Citrus Genotypes Research Articles

Genetic relationships of some Citrus genotypes based on the candidate iron chlorosis genes

Iron is one of the most important elements in plant mineral nutrition. Fe deficiency is a critical abiotic stress factor for Mediterranean citriculture; the development of marker-assisted selection for this trait would greatly enhance rootstock breeding. In this study, DNA sequencing and single-stranded conformation polymorphism (SSCP) analyses were performed to determine the allelic diversity of genes associated with tolerance to iron chlorosis in citrus. Two candidate iron chlorosis tolerance genes were selected from existing Citrus EST databases and Arabidopsis thaliana genome databases. Ferritin-3 chloroplast precursor and putative membrane transporter candidate gene sequences were used to define primers in conserved regions. Six citrus genotypes from the basic taxon of Citrus were used to identify polymorphic regions in the genes. Direct sequencing of the amplified DNA fragments from the candidate genes was performed, and single nucleotide polymorphisms (SNPs) and insertions/deletions (indels) were identified after sequence alignment. Based on the DNA sequencing analysis, a total of 6840 nucleotides of DNA were sequenced to identify SNPs and indels. In total, 263 SNPs and 15 indels were identified for both genes. We detected 38.45 SNPs and 2.19 indels for each 1000 b on average from the DNA sequencing results. New primers were designed in conserved areas flanking polymorphic ones for SSCP analysis. SSCP-PCR analysis was performed with 25 citrus genotypes. The neighbor-joining method was used for cluster analysis. Trifoliate genotypes and their hybrids (known to be sensitive to iron chlorosis) clustered together, whereas genotypes tolerant to iron chlorosis were more spread out on the dendrogram. Mandarins also showed high diversity for both genes according to SSCP results. Differences were found among sour orange genotypes known to have differential tolerance behavior to iron chlorosis.

Rapid Differentiation of Closely Related <i>Citrus Genotypes</i> by Fluorescence Spectroscopy

The differentiation of closely related Citrus genotypes is a meticulous, laborious, and time-consuming task that involves the assessment of complex traits such as growth, tolerance to stress, photosynthetic efficiency, yield, and many others. Such a task is generally accomplished either by analyzing specific features of adult plants or by applying molecular markers to young trees. On one hand, only after plants start yielding can distinct genotypes be differentiated by comparing their fruit sizes, shapes, taste, and the number of seeds. On the other hand, molecular markers are expensive, and demand expertise and time for the analysis of a larger number of plants. For these reasons, the development of techniques that could assist in an early, quick and accurate differen-tiation of closely related Citrus varieties is of utmost importance. In this context, laser-induced fluorescence spectroscopy (LIFS) is a promising technique, since it is rapid, highly sensitive, and inexpensive. Previous studies showed that LIFS can differentiate a variety of sweet orange. However, this new study aimed to determine LIFS accuracy in the differentiation and grouping of very closely varieties of four Sunki mandarin selections: Comum, Florida, Tropical, and Maravilha. Furthermore we compared the results with ISSR and SSR molecular markers for the same varieties. LIFS technique distinguished the four selections with accuracy greater than 70%. Only with molecular markers was possible distinguishing clearly Tropical from Maravilha, but not Comum from Florida selections. In this way the results suggest that LIFS may be a sound tool for helping the identification of closely Citrus varieties.

Avaliação da resistência de variedades de Citrus spp. à Xanthomonas citri subsp. citri na região Noroeste Paranaense, em condições de campo

A identificação de cultivares com determinados níveis de resistência ao cancro cítrico, com características agronômicas e comerciais desejáveis, assume grande importância para o manejo desta doença. A avaliação de genótipos promissores pode ser fetuada em condições controladas ou em campo. O trabalho teve como objetivo avaliar genótipos de laranja doce (Citrus sinensis), tangerinas (Citrus reticulata) e híbridos quanto à resistência ao cancro cítrico, em diferentes épocas de avaliação. O experimento foi conduzido em pomar experimental no município de Maringá, Paraná. Para a determinação da severidade média da doença, foram utilizadas escalas diagramáticas, sendo avaliadas quatro plantas por bloco e amostrados ramos na porção mediana de cada planta. Além disso, foi calculada a Área Abaixo da Curva de Progresso da Severidade (AACPS). De acordo com os dados obtidos, ocorreram diferenças significativas na severidade e na AACPS, ao longo das avaliações, sendo que a severidade variou de 0,25% a 4,88% e a AACPS de 49,80 a 742,65. As variedades cítricas Tang. Ponkan, Rosehaugh Nartjee, Harris, De wildt, Dekopon, Lee, África do Sul, Tarocco#23, Solid Scarlet, Satsuma Okitsu SPA 29, Cristalina, Shamouti e Pêra Olimpia se destacaram e apresentaram as menores médias de severidade, sendo essas classificadas como resistentes. Os resultados sugerem que esses genótipos podem ser importantes ferramentas nos programas de melhoramento de genótipos de citros, visando resistência a Xanthomonas citri subsp. citri.

