Efficient exploration and SSR identification of autotetraploids from the seedlings of thirteen apomictic Citrus genotypes北大核心CSCD

【Objective】Citrus is a crucial part of Chinese fruit crops. There are abundant citrus germplasm resources in China, but many excellent local varieties are gradually eliminated by the market due to the problem of numerous seeds within the fruit. The fruits of triploid plants are generally seedless because of their sterile male and female gametes. Therefore, triploid production is a promising strategy to breed seedless cultivars in citrus. Triploids can be obtained by interploidy crossing between diploids and tetraploids. However, the tetraploid germplasm is rare, which limits the application of this strategy. Exploration of tetraploids is an important prerequisite for triploid production with the aim to improve the seedy local cultivars in our country. For the rootstock improvement, tetraploid plants are also valuable resources because of their higher metabolite content, and better resistance than their diploid parents. In this study, we planned to explore tetraploid plants from 13 local cultivars in our country by using the traits of spontaneous doubling of the nucellar cells in polyembryonic citrus varieties. The exploration of tetraploids from the above 13 local cultivars will not only provide excellent tetraploid parents for the production of triploid plants, but also lay the foundation for the basic research about the effect of genome duplication on some important trait change, such as dwarfing, extensive adaptivity and higher medicinal value in tetraploids.【Methods】After the mature fruits were harvested, the seeds were extracted and the seed coats were peeled off, and then they were placed in a thermostat to accelerate germination. When the seeds germinated, they were sown in pots and cultivated in a plant growth chamber. After the seedlings grew up with three or more leaves, putative polyploids were screened according to the morphological feature showing lower height, shorter taproots, less lateral roots, thicker and rounder leaves and declined oil gland density. The ploidy levels of these putative polyploids were further confirmed by flow cytometric analysis and the observation on root tip chromosome numbers. After determination of the ploidy level, some morphological traits, including plant height, root length and diameter, lateral root number, stem diameter, leaf thickness and shape index of the tetraploids and their corresponding diploid parents were measured at the same developmental stage. SSR analysis was used to identify the genetic origin of the explored tetraploids with at least three pairs of SSR primers selected for each cultivar.【Results】The polyembryonic degree of seeds from each cultivar was firstly determined and it showed that the seeds of all 13 cultivars were polyembryonic. Among them, Qu tangerine had the highest number of embryos with an average of 9.4 embryos per seed and Bingtang sweet orange had the lowest number of embryos with an average of 2.2 embryos per seed. Based on the morphological trait screening, we identified 2, 1, 3, 2, 7, 3, 1, 3, 1, 3, 17, 1 and 2 putative polyploids respectively from 343, 499, 892, 385, 519, 290, 457, 241, 119, 690, 828, 114 and 129 seedlings of Qianshanhong tangerine, Bayue tangerine, Qu tangerine, Zao tangerine, Bianping tangerine, Ougan tangerine, Shitougan, Bingtang sweet orange, Jinmi sweet orange, Moping Xiangcheng, Japanese Xiangcheng, Zhique and Youpi kumquat. After further confirmation of ploidy levels concerning above putative tetraploids, we obtained 45 tetraploids and one hexaploid plant from Qu tangerine, with an average occurrence rate of 0.85%, among which the rate of Japanese xiangcheng was the highest with 2.05% and the rate of Bayue tangerine was the lowest with 0.20%. The exploration time from seed germination to obtaining tetraploid seedlings varied among cultivars, with the longest time (42 days) used in Youpi kumquat and the shortest time (23 days) in Shitougan. The morphological traits of tetraploids and their corresponding diploid seedlings from nine cultivars of Qianshanhong tangerine, Qu tangerine, Zao tangerine, Bianping tangerine, Ougan tangerine, Bingtang sweet orange, Moping Xiangcheng, Japanese Xiangcheng and Youpi kumquat were measured. For plant height, tap root length, lateral root numbers and leaf thickness, the tetraploid seedlings of seven cultivars showed significant differences with their diploid parents. For taproot and stem diameter, only the tetraploid seedlings explored from Bingtang sweet orange and Japanese Xiangcheng had significant difference with their diploid parents. For leaf shape index, the tetraploid seedings from Bianping tangerine and Moping Xiangcheng exhibited significant differences with their diploid seedlings. In conclusion, most tetraploid seedlings of all nine cultivars showed lower plant height, shorter and thicker taproot, less lateral root number, thicker and rounder leaves than those of their diploid parents. These results provide supports for the screening of putative tetraploids based on morphological trait observation. For analyzing the genetic origin of the tetraploids obtained in this study, at least three SSR markers were used in each genotype. The results showed that the bands of all 45 tetraploids were identical with those of their corresponding diploids, indicating that all the 45 tetraploids might originate from the spontaneous chromosome doubling of nucellar cells of their corresponding diploids. In addition, the bands of the hexaploid from Qu tangerine were also identical with their diploid parent. We speculated that it might derive from chromosome doubling of a triploid zygotic cell, which formed by selfing of a FDR-type 2n gamete with a normal n gamete, and both gametes were produced by Qu tangerine.【Conclusion】This study verified that morphological screening combined with flow cytometry ploidy determination and SSR analysis is an efficient approach to exploring polyploid seedlings from apomictic citrus. Using this method, 45 autotetraploid and one hexaploid plants were obtained from 13 apomictic citrus genotypes. These newly discovered tetraploids are potentially valuable for not only genetic improvement of some elite local citrus cultivars with seeds produced by triploids using interploidy hybridization, but also selection of the promising rootstocks with dwarf, multi-resistance and broad adaptability characteristics to improve the ability to resist various abiotic and biotic stresses. © 2023 Journal of Fruit Science. All rights reserved.     

