Assessment of the Genetic Diversity of Pummelo Germplasms Using AFLP and SSR Markers

The genetic diversities of 110 pummelo germplasms and 12 of their relatives were analyzed by SSR and AFLP methods. Approximately 99.1% of the 335 SSR loci were polymorphic, and 9.85 alleles per SSR locus were identified. The gene diversity values changed from 0.1939 to 0.9073, and 46 SSR polymorphic bands were scored. 72% of the 343 AFLP loci were polymorphic, and 82 polymorphic loci per AFLP were identified. Heterozygosity changed from 0.21863 to 0.28445, and 44 AFLP polymorphic bands were scored. The UPGMA result showed that 122 pummelo genotypes and their relatives could be divided into eight groups, and the pummelo genotypes composed mainly of Shatian pummelo varieties group, Wendan pummelo vareties group and a huge hybrid pummelo varieties group. The classification result was expected to widen the genetic background of pummelos using various target varieties.

Induction and Genetic Identification of Embryogenic Calli from Hybrids of Shatian Pummelo

Shatian pummelo （Citrus grandis L. Osbeck cv. Shatian） is an elite variety in China, and the regeneration of the embryogenic callus is difficult. Diploid Shatian pummelo was used as the female and crossed with the allotetraploid somatic hybrid NS （Nova Tangelo ＋ Succari Sweet orange）, [ （ C reticulata Blanco x C. paradisi Macf.） cv. Nova ＋ C sinensis L. Osbeck cv. Succari]. About 90 days after pollination, the embryos obtained from crosses were cultured on the solid media of MT ＋ ME （malt extraction, 500 mg L^-1） and MT ＋ GA3 （1 mg L^-1）. The embryogenic callus was initiated from the embryoids and plantlets＇ hypocotyls and could be subcultured. Flow cytometry and SSR analysis verified that the callus was from the triploid hybrids. The callus had embryogenesis capacity and produced a large number of embryoids on MT ＋Lactose （50 g L^-1） medium after being subcultured for two years. It is comparatively easier to obtain the callus from the hybrid embryo than from Shatian pummelo itself. The callus is valuable for the conservation and utilization of Shatian pummelo.

Quality and Nutrition of Pummelo as Influenced by Potassium

This study aimed to determine the influence of different rates of K （potassium） and K foliar application on the quality and plant nutrition condition of 13-year old ＇Magallanes＇ pummelo trees [Citrus maxima （Burm. ex Rumph.） Merr.]. The experiment was conducted at SODACO （South Davao Corporation） farm, Davao City, Philippines from March to October 2010. Five treatments with increasing K levels were applied per tree： control, no K, 150 g K basal, 225 g K basal and 225 g K basal ＋ foliar application. Application of 225 g K rates positively influenced fruit quality of pummelo. Fruit pulp diameter and juice weight increased while peel thickness decreased. The 225 g K rates also increased juice pH, TA （titrable acidity） and TSS （total soluble solids） per tree. The yield of total phenol, flavonoid, vitamin C and oil per tree increased with 225 g K application. Results of the soil analysis before and after the conduct of the study showed an evidence of fertilizer absorption by the trees. It was also observed that foliar fertilization reduced soil acidity. This indicates the important role of K in improving the nutrient utilization and quality of ＇Magallanes＇ pummelo.

Phytochemical Composition,Antioxidant and Antibacterial Properties of Pummelo(Citrus maxima(Burm.))Merr.against Escherichia coli and Salmonella typhimurium

The antioxidant and antibacterial activities of the phytochemical constituents of the pericarp, mesocarp and segment membrane crude ethanolic extracts of Pummelo (Citrus maxima (Burm.)) fruit were were tested against Escherichia coli and Salmonella typhimurium. Preliminary phytochemical test revealed the presence of phenols, tannins, saponins expressed as catechine quivalent (CE)/100ml and flavonoid expressed as gallic acid equivalent (GAE)/100ml. The order of which was as follows pericarp > segment membrane > mesocarp. The strongest antioxidant activity was obtained by the pericarp extract (29.64 expressed as % lipid peroxidation). The differences in the measured amount of phytochemicals and antioxidant activity among the three sample extracts were found to be significant. In terms of antimicrobial activity, the pericarp, mesocarp and segment membrane extracts generated zone of inhibitions measuring 17.10, 18.00 and 17.03 mm for S. typhimurium, respectively at 100% concentration. E. coli was noted to be inactive in all three sample extracts at 100% concentration. The capacity of E. coli to counteract the inhibitory effect of the phytochemicals contained in the pummelo extracts may be attributed to its rough corrugated cell wall and thick periplasmic space as opposed to the smooth curved and barely seen periplasmic space of S. typhimurium. However, no significant correlation was detected among the phytochemical content, antioxidant and in vitro antimicrobial activities of the sample extracts against S. typhimurium.

