Systematic analysis and functional verification of citrus glutathione S-transferases reveals that CsGSTF1 and CsGSTU18contribute negatively to citrus bacterial canker

Citrus bacterial canker(CBC) is resulted from Xanthomonas citri subsp. citri(Xcc) infection and poses a significant threat to citrus production.Glutathione S-transferases(GSTs) are critical in maintaining redox homeostasis in plants, especially in relation to abiotic and biotic stress responses. However, the function of GSTs in resisting CBC remains unclear. Here, citrus glutathione S-transferases were investigated applying a genome-wide approach. In total, 69 CsGSTs belonging to seven classes were identified, and the phylogeny, chromosomal distribution, gene structures and conserved motifs were analyzed. Several CsGSTs responded to Xcc infection, as observed in the upregulation of CsGSTF1 and CsGSTU18 in the CBC-sensitive ‘Wanjincheng' variety but not in the resistant ‘Kumquat' variety. CsGSTF1 and CsGSTU18 were localized at the cytoplasm. Transient overexpression of CsGSTF1 and CsGSTU18 mediated reactive oxygen species(ROS) scavenging, whereas the virus-induced gene silencing(VIGS) of CsGSTF1 and CsGSTU18 caused strong CBC resistance and ROS burst. The present study investigated the characterization of citrus GST gene family, and discovered that CsGSTF1 and CsGSTU18 negatively contributed to CBC through modulating ROS homeostasis. These findings emphasize the significance of GSTs in infection resistance in plants.

CsABF3-activated CsSUT1 pathway is implicated in pre-harvest water deficit inducing sucrose accumulation in citrus fruit

Pre-harvest water deficit(PHWD)plays an important role in sugar accumulation of citrus fruit.However,the mechanism is not known well.Here,it was confirmed that PHWD promoted sucrose accumulation of citrus fruit,but had limited effect on fructose,glucose and total acid.A sucrose transporter,Cs SUT1,which localizes to the plasma membrane,was demonstrated to function in sucrose transport induced by PHWD.Compared to wild-type,Cs SUT1 overexpression in citrus calli stimulated sucrose,fructose and glucose accumulation,while its silencing in juice sacs reduced sucrose accumulation.Increased sugar accumulation in transgenic lines enhanced plant drought tolerance,and resulted in decreased electrolyte leakage,malondialdehyde and hydrogen peroxide contents,as well as increased superoxide dismutase activity and proline contents.An abscisic acid(ABA)-responsive transcription factor,Cs ABF3,was found with a same expression pattern with Cs SUT1 under PHWD.Yeast one-hybrid,electrophoretic mobility shift assay and dual-luciferase assays all revealed that Cs ABF3 directly bound with the Cs SUT1 promoter by ABA responsive elements.When Cs ABF3 was overexpressed in citrus calli,the sucrose,fructose and glucose concentration increased correspondingly.Further,transgenic studies demonstrated that Cs ABF3 could affect sucrose accumulation by regulating Cs SUT1.Overall,this study revealed a regulation of Cs ABF3 promoting Cs SUT1 expression and sucrose accumulation in response to PHWD.Our results provide a detail insight into the quality formation of citrus fruit.

Crc1,6RhaT is involved in the synthesis of hesperidin of the main bioactive substance in the Citrus reticulata ‘Chachi' fruit

Hesperidin is a dihydroflavonoids, accounting for more than 50% of the total flavonoids in Citri Reticulatae Pericarpium(CRP) of traditional Chinese medicine. It is an effective antioxidant and free radical scavenger that has anti-inflammatory, antiviral and hypoglycemic properties.The latest studies reported that hesperidin has a potential for novel coronavirus resistance. However, little is known about the synthesis regulation and accumulation site of hesperidin in plants. In this study, hesperidin synthase gene Crc1,6RhaT was cloned, and the protein can be completely transformed flavanone-7-O-glucoside into hesperidin in vitro and in vivo. Studies on biological characteristics of ovary walls and exocarps showed that the relative expression levels of the Crc1,6RhaT gene and protein decreased gradually with the development of citrus fruits, and the relative content of hesperidin firstly increased, then sequentially decreased. In situ hybridization results further revealed that Crc1,6RhaT transcription was mainly concentrated in the secretory cavity cells, which are revealed to be the site of flavonoid synthesis.Immunocytochemistry localization results showed that the Crc1,6RhaT was mainly located in the endoplasmic reticulum, nucleus and vacuole of secretory cells. We inferred that the Crc1,6RhaT was synthesized in the endoplasmic reticulum, then was transported into the vacuoles through enlarged vesicles at the end of the endoplasmic reticulum. Our results not only revealed that Crc1,6RhaT may be involved in the synthesis of hesperidin of the main bioactive substance in the medicinal plant Citrus reticulata ‘Chachi' fruit, but also provided novel insights into the main subcellular sites of hesperidin biosynthesis in vacuoles.