Expression profiling of two stress-inducible genes encoding for miraculin-like proteins in citrus plants under insect infestation or salinity stress

The expression of two genes, namely Mir1 and Mir3 and the abundance of their encoded proteins, the putative miraculin-like proteins, MLP1 and MLP3, showing similarity to the Kunitz family of protease inhibitors, were monitored in the leaves of the citrus variety, ‘Clementine’ after Tetranychus urticae infestation and elicitor treatments, or in the leaves of three other diploid citrus: ‘Willow leaf’, ‘Cleopatra’ mandarins and ‘Trifoliate’ orange, as well as their respective doubled diploids and the allotetraploid somatic hybrid ‘FLHORAG1’ under salt stress. RT-PCR and 2-DE indicated that Mir1 and Mir3 and their products were present at low-basal expression in all citrus genotypes. Both genes and products were induced in the ‘Clementine’ leaves infested by T. urticae, but a contrasting profile was observed under elicitor treatments. Under salt stress, the two genes showed an expression pattern contrasting each other and depending on the genotypes. ‘Cleopatra’ mandarin, ‘Trifoliate’ orange and ‘FLHORAG1’ presented overexpression of Mir3 and MLP3 and decreased levels of Mir1 and MPL1. The opposite behaviour was found in ‘Willow leaf’ mandarin. The positive correlation of the expression profile of the two genes with that of a gene encoding a putative apoplastic cysteine protease (CysP) might suggest a possible interaction of the respective encoded proteins during the response to biotic stress. Under salt stress, CysP and Mir 1 showed a similar expression pattern but only at transcript level. The possible occurrence of post-translational CysP regulation is discussed.

Cluster analysis of citrus genotypes using near-infrared spectroscopy

There are many genotypes and varieties in the citrus family. Currently, citrus classification systems have significant divergences in varieties of species, and subgenus classification as well. In this study, near-infrared spectroscopy technique was used to acquire spectral information on the surface of citrus fruits. Cluster analysis was consequently conducted to identify citrus genotypes. Results indicated that the combination of 9-point moving average smoothing and multiplicative scattering correction was optimal for preprocessing spectral data. In the spectral range of 1,180–1,220 nm, the cumulative reliability of the first two principal components were greater than 99.4%, and sweet oranges were clustered into an independent class. In 1,280–1,320 nm, systematic clustering performed better than principal component clustering, and all other sour oranges, except Goutoucheng, were clustered into a single clade. With dimensions reduction, the cumulative reliability of first five principle components in full...

Validation of high-throughput real time polymerase chain reaction assays for simultaneous detection of invasive citrus pathogens