Preliminary Study on Quality Standard of Pummelo Pericarp from Guangxi

[Objectives]This paper aimed to study the quality standard of pummelo pericarp from Guangxi.[Methods]The medicinal material was identified by microscopy and thin-layer chromatography,and the contents of moisture,total ash,acid-insoluble ash and extract in the medicinal material were determined according to the methods of the Chinese Pharmacopoeia(2010 edition).[Results]The content limits for moisture,total ash,acid-insoluble ash and extract were determined(moisture≤12%;total ash≤6.0%;acid-insoluble ash≤0.6%;water-soluble extract≥43.0%;naringin≥10.00 mg/g).The micro-characteristics of pummelo pericarp were identified,and a thin-layer chromatography identification and content determination method was established.[Conclusions]The established method can provide an experimental basis for the formulation of quality standard for pummelo pericarp.

Phenolic Composition and Antioxidant Capacities of Chinese Local Pummelo Cultivars' Peel

China is one of the main production areas of pummelo [Citrus grandis(L.) Osbeck.] in the world and has lots of distinctive local cultivars.Systematic research on the detection of phenolics and antioxidant capacity of peels of mature local cultivars pummelo fruits is rare.In the current study,phenolic composition and content in peels(flavedo and albedo) of ten Chinese local pummelo cultivars were determined using Ultraviolet Spectrophotometer and High Performance Liquid Chromatography(HPLC),and their antioxidant capacities were evaluated using 2,2-diphenyl-1-picrylhydrazyl(DPPH) radicals,ferric reducing antioxidant power(FRAP),and 2,2′-azino-bis(3-ethylbenzthiazoline-6)-sulfonic acid(ABTS)methods in the current study.The research not only provides data that support making full use of the resources of Chinese local pummelo cultivars,but also lays the theoretical basis for research of pummelo fruit nutrition and health values.The results showed that the total phenolic(TP)content in albedo was significantly higher than that in flavedo;on the contrary,the total flavonoid(TF) content was lower in the albedo than in the flavedo.C.grandis ‘Hongxinyou' flavedo contained the highest TP contents,C.grandis ‘Liangpingyou 78-8' flavedo contained the highest TF contents,and C.grandis ‘Guanxi Miyou' albedo contained the highest TP and TF contents.Naringenin,hesperetin,diosmin,and gallic acid were the predominant phenolics in the flavedo,whereas hesperetin,diosmin,rutin,chlorogenic acid,and gallic acid were the primary phenolics in the albedo.The flavedo of C.grandis ‘Yubei Shatianyou' and the albedo of C.grandis ‘Dianjiang Baiyou' had the highest antioxidant potency composite(APC) indexes.Overall,the ‘Yubei Shatianyou' flavedo and the ‘Dianjiang Baiyou' albedo are excellent sources of antioxidants and have the greatest potential value for exploitation and utilization.

Identification of a delayed leaf greening gene from a mutation of pummelo

Delayed greening of young leaves is an unusual phenomenon of plants in nature.Citrus are mostly evergreen tree species.Here,a natural mutant of“Guanxi”pummelo(Citrus maxima),which shows yellow leaves at the young stage,was characterized to identify the genes underlying the trait of delayed leaf greening in plants.A segregating population with this mutant as the seed parent and a normal genotype as the pollen parent was generated.Two DNA pools respectively from the leaves of segregating seedlings with extreme phenotypes of normal leaf greening and delayed leaf greening were collected for sequencing.Bulked segregant analysis(BSA)and In Del marker analysis demonstrated that the delayed leaf greening trait is governed by a 0.3 Mb candidate region on chromosome 6.Gene expression analysis further identified a key candidate gene(Citrus Delayed Greening gene 1,CDG1)in the 0.3 Mb region,which showed significantly differential expression between the genotypes with delayed and normal leaf greening phenotypes.There was a 67 bp In Del region difference in the CDG1 promoter and the In Del region contains a TATA-box element.Confocal laser-scanning microscopy revealed that the CDG1-GFP fusion protein signals were co-localized with the chloroplast signals in the protoplasts.Overexpression of CDG1 in tobacco and Arabidopsis led to the phenotype of delayed leaf greening.These results suggest that the CDG1 gene is involved in controlling the delayed leaf greening phenotype with important functions in chloroplast development.

