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.

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.

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.

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.

INTERSPECIFIC SOMATIC HYBRIDS IN ACTINIDIA

Protoplasts isolated from cotyledon-derived calli of Actinidia chinensis var. chinensis (2n = 2x=58) were fused by the PEG method with cotyledon-callus protoplasts of A. deliciosa var. deliciosa (2n = 6x = 174) or with mesophyll protoplasts of A. kolomikta (2n = 2x = 58), respectively. Randomly amplified polymorphic DNA (RAPD) markers and flow cytometry was used to confirm the occurrence of somatic hybrids. RAPD results with some primers surveyed indicated that one clone (A. chinensis + A. deliciosa) and four clones (A. chinensis + A. kolomikta) had RAPD banding patterns which combined the parental banding profiles. Ploidy levels of the (A. chinensis + A. deliciosa) clone were deduced as octoploid (2n = 8x), and the (A. chinensis + A. kolomikta) clones were tetraploid (2n = 4x), triploid (2n = 3x) or pentaploid (2n = 5x). The clones were confirmed as interspecific somatic hybrids in Actinidia.

Plantlets Regeneration via Somatic Embryogenesis from the Nucellus Tissues of Kinnow Mandarin (Citrus reticulata L.)

Studies were initiated to explore the role of nucellus tissues and growth regulators in plantlets regeneration via somatic embryogenesis of Kinnow mandarin [Citrus reticulata L. (Blanco)]. Nucellus tissues were cultured on MS medium supplemented with different concentrations and combinations of auxins, cytokinins and malt extract for primary callus induction. The best response for primary callus induction (90%) was obtained when MS medium was supplemented with 5 mg/l 2,4-D and 500 mg/l malt extract. Best results for embryogenic callus induction (80%) were obtained in C8 medium. The induction of somatic embryos was highest when MS medium was supplemented with 1 mg/l BAP and maturation of somatic embryos occurred when MS medium was supplemented with 5 mg/l 2,4-D and 1 mg/l BAP. Maximum plantlets were regenerated (92%) from the somatic embryos on half strength MS medium with no hormones. The plantlets were successfully acclimatized in different potting mixtures and highest survival rate (100%) was achieved in potting mixture containing sand and peat moss (2:1).

Selection of Tolerant of Some Citrus Hybrids F1 to Calcareous Stress and Identification Sexuall Individuals by SSR Marker

The research was conducted during two seasons 2018-2019 and 2019-2020, at the Scientific Research Center in Lattakia-Syria, where the cross between Sour orange, Cleopatra mandarin and Trifoliate orange. The first season was (Sour orange ♂ × Cleopatra mandarin ♀) and (Cleopatra mandarin ♂ × sour orange ♀) and (sour orange ♂ × trifoliate orange ♀), and the crossing in the second season was (Trifoliate orange ♂ × Sour orange ♀), and (Trifoliate orange ♂ × Cleopatra mandarin ♀). The plants of the first generation F1 were exposed to calcareous stress according to two methods. The first one for a long term (two years) method was grown in plots containing 15% and 35% Calcium Carbonate and the control free of Calcium Carbonate for the first cross, and the second one short term (two months) by watering method was with a solution of 1 mol and 2 mol of Calcium Carbonate for the second cross. The indicators of proline and chlorophyll A, B and T were measured in the first-generation individuals, once at the beginning of the experiment and a second time at the end of study. Seven SSR primers were used to identify sexual individuals, five primers were able to identify sexual F1 individuals are CSM13, CSM17, CSM147, CSM18 and TAA27, F1 individuals in the first season (mixture experiment) gave high values of chlorophyll and were tolerant to calcareous stress from crossing Cleopatra mandarin and Sour orange, where they did not show a deterioration in chlorophyll, but rather increased chlorophyll values, and the Trifoliate orange hybrids were sensitive to this stress, and in the watering experiment the F1 showed significant deterioration in Chlorophyll in all seedlings, and symptoms of Lime-induced chlorosis appeared on the more sensitive seedlings.