湖南安化宜昌橙(Citrus ichangensis Swing.)调查

湖南安化宜昌橙（ＣｉｔｒｕｓｉｃｈａｎｇｅｎｓｉｓＳｗｉｎｇ．）调查＊刘干生易建礼毛峰（湖南省安化县农业局４１３５００安化县地处湘中，属亚热带季风湿润气候。安化宜昌橙资源非常丰富，野生分布于全县山地。我们于１９９２～１９９７年，在大埠溪杉树弯对野生宜...     

江西崇义的野生桔(Citrus reticulata Blanco)

七十年代末期在江西南部的崇义县原始森林中发现的野生桔,其形态特征与已见诸报道的柑桔属中野生种质有明显的差异性。因其产地处于南岭山脉支系的特定环境中,立地条件优越,并有多种近缘植物,应是在长期自然演化和发展过程中形成的原生物种。因此,可以说明南岭山脉地区是我国宽皮类种质起源地。     

Cultivar identification of ‘Yuzu’ (Citrus junos Sieb. ex Tanaka) and related acid citrus by leaf isozymes

Leaf extracts of 27 ‘Yuzu’ and related acid citrus cultivars were analyzed using polyacrylamide gel electrophoresis for isozyme variation of glutamate oxaloacetate transaminase (GOT) and shikimate dehydrogenase (SDH). SDH yielded 12 different isozyme phenotypes and six cultivars were discriminated by this enzyme alone. GOT produced 10 different isozyme phenotypes and four cultivars were separated. When both enzyme systems were taken together, 16 cultivars (59%) were uniquely discriminated and the rest could be classified into four groups of 2–4 cultivars each. Mutation originated cultivars could not be discriminated. Differences between cultivars suggested that isozymes may provide useful markers for cultivar identification.     