A novel histone methyltransferase gene Cg SDG40 positively regulates carotenoid biosynthesis during citrus fruit ripening

The flesh color of pummelo(Citrus maxima)fruits is highly diverse and largely depends on the level of carotenoids,which are beneficial to human health.It is vital to investigate the regulatory network of carotenoid biosynthesis to improve the carotenoid content in pummelo.However,the molecular mechanism underlying carotenoid accumulation in pummelo is not fully understood.In this study,we identified a novel histone methyltransferase gene,CgSDG40,involved in carotenoid regulation by analyzing the flesh transcriptome of typical white-fleshed pummelo,red-fleshed pummelo and extreme-colored F1 hybrids from a segregated pummelo population.Expression of CgSDG40 corresponded to flesh color change and was highly coexpressed with CgPSY1.Interestingly,CgSDG40 and CgPSY1 are located physically adjacent to each other on the chromosome in opposite directions,sharing a partially overlapping promoter region.Subcellular localization analysis indicated that CgSDG40 localizes to the nucleus.Overexpression of CgSDG40 significantly increased the total carotenoid content in citrus calli relative to that in wild type.In addition,expression of CgPSY1 was significantly activated in overexpression lines relative to wild type.Taken together,our findings reveal a novel histone methyltransferase regulator,CgSDG40,involved in the regulation of carotenoid biosynthesis in citrus and provide new strategies for molecular design breeding and genetic improvement of fruit color and nutritional quality.

基于Spring MVC的化橘红病虫害防治技术数据库系统的设计

文章设计了化橘红病虫害防治技术数据库系统,并详细分析了设计流程。系统界面设计友好,操作便捷,收集了与化橘红相关的病虫害信息及防治方法,能够为果农提供参考,具有一定的应用价值。

柚和葡萄柚花粉直感对东试早柚果实生长及品质的影响

【目的】针对东试早柚果实个体差异大、品质不稳定等问题,通过异花授粉探究花粉直感对东试早柚果实品质的影响,并对各项指标进行综合评价,旨在筛选出能够有效改善东试早柚果实品质的授粉品种,为生产上授粉品种配置和品质提升提供理论依据。【方法】以23年生东试早柚为母本,柚和葡萄柚不同品种为父本,进行人工授粉,比较不同授粉组合的坐果率、果实生长、果实品质差异,并采用隶属函数法进行综合评价。【结果】通过比较分析发现果实横径和纵径生长动态呈“S”形;异花授粉显著提高东试早柚坐果率,坐果率均高于60%。在果实外在品质方面,Flame授粉对果实单果质量提升效果不明显,但其他授粉组合果实单果质量比自然授粉显著提升27.77%~85.08%;在所有授粉组合中,自然授粉果实横径和纵径最小,Sarawak授粉果实横径和纵径显著高于自然授粉和其余品种授粉果实。异花授粉能够降低果实裂瓣数,增加种子数,且不同品种授粉种子直感存在差异。在果实内在品质方面,Oroblanco授粉能提高可溶性固形物含量,但Sarawak、Tahition、泰国红宝石柚、越南青柚授粉显著降低果实可溶性固形物含量,显著提高总酸含量;不同授粉组合间果实可溶性蛋白含量无显著差异,但可溶性糖、维生素C以及木质素、柚皮苷含量存在差异,表现出明显花粉直感效应。果实种子数与果实横径呈显著正相关,裂瓣数与果实横径和种子数呈显著负相关;外在品质与内在品质主要指标间呈显著负相关。通过主成分分析法和隶属函数法综合评价结果显示,Genetic Dwarf、Oroblanco授粉果实综合得分为0.68、0.67,高于自然授粉0.66。【结论】异花授粉能够提高坐果率,同时对果实生长发育和果实品质有显著影响。Genetic Dwarf和Oroblanco授粉在一定程度上能够改善东试早柚果实综合品质,可为生产上授粉品种配置、提高果实品质提供参考依据。