Isozyme analysis on progenes of somatic hybrids of Oryza sativa L.and O.officinalis

Some wild species of Oryza possess agronomi-cally useful traits such as disease/insect resis-tance and salt tolerance.However,the use ofwild Oryza species for rice improvement hasbeen hampered by the sexual incompatibility orsterility of hybrids obtained by conventionalmethods.We have used protoplast fusion as amethod to incorporate useful trait of wild O-ryza species into cultivated rice and~ obtainedsomatic hybrid plants between O.sativa and

Abortive Process of a Novel Rapeseed Cytoplasmic Male Sterility Line Derived from Somatic Hybrids Between Brassica napus and Sinapis alba

Somatic hybridization is performed to obtain significant cytoplasmic male sterility （CMS） lines, whose CMS genes are derived either from the transfer of sterile genes from the mitochondrial genome of donor parent to the counterpart of receptor or production of new sterile genes caused by mitochondrial genome recombination of the biparent during protoplast fusion. In this study, a novel male sterile line, SaNa-IA, was obtained from the somatic hybridization between Brassica napus and Sinapis alba. The normal anther development of the maintainer line, SaNa-IB, and the abortive process of SaNa-IA were described through phenotypic observations and microtome sections. The floral organ of the sterile line SaNa-IA was sterile with a shortened filament and deflated anther. No detectable pollen grains were found on the surface of the sterile anthers. Semi-thin sections indicated that SaNa-IA aborted in the pollen mother cell （PMC） stage when vacuolization of the tapetum and PMCs began. The tapetum radically elongated and became highly vacuolated, occupying the entire locule together with the vacuolated microspores. Therefore, SaNa-IA is different from other CMS lines, such as ogu CMS, pol CMS and nap CMS as shown by the abortive process of the anther.

Intermediate fertile Triticum aestivum (+) Agropyron elongatum somatic hybrids are generated by low doses of UV irradiation

We report the production and characterization of somatic hybrids between Triticum aestivum L. and Agropyron elongatum (Host) Nevishi (the synonym is Thinopyrum ponticum). Asymmetric protoplast fusion was performed between Agropyron elongatum protoplasts irradiated with a low UV dose and protoplasts of wheat taken from nonregenerable suspension cultures. More than 40 green plantlets were obtained from 15 regenerated clones and one of them produced seeds. The phenotypes of the hybrid plants and seeds were intermediate between wheat and Agropyron eIongatum. All of the regenerated calli and plants were verified as intergeneric hybrids on the basis of morphological observation and analysis of isozyme, cytological, 5SrDNA spacer sequences and random amplified polymorphic DNA (RAPD). RFLP analysis of the mitochondrial genome revealed evidence of random segregation and recombination of mtDNA.

Synthesis of a Wheat/Maize Hybrid CENH3 Gene, the Genetic Transformation of Wheat, Its Chromosomal Localization and Effects on Chromosome Behaviors in Wheat/Maize Somatic Hybrids

Centromere-specific histone H3 (CENH3) replaces the canonical histone H3 in nucleosomes of functional centromeres, and plays important roles in faithful chromosome segregation during cell division. CENH3 is also important in the recognition of alien centromeres and determines the accommodation or elimination of alien chromosomes in interspecific or intergenic hybridization. In this study, a maize full length CENH3 with a yellow fluorescent protein (YFP) tag at C-terminus (ZmCENH3-YFP) and a synthetic hybrid wmCENH3 with the N-terminus from wheat CENH3 and the histone fold domain (HFD) from maize tagged with a red fluorescent protein (RFP) at the C-terminus (wmCENH3-RFP) were transformed to wheat by biolistics transformation. Transgenic wheat plants with both ZmCNEH3-YFP and wmCENH3-RFP genes were identified by PCR. The expression of ZmCENH3-YFP was not observed, while the expression of wmCENH3-RFP could be detected by RT-PCR, direct fluorescence microscopy, and immunostaining with anti-RFP antibody. The expressed wmCENH3-RFP was localized to nuclei as dotted patterns, indicating its targeting to wheat centromeres. Somatic hybridization was performed between wmCENH3-RFP transgenic wheat and transgenic maize that expressed a ZmCENH3-YFP gene to investigate chromosome behaviors in somatic hybrids. Cytological and FISH analyses of somatic hybrid cells showed the formation of micronuclei and lagging chromatin in both somatic hybridizations with or without the wmCENH3-RFP transgene, indicating that ectopically expressed wmCENH3 could not overcome chromosome elimination in wheat/maize somatic hybrids. Immunostaining of wmCENH3-RFP and ZmCENH3-YFP in early stage somatic hybrid cells indicated that both wmCENH3-RFP and ZmCENH3-YFP proteins were expressed, but their binding patterns changed from the commonly observed dotted patterns to diffused ones, suggesting that the inactivation of CENH3 might be a factor for chromosome elimination in wheat/maize somatic hybridization.

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 .

