Citrus Genotypes Research Articles

Analysis of genetic relationship between ‘Bakraee’ (Citrus sp.) and some known Citrus genotypes through SSR and PCR-RFLP markers

Phylogenic relationships and genetic diversity in citrus are considered to be important in clarifying their genetic relationships, germplasm characterization and the registration of new varieties. The ‘Bakraee’ (Citrus sp.) is an unknown natural biotype which is being used widely as rootstock as well as edible fruit in some regions of Iran. In the present research work, phylogenic relationships among ‘Bakraee’ and some commercially important citrus varieties were investigated through SSR and PCR-RFLP molecular markers. Two UPGMA dendrograms were generated using SSR and PCR-RFLP data. SSR data showed 0.87 and 0.65 genetic similarities between ‘Bakraee’ and rough lemon (Citrus jambhiri Lush.) and sweet lime (Citrus limettioides Tan.), respectively. PCR-RFLP analysis demonstrated that all of the amplified fragments in ‘Bakraee’ are also present in rough lemon. Our study confirmed a close relationship between ‘Bakraee’ and rough lemon and support the possibility of rough lemon as maternal parent in the hybrid origin of ‘Bakraee’.

A nuclear phylogenetic analysis: SNPs, indels and SSRs deliver new insights into the relationships in the ‘true citrus fruit trees’ group (Citrinae, Rutaceae) and the origin of cultivated species

Despite differences in morphology, the genera representing 'true citrus fruit trees' are sexually compatible, and their phylogenetic relationships remain unclear. Most of the important commercial 'species' of Citrus are believed to be of interspecific origin. By studying polymorphisms of 27 nuclear genes, the average molecular differentiation between species was estimated and some phylogenetic relationships between 'true citrus fruit trees' were clarified. Sanger sequencing of PCR-amplified fragments from 18 genes involved in metabolite biosynthesis pathways and nine putative genes for salt tolerance was performed for 45 genotypes of Citrus and relatives of Citrus to mine single nucleotide polymorphisms (SNPs) and indel polymorphisms. Fifty nuclear simple sequence repeats (SSRs) were also analysed. A total of 16 238 kb of DNA was sequenced for each genotype, and 1097 single nucleotide polymorphisms (SNPs) and 50 indels were identified. These polymorphisms were more valuable than SSRs for inter-taxon differentiation. Nuclear phylogenetic analysis revealed that Citrus reticulata and Fortunella form a cluster that is differentiated from the clade that includes three other basic taxa of cultivated citrus (C. maxima, C. medica and C. micrantha). These results confirm the taxonomic subdivision between the subgenera Metacitrus and Archicitrus. A few genes displayed positive selection patterns within or between species, but most of them displayed neutral patterns. The phylogenetic inheritance patterns of the analysed genes were inferred for commercial Citrus spp. Numerous molecular polymorphisms (SNPs and indels), which are potentially useful for the analysis of interspecific genetic structures, have been identified. The nuclear phylogenetic network for Citrus and its sexually compatible relatives was consistent with the geographical origins of these genera. The positive selection observed for a few genes will help further works to analyse the molecular basis of the variability of the associated traits. This study presents new insights into the origin of C. sinensis.

Volatile Profiles of Young Leaves of Rutaceae spp. Varying In Susceptibility to the Asian Citrus Psyllid (Hemiptera: Psyllidae)

require the newly expand-ing terminal leaves (flush) of citrus and related species in order to complete their development (Shavankar et al. 2000; Michaud 2004). The study presented here examined host plant vola-tiles from uninfested flush and young leaves of citrus genotypes that were reported to vary in susceptibility as measured by colonization by

High Resolution Melting Analysis Is a More Sensitive and Effective Alternative to Gel-Based Platforms in Analysis of SSR – An Example in Citrus