Antiglycating effects of citrus flavonoids and associated mechanisms

Glycation of proteins and DNA forms advanced glycation end products(AGEs)causing cell and tissue dysfunction and subsequent various chronic diseases,in particular,metabolic and age-related diseases.Targeted AGE inhibition includes scavengers of reactive carbonyl species(RCS)such as methylglyoxal(MG),glyoxalase-1 enhancers,Nrf2/ARE pathway activators,AGE/RAGE formation inhibitors and other antiglycatng agents.Citrus flavonoids have demonstrated antioxidant and anti-inflammatory effects and are also found to be effective antiglycating agents.Herein,we reviewed the up to date progress of the antiglycation effects of citrus flavonoids and associated mechanisms.Major citrus flavonoids,hesperedin and its aglycone,hesperetin,inhibited glycation by scavenging MG forming mono-or di-flavonoid adducts with MG,enhanced the activity of glyoxase-1,activated Akt/Nrf2 signal pathway while inhibiting AGE/RAGE/NF-κB pathway,reduced the formation of Nε-(carboxylmethyl)lysine(CML)and pentosidine,inhibited aldol reductase activity and decreased the levels of fructosamine.The antiglycating activity and mechanisms of other flavonoids was also summarized in this review.In conclusion,citrus flavonoids possess effective antiglycating activity via different mechanisms,yet there are many challenging questions remaining to be studied in the near future such as in vivo testing and human study of citrus flavonoids for efficacy,effectiveness and adverse effects of citrus flavonoids as a functional food in managing high levels of AGEs and controlling AGE-induced chronic diseases,diabetic complications in particular.

Improved observation of colonized roots reveals the regulation of arbuscule development and senescence by drought stress in the arbuscular mycorrhizae of citrus

Citrus is the typical mycorrhizal fruit tree species establishing symbiosis with arbuscular mycorrhizal (AM) fungi. However, arbuscule development and senescence in colonized citrus roots, especially in response to drought stress, remain unclear, which is mainly due to the difficulty in clearing and staining lignified roots with the conventional method. Here, we improved the observation of colonized roots of citrus plants with the sectioning method, which enabled the clear observation of AM fungal structures. Furthermore, we investigated the effects of one week of drought stress on arbuscule development and senescence with the sectioning method. Microscopy observations indicated that drought stress significantly decreased mycorrhizal colonization (F%and M%) although it did not affect plant growth performance. Fluorescence probes (WGA 488 and/or Nile red) revealed that drought stress inhibited arbuscule development by increasing the percentage of arbuscules at the early stage and decreasing the percentages of arbuscules at the midterm and mature stages. Meanwhile, drought stress accelerated arbuscule senescence, which was characterized by the increased accumulation of neutral lipids. Overall, the sectioning method developed in this study enables the in-depth investigation of arbuscule status, and drought stress can inhibit arbuscule development but accelerate arbuscule senescence in the colonized roots of citrus plants. This study paves the way to elaborately dissecting the arbuscule dynamics in the roots of fruit tree species in response to diverse abiotic stresses.

Eureka lemon zinc finger protein ClDOF3.4 interacts with citrus yellow vein clearing virus coat protein to inhibit viral infection

Citrus yellow vein clearing virus(CYVCV)is a new citrus virus that has become an important factor restricting the development of China’s citrus industry,and the CYVCV coat protein(CP)is associated with viral pathogenicity.In this study,the Eureka lemon zinc finger protein(ZFP)ClDOF3.4 was shown to interact with CYVCV CP in vivo and in vitro.Transient expression of ClDOF3.4 in Eureka lemon induced the expression of salicylic acid(SA)-related and hypersensitive response marker genes,and triggered a reactive oxygen species burst,ion leakage necrosis,and the accumulation of free SA.Furthermore,the CYVCV titer in ClDOF3.4 transgenic Eureka lemon plants was approximately 69.4%that in control plants 6 mon after inoculation,with only mild leaf chlorotic spots observed in those transgenic plants.Taken together,the results indicate that ClDOF3.4 not only interacts with CP but also induces an immune response in Eureka lemon by inducing the SA pathways.This is the first report that ZFP is involved in the immune response of a citrus viral disease,which provides a basis for further study of the molecular mechanism of CYVCV infection.