A number of important citrus pathogens are spread by graft propagation, arthropod vector transmission and inadvertent import and dissemination of infected plants. For these reasons, citrus disease management and clean stock programs require pathogen detection systems which are economical and sensitive to maintain a healthy industry. To this end, multiplex quantitative real-time PCR (qPCR) assays were developed allowing high-throughput and simultaneous detection of some major invasive citrus pathogens. Automated high-throughput extraction comparing several bead-based commercial extraction kits were tested and compared with tissue print and manual extraction to obtain nucleic acids from healthy and pathogen-infected citrus trees from greenhouse in planta collections and field. Total nucleic acids were used as templates for pathogen detection. Multiplex reverse transcription-qPCR (RT-qPCR) assays were developed for simultaneous detection of six targets including a virus, two viroids, a bacterium associated with huanglongbing and a citrus RNA internal control. Specifically, two one-step TaqMan-based multiplex RT-qPCR assays were developed and tested with target templates to determine sensitivity and detection efficiency. The first assay included primers and probes for ‘Candidatus Liberibacter asiaticus’ (CLas) and Citrus tristeza virus (CTV) broad spectrum detection and genotype differentiation (VT- and T3-like genotypes). The second assay contained primers and probes for Hop stunt viroid (HSVd), Citrus exocortis viroid (CEVd) and the mitochondrial NADH dehydrogenase (nad5) mRNA as an internal citrus host control. Primers and TaqMan probes for the viroids were designed in this work; whereas those for the other pathogens were from reports of others. Based on quantitation cycle values, automated high-throughput extraction of samples proved to be as suitable as manual extraction. The multiplex RT-qPCR assays detected both RNA and DNA pathogens in the same dilution series as singleplex assays and yielded similar quantitation cycle values. Taken together, high throughput extraction and multiplex RT-qPCR assays reported in this study provided a rapid and standardized method for routine and simultaneous diagnosis of different RNA and DNA citrus pathogens.

Arbuscular mycorrhizal fungi induce sucrose cleavage for carbon supply of arbuscular mycorrhizas in citrus genotypes

Arbuscular mycorrhizas (AMs) must gain carbohydrates (mainly glucose) through sucrose cleavage from their plant hosts. Using the AM fungus Funneliformis mosseae inoculated or non-inoculated citrus genotypes [red tangerine (Citrus tangerina Hort. ex Tanaka), kumquat (Fortunella margarita Lour. Swingle), and trifoliate orange (Poncirus trifoliata L. Raf.)], we tested the hypothesis that the sucrose cleavage would depend on sucrose-cleaving enzyme types and/or host genotypes for carbon supply to the AM development. Mycorrhizal inoculation significantly increased biomass production and glucose contents of leaf, root, and total plant, irrespectively of citrus genotypes. In contrast, under mycorrhization similar leaf sucrose contents were observed between these three citrus genotypes, whereas significantly higher sucrose contents were in roots of red tangerine and kumquat, and in total plant (leaf plus root) of red tangerine and trifoliate orange. Root AM colonization was significantly positively correlated with leaf and total plant glucose but not with root glucose or with sucrose in either leaf and/or root. Root AM colonization increased the activity of leaf acid invertase (AI) and sucrose synthase (SS) and root neutral invertase (NI) in trifoliate orange, leaf SS and root AI in kumquat, and root NI in red tangerine, but decreased leaf NI in all three citrus genotypes, leaf SS in red tangerine, root AI in red tangerine and trifoliate orange, and root SS in kumquat and trifoliate orange. These results support our hypothesis that the sucrose cleavage highly depended on both citrus genotypes and sucrose-cleaving enzyme types/activity, and thus may provide higher glucose as carbon source to support their fungal partners.

The effect of sodium bicarbonate on plant performance and iron acquisition system of FA-5 (Forner-Alcaide 5) citrus seedlings

This work studies the effect of bicarbonate on plant performance and the iron acquisition system of Forner-Alcaide 5 (FA-5) seedlings, a citrus genotype known for its tolerance to calcareous soils. Plants were irrigated for 6 weeks with or without 10 mM NaHCO3. Treatment significantly decreased shoot growth, photosynthetic levels and iron concentration in shoots and roots. o,o-57FeEDDHA experiments indicated that 57Fe uptake by roots was inhibited in treated plants. Moreover, those seedlings accumulated more 57Fe in roots, and enhanced mRNA accumulation of ferric reductase genes FRO1 and FRO2 and FC-R activity in roots. H+-ATPase activity and HA1 gene expression were also increased, while HA2 was not affected. In addition, expression of the iron transporter gene IRT1 was increased, while IRT2 was not significantly affected. Finally, according to PEPC enzymatic activity, PEPC1 gene expression was higher in treated roots. In conclusion, it appears that bicarbonate prevents medium acidification by roots, thus reducing Fe2+ uptake. Accordingly, Fe deficiency enhanced the expression of some genes related with the Fe acquisition system (IRT1, FRO1, FRO2, HA1 and PEPC1) and the activity of the corresponding enzymes, which appear to constitute an adaptive mechanism of FA-5 in these soils.

