Induction Of Citrus Homozygous Lines, And Metabolomic, Transcriptomic Comparison Between DH Line And Diploid Of ‘Early Gold’ Sweet Orange

Homozygous genotypes play an important role in plant breeding and genomic studies. However, citrus homozygous lines can hardly be developed through conventional methods due to the high heterozygosity, long juvenility, large size, nucellar embryo and sexual incompatibility in most citrus species. Gametic embryogenesis, including anther culture, isolated microspore culture, inducing parthenogenesis and in vitro ovary culture, serves as an effective single-step approach to produce homozygous lines with less time and labors. Since the 1970 s, a few citrus homozygous lines have regenerated in several major cultivated species, however, the majority of which have the similar genetic background. And it seems that all of them occurred with drastically reduced growth and difficultly developed into whole plants, which limit their application in the researches. In this study, we recovered homozygous lines through anther culture and parthenogenetic induction to extend the homozygous genotypes in citrus. And we also analyzed the differences of aromatic hydrocarbon metabolites and RNA-seq between DH line and diploid of ’Early Gold’ sweet orange. The results are as follows:1. The anthers of 15 citrus cultivars at the uninucleate stage were cultured and induced using four previously reported mediums. Fifteen haploid lines, ten DH lines, two tetraploid lines of ’Early Gold’ sweet orange, one haploid line of ?Rohde Red’ Valencia sweet orange, two haploid callus lines of ?An Liu’ sweet orange and one haploid callus line of ?Cocktail’ grapefruit were obtained, as identified by ploidy and simple sequence repeats(SSRs) analysis. Among them, plants regenerated from two DH lines of ?Early Gold’ sweet orange grew vigorously in the greenhouse. Sixteen homozygous lines of ?Early Gold’ sweet orange were confirmed to be fully homozygous by 31 SSR markers which distributed evenly on each of the chromosomes. In 16 out of 29 primers with two bands, the allele segregation of microspores conformed to the law of the 1:1 ratio of two alleles.2.Parthenogenetic induction was carried out to develop haploid plants in ?Hirado Buntan’ pummelo(Citrus grandis Osbeck), ?Gao Ban’ pummelo(C. grandis Osbeck), ?Feng Huang’ pummelo(C. grandis Osbeck), ?Huanong Red’ pummelo(C. grandis Osbeck), ?Qiu Hui’ hybrid([C. reticulata Blanco×(C. paradis Macf.×C. reticulata Blanco)]×(C. reticulata Blanco×C. sinensis Osbeck)) and Eureka lemon(C. limon Burm. f.) through pollination with irradiated pollens of trifoliata orange(255, 325 and 500 Gy) and ?Tongshui 72-1 Jincheng’ sweet orange(255, 395 and 500 Gy). There were no significant differences between irradiated pollens and normal pollens in pollen stainability and pollen tube behavior after pollination. The seeds extracted from the fruits were rescued by in vitro embryo culture, and the ploidy level of the obtained plantlets was determined by flow cytometry. The results showed that two haploid plants and one triploid plant were obtained from the cross-pollination of ?Hirado Buntan’ pummelo and irradiated pollens of trifoliata orange; two haploid plants were obtained from the cross-pollination of ?Hirado Buntan’ pummlo and irradiated pollens of ?Tongshui 72-1 Jincheng’ sweet orange. The parthenogenic origin of the haploid plants was further confirmed by chromosomal cytogenetic and SSR analysis.3. Citrus monoembryonic cultivars including ?Hirado Buntan’ pummelo, ?Gao Ban’ pummelo, ?Huanong Red’ pummelo, ?Shatian’ pummelo and ?Qiu Hui’ hybrid were pollinated with triploid pollen of Melgold(C. paradis Macf.) and Oroblanco(C. paradis Macf.) to induce parthegenesis to develop haplid lines. Though embryo rescue and ploidy analysis, seven triploid, one triploid and one tetraploid were identified from the cross HB × Melgold, ?Gao Ban’ pummelo × Melgold and ?Qiu Hui’ × Oroblanco respectively.4. Metabolomic and transcriptomic comparison between DH and diploid of ?Early Gold’ sweet orange. Morphological analysis showed that DH line had significant differences with diploid in leaf area, wing leaf area, ratio of leaf area and wing leaf area and lesf shape index. A total of 45 aromatic hydrocarbon metabolites were identified though GC-MS. Among them, the levels of 12 aromatic hydrocarbons in DH leaves were significantly higher than those in diploid and 30 were significantly lower, among the 30 down-regulated aromatic hydrocarbon, 20 aromatic hydrocarbons have not idengtified in DH leaves. By RNA-seq analysis, 1863 genes(5.77% of detected genes) are found significantly differentially expressed between DH and diploid of ?Early Gold’ sweet orange. Of all DEGs, 506 genes(27.16%) were up-regulated and 1357 genes(72.84%) were down-regulated in DH line, relative to diploid. Notably, those genes were highly related to stress-response functions, defense-response functions and cell death.