High resolution melting curve analysis (HRM) has been used as an efficient, accurate and cost-effective tool to detect single nucleotide polymorphisms (SNPs) or insertions or deletions (INDELs). However, its efficiency, accuracy and applicability to discriminate microsatellite polymorphism have not been extensively assessed. The traditional protocols used for SSR genotyping include PCR amplification of the DNA fragment and the separation of the fragments on electrophoresis-based platform. However, post-PCR handling processes are laborious and costly. Furthermore, SNPs present in the sequences flanking repeat motif cannot be detected by polyacrylamide-gel-electrophoresis based methods. In the present study, we compared the discriminating power of HRM with the traditional electrophoresis-based methods and provided a panel of primers for HRM genotyping in Citrus. The results showed that sixteen SSR markers produced distinct polymorphic melting curves among the Citrus spp investigated through HRM analysis. Among those, 10 showed more genotypes by HRM analysis than capillary electrophoresis owing to the presence of SNPs in the amplicons. For the SSR markers without SNPs present in the flanking region, HRM also gave distinct melting curves which detected same genotypes as were shown in capillary electrophoresis (CE) analysis. Moreover, HRM analysis allowed the discrimination of most of the 15 citrus genotypes and the resulting genetic distance analysis clustered them into three main branches. In conclusion, it has been approved that HRM is not only an efficient and cost-effective alternative of electrophoresis-based method for SSR markers, but also a method to uncover more polymorphisms contributed by SNPs present in SSRs. It was therefore suggested that the panel of SSR markers could be used in a variety of applications in the citrus biodiversity and breeding programs using HRM analysis. Furthermore, we speculate that the HRM analysis can be employed to analyse SSR markers in a wide range of applications in all other species.

Physiological analysis of salt stress behaviour of citrus species and genera: Low chloride accumulation as an indicator of salt tolerance

Tolerant citrus rootstocks are defined as Cl− excluders. However, little is known about the salt tolerance of cultivars used as scions, particularly the tolerance of monoembryonic citrus genotypes. To enhance the genetic resources for generating improved hybrid rootstocks, the evaluation of large samples of citrus species, including both monoembryonic and polyembryonic genotypes, is necessary. In this study, 12 citrus genotypes representing the major Citrus species and all the three genera of the Rutaceae family were subjected to moderate salt stress (75mM) for 12weeks to characterise their physiological response to salt stress. Various symptoms and physiological parameters were evaluated to characterise their salt sensitivity. These included plant growth (stem diameter), leaf chlorophyll content, leaf flavonoid content, maximum quantum yield of PSII [(Fm−F0)/Fm)], net photosynthesis, stomatal conductance and leaf Na+ and Cl− contents. The results clearly demonstrated that the most salt sensitive genotypes accumulated high concentrations of Na+ and Cl− and maintained a fair growth and photosynthetic rate. By contrast, salt-tolerant genotypes accumulated less Na+ and Cl− and decreased their growth and gas exchange. Poncire commun citron and Marumi kumquat were the most sensitive species, while mandarins, pummelo and Australian sour orange were the most tolerant species. Among the genotypes, Engedi pummelo presented a specific trait for salt tolerance that has not been previously reported. Taken together, the results suggest that low leaf chloride content can be used as an indicator of salt stress tolerance in citrus genotypes. Exploitation of this indicator will enable the improved evaluation of citrus genetic resources and should lead to the identification of new sources of tolerance for rootstock breeding.

Effects of arbuscular mycorrhizal fungi on resistance to Phytophthora parasitica of citrus seedlings and on growth of Thai honey tangerine scions on citrus rootstocks

