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The Gibberellic Acid Stimulated Arabidopsis (GASA) proteins were investigated in the study to help understand their possible roles in fruit trees, particularly in Citrus. A total of 18 CcGASA proteins were identified and characterized in Citrus clementina via a genome-wide approach. It was shown that the CcGASA proteins structurally shared a conserved GASA domain but varied considerably in primary sequences and motif compositions. Thus, they could be classified into three major phylogenetic groups, G1~G3, and two groups, G1 and G3 could be further classified into subgroups. The cis- elements on all CcGASA promoters were identified and categorized, and the associated transcription factors were predicted. In addition, the possible interactions between the CcGASA proteins and other proteins were predicted. All the clues suggested that these genes should be involved in defense against biotic and abiotic stresses and in growth and development. The notion was further supported by gene expression analysis that showed these genes were more or less responsive to the treatments of plant hormones (GA 3 , SA, ABA and IAA), and infections of citrus canker pathogen Xanthomonas citri. It was noted that both the segmental and the tandem duplications had played a role in the expansion of the CcGASA gene family in Citrus. Our results showed that the members of the CcGASA gene family should have structurally and functionally diverged to different degrees, and hence, the representative group members should be individually investigated to dissect their specific roles., (© 2021. The Author(s).)

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.

Background: Citrus breeding programs have many limitations associated with the species biology and physiology, requiring the incorporation of new biotechnological tools to provide new breeding possibilities. Diversity Arrays Technology (DArT) markers, combined with next-generation sequencing, have wide applicability in the construction of high-resolution genetic maps and in quantitative trait locus (QTL) mapping. This study aimed to construct an integrated genetic map using full-sib progeny derived from Murcott tangor and Pera sweet orange and DArTseq™ molecular markers and to perform QTL mapping of twelve fruit quality traits. A controlled Murcott x Pera crossing was conducted at the Citrus Germplasm Repository at the Sylvio Moreira Citrus Centre of the Agronomic Institute (IAC) located in Cordeirópolis, SP, in 1997. In 2012, 278 F 1 individuals out of a family of 312 confirmed hybrid individuals were analyzed for fruit traits and genotyped using the DArTseq markers. Using OneMap software to obtain the integrated genetic map, we considered only the DArT loci that showed no segregation deviation. The likelihood ratio and the genomic information from the available Citrus sinensis L. Osbeck genome were used to determine the linkage groups (LGs)., Results: The resulting integrated map contained 661 markers in 13 LGs, with a genomic coverage of 2,774 cM and a mean density of 0.23 markers/cM. The groups were assigned to the nine Citrus haploid chromosomes; however, some of the chromosomes were represented by two LGs due the lack of information for a single integration, as in cases where markers segregated in a 3:1 fashion. A total of 19 QTLs were identified through composite interval mapping (CIM) of the 12 analyzed fruit characteristics: fruit diameter (cm), height (cm), height/diameter ratio, weight (g), rind thickness (cm), segments per fruit, total soluble solids (TSS, %), total titratable acidity (TTA, %), juice content (%), number of seeds, TSS/TTA ratio and number of fruits per box. The genomic sequence (pseudochromosomes) of C. sinensis was compared to the genetic map, and synteny was clearly identified. Further analysis of the map regions with the highest LOD scores enabled the identification of putative genes that could be associated with the fruit quality characteristics., Conclusion: An integrated linkage map of Murcott tangor and Pera sweet orange using DArTseq™ molecular markers was established and it was useful to perform QTL mapping of twelve fruit quality traits. The next generation sequences data allowed the comparison between the linkage map and the genomic sequence (pseudochromosomes) of C. sinensis and the identification of genes that may be responsible for phenotypic traits in Citrus. The obtained linkage map was used to assign sequences that had not been previously assigned to a position in the reference genome.