Citrus aurantium L. extract alleviate depression by inhibiting gut microbiota-mediated inflammation in mice

Gut microbiota plays a crucial role in the pathophysiology of depression.This study aimed to explore the antidepressant effect of mature whole Citrus aurantium fruit extract(FEMC)in the chronic unpredictable mild stress(CUMS)model.The behavioral tests were applied to assess antidepressant effect and 16S rRNA sequencing was used to analyze the changes of gut microbiota.The results showed that the major components of FEMC were naringin and neohesperidin and significantly increased the sucrose preference index of the mice.FEMC also could reduce the feeding latency in an open field test and the rest time in a novelty suppressed feeding test.In addition,FEMC could increase CUMS-induced reduction in the levels of BDNF,PSD95,and SYN in the hippocampus.Moreover,FEMC intervention slightly decreased the ratio of Firmicutes to Bacteroidota.Meanwhile,FEMC reduced the abundance of the Prevotellaceae_Ga6A1_group,[Ruminococcus]_torques_group,which have been reported to be closely related to inflammation.Bioinformatics analysis revealed that mitogen-activated protein kinase(MAPK)signaling pathway and lipopolysaccharide biosynthesis were involved in the anti-inflammatory effect of FEMC in the CUMS animal model.Finally,the ELISA results showed that FEMC could significantly reduce the expression of pro-inflammatory cytokines IL-6 and TNF-αin the serum of depressive mice.Our results suggest FEMC can am eliorate depressive behavior by i nhibiting gut microbiota-mediated inflammation in mice.

High-throughput screening system of citrus bacterial cankerassociated transcription factors and its application to the regulation of citrus canker resistance

One of the main diseases that adversely impacts the global citrus industry is citrus bacterial canker(CBC),caused by the bacteria Xanthomonas citri subsp.citri(Xcc).Response to CBC is a complex process,with both proteinDNA as well as protein–protein interactions for the regulatory network.To detect such interactions in CBC resistant regulation,a citrus high-throughput screening system with 203 CBC-inducible transcription factors(TFs),were developed.Screening the upstream regulators of target by yeast-one hybrid(Y1H)methods was also performed.A regulatory module of CBC resistance was identified based on this system.One TF(CsDOF5.8)was explored due to its interactions with the 1-kb promoter fragment of CsPrx25,a resistant gene of CBC involved in reactive oxygen species(ROS)homeostasis regulation.Electrophoretic mobility shift assay(EMSA),dual-LUC assays,as well as transient overexpression of CsDOF5.8,further validated the interactions and transcriptional regulation.The CsDOF5.8–CsPrx25 promoter interaction revealed a complex pathway that governs the regulation of CBC resistance via H2O2homeostasis.The high-throughput Y1H/Y2H screening system could be an efficient tool for studying regulatory pathways or network of CBC resistance regulation.In addition,it could highlight the potential of these candidate genes as targets for efforts to breed CBC-resistant citrus varieties.

Effect of Various Citrus Rootstock-scion Combinations on Tree Growth,Fruit Quality,and Photosynthetic Characteristics under Red Loam Soil Conditions