Thai honey tangerine (Sainamphueng tangerine) is generally grown by grafting on rootstocks of another variety of tangerine or citrus species which may differ in their reaction to beneficial and pathogenic soil organisms. The objectives of this study were to evaluate responses to arbuscular mycorrhizal (AM) fungi and Phytophthora parasitica of different citrus genotypes and the effect of AM fungi on the growth of scions of Thai honey tangerine grafted on different citrus rootstocks including Cleopatra tangerine, lime, pomelo, Swingle citrumelo and Troyer citrange. Significant differences were found among citrus species in the percentage of root colonization by the AM fungi and in the severity of root rot disease when inoculated with P. parasitica alone. Thai honey tangerine was most susceptible and Cleopatra tangerine was most resistant to P. parasitica . Inoculation with AM fungi could reduce disease severity of all the citrus plants from P. parasitica . AM fungi enhanced the growth of seedling to the greatest extent in lime. Variation in the response to AM fungi was found among the scions of Thai honey tangerine on different citrus rootstocks. The scion of Thai honey tangerine grew best on the lime rootstock inoculated with AM fungi. Key words: Phytophthora parasitica , citrus, rootstock, arbuscular mycorrhizal (AM) fungi, root rot.

Extensive citrus triploid hybrid production by 2x × 4x sexual hybridizations and parent-effect on the length of the juvenile phase

The citrus fresh market demands the production of seedless citrus fruits, as seedy fruits are not accepted by consumers. The recovery of triploid plants has proven to be the most promising approach to achieve this goal, since triploids have very low fertility, are generally seedless and do not induce seeds in other cultivars by cross pollination. Triploid plants can be recovered by 2x×4x sexual hybridization. In this work, we present an effective methodology to recover triploid plants from 2x×4x hybridizations based on in vitro embryo rescue, ploidy level analysis by flow cytometry and genetic origin of triploid plants. The pollen viability of diploid and tetraploid citrus genotypes was analyzed by comparing the pollen germination rate in vitro. The pollen viability of tetraploid (doubled-diploid) genotypes is generally reduced but sufficient for successful pollination. Triploid embryos were identified in normal and undeveloped seeds that did not germinate under greenhouse conditions. The influence of parents and environmental conditions on obtaining triploid plants was analyzed and a strong interaction was noted between the parents and environmental conditions. The parental effect on the length of the juvenile phase was also demonstrated through observations of a large number of progeny over the last 15 years. The juvenile phase length of the triploid hybrids obtained with 'Fortune' mandarin as female parent and tetraploid 'Orlando' tangelo as male parent was shorter than the juvenile phase obtained with a clementine as female parent and tetraploids of 'Nova', 'W. Leaf' and 'Pineapple' male parents. Effective methodology to recover citrus triploid plants from 2x×4x sexual hybridizations and the parental effect on the length of the juvenile phase.

Comprehending crystalline β-carotene accumulation by comparing engineered cell models and the natural carotenoid-rich system of citrus

Genetic manipulation of carotenoid biosynthesis has become a recent focus for the alleviation of vitamin A deficiency. However, the genetically modified phenotypes often challenge the expectation, suggesting the incomplete comprehension of carotenogenesis. Here, embryogenic calli were engineered from four citrus genotypes as engineered cell models (ECMs) by over-expressing a bacterial phytoene synthase gene (CrtB). Ripe flavedos (the coloured outer layer of citrus fruits), which exhibit diverse natural carotenoid patterns, were offered as a comparative system to the ECMs. In the ECMs, carotenoid patterns showed diversity depending on the genotypes and produced additional carotenoids, such as lycopene, that were absent from the wild-type lines. Especially in the ECMs from dark-grown culture, there emerged a favoured β,β-pathway characterized by a striking accumulation of β-carotene, which was dramatically different from those in the wild-type calli and ripe flavedos. Unlike flavedos that contained a typical chromoplast development, the ECMs sequestered most carotenoids in the amyloplasts in crystal form, which led the amyloplast morphology to show a chromoplast-like profile. Transcriptional analysis revealed a markedly flavedo-specific expression of the β-carotene hydroxylase gene (HYD), which was suppressed in the calli. Co-expression of CrtB and HYD in the ECMs confirmed that HYD predominantly mediated the preferred carotenoid patterns between the ECMs and flavedos, and also revealed that the carotenoid crystals in the ECMs were mainly composed of β-carotene. In addition, a model is proposed to interpret the common appearance of a favoured β,β-pathway and the likelihood of carotenoid degradation potentially mediated by photo-oxidation and vacuolar phagocytosis in the ECMs is discussed.