柚Citrus grandis(L.) Osbeck种质多样化中心的探讨

柚的起源中心众说纷纭，有人认为在印度，亦有人认为在东南亚地区或中国。根据柚的种质多样性、栽培历史以及物种演化的原始先头种——红河大翼橙的存在，作者提出柚的起源中心当在中国。随着这一物种的扩散，形成若干次级多样化中心，主要有日本、东南亚地区和印度。     

柑橘黑斑病（Citrus Black Spot）发生危害现状和研究进展

柑橘黑斑病（Citrus Black Spot,CBS）主要危害果实,在果皮上形成各种病斑而影响果实的外观品质和销售价格,严重时可造成田间落果和贮藏期果实腐烂。同时,黑斑病被欧盟列为A1类外来有害生物,美国对此也有严格的限制。因此,该病害影响柑橘鲜果的国际贸易。黑斑病的病原属球座菌（Guignardia）,其无性态为叶...     

葡萄柚(Citrus paradisi Macf)及其在我国的发展前景

葡萄柚（ＣｉｔｒｕｓｐａｒａｄｉｓｉＭａｃｆ）及其在我国的发展前景叶荫民（中国农业科学院柑桔研究所重庆４００７１２）葡萄柚营养价值高，风味独特，是世界柑桔类四大类群之一（甜橙类、宽皮柑桔类、柠檬来檬类、葡萄柚和柚类）。８０年代初至９０年代初的１０年...     

Foliar Spray with Different Agrochemicals on Fruit Quality and Exportability of ‘Washington’ Navel Orange Fruit (Citrus sinensis L.)

The present study was carried out to improve fruit set, yield, quality, marketability, and exportability of ‘Washington’ Navel orange. Pre-harvest foliar sprays of GA₃, K, and Ca, either alone or combined, were applied at full bloom. The following treatments were applied: {control (T₁), 25 mg/L GA₃ (T₂), 2% K₂SO₄ (T₃), 25 mg/L GA₃ + 2% K₂SO₄ (T₄), 25 mg/L GA₃ + 2% Ca Cl₂ (T₅), 2% K₂SO₄ + 2% Ca Cl₂ (T₆), and 25 mg/L GA₃ + 2% K₂SO₄ + 2% Ca Cl₂ (T₇). All treatments improved fruit set, yield, physical and chemical characteristics, mineral content of leaves and fruit rind, and the percentage of exportable fruit in comparison to the control. Fruit set, fruit detachment force, peel firmness, and the mineral content of leaves and fruit rind were enhanced significantly with the combination of GA₃ + K₂SO₄ + Ca Cl₂ (T₇) in comparison to all other treatments and the control. Results indicated that all foliar combination treatments improved fruit quality and marketability, and increased the percentage of exportable fruit and reduced the defects of non-exportable fruit.     

宜昌橙(Citrus ichangensis)体细胞中期染色体CMA荧光显带分析

【目的】宜昌橙是柑橘属抗逆性较强的一类重要野生种质资源,且具有特殊的体细胞中期染色体联会现象,染色体研究对其起源与进化、重要基因的发掘与应用以及染色体结构与组成等研究具有重要意义。宜昌橙体细胞中期染色体CMA荧光显带分析可为丰富其染色体特征、遗传差异与亲缘关系以及体细胞染色体联会现象发生机制探讨等提供理论与技术支持。【方法】选取8个不同类型的宜昌橙为试验材料,采集幼嫩茎尖制备体细胞中期染色体标本,利用CMA荧光显带技术对其进行荧光带型分析。【结果】8个类型宜昌橙均显示出了不同的CMA(+)带型,根据带型特征并结合前人研究将其分为4个不同类型:C型:两臂的端部具有CMA(+)荧光带;D型:长臂或短臂的端部具有CMA(+)荧光带;E型:没有荧光带或较弱;Bst型:宜昌橙随体染色体荧光带型,呈现为一个端部、近着丝粒区域及整个随体具有CMA(+)荧光带。【结论】各供试宜昌橙CMA带型呈现明显的多态性与差异性,且部分同源染色体表现出CMA(+)条带杂合性;CMA显带分析可初步对宜昌橙不同种质进行遗传分类;宜昌橙遗传差异与CMA荧光显带技术在宜昌橙与柑橘属植物染色体特征分析以及系统分类方面具有一定的应用前景。     