EST-SNP genotyping of citrus species using high-resolution melting curve analysis

Citrus taxonomy is very complex mainly due to specific aspects of its reproductive biology. A number of studies have been performed using various molecular markers in order to evaluate the level of genetic variability in Citrus. SNP markers have been used for genetic diversity assessment using a variety of different methods. Recently, the availability of EST database and whole genome sequences has made it possible to develop more markers such as SNPs. In the present study, the high-resolution melting curve analysis (HRM) was used to detect SNPs or INDELs in Citrus genus for the first time. We aimed to develop a panel of SNPs to differentiate Citrus genotypes which can also be applied to Citrus biodiversity studies. The results showed that 21 SNP containing markers produced distinct polymorphic melting curves among the Citrus spp. investigated through HRM analysis. It was proved that HRM is an efficient, cost-effective, and accurate method for discriminating citrus SNPs as well as a method to analyze more polymorphisms in a single PCR amplicon, representing a useful tool for genetic, biodiversity, and breeding studies. SNPs developed based on Citrus sinensis EST database showed a good transferability within the Citrus genus. Moreover, HRM analysis allowed the discrimination of citrus genotypes at specific level and the resulting genetic distance analysis clustered these genotypes into three main branches. The results suggested that the panel of SNP markers could be used in a variety of applications in citrus biodiversity assessment and breeding programs using HRM analysis.

Salt-stress induced changes in the leaf proteome of diploid and tetraploid mandarins with contrasting Na+ and Cl− accumulation behaviour

To understand the genotypic variation of citrus to mild salt stress, a proteomic approach has been carried out in parallel on two citrus genotypes (‘Cleopatra’ and ‘Willow leaf’ mandarins), which differ for Na+ and Cl− accumulation, and their cognate autotetraploids (4×). Using two-dimensional electrophoresis approximately 910 protein spots were reproducibly detected in control and salt-stressed leaves of all genotypes. Among them, 44 protein spots showing significant variations at least in one genotype were subjected to mass spectrometry analysis for identification. Salt-responsive proteins were involved in several functions, including photosynthetic processes, ROS scavenging, stress defence, and signalling. Genotype factors affect the salt-responsive pattern, especially that of carbon metabolism. The no ion accumulator ‘Cleopatra’ mandarin genotype showed the highest number of salt-responsive proteins, and up-regulation of Calvin cycle-related proteins. Conversely the ion accumulator ‘Willow leaf’ mandarin showed high levels of several photorespiration-related enzymes. A common set of proteins (twelve spots) displayed higher levels in salt-stressed leaves of 2× and 4× ‘Cleopatra’ and 4× ‘Willow leaf’ mandarin. Interestingly, antioxidant enzymes and heat shock proteins showed higher constitutive levels in 4× ‘Cleopatra’ mandarin and 4× ‘Willow leaf’ mandarin compared with the cognate 2× genotype. This work provides for the first time information on the effect of 8 weeks of salt stress on citrus genotypes contrasting for ion accumulation and their cognate autotetraploids. Results underline that genetic factors have a predominant effect on the salt response, although a common stress response independent from genotype was also found.

Effect of cadmium and calcium treatments on phytochelatin and glutathione levels in citrus plants

Industry residues, phosphate fertilisers and wastewater as a source of irrigation have considerably increased levels of heavy metals in the soil, mainly cadmium (Cd(2+)). To test the effects of a calcium (Ca(2+)) treatment on Cd(2+) accumulation and plant tolerance to this heavy metal, plants of two citrus genotypes, Cleopatra mandarin (CM) and Carrizo citrange (CC), were watered with increasing concentrations of Cd(2+), and phytochelatin (PC) and glutathione (GSH) content were measured. Both genotypes were able to synthesise PCs in response to heavy metal intoxication, although CM seems to be a better Cd(2+) excluder than CC. However, data indicate that CC plants had a higher capacity for regenerating GSH than CM plants. In this context, the effects of Ca(2+) treatment on Cd(2+) accumulation, plant survival and PC, GSH and oxidised glutathione (GSSG) content were assessed. Data indicate that treatment with Ca(2+) had two positive effects on citrus physiology: it reduced Cd(+2) uptake into roots and also increased GSH content (even in the absence of Cd(2+)). Overall, the data indicate that although Cd(2+) exclusion is a powerful mechanism to avoid heavy metal build-up into photosynthetic organs, the capacity to maintain optimum GSH levels to feed PC biosynthesis could also be an important factor in stress tolerance.