琯溪蜜柚疏果黄酮酶法辅助提取工艺优化及其抑制酪氨酸酶活性

本论文旨在探讨β-葡萄糖苷酶法辅助提取琯溪蜜柚疏果黄酮的工艺及提取得到的黄酮对酪氨酸酶抑制作用。以总黄酮提取量为指标,采用单因素实验及响应面试验对β-葡萄糖苷酶酶活力、酶解pH、温度及时间进行优化,并研究总黄酮提取物对酪氨酸酶的抑制作用。结果表明,通过单因素实验和响应面试验优化得到琯溪蜜柚疏果黄酮提取的最优条件是:β-葡萄糖苷酶酶活力0.0128 U/mL、酶解pH4.4、酶解温度35℃、酶解时间4.8 h,在该工艺条件下疏果总黄酮提取量为34.0 mg/g;该黄酮提取物对酪氨酸酶的半抑制浓度(IC50)为0.31 mg/mL。本研究结果说明酶法工艺可显著提高琯溪蜜柚疏果黄酮的提取量,且琯溪蜜柚疏果黄酮具有较强的酪氨酸酶抑制作用,研究结果为琯溪蜜柚疏果黄酮提取及应用提供了参考依据,有利于促进琯溪蜜柚的高值化利用。

重构本草——化橘红

通过古籍文献调研、中医药现代研究成果的梳理,结合中医临床及中药学等学科领域内知名专家学者的认识及应用经验,归纳出化橘红:功效主要为化痰止咳、理气宽中。症靶为咳嗽。现代药理研究发现化橘红及其有效成分具有化痰止咳、抗呼吸道炎症、降糖调脂、抗阿尔茨海默病、防治糖尿病心肌功能损伤等作用。化橘红基本无毒副作用,阴虚燥咳及久嗽气虚者应慎用。临床医家常用剂量为10~30 g,内服时当根据不同疾病,或同一疾病的不同阶段进行辨证使用,并积极探索化橘红用量与症靶、标靶的量效关系构建。

柚果皮中生理活性成分研究进展

对柚果皮中黄酮类化合物、类柠檬苦素、天然色素及香精油的化学组成、分布。

柚皮中柚皮苷的乙醇提取工艺研究

目的 探讨柚皮中柚皮苷的乙醇提取最佳工艺条件。方法 运用正交试验法考察乙醇浓度、提取温度、提取时间和乙醇用量对提取率的影响 ,采用两步结晶法对提取物中的柚皮苷进行精制。结果 柚皮中柚皮苷的乙醇提取最佳工艺条件为 2 5倍原料重的 70 %乙醇、6 0℃条件下保温浸提 1h;两步结晶法所得柚皮苷精制品的纯度为90 .0 1%。结论 采用本实验所确定的提取及精制方法可使柚皮中柚皮苷得到有效提取和分离。

沙田柚杂交后代群体的SSR鉴定与遗传多样性分析

【目的】利用SSR标记对沙田柚杂交后代群体进行杂种鉴定和遗传多样性研究,为柑橘种质创新和遗传改良提供技术、材料和理论支持。【方法】利用SSR技术分析沙田柚两个杂交组合159株后代的杂种性质,采用UPGMA聚类分析法分析后代群体和亲本的遗传关系。【结果】4对引物可以从104株沙田柚×强德勒柚的杂交后代鉴定出103株真杂种,鉴定率达到99.04%,且在引物AGC9的扩增图谱中有70个单株出现了双亲都没有的新条带;5对引物可鉴定出沙田柚×红江橙的全部杂交后代均为真杂种,一个纯合显性标记AAT12被发现,在引物GA18和AGC9的扩增结果中出现了亲本位点的缺失;UPGMA聚类分析结果显示,两个杂交组合后代群体均出现较显著的遗传变异,两个组合的遗传多样性也有较大的差异。【结论】SSR标记适合柑橘杂种的鉴定和遗传多样性分析,杂交是获得柑橘遗传变异株系的有效途径。

柚类种质资源AFLP与SSR遗传多样性分析

结合SSR引物和AFLP分子标记对110份柚类基因型、12份野生近缘种进行遗传多样性研究。结果表明,SSR标记的335个位点中99.1%为多态性位点,每个位点可检测到9.85个等位基因,基因多样度(GD)变幅为0.1939～0.9073,获得了46个特异性SSR标记;AFLP标记的343个位点中72%为多态性位点,3对引物平均期望杂合度变幅为0.21863～0.28445,平均每对引物组合能产生82个多态位点,获得了44个AFLP特异性标记。UPGMA聚类结果显示,122份基因型在相似系数0.61时,分成8个组群,其中柚类主要由沙田柚品种群、文旦品种群与庞大的杂种柚品种群组成。分类结果将有利于更好地利用这些丰富的育种资源。

