthocyanin synthesis in bronze loquat compared to the ‘Ninghaibai’ loquat anthocyanin synthesis.Using the c DNA of bronze loquat as a template,the full length CDS of Ej ANS,Ej C4 H,Ej CHS and Ej F3 H genes were successfully cloned.The analysis results show that the structure and cell location of the above genes are similar to the homologous genes of apple and pear.This study believes that the use of loquat and bronze loquat can obtain interspecific triploid hybrids;in the triploid leaves formed by the combination of the 2n gametes of‘Ninghaibai’ and the n gametes of bronze loquat,the synthesis of anthocyanins is positively regulated.