Citrus (Rutaceae) SNP markers based on Competitive Allele-Specific PCR; transferability across the Aurantioideae subfamily.

• Premise of the study: Single nucleotide polymorphism (SNP) markers based on Competitive Allele-Specific PCR (KASPar) were developed from sequences of three Citrus species. Their transferability was tested in 63 Citrus genotypes and 19 relative genera of the subfamily Aurantioideae to estimate the potential of SNP markers, selected from a limited intrageneric discovery panel, for ongoing broader diversity analysis at the intra- and intergeneric levels and systematic germplasm bank characterization.• Methods and Results: Forty-two SNP markers were developed using KASPar technology. Forty-one were successfully genotyped in all of the Citrus germplasm, where intra- and interspecific polymorphisms were observed. The transferability and diversity decreased with increasing taxonomic distance.• Conclusions: SNP markers based on the KASPar method developed from sequence data of a limited intrageneric discovery panel provide a valuable molecular resource for genetic diversity analysis of germplasm within a genus and should be useful for germplasm fingerprinting at a much broader diversity level.

Chemotaxonomic Study of Citrus, Poncirus and Fortunella Genotypes Based on Peel Oil Volatile Compounds - Deciphering the Genetic Origin of Mangshanyegan (Citrus nobilis Lauriro)

Volatile profiles yielded from gas chromatography-mass spectrometry (GC-MS) analysis provide abundant information not only for metabolism-related research, but also for chemotaxonomy. To study the chemotaxonomy of Mangshanyegan, its volatile profiles of fruit and leaf and those of 29 other genotypes of Citrus, Poncirus, and Fortunella were subjected to phylogenetic analyses. Results showed that 145 identified (including 64 tentatively identified) and 15 unidentified volatile compounds were detected from their peel oils. The phylogenetic analysis of peel oils based on hierarchical cluster analysis (HCA) demonstrated a good agreement with the Swingle taxonomy system, in which the three genera of Citrus, Poncirus, and Fortunella were almost completely separated. As to Citrus, HCA indicated that Citrophorum, Cephalocitrus, and Sinocitrus fell into three subgroups, respectively. Also, it revealed that Mangshanyegan contain volatile compounds similar to those from pummelo, though it is genetically believed to be a mandarin. These results were further supported by the principal component analysis of the peel oils and the HCA results of volatile profiles of leaves in the study.

Differences in oxidative stress, antioxidant systems, and microscopic analysis between regenerating callus-derived protoplasts and recalcitrant leaf mesophyll-derived protoplasts of Citrus reticulata Blanco

It is known that protoplasts derived from either leaves or suspension cultures of a citrus genotype vary greatly in their regeneration capacities; however, the underlying physiological mechanisms are not well known. In this study, oxidative stress and antioxidant systems during in vitro culture of callus-derived protoplasts and leaf mesophyll-derived protoplasts of Ponkan (Citrus reticulata Blanco) were analyzed to gain insights into observed physiological differences. Morphological observations using light microscopy and scanning microscopy have shown that new cell wall materials appeared within 2–3 days, and the integrate cell walls were regenerated approximately after 6 days of culture of the callus protoplasts, whereas no cell wall formation was observed in the mesophyll protoplasts after culture. During the culture, higher levels of H2O2 and malondialdehyde were detected in the mesophyll protoplasts as compared with the callus ones. On the contrary, the callus protoplasts possessed higher activities of antioxidant enzymes (SOD, POD and CAT) and larger amount of glutathione and ascorbic acid (at one time point) than the mesophyll protoplasts during the culture process. The current data indicate that the mesophyll and callus protoplasts displayed remarkable difference in the degree of oxidative stress and the antioxidant systems, suggesting that high levels of antioxidant activities might play an important role in the regeneration of protoplasts.

