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Two deep-coverage Bacterial Artificial Chromosome (BAC) libraries of Citrus sinensis (L.) Osbeck 'Cara Cara' navel orange and Citrus reticulata (L.) Blanco 'Egan No. 1' Ponkan mandarin, which belong to the two most important species of the Citrus genus, have been constructed and characterized to facilitate gene cloning and to analyze variety-specific genome composition. The C. sinensis BAC library consists of 36 000 clones with negligible false-positive clones and an estimated average insert size of 126 kb covering ~4.5 x [10.sup.9] bp and thus providing an 11.8-fold coverage of haploid genome equivalents, whereas the C. reticulata library consists of 21 000 clones also with negligible false-positive clones and an estimated average of 120 kb covering ~2.5 x [10.sup.9] bp representing a 6.6-fold coverage of haploid genome equivalents. Both libraries were evaluated for contamination with high-copy vector, empty pIndigoBAC536 vector, and organellar DNA sequences. Screening has been performed by Southern hybridization of BAC filters, which results in Key words: Citrus, bacterial artificial chromosome (BAC) library, phytoene synthase, genomics, pCUGIBAC1, carotenoid. Deux banques de clones BAC (chromosomes bacteriens artificiels) ont ete produites et offrent une couverture exhaustive des genomes du Citrus sinensis (L.) Osbeck 'Cara Cara' orange navel et du Citrus reticulata (L.) Blanco 'Egan No. 1' mandarine Ponkan, lesquels font partie des deux plus importantes especes au sein du genre Citrus. Ces banques ont ete produites et caracterisees pour faciliter le clonage de genes et pour analyser la composition du genome au niveau de varietes specifiques. La banque du C. sinensis compte 36 000 clones dont un nombre negligeable de faux positifs et des inserts dont la taille moyenne est de 126 kb, ce qui totalise ~4,5 x [10.sup.9] pb et correspond a 11,8 fois le genome haploide. La banque du C. reticulata compte 21 000 clones dont un tres faible nombre de faux positifs et dont la taille moyenne des inserts est de 120 kb, soit un total de ~2,5 x [10.sup.9] pb ou l'equivalent de 6,6 fois le genome haploide. Les deux banques ont ete evaluees pour ce qui est de la contamination avec des sequences du vecteur a grand nombre de copies, du vecteur pIndigoBAC536 vide ou d'ADN des organites. Le criblage a ete realise par hybridation Southern et a revele une contamination Mots-cles : Citrus, banque de clones bacteriens artificiels (BAC), phytoene synthetase, genomique, pCUGIBAC1, carote-noides. [Traduit par la Redaction], Introduction Citrus is one of the most widespread fruit crops globally with great economic and health value. It is an excellent model species for functional genomics research of woody trees [...]

From chestnut rose, a promising fruit crop of the Rosa genus, powdery mildew disease-resistant and susceptible genotypes and their [F.sub.1] progeny were used to isolate nucleotide-binding-site (NBS)-encoding genes using 19 degenerate primer pairs and an additional cloning method called overlapping extension amplification. A total of 126 genes were harvested; of these, 38 were from a resistant parent, 37 from a susceptible parent, and 51 from F1 progeny. A phylogenetic tree was constructed, which revealed that NBS sequences from parents and F1 progeny tend to form a mixture and are well distributed among the branches of the tree. Mapping of these NBS genes suggested that their organization in the genome is a 'tandem duplicated cluster' and, to a lesser extent, a 'heterogeneous cluster.' Intraspecific polymorphisms and interspecific divergence were detected by Southern blotting with NBS-encoding genes as probes. Sequencing on the nucleotide level revealed even more intraspecific variation: for the R4 gene, 9.81% of the nucleotides are polymorphic. Amino acid sites under positive selection were detected in the NBS region. Some NBS-encoding genes were meiotically unstable, which may due to recombination and deletion events. Moreover, a transposon-like element was isolated in the flanking region of NBS genes, implying a possible role for transposon in the evolutionary history of resistance genes.