Gametophytic self-incompatibility causes seedlessness in ‘Kagzi Kalan’ lemon (Citrus limon)

The present study aimed to study the flower and pollen structure, pollen viability, in vitro pollen germination of ‘Kagzi Kalan’ lemon (Citrus limon (L.) Osbeck), and in vivo pollen tube growth in self- and cross pollination to understand the mechanism of seedlessness. The results revealed that Kagzi Kalan had tetra-colporate spherical shaped pollen of uniform size. The results of Acetocarmine and FDA test and in vitro germination indicated normal pollen viability and germinability in Kagzi Kalan. In vivo pollen tube growth showed > 50% pollen germination on stigma in all treatments though a greater number of pollen tubes at the mid style, base of the style, and near the ovule was found in cross pollination. Contrary to this, despite optimum pollen germination (53.51–70.37%) in self-pollination treatments, none of the pollen tubes reached the base of the style due to abnormal growth of pollen tubes, viz. curvatures, inhibition of pollen tube growth, and callose deposition. The inhibition of pollen tubes at the mid style under self-pollination treatments, and production of seeded fruits under cross pollination treatments, indicate the presence of self-incompatibility and the site of inhibition of pollen tubes. Thus, gametophytic self-incompatibility (SI) caused seedlessness in Kagzi Kalan by blocking fertilization in the ovary.     

红肉脐橙(Citrus sinensis L.)果肉中特征色素提取方法探索

红肉脐橙是目前为止惟一果肉呈粉红色或红色的脐橙,其呈色色素尚未见报道。为深入研究其果肉中特征色素的构成及在果实生长发育和贮存过程中色素含量的变化,本研究在对该色素初步定性的基础上对提取方法进行了探索。在分析红肉脐橙果肉中色素萃取液的紫外可见吸收光谱时发现,该色素有类胡萝卜素的特征吸收峰,将萃取液进行薄层层析则进一步证实其内含有番茄红素。在色素提取的前处理方式上,本研究认为冷冻干燥样品的浸提效果较真空干燥的样品稳定(变异系数小),且浸提率相差无几,所以选择冷冻干燥作为合适的样品前处理方式。色素浸提条件的正交实验结果表明,最佳浸提条件为50℃、1.5h内浸提4次,浸提的料液比为1:60。此外,浸提温度、浸提时间、浸提料液比和浸提次数对浸提率都有显著影响,影响最大的因素是浸提温度,最佳温度为50℃,随温度降低浸提率显著降低。其次是侵提时间的长短,最佳浸提时间为1.5h,延长或缩短0.5h都会使浸提率下降。浸提次数对浸提率的影响是最小的。     

航天搭载后福橘（Citrus reticulata Blanco）茎尖无性系有丝分裂行为和细胞超微结构的变化

福橘（Citrus reticulata Blanco）茎尖经‘实践八号’育种卫星搭载后，经组织培养获得其再生植株，以此为材料，结合常规压片法与超薄切片技术，研究航天搭载对福橘当代无性系细胞有丝分裂行为及细胞超微结构的影响。     