Poliembrionia e atributos morfológicos de sementes de porta-enxertos de citros

A poliembrionia é um caráter fundamental no melhoramento genético e na multiplicação comercial de porta-enxertos de citros. Este trabalho avaliou as relações entre poliembrionia e diferentes atributos morfológicos de sementes de 12 genótipos de citros selecionados como porta-enxertos. Foram analisadas 50 sementes por genótipo, quanto a massa, altura, largura, número de embriões, taxa de poliembrionia, grau de dificuldade de extração da testa, coloração da semente, estimada por um matiz das duas notas de cores predominantes, e razão de cor, calculada pela divisão entre a menor e a maior nota do matiz. Os resultados foram submetidos à análise de variância, comparando-se as médias pelo teste de Scott-Knott e determinando-se os coeficientes de correlação de Pearson entre as variáveis (P < 0,05). Os porta-enxertos apresentaram atributos de sementes distintos, podendo ser agrupados em três classes de poliembrionia, destacando-se três seleções de limoeiro 'Cravo' com poliembrionia inferior a 50%, citrandarins como totalmente poliembriônicos e outros híbridos de Poncirus trifoliata com taxas baixas a intermediárias. O grau de dificuldade de extração da testa foi maior nas sementes menores. Na maioria dos genótipos avaliados, o número de embriões por semente correlacionou- se positivamente à massa, altura e largura de sementes e negativamente à razão de cor, sugerindo que a análise de coloração de sementes inteiras pode ser ferramenta alternativa para uma avaliação visual rápida da ocorrência de poliembrionia.

Genetic variation and phylogenetic relationships among Indian Citrus taxa revealed by DAMD-PCR markers

Genetic variation and phylogenetic relationships among 50 wild and cultivated accessions of 19 Indian Citrus genotypes were examined through comparison of Directed Amplification of Minisatellite DNA (DAMD) markers and morphological characters. DAMD-PCR analysis with four primers resulted in amplification of a total of 45 bands, of which 35 (78 %) were polymorphic. Morphometric evaluation using 76 morphological characters showed high level of variability ranging from 0.18 to 1.00 (avg 0.39), whereas the Jaccard’s coefficient values of genetic similarity calculated from DAMD data ranged from 0.41 to 1.00 (avg 0.68), indicating moderate genetic divergence among the accessions studied. UPGMA dendrograms generated separately from morphometric and DAMD data segregated all the accessions of Citrus into four main clusters, each containing a true basic species and their probable hybrids. The grouping of individual accessions/genotypes under respective species or cultivars in DAMD dendrogram was based purely on their genetic relationships rather than geographical origin. There was no absolute congruence between the data and dendrograms generated from morphometric and DAMD analyses. The study demonstrates the resolving power of DAMD markers for discrimination of individual genotypes of Citrus under its respective species, hybrid or cultivar groups and inferring their genetic and phylogenetic relationships as well. This is the first report on application of DAMD markers in Citrus.

Use of an expressed sequence tag-based method for single nucleotide polymorphism identification and discrimination of Citrus species and cultivars

Sweet orange [Citrus sinensis (L.) Osbeck] represents the most important Citrus species, followed by clementine (C. clementina Hort. ex Tan.). Citrus species and genotypes are difficult to recognize as they have a moderate level of diversity due to nucellar selection, vegetative propagation and origin by single spontaneous mutation. Despite the large number of available sequences and the existence of a draft assembly of sweet orange and clementine, there are currently no single nucleotide polymorphism (SNP) databases for Citrus species. For this purpose, the QualitySNP software was used to discover SNPs in 19 Citrus species starting from 540,000 expressed sequence tags (ESTs) assembled in 52,000 contigs. The vast majority of ESTs, contigs and SNPs were found in C. clementina and C. sinensis: 4,400 out of 16,000 contigs (27 %) of C. clementina and 4,100 out of 17,000 contigs (24 %) of C. sinensis contained putative SNPs. A total of 3,634 sequences were associated with enzymes belonging to 121 metabolic KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways, among which the secondary metabolite pathway was the most represented. A total of 163 SNPs from 52 contigs and genes of specific functional categories were validated and 81 polymorphic sites were found. Thirty-seven selected SNPs, validated by Sanger sequencing, confirmed that polymorphisms were mainly between species, while poor within-species variability was discovered. This work provides a collection of 15,879 putative SNP markers that could be exploited by the Citrus community. Furthermore, the validated SNPs associated with specific genes could be used for functional genetic studies in germplasm diversity analysis, mapping and breeding.