A Citrus sinensis R2R3 MYB transcription factor (CsMYB96) has previously been shown to be strongly associated with the expression of many genes related to wax biosynthesis in the fruit. In this study, CsMYB96 was found to alleviate water loss by simultaneously regulating the expression of genes encoding plasma membrane intrinsic proteins (CsPIP s) and wax-related genes. Expression profiling indicated that CsPIP1;1 and CsPIP2;4 had high expression that was representative of other aquaporins, and they were down-regulated in the peel of post-harvest citrus fruit. CsPIP2;4 was further characterized as the predominant CsPIP, with high expression and high-water channel activity. Transient overexpression of CsPIP2;4 accelerated water loss in citrus fruit. In silico analysis further indicated that the expression of CsMYB96 had a significant negative correlation with that of CsPIP s. In vivo and in vitro experiments confirmed that CsMYB96 was able to directly repress the expression of CsPIP s. In addition, CsMYB96 was able to activate wax-related genes and promote wax biosynthesis for defense against water loss. Transient and stable overexpression of CsMYB96 reduced water loss from both citrus fruit and Arabidopsis. [ABSTRACT FROM AUTHOR]

Self-incompatibility (SI) substantially restricts the yield and quality of citrus. Therefore, breeding and analyzing self-compatible germplasm is of great theoretical and practical significance for citrus. Here, we focus on the mechanism of a self-compatibility mutation in 'Guiyou No. 1' pummelo (Citrus maxima), which is a spontaneous mutant of 'Shatian' pummelo (Citrus maxima, self-incompatibility). The rate of fruit set and the growth of pollen tubes in the pistil confirmed that a spontaneous mutation in the pistil is responsible for the self-compatibility of 'Guiyou No. 1'. Segregation ratios of the S genotype in F1 progeny, expression analysis, and western blotting validated that the reduced levels of S2-RNase mRNA contribute to the loss of SI in 'Guiyou No. 1'. Furthermore, we report a phased assembly of the 'Guiyou No. 1' pummelo genome and obtained two complete and well-annotated S haplotypes. Coupled with an analysis of SV variations, methylation levels, and gene expression, we identified a candidate gene (CgHB40), that may influence the regulation of the S2-RNase promoter. Our data provide evidence that a mutation that affects the pistil led to the loss of SI in 'Guiyou No. 1' by influencing a poorly understood mechanism that affects transcriptional regulation. This work significantly advances our understanding of the genetic basis of the SI system in citrus and provides information on the regulation of S-RNase genes. [ABSTRACT FROM AUTHOR]

Abstract Background The transcriptome of the fruit pulp of the sweet orange variety Anliu (WT) and that of its red fleshed mutant Hong Anliu (MT) were compared to understand the dynamics and differential expression of genes expressed during fruit development and ripening. Results The transcriptomes of WT and MT were sampled at four developmental stages using an Illumina sequencing platform. A total of 19,440 and 18,829 genes were detected in MT and WT, respectively. Hierarchical clustering analysis revealed 24 expression patterns for the set of all genes detected, of which 20 were in common between MT and WT. Over 89% of the genes showed differential expression during fruit development and ripening in the WT. Functional categorization of the differentially expressed genes revealed that cell wall biosynthesis, carbohydrate and citric acid metabolism, carotenoid metabolism, and the response to stress were the most differentially regulated processes occurring during fruit development and ripening. Conclusion A description of the transcriptomic changes occurring during fruit development and ripening was obtained in sweet orange, along with a dynamic view of the gene expression differences between the wild type and a red fleshed mutant.

Abstract Background Chestnut rose (Rosa roxburghii Tratt) is a promising wild fruit crop in Southwest China. However, chestnut rose suffers from several important diseases such as powdery mildew and black spot. Cloning and phylogenetic analysis of plant immunity related genes will strengthen the evolutionary knowledge of plant immune system and will facilitate the utilization of candidate genes in disease resistance breeding programs. Findings Serine/threonine kinase (STK) genes, encoding one of the important proteins for defense signal transduction, were cloned from 'chestnut rose'. Fifteen STK sequences were obtained by degenerate PCR. Sequence analysis showed that nine of them have continued open reading frames, and they are separated into five classes based on sequence analysis. Interestingly, one of the classes (STK V) showed less than 40% similarity to any other class, possibly representing new type genes from chestnut rose. Southern blotting analysis revealed that the new type STK V genes are single copy, while all the other genes have several copies in the genome. Phylogenetic analysis of STK genes from chestnut rose and 21 plant species revealed that most chestnut rose genes show close relationship with Rosaceae homologs, while the STK V genes are rather ancient and form a unique clade distantly from plant homologs. Conclusions We cloned nine STK genes from a wild fruit crop 'chestnut rose', of which a new type of STK genes was identified. The new type STK genes exist as single copies in the genome, and they are phylogenetically distant to plant homologs. The polymorphic STK genes, combined with other plant immunity genes, provide plenty of resources to be utilized to defend against pathogens attack.