[Objectives]To investigate the effects of 15 distinct citrus rootstock-scion combinations on tree growth,fruit quality,and photosynthetic characteristics under red loam soil conditions and provide a theoretical foundation for the selection of appropriate citrus rootstock-scion combinations in the Zhaoqing region.[Methods]A total of 15 citrus rootstock-scion combinations were utilized as test materials for a comprehensive analysis of their phenological periods(budding,flowering,and fruiting),tree growth indicators(tree height,crown diameter,and growth),and fruit quality(appearance quality and intrinsic quality).The photosynthetic characteristics of the test materials,including the net photosynthetic rate(Pn),transpiration rate(Tr),water use efficiency(WUE),apparent quantum yield(AQY),and carboxylation efficiency(CE),were analyzed to determine their significance.Additionally,the leaf photosynthetic physiological indicators,such as soluble protein,specific leaf weight,chlorophyll,and carotenoids,were evaluated.[Results]There were notable differences observed in the phenological period,growth potential of trees,fruit quality,and photosynthetic characteristics among various citrus rootstock-scion combinations.The phenological periods exhibited variation contingent on the grafting varieties.In terms of tree growth potential,the Citrus tangerina Tanaka‘Hongju’and C.haniana Hort.‘Suanju’rootstocks demonstrated greater tree height,crown growth,and overall tree strength;however,they were also prone to excessive growth.Conversely,the C.limonia Osbeck‘Hongningmeng’and C.sinensis×P.trifoliata‘Zhicheng’rootstocks displayed medium growth potential,while the Poncirus trifoliate(L)Raf.‘Zhike’rootstock resulted in shorter trees.In terms of fruit quality,the single fruit weight of C.flamea Hort.‘Shatangju’ranged from 33 to 50 g,exhibiting a flat and round shape.The total soluble solids and titratable acid content of‘Shatangju’grafted onto the‘Zhike’rootstock were notably high.In contrast,the single fruit weight of C.haniana Hort.‘Chuntianju’varied between 65 and 81 g,characterized by a high flat round shape.The‘Suanju’rootstock demonstrated a higher sugar and acid content compared to other rootstocks.Additionally,the single fruit weight of C.nobilis Lour.‘Gonggan’ranged from 62 to 145 g,with the fruit shape being either round or oval.The soluble sugar and total soluble solids content associated with the‘Zhike’rootstock was also elevated.In relation to photosynthetic characteristics,the photosynthetic performance of the‘Shatangju’variety was superior when grafted onto the‘Zhike’and‘Hongju’rootstocks.Similarly,the‘Chuntianju’variety exhibited enhanced photosynthetic performance on the‘Zhike’,‘Zhicheng’,and‘Hongju’rootstocks.Furthermore,the‘Gonggan’variety demonstrated improved photosynthetic performance when grafted onto the‘Zhike’and‘Suanju’rootstocks.[Conclusions]Based on the characteristics of the red loam soil in the Zhaoqing region,the rootstocks‘Zhike’and‘Hongju’are conducive to the cultivation of the‘Shatangju’variety.Additionally,the rootstocks‘Zhike’,‘Zhicheng’,and‘Hongju’are optimal for the growth of the‘Chuntianju’variety,while the rootstocks‘Zhike’and‘Suanju’are appropriate for the growth of the‘Gonggan’variety.

Survey and Detection of Disease and Pest Occurrence in Citrus Nurseries

[Objectives]The paper was to ascertain the prevalence of diseases and pests in a range of citrus nurseries situated in Guangdong Province and its neighboring provinces.[Methods]Citrus diseases and pests were systematically investigated,and citrus leaf samples were randomly collected from 15 citrus nurseries across 8 regions in Guangdong Province and its neighboring provinces.Quantitative polymerase chain reaction(qPCR)and reverse transcription polymerase chain reaction(RT-PCR)techniques were employed to detect diseases in the collected samples.Additionally,root and substrate samples were obtained,and root-knot nematodes were isolated using the Baermann funnel method.[Results]The positive detection rate of citrus huanglongbing(HLB)was recorded at 3%,indicating an increase in attention towards this disease compared to 2013.Additionally,the positive detection rate for citrus bacterial canker disease(CBCD)was found to be 16.5%.It was observed that the majority of nurseries with positive samples employed open field rearing practices without the use of mesh chambers,and the primary source of scions was self-propagation.The detection rate of citrus tristeza virus(CTV)was found to be the highest,with a positive detection rate of 63%,and the prevalence in disease-bearing nurseries reached as high as 90%.In comparison to 2013,there had been no improvement in the condition of seedlings affected by CTV.The positive detection rate of citrus yellow vein clearing virus(CYVCV)was found to be 38%,with 70%of the surveyed nurseries exhibiting the disease.The citrus varieties identified as carriers of the disease included‘Qicheng’,‘Shatangju’,‘Wogan’,and‘Gonggan’.Nematodes were isolated from the matrix and roots of seedlings grown in both container and open field environments.The susceptibility of container seedlings to nematodes was found to be 36.4%,while the susceptibility of open field seedlings was 38.6%.Statistical analysis indicated no significant difference in susceptibility between the two groups.[Conclusions]The disease detection rates associated with various seedling rearing methods and citrus varieties exhibited notable variability.Open field seedlings without the protection of mesh chambers demonstrated a higher susceptibility to disease.Additionally,the types of infectious diseases varied among the different citrus varieties.