Mining of haplotype-based expressed sequence tag single nucleotide polymorphisms in citrus

BackgroundSingle nucleotide polymorphisms (SNPs), the most abundant variations in a genome, have been widely used in various studies. Detection and characterization of citrus haplotype-based expressed sequence tag (EST) SNPs will greatly facilitate further utilization of these gene-based resources.ResultsIn this paper, haplotype-based SNPs were mined out of publicly available citrus expressed sequence tags (ESTs) from different citrus cultivars (genotypes) individually and collectively for comparison. There were a total of 567,297 ESTs belonging to 27 cultivars in varying numbers and consequentially yielding different numbers of haplotype-based quality SNPs. Sweet orange (SO) had the most (213,830) ESTs, generating 11,182 quality SNPs in 3,327 out of 4,228 usable contigs. Summed from all the individually mining results, a total of 25,417 quality SNPs were discovered – 15,010 (59.1%) were transitions (AG and CT), 9,114 (35.9%) were transversions (AC, GT, CG, and AT), and 1,293 (5.0%) were insertion/deletions (indels). A vast majority of SNP-containing contigs consisted of only 2 haplotypes, as expected, but the percentages of 2 haplotype contigs varied widely in these citrus cultivars. BLAST of the 25,417 25-mer SNP oligos to the Clementine reference genome scaffolds revealed 2,947 SNPs had “no hits found”, 19,943 had 1 unique hit / alignment, 1,571 had one hit and 2+ alignments per hit, and 956 had 2+ hits and 1+ alignment per hit. Of the total 24,293 scaffold hits, 23,955 (98.6%) were on the main scaffolds 1 to 9, and only 338 were on 87 minor scaffolds. Most alignments had 100% (25/25) or 96% (24/25) nucleotide identities, accounting for 93% of all the alignments. Considering almost all the nucleotide discrepancies in the 24/25 alignments were at the SNP sites, it served well as in silico validation of these SNPs, in addition to and consistent with the rate (81%) validated by sequencing and SNaPshot assay.ConclusionsHigh-quality EST-SNPs from different citrus genotypes were detected, and compared to estimate the heterozygosity of each genome. All the SNP oligo sequences were aligned with the Clementine citrus genome to determine their distribution and uniqueness and for in silico validation, in addition to SNaPshot and sequencing validation of selected SNPs.

Tolerância de genótipos de citros ao estresse hídrico na fase de porta-enxerto

We studied the water stress tolerance of eight genotypes of citrus rootstocks on stage, in an experiment conducted in a greenhouse at UAEAg / CTRN UFCG with the experimental design in blocks with three replications in a factorial of two treatments (EH1 : witnesses irrigated at 100% Field Capacity (CC) throughout the experiment and EH2: limited irrigation at 50% DC for 51 days and then 25% of DC) and three genotypes. Each experimental unit consisted of 10 plants. Observing the effects of water stress in citrus genotypes with the potential rootstocks on growth variables, we highlight the genotype 15 (TSKC CTARG x - 019), with the highest averages. The Rangpur lime was the one most sensitive to stress in all variables, showing sensitivity to water stress.

Produção de fitomassa em genótipos de citros submetidos a estresse hídrico na formação do porta-enxerto

Devido à importância dos porta-enxertos no sucesso da citricultura e sua influência na tolerância a fatores de estresse, desenvolveu-se este estudo com o objetivo de avaliar a tolerância de porta-enxertos de citros ao estresse hídrico. Estudaram-se dois fatores: manejo hídrico ('sem estresse' e 'com estresse', neste último caso, com irrigação baseada em 50 e 25% da capacidade de campo) e porta-enxerto (10 genótipos de citros fornecidos pelo Programa de Melhoramento Genético de Citros da Embrapa Mandioca e Fruticultura Tropical). Adotou-se o delineamento em blocos casualizados com três repetições e 15 plantas por parcela; avaliaram-se as fitomassas da parte aérea, das folhas, do caule, da raiz e total, além da relação raiz/parte aérea, razão de peso foliar e rendimento relativo. O déficit hídrico reduziu a fitomassa total e incrementou, em alguns porta-enxertos, a relação raiz/parte aérea. A fitomassa foliar é a variável mais afetada, negativamente, nos porta-enxertos de citros sob deficiência hídrica. O genótipo 'TSKFL x CTTR-017' possui capacidade de adaptação ao déficit hídrico, com potencial promissor para uso em áreas sujeitas às condições de seca.