Y. J. Cheng

The draft genome of sweet orange (Citrus sinensis)

Oranges are an important nutritional source for human health and have immense economic value. Here we present a comprehensive analysis of the draft genome of sweet orange (Citrus sinensis). The assembled sequence covers 87.3% of the estimated orange genome, which is relatively compact, as 20% is composed of repetitive elements. We predicted 29,445 protein-coding genes, half of which are in the heterozygous state. With additional sequencing of two more citrus species and comparative analyses of seven citrus genomes, we present evidence to suggest that sweet orange originated from a backcross hybrid between pummelo and mandarin. Focused analysis on genes involved in vitamin C metabolism showed that GalUR, encoding the rate-limiting enzyme of the galacturonate pathway, is significantly upregulated in orange fruit, and the recent expansion of this gene family may provide a genomic basis. This draft genome represents a valuable resource for understanding and improving many important citrus traits in the future.

Retro-transposon based genetic similarity within the genus Citrus and its relatives

Retro-transposons are common components of plant genomes, functional at transcription, translation and integration levels. Their abundance and ability to transpose render them good potential markers. Present study was undertaken with the view of IRAP (inter-retrotransposon amplified polymorphism) and REMAP (retrotransposon-microsatellite amplified polymorphism) based DNA marker analysis for genus Citrus and related taxa. Both the IRAP and REMAP exhibited a remarkable variation among the tested genotypes. As a whole, ISSR, IRAP and REMAP analysis generated 113 score able bands ranging from 250 to 2,000 bps. There were 94 polymorphic bands, with an average of eight polymorphic bands per primer combination. The level of polymorphism was found to be 84%. No bands were shared with the ISSR pattern in REMAP analysis. Genetic similarity analysis was performed based on the Dice coefficient, and dendrogram was constructed by using the average linkage methods from combined analysis of IRAP and REMAP. A cophenetic correlation coefficient was also calculated. The clustering approach revealed a good adjustment between matrixes, with correlation coefficient of 0.77. Average similarity for all the genotype pairs was used as a cutoff value for defining the clusters. UPGMA demonstrated eight different clusters. Citrus genus showed wide range of heterogeneity, specially the mandarin group. Genera Fortunella and Poncirus were placed in relatively divergent cluster.

Molecular characterization of the nuclear and cytoplasmic genomes of intergeneric diploid plants from cell fusion between Microcitrus papuana and Rough lemon

Abstract. Simple-sequence-repeat (SSR) and PCR-RFLP were employed to characterize the nuclear and cytoplasmic genomes of intergeneric diploid plants derived from symmetric fusion between Microcitrus papuana Swingle and Rough lemon (Citrus jambhiri Lush). Three out of five SSR primers distinguished the fusion parents from each other and the regenerated plants showed band profiles completely identical to the leaf parent, Rough lemon. Amplified products from the intergenic regions of cpDNA between trnD-trnT were digested with HaeIII and MspI, and those between trnH-trnK were digested with HinfI, and both the regenerated plants and Rough lemon shared the same band patterns, which were different from the embryogenic parent, M. papuana. With mtDNA, only 2 out of 12 primer pair/restriction enzyme combinations (nad4 ex 1–2/TaqI and nad4 ex 1–2/HindIII) revealed polymorphisms between the fusion parents. With the former combination the regenerated plants showed the same fragment distribution as that of the embryogenic parent, M. papuana, whereas with the latter, a novel band absent in the fusion parents was detected in all of the regenerated plants, suggesting a possible rearrangement. The present research indicates that the plants analyzed were putative cybrids containing nuclear DNA and cpDNA from Rough lemon and mtDNA from M. papuana. Presumed mechanisms leading to the regeneration of diploid hybrid plants following symmetric fusion are discussed herein.

Universal chloroplast primer pairs for Simple Sequence Repeat analysis in diverse genera of fruit crops

To provide an alternative method to investigate chloroplast genome (cpDNA) diversity in sub-tropical and tropical fruit crops, where cpDNA sequences are unknown, a universal set of chloroplast simple sequence repeat (cpSSR) primers were developed. Thirty-three cpSSR primers from rice (Oryza sativa), pine (Pinus thunbergii), tobacco (Nicotiana tabacum) and Arabidopsis thaliana were applied to 45 fruit species (or varieties) belonging to 27 genera in 20 families. After being screened, the utility of these primers was verified. Experiments revealed that nine out of 14 primer pairs from N. tabacum, and seven out of 11 from A. thaliana, specifically amplified bands in cpDNA from most species tested, and polymorphisms were observed. While four out of the five primers from O. sativa generated intensive bands in agarose gels, but only a smear in polyacrylamide gels, all cpSSR primers from P. thunbergii failed to generate any specific amplified products in polyacrylamide gels. The results illustrate the feasibility of exploring diversity among unknown cpDNA sequences in exotic minor fruit crops by PCR methods.