Effects of Different Seasons on Cutting Propagation of Five Citrus Rootstocks

[Objectives]To explore the cutting propagation mode of citrus rootstock,improve the survival rooting rate of citrus rootstock,and provide theoretical guidance and technical reference for the rooting research of difficult-to-root plants such as citrus.[Methods]Five citrus rootstocks Citrus tangerina Tanaka‘Hongju’,Citrus haniana Hort‘Suanju’,Citrus limonia Osbeck‘Hongningmeng’,Citrus sinensis×Poncirus trifoliata‘Zhicheng’and Poncirus trifoliate(L)Raf.‘Zhike’were used as experimental materials to select the suitable cutting substrate for citrus rootstocks by measuring the physical properties of the substrate.Cutting was carried out in spring,summer,autumn and winter respectively.After cutting,the morphological changes of cuttings were observed regularly,and the callus rate,germination rate and rooting rate of cuttings were recorded.[Results]The best substrate for citrus rootstock cutting was peat soil,vermiculite and fine river sand(2:1:1).The callus of citrus rootstock in different cutting seasons began to appear in 10-22 d,and the callus rate reached 55%-100%.In terms of budding,the budding time was the earliest in summer and autumn,and slightly later in spring and winter;the germination rate of C.limonia Osbeck‘Hongningmeng’was the highest,and the germination rate of C.sinensis×P.trifoliata‘Zhicheng’and P.trifoliate(L)Raf.‘Zhike’was lower;in terms of rooting,C.limonia Osbeck‘Hongningmeng’had the earliest rooting time and the highest rooting rate and could reach 100%in all seasons;the rooting rate of C.tangerina Tanaka‘Hongju’was 50%-80%;the rooting rate of C.haniana Hort‘Suanju’was 60%-80%;C.sinensis×P.trifoliata‘Zhicheng’and P.trifoliate(L)Raf.‘Zhike’showed the earliest rooting time and the highest rooting rate in summer,and the latest rooting time and the lowest rooting rate in winter,which were only 14.5%.Therefore,different citrus rootstock varieties should choose the appropriate cutting time according to their own characteristics.[Conclusions]The results of this study can provide a scientific basis for a large number of cutting propagation of different citrus rootstocks,and have practical guiding significance for large-scale planting.

Regeneration of Diploid Intergeneric Somatic Hybrid Plants Between Microcitrus and Citrus via Electrofusion

Leaf-derived protoplasts of Rough lemon (Citrus jambhiri Lush, 2n = 2x = 18) were electrofused with embryogenic suspension protoplasts of its relative, Microcitrus papuana Swingle (2n = 2x = 18), with an intention of creating novel germplasm. Six plants were regenerated following protoplasts fusion. Cytological examination demonstrated that they were diploids with 18 chromosomes (2n = 2x = 18). RAPD (random amplified polymorphic DNA) analyses with six arbitrary 10-mer primers showed that the regenerated plants had identical band patterns to those of Rough lemon for primers OPA-07, OPAN-07, OPE-05 and OPA-08, Whereas for the other two primers, OPA-04 and OPS-13, bands specific to M. papuana could be detected in the regenerated plants. Cytological and RAPD analysis revealed that the regenerated plants were diploid somatic hybrids between M. papuana and Rough lemon. The putative hybrids were morphologically similar to Rough lemon. This is the first report on production of diploid somatic hybrid plants between citrus with its related genus via symmetric fusion.

Observation on the Embryonic Development in Citrus after Cross Pollination

Embryonic development was studied in six cross combinations ofCitrus sinensis x C. tangerina, C. sinensis x C. reticulata, C. sinensis x (C. tangerina + C.reticulata), C. sinensis x Poncirus trifoliate, C.reticulata x C grandis and C. grandis xPoncirus trifoliate. The results showed that on the 30th day after pollination thezygote remained a single cell. On the 40th day, the zygote began to divide. On the50th day, zygotic embryo became globular-shaped while nucellar embryos had notinvaded the embryo sac. On the 55th day, a few nucellar embryos began to invadethe embryo sac. On the 60th day, the zygotic embryo became heart-shaped, and atthe same time, a large number of nucellar embryos invaded the embryo sac. On the80th day after pollination, the zygotic embryo was surrounded by nucellar embryosand it was not easy to distinguish these embryos. The cross combination affected thedevelopments of zygotic embryos, ovules and fruits, which were mainly determined bythe cross parents. As compared with interspecies crossing, the zygotic division ofintergenus crossing began later, the zygotic embryos developed slowlier and theinvading time of nucellar embryos was also delayed.