Generation, functional analysis and utility of Citrus grandis EST from a flower-derived cDNA library

Pummelo (Citrus grandis) is one of the most important species found in the genus Citrus and one of the ancestors of sweet oranges. We used flower buds at different developmental stages to construct the first cDNA library for this species. A total of 3,758 EST sequences were generated from the cDNA library and clustered into 2,228 unigenes, comprising 451 contigs and 1,777 singletons. Among these unigene sequences, 1,266 have significant homology to the non-redundant protein database, from which 891 were assigned to one or more gene ontology categories. Functional categorization of the annotated unigenes showed that 760 genes were involved in molecular function, 1,189 in biological processes and 1,154 in cellular component categorization. Homologs of genes regulating many aspects of flower development were also identified, including those for organ development, cell-cycle control and cell and tissue differentiation. The majority of these genes (e.g., embryo relatives, YABBY-like, MAD Box, SKP-like and SRNAs) are the first representatives in Citrus, providing an opportunity to explore the cause of self incompatibility and embryo development in Citrus. Patterns of transcript accumulation were characterized by real-time qPCR for 13 of these genes. Many potential molecular markers were also identified in this EST data set; 212 Simple Sequences Repeats (SSRs), 717 transposon elements and 115 candidate single nucleotide polymorphisms (SNPs) were found. An assessment of a set of 212 SSR primer pairs on 16 citrus genotypes showed polymorphism with 122 (57.82%) markers. Similarly, a set of eight contigs were used to confirm in silico predicated SNPs in a set of five genotypes using wet lab experiments, three contigs were generated as scorable and sequenceable amplicons and no PCR amplicons were obtained from five contigs. The outcome of this study could aid in the discovery of genes involved in reproductive developments. Identified candidate genes can be experimentally tested for their functions in various important processes. SSR, SNP and transposon element-containing data sets may facilitate marker development and can be used for citrus molecular breeding, linkage map construction, evolutionary, phylogenetic and population genetic studies.

Polyembryony and seedling emergence traits in apomictic citrus

Highest polyembryony, range of embryo, clutch size and frequency of quintuplets, sextuplets and heptuplets were observed in Citrus jambhiri. However, quadruplet and triplet embryos were more frequent regardless the genotypes. Embryos were mainly arranged at micropylar region which were characterized by asynchronous developments. Moreover, their biomass partitioning significantly varied with morphotypes. C. jambhiri also had highest CVE and uncertainty of emergence. However, it had minimal emergence time (34.4±1.8 days) and synchronization index. Additionally, it had highest frequency of triplet and quadruplet seedlings and maximal embryo to seedling conversion rate. Synchronization index showed significantly negative correlation with the number of seedlings and frequency of multiple seedlings. Emergence rate and time were not influenced with the intensity of embryony in citrus genotypes. Moreover staggered seedling emergence gives an indication of bet-hedging strategy which helps in better seedling survival in a temporally variable environment.

Reference genes for accurate transcript normalization in citrus genotypes under different experimental conditions.

Real-time reverse transcription PCR (RT-qPCR) has emerged as an accurate and widely used technique for expression profiling of selected genes. However, obtaining reliable measurements depends on the selection of appropriate reference genes for gene expression normalization. The aim of this work was to assess the expression stability of 15 candidate genes to determine which set of reference genes is best suited for transcript normalization in citrus in different tissues and organs and leaves challenged with five pathogens (Alternaria alternata, Phytophthora parasitica, Xylella fastidiosa and Candidatus Liberibacter asiaticus). We tested traditional genes used for transcript normalization in citrus and orthologs of Arabidopsis thaliana genes described as superior reference genes based on transcriptome data. geNorm and NormFinder algorithms were used to find the best reference genes to normalize all samples and conditions tested. Additionally, each biotic stress was individually analyzed by geNorm. In general, FBOX (encoding a member of the F-box family) and GAPC2 (GAPDH) was the most stable candidate gene set assessed under the different conditions and subsets tested, while CYP (cyclophilin), TUB (tubulin) and CtP (cathepsin) were the least stably expressed genes found. Validation of the best suitable reference genes for normalizing the expression level of the WRKY70 transcription factor in leaves infected with Candidatus Liberibacter asiaticus showed that arbitrary use of reference genes without previous testing could lead to misinterpretation of data. Our results revealed FBOX, SAND (a SAND family protein), GAPC2 and UPL7 (ubiquitin protein ligase 7) to be superior reference genes, and we recommend their use in studies of gene expression in citrus species and relatives. This work constitutes the first systematic analysis for the selection of superior reference genes for transcript normalization in different citrus organs and under biotic stress.

