Creation of polyploids and inheritance analysis of mono-/polyembryony in the hybrid progeny from Citrus ‘Beni Madona’×Citrus paradisi‘Cocktail’

【Objective】Citrus is the most economically important and widely cultivated fruit crop and encompasses a diversity of wild and domesticated germplasms. The growing consumer demand for better-tasting and seedless fruit has driven the development of new varieties through interspecific hybridization of citrus. Parthenocarpy, almost present in all species of citrus germplasm, is an essential trait for seedless fruit production. Recovery of the sexual hybrid is challengeable when the polyembryonic genotype is used as seed parent due to the occurrence of nucellar embryos. Monoembryonic parent can promise an effective harvest of true hybrids. Citrus‘Beni Madona’is a citrus hybrid cultivar competent for the maternal parent in hybridization due to the excellent fruit quality, monoembryony, and strong ability of parthenocarpy yielding seedless fruits. Citrus paradisi‘Cocktail’is a citrus hybrid cultivar producing seedy fruits and distinctive juices riched in flavonoids with bitter taste. The purpose of this research was to develop new genotypes through an interspecific cross between Beni Madona and Cocktail to improve the fruit quality and reduce the seed number.【Methods】Pollination was performed during the flowering period in mid-April, using Beni Madona as the female parent and Cocktail grapefruit as the pollen donor. Mature fruits were harvested and seeds were collected to regenerate plantlets via tissue culture on Murashige and Tucker (MT) basal medium or MT medium supplemented with 1 mg·L-1 GA3 under the condition of 26±1 ℃, 70% relative humidity and 8h (night)/16 h (day) photoperiod. Regenerated plantlets with 4-5 functional leaves were acclimatized and transferred to a greenhouse. Ploidy analysis were performed on all recovered seedlings by a flow cytometer. Approximately 0.5 cm2 of young leaf tissue was chopped in a nuclei extraction buffer, stained with DAPI, filtered, and analyzed. Genomic DNA was extracted from young leaves of parents and progeny using a commercial plant DNA extraction kit. The genetic origin of all recovered seedlings was analyzed using two Simple Sequence Repeats (SSR) markers, which could display different profiles in Beni Madona and Cocktail. The embryony genotype (mono- vs. polyembryonic) was determined using the co-dominant PCR marker mite_p2, which detects the presence or absence of a Miniature Inverted-repeat Transposable Element (MITE) insertion in the promoter region of the CitRWP gene, a key regulator of nucellar embryony. The PCR products were separated on agarose gels, and the segregation ratio of the bands represent monoembrynic and polyembryonic trait were calculated within the progeny of diploid seedlings and statistically evaluated using a Chi- square (χ2 ) test against the expected Mendelian ratio. The leaf morphological traits (leaf length, width, and leaf index) of recovered seedlings of different ploidy levels were measured and compared.【Results】A total of 76 seeds were obtained from 11 fruits harvested at maturation, and 47 independent seedlings were recovered in vitro. Flow cytometric analysis revealed that 45 seedlings were diploid (2x), one was triploid (3x), and one was tetraploid (4x), corresponding to the occurrence frequency of 95.74%, 2.13%, and 2.13%, respectively. The hybrid nature of all 47 seedlings were confirmed by the SSR marker of F14 and SSR15. The triploid seedling presented two alleles of Beni Madona and one allele of Cocktail, indicating a possible origin from an unreduced (2n) female gamete of Beni Madona fertilized by a reduced (n) male gamete of Cocktail. The tetraploid exhibit two allels of maternal and parternal parent, respectively, suggesting a union of unreduced gametes (2n + 2n) from both parents. Genotyping with the mite_p2 marker showed that the maternal parent Beni Madona was identified as monoembryonic genotype due to missing the band of MITE insertion, while the paternal parent Cocktail was identified as polyembryonic genotype because of the insertion presence. Among the 45 diploid hybrids, 21 seedlings were genotyped as monoembryonic and 24 seedlings as polyembryonic, exhibiting a 21∶24 segregation ratio. Chi-square analysis indicated this ratio did not deviate significantly from the expected 1∶1 ratio (χ2 =0.20＜χ2 0.05=3.84, df=1), suggesting the single dominant allele of polyembryony. One triploid plant was genotyped as polyembryonic, and one tetraploid plant was genotyped as monoembryonic. Morphological analysis of leaves revealed that the leaf index decreased significantly with increasing ploidy level, indicating a trend towards broader and rounder leaves in polyploids, which provides phenotypic clues for early ploidy screening.【Conclusion】A batch of novel citrus hybrids recovered from the cross between Beni Madona and Cocktail grapefruit were obtained, of which there were 45 diploid seedlings, one triploid seedling and one tetraploid seedling. Twenty-one diploid seed-lings and one tetraploid seedling were inferred as monoembryonic via early evaluation of MITE markers. These hybrid progenies contribute to the further selection of new citrus varieties, and the triploid and tetraploid progeny provides vital germplasm for seedless breeding in citrus.