Expression of Ferric Chelate Reductase Gene in Citrus junos and Poncirus trifoliataTissues

It has been hypothesized that under iron stress high ferric chelate reductase (FCR) activity in the absorptive root of plants tolerant to iron_deficiency will be induced and result in subsequent Fe 2+ transport across the plasmalemma. The activity of FCR and expression of FCR gene (FRO2) in Citrus junos Sieb. ex Tanaka tolerant to iron_deficiency and Poncirus trifoliata (L.) Raf. susceptible to iron_deficiency were determined to elucidate the physiological difference which causes the different tolerance of the two citrus rootstocks to iron stress. The activity of FCR was detectable in excised roots and was stimulated about 20_times in C. junos and only about 3_times in P. trifoliata under iron deficiency for four weeks. The FRO2 of Arabidopsis was used as a probe, the tissue print technique was used to ascertain the expression of the FCR gene in C. junos and P. trifoliata under iron stress. High_level transcripts were observed in the absorptive root, young green stem as well as new leaf of C. junos under iron stress for two weeks, and the transcripts were accumulated only slightly in P. trifoliata at the same time. The results showed that the obvious increase of FCR activity was an important reason for the tolerance of C. junos to iron_deficiency, and the regulation of FCR activity seemed to be at the transcriptional level, and the expression of FRO2 occurred in the root, stem and leaf.

两类柑桔杂种砧木对脐橙(Citrus sinensis Osbeck)幼树生长和叶片糖营养含量年变化的影响

以体细胞杂种红桔+枳和红桔+粗柠檬、有性杂种Troyer枳橙和Swingle枳柚作砧木的耐湿脐橙(CitrussinensisOsbeck)2年生嫁接苗为试材,通过盆栽试验研究了砧木对苗木生长和叶片糖营养含量年变化动态的影响。结果表明:红桔+枳的生长势和花量明显地强于和大于其它砧木,红桔+粗柠檬的生长和花量表现介于2种有性杂种之间。叶绿素a/b和类胡萝卜素含量在不同砧木间无显著差异,而不同砧木显著或极显著地影响叶绿素含量各指标。砧木影响叶片可溶性糖和淀粉含量年变化动态的基本特征。在休眠期,红桔+枳的可溶性糖和淀粉含量都高于2种有性杂种砧木,而红桔+粗柠檬则反之;在生长期,红桔+粗柠檬的可溶性糖和淀粉含量表现出高于有性杂种砧木的趋势,而红桔+枳则介于2种有性杂种砧木之间。同时还重点讨论了2种体细胞杂种砧木的利用价值。

Identification and Expression Analysis of Salinity-induced Genes in Rangpur lime(Citrus limonia)

Among the economically important horticultural crops,citrus is one of the most vulnerable crops to soil salinity.Rangpur lime is more tolerant to high soil salinity than other commonly used citrus rootstocks.However,the molecular mechanism involved in salinity tolerance has not been explored in Rangpur lime.In this study,a cDNA library was constructed from leaves of Rangpur lime watered for 30 days with 100mmol·L^−1 NaCl in tap water or tap water using suppression subtractive hybridization(SSH).Two hundred cDNA clones randomly selected from this library were sequenced,and an average of 569 bp was obtained from the majority of clones.Fifty-six salinity-induced genes,showing 2-to 6-fold increases in their expression levels,were identified by macroarray hybridizations.Salinity-induced genes were associated with transcription(5.36%),stress response and signaling(21.43%),metabolism(16.07%),transport facilitation(10.71%),photosynthesis(10.71%),protein synthesis and fate(19.64%)and cellular biogenesis(3.57%).Stress response-and signaling-related genes constituted the largest functional group,associated with the production of compatible solutes,regulation of stomatal movement,lipid modification,oxidative stress,antioxidant defense,and stress signaling.Expression levels of 13 identified genes were induced 1.8-to 3.1-fold,which were validated in salt-treated and untreated Rangpur lime.The functions of salinity-induced,stress-related genes and their potential roles in salinity tolerance in Rangpur lime were discussed.