Inhibition of foodborne pathogen bacteria by essential oils extracted from citrus fruits cultivated in Sicily

The antagonistic activity of the essential oils (EOs) extracted by hydrodistillation from the fruit peel of several citrus genotypes (pummelo, grapefruit, orange, kumquat, mandarin and lemon) was evaluated against foodborne pathogen bacteria (43 strains of Listeria monocytogenes, 35 strains of Staphylococcus aureus and 14 strains of Salmonella enterica). Five commercial EOs were used for comparison. Most of the EOs were more effective against the Gram-positive bacteria rather than Salmonella. EOs of lemon genotypes 14 and 15 showed the best results in terms of number of strains inhibited and width of the inhibition zone. The most susceptible strain of each species (L. monocytogenes 133, St. aureus 473 and Salmonella Newport 50404) was used as indicator for the evaluation of the minimum inhibition concentration (MIC) of the EOs 14 and 15. The last two EOs, EO 16 (showing the weakest inhibition power among lemon EOs), and the commercial EO L (lemon) were analysed for their chemical composition. A total of 30 major compounds were identified by gas chromatography/mass spectrometry (GC/MS) and the oxygenated monoterpenes were suggested to be implicated in the process of bacterial inhibition by citrus EOs.

Simple Sequence Repeat (SSR) profiling of cultivated Limau Madu (Citrus reticulataBlanco) in Malaysia

Introduction . In Malaysia, Limau Madu (Citrus reticulata Blanco; Citrus suhuiensis Hort. ex Tanaka) is commercially cultivated for local consumption. It is a loose-peel mandarin with fruits that are spherical in shape with shiny green or greenish yellow peel. In Malaysia, Limau Madu is vegetatively propagated, thus it is presumed to exhibit minimal variability. In our study, the genetic variability and genetic relatedness among cultivated samples collected from different states in Malaysia were assessed using Simple Sequence Repeat (SSR) primers. Materials and methods . Thirty pairs of SSR primers derived from Citrus unshiu were screened and 22 SSR primer pairs were utilized to assess genetic variability and relatedness among 118 cultivated samples. Results and discussion . The percentage of SSR transferability from C. unshiu to C. reticulata (Limau Madu) was 73.33%, which indicated that the primer sequences flanking simple sequence repeats were conserved among these Citrus species. Most SSR loci revealed a large excess of heterozygotes. In our study, low allelic diversity was shown, with an average of 5.227 alleles per locus. Polymorphic information content (PIC) ranged from 0.048 to 0.674. Based on UPGMA clustering analysis, four groups were identified from these citrus genotypes with a mean genetic distance of 0.170. Low genetic variability within species was probably due to vegetative propagation or inability to detect differences among samples using these primers.