Antibacterial Effect of Nano-scale TiO_2 on Parasitic Bacterium of Nanfeng Citrus in Storage Period

[Objective] The aim was to observe the antibacterial effect of nano-scale Titanium dioxide on parasitic bacterium of Nanfeng Citrus in storage period.[Method] Nano-scale Titanium dioxide was prepared by dibutyl phthalate through sol-gel method under anhydrous conditions,and orthogonal experiment was used to determine optimum conditions for nano-scale Titanium dioxide preparation,and structure characterization of nano-scale Titanium dioxide was carried out by X-Ray diffractometer.Oxford cup method was used to explore inhibition effect of nano-scale Titanium dioxide suspension on the activity of normal parasitic bacterium of Nanfeng Citrus.Simultaneously,the empirical preservation test was carried out.[Result] The average diameter of nano-scale Titanium dioxide powder attained to 14.6 nm,actual average yield could reach 90.83% with RSD（Relative Standard Deviation）of 0.86%.[Conclusion] Nano-scale Titanium dioxide had good antibacterial effect on the parasitic bacterium of Nanfeng Citrus in storage period.

Intergeneric Somatic Hybrid Plants Between Citrus and Poncirus trifoliata and Evaluation of Their Root Rot Resistance

Protoplasts of Page tangelo (Citrus reticulata Blanco×C. paradisi Macf.) cell suspension culture were electrically fused with mesophyll protoplasts isolated from trifoliate orange (Poncirus trifoliata (L.) Raf.). More than 150 plantlets regenerated after 4-5 months of culture. The regenerated plants were trifoliate with well developed root systems. Root_tip chromosome counting of more than 20 randomly selected plants revealed that they were all tetraploids (2n=4x=36). RAPD analysis of 7 randomly selected plants verified their hybridity. Inoculation of citrus Phytophthora parasitica Dastar toxin on leaves of somatic hybrids and both parental genotypes showed that Page tangelo was moderately susceptible, and trifoliate orange was highly resistant while the somatic hybrids were resistant. The potential of this somatic hybrid as rootstock is also discussed.

Analysis of Meteorological Conditions of Freeze Damage to Citrus in Southern Shaanxi in the Winter of 2010 and Defensive Countermeasures

In order to take advantage of the climate resources more effectively ac- cording to the local circumstances and to plan and develop the citrus industry in Southern Shaanxi more reasonably. On the basis of the investigation of freeze dam- age to citrus occurring in Southern Shaanxi in the winter of 2010, the climatic back- ground for the formation of this freeze damage was analyzed. In combination with the freeze damage indicators during the overwintering period and the harmful accu- mulated cold during the cold wave, indexes for grading the freeze damage in southern Shaanxi were analyzed and verified, and the perspective of grading the freeze damage using the harmful accumulated cold during the cold wave was also presented. Through analyzing the extremely lowest temperature and the harmful ac- cumulated cold in the winter of 2010 and in history at 12 citrus growing counties （districts） in Ankang area and Hanzhong area, the reasons why the freeze damage to citrus during the overwintering period was severer in the west than in the east of Southern Shaanxi were discussed, and the results obtained were basically consistent with the actual situation observed from investigation. Finally, defensive countermea- sures against the freeze damage to citrus during the overwintering period were put forward from several aspects.

cpSSR: a New Tool to Analyze Chloroplast Genome of Citrus Somatic Hybrids

Chloroplast simple sequence repeat (cpSSR) markers in Citrus were developed and successfully used to analyze chloroplast genome inheritance of Citrus somatic hybrids. Twenty-two previously reported cpSSR primer pairs from pine (Pinus thunbergii Parl.), rice (Otyza sativa L.) and tobacco (Nicotiana tabacum L.) were tested in Citrus, nine of which could amplify intensive PCR products by agarose gel electrophoresis. Chloroplast genome inheritance of Citrus somatic hybrids from nine fusions was then analyzed, and five of the nine pre-screened primer pairs showed polymorphisms by polyacrylamide gel electrophoresis. The results revealed the random inheritance nature of chloroplast genome in all analyzed Citrus somatic hybrids, which was in agreement with previous reports based on RFLP or CAPS analyses. It was also shown that cpSSR is a more efficient tool in chloroplast genome analyses of somatic hybrids in higher plants, compared with the conventional RFLP or CAPS analyses.