Charaterization of Citrus Hybrid ＂Huangguogan＂ through the Combination of Morphological and Molecular Markers

Huangguogan, an obvious Citrus hybrid, is suitable for transportation and export and ripens in March or April. Because of late season, it may playa significant role in fruit market. However, its origin is still unconfirmed. The aim of this study was to clarify the possible parentage of Huangguogan via the combination of morphological and molecular markers including simple sequence repeat （SSR） and chloroplast simple sequence repeat （cpSSR）. Analysis of morphological traits including leaf stalk length, phylliform index and fruit shape index indicated that Huangguogan had similarities in morphology with Sweet orange. The SSR Cluster Analysis showed that Huangguogan was clustered together with Hongju tangerine and revealed -80% genetic similarity. They illustrated a close genetic distance between Huangguogan and Hongju tangerine. In addition, the bands of2 polymorphic cpSSR were identical in Huangguoggan and Sweet orange. Consequently, it is likely that its female parentage was the sweet orange （Citrus sinensis （L.） Osbeck） and its male parentage was the tangerine （Citrus reticulata Blanco）.

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。

Mitochondrial Genome of Callus Protoplast Has a Role in Mesophyll Protoplast Regeneration in Citrus: Evidence From Transgenic GFP Somatic Homo-Fusion

Protoplast fusion has great potential in citrus improvement. Although citrus mesophyll protoplasts usually cannot divide and regenerate,symmetric protoplast fusion of embryogenic callus protoplast + mesophyll protoplast sometimes results in the regeneration of mesophyllparent-type cybrids. It suggested that mitochondrial DNA(mt DNA) from protoplasts of embryogenic callus parent plays an important role in stimulating division and regeneration of mesophyll protoplasts. Herein, somatic fusion was conducted via electrofusion between callus protoplasts isolated from Valencia orange [Citrus sinensis(L.) Osbeck] cell suspension cultures and transgenic GFP-tagged mesophyll protoplasts from the same genotype, i.e. transgenic Valencia orange plants containing the green fluorescent protein(GFP) gene, in an effort to elucidate whether mt DNA of callus line could stimulate the division and regeneration of mesophyll protoplasts from the same genotype. Two embryoids and one plantlet with GFP expression were successfully obtained and subsequent ploidy analysis by flow cytometry indicated that they were all diploids. The regenerated diploid embryoids and plantlet with GFP expression could be considered as ‘cybrids' with mt DNA from the callus protoplasts of Valencia orange. The result indicated that citrus mesophyll-parent-type cybrid regeneration needed the stimulation of mt DNA from protoplasts of embryogenic callus parent regardless of their origin either from another genotype or the same genotype as the mesophyll parent.

RAPD-based genetic analysis of offsprings from the sexual cross using allotetraploid citrus somatic hybrid as pollen parent

Thirty-one polymorphic decamer primers were selected to genotype 92 progenies from the cross be- tween Yiben No.4, a monoembryonic diploid F1 hybrid of Citrus reticulata Blanco cv Huanongbendizao tangerine and C. ichangensis Swingle, and [Hamlin sweet orange + Rough lemon], an allotetraploid somatic hybrid of Citrus sinensis Osbeck cv. Hamlin and C. jambhiri Lush cv. Rough Lemon. χ2 (Chi-square) analysis of RAPD markers in the progenies indicated they were randomly transmitted from the four donor parents, without significant difference between the diploids and triploids. However, these progenies were clustered into three major groups using dendrogram constructed by UPGMA, skewed to three parents in certain degrees, 15 (13 triploids and 2 diploids) to Hamlin, 16 (9 and 7) to Yiben No. 4, and 61 (57 and 4) to [Hamlin sweet orange + Rough Lemon] from which genomic contribu- tion was predominant in progenies, respectively.

Regeneration of intergeneric somatic hybrids by protoplast fusion between Onobrychis viciaefolia and Medicago sativa

Protoplast fusion was induced between sainfoin and alfalfa by an improved polyethyleneglycol (PEG) method. The intergeneric somatic calluses were selected based on complementation of hydroxyproline-resistance of sainfoin and hormone autonomy growth of alfalfa transformation cell line. 17 somatic hybrid plantlets were regenerat-ed. PEG could induce the tight agglutination of protoplasts. During diluting and washing process, cyclization of the linked membrane and formation of vesicle-like structures were observed, resulting in protoplast fusion. 5%-10% glycerol supplemented in the fusion inducing solution markedly increased the frequency of heterogeneous fusion. Better fusion results were obtained when mixed protoplast suspension was dripped in petri dishes in which PEG solution was previously placed. Chromosome number of regenerated hybrid buds varied from 30 to 60. The genome of hybrids in-cluded the small chromosome from sainfoin and two chromosomes with two clear constrictions from alfalfa. The hybrid-ity of obtained hybrid calluses was confirmed by their isozyme banding patterns and their nopaline synthetase activity.