Background: Xanthophylls are oxygenated carotenoids and fulfill critical roles in plant growth and development. In plants, two different types of carotene hydroxylases, non-heme di-iron and heme-containing cytochrome P450, were reported to be involved in the biosynthesis of xanthophyll. Citrus fruits accumulate a high amount of xanthophylls, especially β,β-xanthophylls. To date, however, the roles of carotene hydroxylases in regulating xanthophyll content and composition have not been elucidated., Results: In the present study, the roles of four carotene hydroxylase genes (CitHYb, CitCYP97A, CitCYP97B, and CitCYP97C) in the biosynthesis of xanthophyll in citrus fruits were investigated. Phylogenetic analysis showed that the four citrus carotene hydroxylases presented in four distinct clusters which have been identified in higher plants. CitHYb was a non-heme di-iron carotene hydroxylase, while CitCYP97A, CitCYP97B, and CitCYP97C were heme-containing cytochrome P450-type carotene hydroxylases. Gene expression results showed that the expression of CitHYb increased in the flavedo and juice sacs during the ripening process, which was well consistent with the accumulation of β,β-xanthophyll in citrus fruits. The expression of CitCYP97A and CitCYP97C increased with a peak in November, which might lead to an increase of lutein in the juice sacs during the ripening process. The expression level of CitCYP97B was much lower than that of CitHYb, CitCYP97A, and CitCYP97C in the juice sacs during the ripening process. Functional analysis showed that the CitHYb was able to catalyze the hydroxylation of the β-rings of β-carotene and α-carotene in Escherichia coli BL21 (DE3) cells. Meanwhile, when CitHYb was co-expressed with CitCYP97C, α-carotene was hydroxylated on the β-ring and ε-ring sequentially to produce lutein., Conclusions: CitHYb was a key gene for β,β-xanthophyll biosynthesis in citrus fruits. CitCYP97C functioned as an ε-ring hydroxylase to produce lutein using zeinoxanthin as a substrate. The results will contribute to elucidating xanthophyll biosynthesis in citrus fruits, and provide new strategies to improve the nutritional and commercial qualities of citrus fruits.

Background: In natural environments, several adverse environmental conditions occur simultaneously constituting a unique stress factor. In this work, physiological parameters and the hormonal regulation of Carrizo citrange and Cleopatra mandarin, two citrus genotypes, in response to the combined action of high temperatures and water deprivation were studied. The objective was to characterize particular responses to the stress combination., Results: Experiments indicated that Carrizo citrange is more tolerant to the stress combination than Cleopatra mandarin. Furthermore, an experimental design spanning 24 h stress duration, heat stress applied alone induced higher stomatal conductance and transpiration in both genotypes whereas combined water deprivation partially counteracted this response. Comparing both genotypes, Carrizo citrange showed higher phostosystem-II efficiency and lower oxidative damage than Cleopatra mandarin. Hormonal profiling in leaves revealed that salicylic acid (SA) accumulated in response to individual stresses but to a higher extent in samples subjected to the combination of heat and drought (showing an additive response). SA accumulation correlated with the up-regulation of pathogenesis-related gene 2 (CsPR2), as a downstream response. On the contrary, abscisic acid (ABA) accumulation was higher in water-stressed plants followed by that observed in plants under stress combination. ABA signaling in these plants was confirmed by the expression of responsive to ABA-related gene 18 (CsRAB18). Modulation of ABA levels was likely carried out by the induction of 9-neoxanthin cis-epoxicarotenoid dioxygenase (CsNCED) and ABA 8'-hydroxylase (CsCYP707A) while conversion to ABA-glycosyl ester (ABAGE) was a less prominent process despite the strong induction of ABA O-glycosyl transferase (CsAOG)., Conclusions: Cleopatra mandarin is more susceptible to the combination of high temperatures and water deprivation than Carrizo citrange. This is likely a result of a higher transpiration rate in Carrizo that could allow a more efficient cooling of leaf surface ensuring optimal CO2 intake. Hence, SA induction in Cleopatra was not sufficient to protect PSII from photoinhibition, resulting in higher malondialdehyde (MDA) build-up. Inhibition of ABA accumulation during heat stress and combined stresses was achieved primarily through the up-regulation of CsCYP707A leading to phaseic acid (PA) and dehydrophaseic acid (DPA) production. To sum up, data indicate that specific physiological responses to the combination of heat and drought exist in citrus. In addition, these responses are differently modulated depending on the particular stress tolerance of citrus genotypes.

