Preliminary Studies on CPG/Hinf Ⅰ RFLP Groups of Citrus tristeza virus Infected Sweet Oranges in China

Citrus tristeza virus （CTV） causes economically important losses to the citrus industry worldwide. Mild strain cross protection （MSCP） against tristeza has hardly been practised due to mixed infection of different CTV-strains and little background of its molecular biology in China. For better cognition on CTV, 192 sweet orange samples collected from eight provinces （Chongqing, Sichuan, Fujian, Hunan, Guangxi, Yunnan, Guangdong and Jiangxi） were tested by direct tissue blot immuno-assay （DTBIA）, and 158 of them were tested positively, which therefore were subjected to coat protein gene （CPG）/Hinf Ⅰ restriction fragment length polymorphism （RFLP） analysis. Sample bulks were compared between Chongqing and Fujian by some statistical data, including ratios of single infection and mixed infection to local samples, proportions of CTV isolates with single RFLP groups, and rates of each RFLP group. The simplified analysis of samples from the other six provinces were then conducted. This study suggests that CTV isolates with CPG/Hinf Ⅰ RFLP groups Ⅲ and Ⅰ are the main epidemic ones in China, and mixed infection of CTV in fields are popular. Based on observation of severity of stem-pitting symptom in field trees, CTV isolates with CPG/Hinf Ⅰ RFLP groups Ⅲ and Ⅰ caused severe stem-pittings in sweet oranges in China.

Production of Transgenic Anliucheng Sweet Orange(Citrus sinensis Osbeck) with Xa21 Gene for Potential Canker Resistance

Citrus canker, an epidemic quarantine disease caused by Xanthomonas axonopodis pv. citri, has brought a great damage in citrus production worldwide. Herein, a rice PRR （pattern recognition receptor） gene Xa21 together with GUS reporter gene and hygromycin phosphotransferase gene （HPT） was introduced into Anliucheng sweet orange （Citrus sinensis Osbeck） via Agrobacterium-mediated transformation of embryogenic callus. The transgenic calluses were screened on MT basal medium containing hygromycin （HYG） and detected by histochemical GUS staining. The transgenic plantlets were recovered through somatic embryogenesis pathway. The regenerated plantlets were accustomed to and maintained in the greenhouse. The transgene integration of recovered plantlets was identiifed by PCR and Southern blot hybridization. It showed that all the transgenic plantlets tested had undergone single copy integration, the expression of Xa21 in eight different transgenic lines detected by qRT-PCR can be divided into three grades, high for T5 and T6, middle for T4 and low for the rest. The tolerance to citrus canker disease of the three recovered transgenic lines T2, T4 and T6 was assessed by in vitro pin-puncture inoculation. The results showed that all the three transgenic lines conferred improved resistance to citrus canker bacterium infection and the T4 transgenic line displayed the highest resistance. The mechanism and feasibility of rice Xa21 in triggering innate immunity in citrus was brielfy discussed.

Proteomic Comparison Between Leaves from a Red-Flesh Mutant and Its Wild-Type in Sweet Orange

The red-flesh mutant Hong Anliu sweet orange is of high nutritional value due to its lycopene accumulation.Our previous studies on this mutant fruits suggested that photosynthesis and oxidative stress could promote the formation of mutation trait.However,leaf rather than fruit is the major part for some important biological processes such as photosynthesis.In this study,we analyzed the proteomic alteration in leaves of the red-flesh mutant Hong Anliu vs.its wild type （WT）.Ten differentially expressed proteins were identified,of which two were involved in photosynthesis,three in oxidative stress,two in defense,and three in metabolism.The high up-regulation of photosynthetic proteins proved the hypothesis that enhanced photosynthesis could provide and transport more substrates into mutant fruits for carotenoid biosynthesis.Similar to the previous results in fruits,anti-oxidative proteins were highly up-regulated in leaves,suggesting the whole plant of Hong Anliu suffered from enhanced oxidative stress.Proteins involved in defense and metabolism were also identified,and their possible roles in the mutation were discussed.

International Competitiveness of Export of China’s Sweet Orange

Selecting the market share,trade competitiveness index,revealed comparative advantage( RCA),and revealed competitive advantage index( CA),this paper compared the competitiveness of China and large sweet orange exporters,and made an empirical analysis. Results indicate that in terms of the market share,Spain has prominent advantage in competitiveness of sweet orange; in terms of the trade competitiveness index,Egypt and South Africa have higher competitiveness in the export of sweet orange; in terms of RCA and CA,Egypt has absolute international advantage in the export of sweet orange. China’s export competitiveness of sweet orange is much lower than other large sweet orange producers such as Spain,Egypt,and South Africa,and the export capacity is much lower than other large sweet orange producers. In view of current situations,it came up with recommendations for improving export competitiveness of China’s sweet orange,including expanding the market,diversifying the varieties,improving the quality,reducing the costs,strengthening cooperation of fruit farmer cooperatives,and enhancing government support.

Molecular cytogenetic map visualizes the heterozygotic genome and identifies translocation chromosomes in Citrus sinensis

Citrus sinensis is the most cultivated and economically valuable Citrus species in the world,whose genome has been assembled by three generation sequencings.However,chromosome recognition remains a problem due to the small size of chromosomes,and difficulty in differentiating between pseudo and real chromosomes because of a highly heterozygous genome.Here,we employ fluorescence in situ hybridization(FISH)with 9 chromosome painting probes,30 oligo pools,and 8 repetitive sequences to visualize 18 chromosomes.Then,we develop an approach to identify each chromosome in one cell through single experiment of oligo-FISH and Chromoycin A3(CMA)staining.By this approach,we construct a high-resolution molecular cytogenetic map containing the physical positions of CMA banding and 38 sequences of FISH including centromere regions,which enables us to visualize significant differences between homologous chromosomes.Based on the map,we locate several highly repetitive sequences on chromosomes and estimate sizes and copy numbers of each site.In particular,we discover the translocation regions of chromosomes 4 and 9 in C.sinensis“Valencia.”The high-resolution molecular cytogenetic map will help improve understanding of sweet orange genome assembly and also provide a fundamental reference for investigating chromosome evolution and chromosome engineering for genetic improvement in Citrus.