Protoplast Technology and Citrus Improvement

Citrus is one most important fruit crop in China and worldwide. Protoplast fusion has been an effective and successful technique for citrus improvement by circumventing reproductive barriers such as nucellar polyembryony, pollen/ovule sterility, sexual incompatibility and long juvenility encountered in conventional breeding. Protoplasts isolated from embryogenic callus line, as source material are convenient and available at any time for genetic transformation of seedless citrus cultivars since most commercial cultivars are seedless, and routinely used epicotyl seedling segments are only available for seedy cultivars. Strategies and recent progress by protoplast fusion and protoplast transformation in our citrus improvement program will be provided.

Regeneration and molecular characterisation of two interspecific somatic hybrids of Citrus for potential rootstock improvement

Summary To produce allotetraploid somatic hybrid citrus rootstocks, protoplasts isolated from ‘Dancy’ tangerine or ‘Kinnow’ mandarin (Citrus reticulata Blanco) were electrically fused with mesophyll protoplasts of ‘Goutou’ sour orange (C. aurantium L.). Three and six plants were regenerated from these two fusions, respectively. They were confirmed to be tetraploids and diploids by flow cytometry. Nuclear DNA simple sequence repeats (SSR) analysis verified that they were true hybrids, with novel bands present. Chloroplast DNA SSR analysis showed random inheritance of chloroplast DNA (cpDNA) in the ‘Dancy’ + ‘Goutou’ hybrids, while all the cpDNA of the ‘Kinnow’ + ‘Goutou’ hybrids was derived from ‘Kinnow’, the callus parent. Cleaved amplified polymorphic sequence (CAPS) analysis using mitochondria-specific DNA (mtDNA) primer pairs, followed by restriction endonuclease digestion, showed that all the mtDNA of the ‘Dancy’ + ‘Goutou’ somatic hybrids was from ‘Dancy’, the callus parent; while that of the ‘Kinnow’ + ‘Goutou’ diploid hybrids was not distinguishable using various primer/endonuclease combinations. The potential of these two somatic hybrids for the improvement of Citrus rootstocks is discussed.

Copia-like retrotransposons in a precocious mutant of trifoliate orange [Poncirus trifoliata (L.) Raf]

Summary Retrotransposons are known to be involved in genetic instability in Citrus. The presence of copia-like retrotransposons was investigated in a precocious trifoliate orange (PTO) accession, a mutant that can flower and set fruit in 1-year-old seedlings. Ten copia-like reverse transcriptase (RT) fragments were cloned from its genome. The predicted amino acid sequences of these fragments showed that they shared the typical conservative and diverse properties of copia-like retrotransposons, and were divided into two sub-groups. Genomic hybridisation using one of the fragments (RT7) as a probe showed an extra copy was present exclusively in PTO. Inverse sequence tagged repeat (ISTR) analysis based on copia-like retrotransposon fingerprints detected two extra bands, and one band missing, in PTO. Despite the lack of transcriptional activity in the flowers and leaves of PTO, our results suggest that new transposition of copia-like retrotransposons has occurred in PTO, which might be related to the precocious trait of this mutant.

Inter-generic mitochondrial genome recombination in somatic hybrids between Citrus and Poncirus

Summary The genetic composition of somatic hybrids between ‘Page’ tangelo (Citrus reticulata × C. paradisi) and trifoliate orange (Poncirus trifoliata) were analysed in fifteen stochastically selected, fusion-derived plants. Flow cytometry revealed that all plants were tetraploids, and nuclear simple sequence repeats (SSR) showed that all plants possessed SSR loci from both fusion parents. Organellar genome compositions were examined using cleaved amplified polymorphic sequences (CAPS) and restriction fragment length polymorphism (RFLP) analyses. Mitochondrial genome (mtDNA) analyses indicated that all 15 hybrids had inherited regions of mtDNA common to both parents, and some specific bands from both fusion parents were also present in the hybrids, indicating the occurrence of mtDNA recombination in these somatic hybrids. No chloroplast genome (cpDNA) polymorphisms were detected by RFLP or CAPS analyses; however, random segregation of cpDNA was revealed by chloroplast simple sequence repeat (cpSSR) analyses. To evaluate genetic variation in mtDNA among the hybrids and both fusion parents, a dendrogram based on the RFLP data for 15 polymorphic mtDNA markers was generated by the Unweighted Pair Group Method with Arithmetic mean. The results showed that the 15 hybrids, and both parents, were located in three distinct clusters, and no variation was detected among these hybrids. The hybrids showed closer genetic similarity coefficients to the suspension cell parent than to the mesophyll cell parent, which demonstrated biased transmission of mtDNA. This is the first report of mtDNA recombination between Citrus and Poncirus by somatic fusion.