Genetic Diversity Analysis of Shatangju Mandarin(Citrus reticulata) by SCoT-PCR

[Objective] This study aimed to optimize the SCoT-PCR system for Citrus.Under the optimized SCoT-PCR amplification conditions, the genetic DNA of Youzishatangju and Wuzishatangju were amplified. [Method] Orthogonal design was adopted to optimize the five influencing factors on amplification effect of SCoT-PCR,including the DNA template, Mg2＋, primers, dNTPs and Taq DNA polymerse concentrations. With the optimized SCoT-PCR system, the genomic DNA of Youzishatangju and Wuzishatangju were amplified by 60 SCoT primers, respectively, and the specific bands for Youzishatangju or Wuzishatangju were selected for SCAR transformation. [Result] The optimized SCoT-PCR reaction system（20 μl） was as follows： Mg2＋1.5 mmol/L, d NTPs 0.35 mmol/L, primer 0.25 μmol/L, Taq enzyme 0.5 U, DNA template 30 ng. The optimum annealing temperature was 50.6 °C. With the optimized SCoT-PCR system, the genomic DNA of Youzishatangju and Wuzishatangju were amplified respectively by 60 SCoT primers, and a total of 42 pairs of primers were screened. Among the 42 pairs of primers, only one primer amplified different band pattern between Youzishatangju and Wuzishatangju. [Conclusion] It indicates that there is certain difference between Youzishatangju and Wuzishatangju at genomic DNA level.

Regeneration and Characterization of Plants Derived from Asymmetric Protoplast Fusion in Citrus

Protoplasts of Valencia sweet orange ( Citrus sinensis Osb.),irradiated by X_ray with a dose rate of 3.8 krad/min for 45 min, were electrically fused with protoplasts of Murcott tangor ( C. reticulata×C. sinensis ) that were treated with 0.25 mmol/L iodoacetic acid for 15 min. It took nearly 15 months for the fusion_derived calli to develop into embryoids that were only originated in the medium of MT supplemented with 2% glycerol. The shoots were recalcitrant to rooting in the root_induction medium. In vitro grafting was employed to produce whole plants though one self_rooting plant was obtained. Cytological determination of root and shoot tips showed mainly diploid and aneuploid cells, together with few tetraploid cells in some plants. RAPD (random amplified polymorphism DNA) analysis with 10_mer primers demonstrated that bands specific to the fusion parents were detected in the regenerated plants, indicating that interspecific somatic hybrids have been obtained via protoplast asymmetric fusion in Citrus .

Identification of Genetic Variation in Citrus sinensis from Hunan Based on Start Codon Targeted Polymorphism

[Objective] The aim was to identify genetic variation in Citrus sinensis （sweet orange） germplasm from Hunan Province according to the Start Codon Targeted （SCoT） Polymorphism. [Method] The reaction system for SCoT amplification from sweet orange was first optimized, and then the SCoT fragments were amplified from 24 sweet orange cultivars collected in Hunan Province and sequenced for genetic variation analysis. [Result] The optimum reaction system for SCoT markers amplification was 2.0 μl containing 80 ng of template DNA, 0.3 mmol/L dNTPs, 0.2 μmol/L primer, 1.6 mmol/L Mg2＋, 1.6 U of Taq DNA polymerase and 10×PCR buffer. By using this reaction system, the PCR products from the sweet orange cultivars produced clear and reproducible bands at 100-2 000 bp through electrophoresis. The SCoT fragments of the 24 sweet orange cultivars were 1 090-1 091 bp, with the homology of 99.84% and nucleotide deletion and substitution. After being sequenced, the SCoT polymorphisms could distinguish 12 sweet orange cultivars. In addition, the BLAST result showed that part of the SCoT fragments coding region shared high homology with ribosomal protein S3 N superfamily. [Conclusion] This study will provide a theoretical basis for breeding sweet orange cultivars.