Volatile composition changes in lemon during fruit maturation by HS-SPME-GC-MS.

BACKGROUND: Volatiles are determinants of fruit aroma and flavor characteristics and also provide valuable information for lemon as ingredient for the food and drinks industry. Volatiles in 'Eureka' lemon and 'Xiangshui' lemon pulps from 130 to 186 days after flowering were enriched by headspace-solid-phase microextraction (HS-SPME), and analyzed by gas chromatography-mass spectrometry (GC-MS). RESULTS: Seventy-seven volatiles of two lemon cultivars at the different ripening stages were identified and divided into six categories. Varieties and ripening stages had significant effects on individual volatiles in each category. The proportion of monoterpenes was found to be higher in 'Eureka' lemon, while 'Xiangshui' lemon had a higher proportion of sesquiterpenes, aldehydes and alcohols. The proportion of monoterpene fluctuation decreased during fruit ripening, while fluctuation of sesquiterpenes, alcohols, aldehydes and esters increased. Among the hydrocarbons, monoterpenes decreased their relative abundance from 91.67% to 81.04% in 'Eureka' lemon, and from 83.01% to 60.04% in 'Xiangshui' lemon; conversely, sesquiterpenes increased from 0.73% to 2.89% in 'Eureka' lemon, and from 3.21% to 8.48% in 'Xiangshui' lemon. Among the oxygenated volatiles, the proportions of alcohols, aldehydes and esters were higher at 186 days after flowering in both two cultivars. CONCLUSION: The volatile organic compounds during fruit ripening of lemon varieties with different resistance were elucidated. The proportion of oxygenated volatiles increased during fruit ripening, and disease-resistant varieties had a higher proportion. These results provided important theoretical support for the utilization of lemon fruits and the innovation of disease-resistant germplasm resources. © 2021 Society of Chemical Industry.

Molecular cytogenetic analysis of genome-specific repetitive elements in Citrus clementina Hort. Ex Tan. and its taxonomic implications.

BACKGROUND: Clementine mandarin (Citrus clementina Hort. ex Tan.) is one of the most famous and widely grown citrus cultivars worldwide. Variations in relation to the composition and distribution of repetitive DNA sequences that dominate greatly in eukaryote genomes are considered to be species-, genome-, or even chromosome-specific. Repetitive DNA-based fluorescence in situ hybridization (FISH) is a powerful tool for molecular cytogenetic study. However, to date few studies have involved in the repetitive elements and cytogenetic karyotype of Clementine. RESULTS: A graph-based similarity sequence read clustering methodology was performed to analyze the repetitive DNA families in the Clementine genome. The bioinformatics analysis showed that repetitive DNAs constitute 41.95% of the Clementine genome, and the majority of repetitive elements are retrotransposons and satellite DNAs. Sequential multicolor FISH using a probe mix that contained CL17, four satellite DNAs, two rDNAs and an oligonucleotide of (TTTAGGG)3 was performed with Clementine somatic metaphase chromosomes. An integrated karyotype of Clementine was established based on unequivocal and reproducible chromosome discriminations. The distribution patterns of these probes in several Citrus, Poncirus and Fortunella species were summarized through extensive FISH analyses. Polymorphism and heterozygosity were commonly observed in the three genera. Some asymmetrical FISH loci in Clementine were in agreement with its hybrid origin. CONCLUSIONS: The composition and abundance of repetitive elements in the Clementine genome were reanalyzed. Multicolor FISH-based karyotyping provided direct visual proof of the heterozygous nature of Clementine chromosomes with conspicuous asymmetrical FISH hybridization signals. We detected some similar and variable distribution patterns of repetitive DNAs in Citrus, Poncirus, and Fortunella, which revealed notable conservation among these genera, as well as obvious polymorphism and heterozygosity, indicating the potential utility of these repetitive element markers for the study of taxonomic, phylogenetic and evolutionary relationships in the future.

Low Female Gametophyte Fertility Contributes to the Low Seed Formation of the Diploid Loquat [Eriobotrya Japonica (Thunb.) Lindl.] Line H30-6.