Applications of Plant Growth Regulators to Container-grown Citrus Trees Affect the Biology and Behavior of the Asian Citrus Psyllid

Asian citrus psyllid [ACP (Diaphorina citri)] is an important pest of citrus (Citrus sp.) in many citrus-growing regions of the world because of its status as the vector of huanglongbing disease [HLB (citrus greening)]. There are currently no HLB-resistant citrus genotypes and no proven treatments for the disease; thus, vector control through the use of frequent prophylactic pesticide applications is key to managing the spread of this disease. However, this practice is unsustainable and other means of altering ACP biology or reducing populations are needed. To this end, six plant growth regulators (PGRs) were tested to determine their effect on citrus tree vegetative growth and the subsequent impact on the biology of ACP. In greenhouse and growth chamber experiments, ACP reared on trees treated with prohexadione calcium and mefluidide exhibited significant reductions in both fecundity and survivorship, whereas uniconazole affected only fecundity and paclobutrazol affected only survivorship. No significant effects of PGRs on adult ACP weight were observed except on uniconazole-treated trees. No eggs were laid on dikegulac sodium-treated trees; however, this was likely the result of severe phytotoxicity rather than a true PGR effect. Oviposition rate was lower on all the PGR-treated trees, except chlormequat chloride under greenhouse conditions, compared with untreated control trees. In general, oviposition was delayed on PGR-treated trees compared with untreated controls. The observed changes in ACP biology and behavior after PGR treatment were not the result of a reduction in the number of suitable oviposition sites (i.e., growth reduction) or toxicity of the PGRs to ACP, suggesting there were PGR-induced plant biochemical changes that altered host plant quality. Leaf nutrient analyses and photosynthesis indicated that there were no correlative changes in plant nutrient status or carbon assimilation that led to the changes in ACP behavior, although it is possible that phloem-specific nutrient or carbohydrate changes could have occurred that were not detected in our whole-leaf analyses. These results support previous studies in which the fitness of various insect species has been affected by PGR applications, but more research is needed to understand the changes in plant chemistry that are responsible.

Pollen competition as a reproductive isolation barrier represses transgene flow between compatible and co-flowering citrus genotypes.

Background/ObjectiveDespite potential benefits granted by genetically modified (GM) fruit trees, their release and commercialization raises concerns about their potential environmental impact, and the transfer via pollen of transgenes to cross-compatible cultivars is deemed to be the greatest source for environmental exposure. Information compiled from field trials on GM trees is essential to propose measures to minimize the transgene dispersal. We have conducted a field trial of seven consecutive years to investigate the maximum frequency of pollen-mediated crop-to-crop transgene flow in a citrus orchard, and its relation to the genetic, phenological and environmental factors involved.Methodology/Principal FindingsThree different citrus genotypes carrying the uidA (GUS) tracer marker gene (pollen donors) and a non-GM self-incompatible contiguous citrus genotype (recipient) were used in conditions allowing natural entomophilous pollination to occur. The examination of 603 to 2990 seeds per year showed unexpectedly low frequencies (0.17–2.86%) of transgene flow. Paternity analyses of the progeny of subsets of recipient plants using 10 microsatellite (SSR) loci demonstrated a higher mating competence of trees from another non-GM pollen source population that greatly limited the mating chance of the contiguous cross-compatible and flowering-synchronized transgenic pollen source. This mating superiority could be explained by a much higher pollen competition capacity of the non-GM genotypes, as was confirmed through mixed-hand pollinations.Conclusions/SignificancePollen competition strongly contributed to transgene confinement. Based on this finding, suitable isolation measures are proposed for the first time to prevent transgene outflow between contiguous plantings of citrus types that may be extendible to other entomophilous transgenic fruit tree species.