十种柚类及柚杂种果实中类黄酮含量的超高效液相色谱分析

【目的】利用超高效液相色谱法同时检测10种柚及柚杂种果实中11种类黄酮含量,测定分析各品种果实类黄酮的种类及其含量变化规律。【方法】超高效液相色谱法,色谱柱:ACQUITY UPLC BEH C18分析柱(2.1 mm×100 mm,1.7μm);流动相:0.1%乙酸水溶液(A)和甲醇(B),采用梯度洗脱,洗脱程序为:(1)0—5 min,90%—80%A;(2)5—10 min,80%—30%A;(3)10—15 min,30%—20%A;(4)15—16 min,20%—90%A;柱温:35℃;流速:0.3 mL.min-1。【结果】类黄酮组分及含量与柑橘的遗传基因型有关,柚果实中类黄酮以柚皮苷为主,葡萄柚果实中以柚皮苷和新橙皮苷为主。柚皮苷在瑞红中含量最高(果皮:27.5 mg.g-1DW,果肉:2.73 mg.g-1DW),新橙皮苷在鸡尾中含量最高(果皮:14.93 mg.g-1DW,果肉:0.70 mg.g-1DW)。杂种后代类黄酮累积受其亲本影响。【结论】超高效液相色谱法是一种定性定量柑橘果实类黄酮的有效方法,柚果实中类黄酮含量丰富且各种类之间组分含量差异显著。

中国柚类生态分布多样性研究

利用分子标记AFLP技术分析了我国柚类生态分布遗传多样性,根据我国自然形成的西南及长江流域、东南沿海、华南柚产区等3个分布带,其中种群内平均预期杂合度为0.210 4,种群间平均预期杂合度为0.006 8,群体内遗传分化程度Fst为0.031 1,即种群内的遗传多样性所占的比例为96.87%,种群间变异占总变异的3.13%,3个中心产区间基因流Nm为7.79。以西南及长江流域遗传多样性最高,其次为东南沿海,华南柚产区遗传多样性最小。按照内陆性与海洋性两大系统进行柚类遗传多样性分析,所有位点总的预期杂合度为0.224 7,其中种群内平均预期杂合度为0.219 1,种群间平均预期杂合度为0.005 7,群体内遗传分化程度Fst为0.025 1,即种群内的遗传多样性所占的比例为97.51%,种群间变异占总变异的2.5%,2个系统间基因流Nm为9.7。预期杂合度内陆性系统(0.229)高于海洋性系统(0.209)。

四季柚生育期叶片和果实中矿质元素含量变化的研究

本研究以四季柚为试材,定期测定果实发育周期内叶片与果实的矿质元素含量,研究四季柚叶片和果实矿质养分需求特征与相关性,为树体营养科学调控,优质高效施肥提供理论依据。结果显示,叶片生长过程中N、P元素含量呈逐渐下降趋势,K、Ca、Mg、B、Zn含量呈先增后降趋势,S含量总体保持上升趋势,Fe含量呈现先降后稳再上升趋势,Cu含量总体较稳定,Mn含量呈现出先上升后下降再上升趋势;果实生长过程中,N、P、K、Ca、S含量呈下降趋势,Mg含量呈现先升后降的趋势,微量元素中Fe、Mn元素含量变化幅度较小,B、Zn、Cu元素含量变化趋势略有不同。叶片与果实之间矿质元素协同吸收作用较弱,而果实与果皮之间作用较强。研究表明,萌芽前应适当增施氮、磷肥,6月中旬增施钙肥,7月中旬增施钾肥和镁肥,以及重视微量元素肥料的应用。

‘琯溪蜜柚’贮藏期间汁胞粒化与木质素代谢的关系

【目的】为了探讨‘琯溪蜜柚’贮藏过程中汁胞粒化与木质素的关系,以及外源激素对木质素合成与汁胞粒化的影响,【方法】以‘琯溪蜜柚’果实为材料,利用水杨酸(SA)处理低温贮藏的‘琯溪蜜柚’果实,测定了贮藏过程中汁胞粒化指数、木质素含量以及木质素合成途径中关键酶的活性。【结果】结果表明,汁胞粒化指数、木质素含量、木质素合成关键酶的活性在整个贮藏过程中逐渐升高,SA处理的木质素合成的关键酶PAL、CAD和POD的活性均显著低于对照,木质素含量和粒化指数也显著低于对照。木质素含量与粒化指数的相关系数为0.943,木质素合成酶的活性与木质素含量的相关系数为0.981。【结论】研究认为,‘琯溪蜜柚’采后贮藏期间汁胞粒化程度与汁胞中木质素含量为正相关的关系,适宜浓度的SA对汁胞粒化的抑制作用主要是通过抑制木质素合成途径中关键酶的活性,减少木质素的合成,从而减轻汁胞粒化的程度。