利用SNP分子标记分析鉴定不同形态的化橘红(Citrus grandis var.tomentosa)单株

【目的】在对比化橘红不同品种形态特征的基础上,利用SNP(Single nucleotide polymorphism)分子标记分析其遗传差异,以期获得能准确鉴定化橘红不同品种的有效方法。【方法】依据叶片及果实茸毛特征,从不同果园采集9个代表性的化橘红单株,分别对果实及叶片进行测定。采用14个SNP位点,结合高分辨率熔解曲线分型(High resolution melting analysis,HRMA)技术对供试材料进行遗传多样性分析。【结果】同一采样地‘光青’单株8和9,仅叶片宽度存在显著差异,果实及叶片的其他特性差异不明显;同一采样地单株样4(‘正毛’)与样7(‘副毛’)的叶形指数及果形指数差异不显著,但叶片及果实的其他特性差异显著;同一果园不同品种的单株叶片及果实部分特性差异不显著。不同果园的同一品种单株,因栽培条件及管理等方面的差异,叶片及果实的部分特征存在显著差异。采用14个SNP位点可将9个化橘红样本有效区分,平均PIC(Polymorphism information content)值为0.29,所有SNP位点的平均自交系数为-0.09。【结论】仅凭形态特征很难区分化橘红不同品种的单株,而采用14个SNP位点能有效鉴定化橘红不同品种的单株。     

Biochemical and physiological changes during chlorophyll degradation in lime (Citrus aurantifolia Swingle cv. ‘Paan’)

Summary

Lime (Citrus aurantifolia Swingle cv. ‘Paan’) native to South East Asia, has a distinct flavour and quality characteristics. Maintenance of the green colour in the peel of lime is a desirable quality attribute during storage. Post-harvest chlorophyll degradation in lime was studied in fruit stored at room temperature (30°C) at a relative humidity (RH) of 70 – 85%. Within 7 d of storage, the total chlorophyll content decreased to 53.9% of its initial level. The highest chlorophyllase activity (1.68 units mg^–1 protein) was observed after 4 d of storage and declined thereafter. Peroxidase activity differed from chlorophyllase activity and increased significantly to 6.25 units mg^–1 protein after 9 d of storage, (i.e., at the late maturity stage). Respiration rate and 1-aminocyclopropene-1-carboxylic acid (ACC) oxidase activity did not significantly affect the chlorophyll degradation process in lime.The total nitrogen content of lime peel was inversely correlated with its chlorophyll content. A higher soluble protein content was observed in yellow peel than in mature green peel. A colour index chart was developed for maturity stages 1–4 in lime based on peel colour changes from mature green to full-yellow, and its correlation with chlorophyll content and fruit quality attributes.     

Introduction of Xa21, a Xanthomonas-resistance gene from rice,into ‘Hamlin’ sweet orange [Citrus sinensis (L.) Osbeck] using protoplast-GFP co-transformation or single plasmid transformation

Summary

Plasmid DNA (pARS108) containing the non-destructive selectable marker Green Fluorescent Protein (GFP) gene, and a plasmid containing a cDNA of the Xa21 gene from rice (pXa21-mtaq) were co-transformed into ‘Hamlin’ orange protoplasts using polyethylene glycol (PEG). Alternatively, plasmid DNA (pAO3), containing both genes (GFP and Xa21) was directly transformed into ‘Hamlin’ orange protoplasts. Over 1,000 transgenic plantlets were regenerated from approx. 80 independent transformation events. The transgenic plants showed normal growth and stable GFP expression over more than 2 years in the greenhouse. This is the first report of a large population of transgenic ‘Hamlin’ sweet orange plants containing one or more target gene(s), using a protoplast-GFP transformation system. Polymerase chain reaction (PCR) revealed the presence of the Xa21 cDNA and the GFP genes in all single plasmid transformed plants, and in 35% of the co-transformed plants. Southern blot analysis showed the integration of the cDNA into one-to-five different sites per plant.Western blot analysis showed the accumulation of the rice XA21 protein in the transgenic sweet orange plants. This is the first time that a gene from rice has been stably integrated and expressed in sweet orange plants. Using the protoplast-GFP transformation system, it is possible to avoid the use of Agrobacterium, antibiotic resistance genes, and destructive assay systems.     

枸头橙(Citrus aurantium)茎陷点病毒(Stem—pitting tristeza)的鉴定(初报)

在浙江黄岩桔区特有的优良柑桔砧木品种枸头橙上发现了茎陷点病,经指示植物生物鉴定结果有茎陷点—衰退病毒存在。