Background: Bhutan is a small Himalayan country lying within the region considered to be the origin of citrus. Diverse citrus wild types grow naturally in different climates, elevations and edaphic conditions, but only mandarin is cultivated commercially. The first report of Huanglongbing (also known as greening disease) in Bhutan in 2003, and the threat it posed to the country's citrus orchards prompted the collection of mandarin germplasm from across the country. This paper describes the genetic diversity of mandarin accessions in Bhutan using amplified fragment length polymorphic (AFLP) markers., Results: Twenty three accessions of Bhutanese mandarin were analyzed using AFLP markers to assess the genetic variability that is believed to exist only in Bhutan and some parts of North East India and South China. Five primer pairs (E-ACA/M-CAG, E-ACG/M-CAT, E-ACC/M-CTT, E-AAG/M-CAA and E-ACA/M-CTC) were identified (based on the number and quality of polymorphic bands produced) and used for the analyses. A total of 244 bands were scored visually of which 126 (52%) were polymorphic with an average polymorphism information content of 0.95 per marker. A cluster dendrogram based on multiscale bootstrap sampling categorized twenty three accessions into two broad groups containing eight and 14 accessions, respectively. Group A consisted accessions (Tsirang1, Tsirang3, Sarpang1, Dagana4, Samtse4, Dagana1, and Trongsa2) from five districts (Tsirang, Sarpang, Samtse, Dagana and Trongsa) and their grouping was strongly supported by bootstrap analysis (B p-value = 96%, AU p-value = 86%). Cluster B consisted of 14 accessions divided into three sub-groups (1, 2 and 3). However, bootstrap value supported significantly for subgroup1 (containing accessions: Tsirang4, Sarpang5, and Tsirang2) and subgroup3 (with accessions - Zhemgang2, Zhemgang3 and Zhemgang4)., Conclusion: This study indicates that Bhutanese mandarin germplasm collected from across the country are genetically diverse although the level of variability differed among the accessions assessed. The variation in genetic variability was observed irrespective of where the accessions were collected suggesting that phenotype and geographical location can serve a basis for future germplasm collection in Bhutan. Further, five primer pair combinations could separate 23 mandarins accessions considered in this study, suggesting that AFLP markers can be a useful tool for future identification.

Background: With the increasing availability of EST databases and whole genome sequences, SNPs have become the most abundant and powerful polymorphic markers. However, SNP chip data generally suffers from ascertainment biases caused by the SNP discovery and selection process in which a small number of individuals are used as discovery panels. The ongoing International Citrus Genome Consortium sequencing project of the highly heterozygous Clementine and sweet orange genomes will soon result in the release of several hundred thousand SNPs. The primary goals of this study were: (i) to estimate the transferability within the genus Citrus of SNPs discovered from Clementine BACend sequencing (BES), (ii) to estimate bias associated with the very narrow discovery panel, and (iii) to evaluate the usefulness of the Clementine-derived SNP markers for diversity analysis and comparative mapping studies between the different cultivated Citrus species., Results: Fifty-four accessions covering the main Citrus species and 52 interspecific hybrids between pummelo and Clementine were genotyped on a GoldenGate array platform using 1,457 SNPs mined from Clementine BES and 37 SNPs identified between and within C. maxima, C. medica, C. reticulata and C. micrantha. Consistent results were obtained from 622 SNP loci. Of these markers, 116 displayed incomplete transferability primarily in C. medica, C. maxima and wild Citrus species. The two primary biases associated with the SNP mining in Clementine were an overestimation of the C. reticulata diversity and an underestimation of the interspecific differentiation. However, the genetic stratification of the gene pool was high, with very frequent significant linkage disequilibrium. Furthermore, the shared intraspecific polymorphism and accession heterozygosity were generally enough to perform interspecific comparative genetic mapping., Conclusions: A set of 622 SNP markers providing consistent results was selected. Of the markers mined from Clementine, 80.5% were successfully transferred to the whole Citrus gene pool. Despite the ascertainment biases in relation to the Clementine origin, the SNP data confirm the important stratification of the gene pools around C. maxima, C. medica and C. reticulata as well as previous hypothesis on the origin of secondary species. The implemented SNP marker set will be very useful for comparative genetic mapping in Citrus and genetic association in C. reticulata.