Abundance of Citrus Leafminer Larvae on Citrus and Citrus-related Germplasm

The citrus leafminer, Phyllocnistis citrella Stainton (Lepidoptera: Gracillariidae), is a key pest in most citrus-growing regions worldwide. Adult citrus leafminers oviposit primarily on young elongating flush of Citrus as well as other Rutaceae and some ornamental plants. Larvae feed on the epidermal cell layer of developing leaves and injury to leaves provides a pathway for infection by the bacterium Xanthomonas citri subsp. citri (Hasse), the causal agent of Asiatic citrus canker. In this study, we quantified abundance of citrus leafminer larvae on progeny of 87 seed parent genotypes of Citrus and Citrus relatives (family Rutaceae) in the field in East–central Florida to identify those that have low abundance of leafminers. Progeny from the 87 parent genotypes varied in abundance of the leafminer. Progeny of 15 parent genotypes had a high mean abundance of more than six leafminers per flush shoot. All but one of these genotypes were in the Citrus genus. Progeny of 16 parent genotypes had zero, or nearly zero, leafminers, but none were from the Citrus genus. However, many of these 16 genotypes were from genera closely related to true citrus (subtribe Citrinae) and are sexually compatible with Citrus. Progeny of two parent genotypes in the subfamily Toddalioideae and Glycosmis pentaphylla (Retz.) Corr. also had a low abundance of leafminer. Glycosmis pentaphylla also is a poor host for the Asian citrus psyllid, Diaphorina citri Kuwayama, and has biochemical resistance to the citrus weevil, Diaprepes abbreviatus (L.), so this genotype as well as others identified as poor hosts for the leafminer may prove useful in breeding programs aimed at reducing the abundance of multiple insect pests on citrus.

Resistance of citrus genotypes to Phyllocnitis citrella Stainton (Lepidoptera: Gracillariidae)

The development and reproduction of the citrus leafminer (CLM), Phyllocnistis citrella Stainton, were evaluated in six citrus genotypes in order to identify genotypes with resistance traits that could be applied in a program for the development of citrus varieties resistant to the citrus leafminer. Tests were conducted under controlled laboratory conditions (25 ± 1ºC, 70 ± 10% RH, and 14h photophase). Seedlings of each genotype tested were infested with eggs obtained from a stock colony of CLM maintained on 'Cravo' lemon (Citrus limonia L. Osbeck), and the duration and survival of the eggs, larval and pupal stages, pupal size and weight, fecundity and longevity of adults, and sex ratio were evaluated. No influence was observed on the duration and survival of eggs, larvae and pupae of P. citrella. However, pupae obtained in the hybrid C x R(4) were significantly smaller and lighter than pupae from the remaining treatments. Adult females from the hybrids C x R(4) and C x R(315) were the least fecund. However, the lowest value for the corrected reproductive potential (CRP) was recorded in the hybrid C x R(315), suggesting that this genotype is the least favorable for the development and reproduction of CLM. On the other hand, the highest CRP value obtained in the 'Rugoso' lemon confirms the susceptibility of this genotype, indicating it as the most suitable for CLM.

Light effect on carotenoids production and expression of carotenogenesis genes in citrus callus of four genotypes

Due to their theoretically identical genetic background, citrus callus and other plant tissues may share some mechanisms in the regulation of carotenogenesis. Thus, in order to gain further information on light regulation of carotenoids biosynthesis in citrus, the carotenoids and expression profiles of carotenogenesis in calluses of four citrus genotypes treated with light or dark were investigated. As a response to white light, results showed that carotenoids biosynthesis in callus of Red Marsh grapefruit (Citrus paradisi Macf.) was hampered, whereas callus of Tarocco blood orange (C. sinensis (L.) Osbeck) was sensitive to light by accumulating over 55% more carotenoids on average. Among the detected carotenoids, the biosynthesis of carotenes seemed to be more sensitive than that of xanthophylls. Expression profiles of eight carotenogenesis genes encoding phytoene synthase (PSY), phytoene desaturase (PDS), ζ-carotene desaturase (ZDS), carotenoids isomerase (CRTISO) etc. were investigated. Results revealed that PSY was up regulated in calluses of two sweet oranges, and down regulated in callus of Murcott tangor (C. reticulata × C. sinensis). Biochemical data in the three genotypes emphasized the PSY as a rate-limiting gene in the carotenogenesis. However, in the callus of Red Marsh grapefruit, PDS and ZDS might be the rate-limiting genes, and their transcripts were apparently inhibited by light, led to significant decreases in contents of β-carotene and total carotenoids irrelevant to transcription levels of PSY. Expression of CRTISO was light-induced, especially in the callus of Murcott tangor, and increased by nearly 12-fold. In conclusion, light regulates the expression of several carotenogenesis genes in citrus callus, but may not necessarily result in significant changes in carotenoids production.