Loquat is a widely grown subtropic fruit because of its unique ripening season, nutrient content, and smooth texture of its fruits. However, loquat is not well-received because the fruits contain many large seeds. Therefore, the development of seedless or few-seed loquat varieties is the main objective of loquat breeding. Polyploidization is an effective approach for few-seed loquat breeding, but the resource is rare. The few-seed loquat line H30-6 was derived from a seedy variety. Additionally, H30-6 was systematically studied for its fruit characteristics, gamete fertility, pollen mother cell (PMC) meiosis, stigma receptivity, in situ pollen germination, fruit set, and karyotype. The results were as follows. (1) H30-6 produced only 1.54 seeds per fruit and the fruit edible rate was 70.77%. The fruit setting rate was 14.44% under open pollination, and the other qualities were equivalent to those of two other seedy varieties. (2) The in vitro pollen germination rate was only 4.04 and 77.46% of the H30-6 embryo sacs were abnormal. Stigma receptivity and self-compatibility in H30-6 were verified by in situ pollen germination and artificial pollination. Furthermore, the seed numbers in the fruits of H30-6 did not significantly differ among any of the pollination treatments (from 1.59 ±0.14 to 2 ± 0.17). (3) The chromosome configuration at meiotic diakinesis of H30-6 was 6.87I + 9.99II + 1.07III +0.69IV +0.24V (H30-6), and a total of 89.55% of H30-6 PMCs presented univalent chromosomes. Furthermore, chromosome lagging was the main abnormal phenomenon. Karyotype analysis showed that chromosomes of H30-6 had no recognizable karyotype abnormalities leading to unusual synapsis on the large scale above. (4) The abnormal embryo sacs of H30-6 could be divided into three main types: those remaining in the tetrad stage (13.38%), those remaining in the binucleate embryo sac stage (1.41%), and those without embryo sacs (52.82%). Therefore, we conclude that the loquat line H30-6 is a potential few-seed loquat resource. The diploid loquat line H30-6 was with low gametophyte fertility, which may be driven by abnormal meiotic synapses. The low female gamete fertility was the main reason for the few seeds. This diploid loquat line provides a new possibility for breeding a few-seed loquat at the diploid level.

Karyotype Analysis of Diploid and Spontaneously Occurring Tetraploid Blood Orange [Citrus sinensis (L.) Osbeck] Using Multicolor FISH With Repetitive DNA Sequences as Probes.

Blood orange [Citrus sinensis (L.) Osbeck] has been increasingly appreciated by consumers worldwide owing to its brilliant red color, abundant anthocyanin and other health-promoting compounds. However, there is still relatively little known about its cytogenetic characteristics, probably because of the small size and similar morphology of metaphase chromosomes and the paucity of chromosomal landmarks. In our previous study, a naturally occurring tetraploid blood orange plant was obtained via seedling screening. Before this tetraploid germplasm can be manipulated into a citrus triploid seedless breeding program, it is of great importance to determine its chromosome characterization and composition. In the present study, an integrated karyotype of blood orange was constructed using sequential multicolor fluorescence in situ hybridization (FISH) with four satellite repeats, two ribosomal DNAs (rDNAs), a centromere-like repeat and an oligonucleotide of telomere repeat (TTTAGGG)3 as probes. Satellite repeats were preferentially located at the terminal regions of the chromosomes of blood orange. Individual somatic chromosome pairs of blood orange were unambiguously identified by repetitive DNA-based multicolor FISH. These probes proved to be effective chromosomal landmarks. The karyotype was formulated as 2n = 2x = 18 = 16m+2sm (1sat) with the karyotype asymmetry degree belonging to 2B. The chromosomal distribution pattern of these repetitive DNAs in this spontaneously occurring tetraploid was identical to that of the diploid, but the tetraploid carried twice the number of hybridization sites as the diploid, indicating a possible pathway involving the spontaneous duplication of chromosome sets in nucellar cells. Our work may facilitate the molecular cytogenetic study of blood orange and provide chromosomal characterization for the future utilization of this tetraploid germplasm in the service of seedless breeding programs.

A modified method for preparing meiotic chromosomes based on digesting pollen mother cells in suspension.

BACKGROUND: Meiotic chromosome preparation is a key step in plant meiotic research. Pollen mother cell (PMC) wall elimination is beneficial to cytogenetic experimental procedures. Without wall interference, these procedures are easier and more successful. In existing methods it is difficult to eliminate PMC walls completely and uniformly. In this paper, we present an improved method for digesting PMC walls, and one for providing massive chromosomal spreads on a slide for other cytogenetic experimental procedures. RESULTS: Three plants were selected to exhibit the modified meiotic chromosome preparation method. PMCs were dispersed as single cells and incubated in a mixed enzyme solution (3 % cellulose + 0.3 % pectinase + 1 % snailase) for 1.5-2.5 h. In total, 28.28 % cells were lost during this process. There were 800-1900 spreads on every slide and no PMC wall interference was found on any of the slides. The spreads were also evenly distributed on the slides. More spreads were obtained when PMC and protoplast densities in the suspension were increased. All three plants' spreads were successfully used to locate a 5 s rDNA conserved sequence. The Nicotiana hybrid's spreads were successfully used to identify the hybrid's parental genome. CONCLUSION: This is an alternative method for meiotic chromosome preparation. Through this method, PMC walls can be completely and uniformly eliminated, and hundreds of spreads on every slide can be obtained. These spreads can be successfully used for DNA in situ hybridization.