Selection of reference genes for quantitative real-time RT-PCR analysis in citrus

Quantitative real-time reverse transcription polymerase chain reaction (qPCR) has become the preferred method for studying low-abundant mRNA expression. Appropriate application of qPCR in such studies requires the use of reference gene(s) as an internal control in order to normalize the mRNA levels between different samples for an exact comparison of gene expression levels. Expression of the reference gene should be independent from development stage, cell/tissue types, treatments and environmental conditions. Recognizing the importance of reference gene(s) in normalization of qPCR data, various reference genes have been evaluated for stable expression under specific conditions in various organisms. In plants, only a few of them have been investigated, and very few reports about such reference genes in citrus. In the present study, seven candidate reference genes (18SrRNA, ACTB, rpII, UBQI, UBQ10, GAPDH and TUB) were tested, and three of them (18SrRNA, ACTB and rpII) proved to be the most stable ones among six leaf samples of different citrus genotypes. The three candidate reference genes were further analyzed for their stability of expression in five different tissues, and the results indicated that they were not completely stable. It is commonly accepted that gene expression studies should be normalized using more than one reference gene. Based on our results, we propose the use of the mean result rendered by18SrRNA, ACTB and rpII as reference genes to normalize mRNA levels in qPCR analysis of diverse cultivars and tissues of citrus. These results may provide a guideline for future works on gene expression in citrus by using qPCR.

Growth, root morphology and boron uptake by citrus rootstock seedlings differing in boron-deficiency responses

Boron (B) is an essential microelement for higher plants and has important physiological functions in plant growth and development. Citrus plants are frequently exposed to B-deficiency, but knowledge regarding the effects of B-deficiency on rootstock growth, root morphology and genotypic variations in citrus is limited. To evaluate the variations in plant-growth parameters in response to B-deficiency, five citrus rootstocks seedlings that grown in modified 1/2-strength Hoagland's solution that contained 0.25 μM H 3BO 3 (moderate B-level, Control) or 0 μM H 3BO 3 (B-deficiency, −B) were investigated before and 90 days after treatment. There are significant genotypic variations in plant-growth parameters (dry mass, leaf area and seedling height), root-morphological traits and B-concentrations. B-deficiency inhibited plant dry mass and leaf area expansion of Fragrant citrus, Sour orange and Trifoliate orange seedlings significantly ( P < 0.05), whereas no effect was found on the total dry mass of Carrizo citrange and Red tangerine seedlings. Further, B-deficiency reduced the root number, root length and root surface area in Fragrant citrus and Trifoliate orange significantly ( P < 0.05); however, no significant effect was detected on these root morphological traits of Carrizo citrange and Red tangerine ( P > 0.05). In addition, B-deficiency reduced the B-concentration in the leaves of all the five genotypes and the B-concentration in the roots of Fragrant citrus and Sour orange, none effect was observed on the B-concentration in stems of all studied genotypes and roots. However, there were significant relationships between the root-morphological traits and B-uptake efficiency. These results showed that Carrizo citrange and Red tangerine are B-efficient, Trifoliate orange is the moderate B-efficient genotypic rootstocks, whereas Fragrant citrus and Sour orange are B-inefficient genotypic rootstocks. In addition, Trifoliate orange was the moderate B-efficient rootstocks of five genotypes. The root-to-shoot dry mass ratio ( R/ S) and longer root numbers were higher in the Carrizo citrange and Red tangerine than that of Trifoliate orange, Fragrant citrus and Sour orange genotypes. That is to say, maintaining higher R/ S and numerous longer lateral roots is very important in improving the B-efficiency and thus contribute much to the resistance of seedlings to B-deficiency in the Carrizo citrange and Red tangerine genotypes.