Effect of salt-stress on gene expression in citrus roots revealed by RNA-seq.

Citrus, as one of the most economically important fruits worldwide, is adversely affected by salinity stress. However, its molecular mechanisms underlying salinity tolerance are still not clear. In this study, next-generation RNA-seq technology was applied to analyze the gene expression profiling of citrus roots at 3 time points over a 24-h period of salt treatment. A total of 1831 differentially expressed genes (DEGs) were identified. Among them, 1195 and 1090 DEGs were found at 4 and 24 h, of which 454 were overlapped. Based on functional annotation, the salt overly sensitive (SOS) and reactive oxygen species (ROS) signaling pathways were found to be involved. Meanwhile, we found that hormone metabolism and signaling played important roles in salt stress. In addition, a multitude of transcription factors (TFs) including WRKY, NAC, MYB, AP2/ERF, bZIP, GATA, bHLH, ZFP, SPL, CBF, and CAMTA were identified. The genes related to cell wall loosening and stiffening (xyloglucan endotransglucosylase/hydrolases, peroxidases) were also involved in salt stress. Our data not only provided a genetic resource for discovering salt tolerance-related genes, but also furthered our understanding of the molecular mechanisms underlying salt tolerance in citrus.

Comprehensive analysis of SAUR gene family in citrus and its transcriptional correlation with fruitlet drop from abscission zone A.

Small auxin-up RNA (SAUR) gene family is large, and the members of which can be rapidly induced by auxin and encode highly unstable mRNAs. SAUR genes are involved in various developmental and physiological processes, such as leaf senescence, fruitlet abscission, and hypocotyl development. However, their modes of action in citrus remain unknown. Hereby, a systematic analysis of SAUR gene family in citrus was conducted through a genome-wide search. In this study, a total of 70 SAUR genes, referred to as CitSAURs, have been identified in citrus. The evolutionary relationship and the intro-exon organization were analyzed, revealing strong gene conservation and the expansion of particular functional genes during plant evolution. Expression analysis showed that the major of CitSAUR genes were expressed in at least one tissue and showed distinctive expression levels, indicating the SAUR gene family play important roles in the development and growth of citrus organs. However, there were more than 20 CitSAUR genes such as CitSARU36, CitSAUR37, and CitSAUR54 exhibiting very low expression level in all tissue tested. Twenty-three out of 70 CitSAUR genes were responded to indole-3-acetic acid (IAA) treatment, of which just CitSAUR19 was down-regulated. Additionally, 14 CitSAUR genes exhibited distinct changes during fruitlet abscission, however just 5 of them including CitSAUR06, CitSAUR08, CitSAUR44, CitSAUR61, and CitSAUR64 were associated with fruitlet abscission. The current study provides basic information for the citrus SAUR gene family and will pave the way for deciphering the precise role of SAURs in citrus development and growth as well as fruitlet abscission.

The ARF, AUX/IAA and GH3 gene families in citrus: genome-wide identification and expression analysis during fruitlet drop from abscission zone A.

Completion of the whole genome sequencing of citrus enabled us to perform genome-wide identification and functional analysis of the gene families involved in agronomic traits and morphological diversity of citrus. In this study, 22 CitARF, 11 CitGH3 and 26 CitAUX/IAA genes were identified in citrus, respectively. Phylogenetic analysis revealed that all the genes of each gene family could be subdivided into three groups and showed strong evolutionary conservation. The GH3 and AUX/IAA gene families shrank and ARF gene family was highly conserved in the citrus genome after speciation from Arabidopsis thaliana. Tissue-specific expression profiles revealed that 54 genes were expressed in at least one tissue while just 5 genes including CitARF07, CitARF20, CitGH3.04, CitAUX/IAA25 and CitAUX/IAA26 with very low expression level in all tissues tested, suggesting that the CitARF, CitGH3 and CitAUX/IAA gene families played important roles in the development of citrus organs. In addition, our data found that the expression of 2 CitARF, 4 CitGH3 and 4 AUX/IAA genes was affected by IAA treatment, and 7 genes including, CitGH3.04, CitGH3.07, CitAUX/IAA03, CitAUX/IAA04, CitAUX/IAA18, CitAUX/IAA19 and CitAUX/IAA23 were related to fruitlet abscission. This study provides a foundation for future studies on elucidating the precise role of citrus ARF, GH3 and AUX/IAA genes in early steps of auxin signal transduction and open up a new opportunity to uncover the molecular mechanism underlying citrus fruitlet abscission.

[Identification of Pummelo Cultivars Based on Hyperspectral Imaging Technology].

Existing methods for the identification of pummelo cultivars are usually time-consuming and costly, and are therefore inconvenient to be used in cases that a rapid identification is needed. This research was aimed at identifying different pummelo cultivars by hyperspectral imaging technology which can achieve a rapid and highly sensitive measurement. A total of 240 leaf samples, 60 for each of the four cultivars were investigated. Samples were divided into two groups such as calibration set (48 samples of each cultivar) and validation set (12 samples of each cultivar) by a Kennard-Stone-based algorithm. Hyperspectral images of both adaxial and abaxial surfaces of each leaf were obtained, and were segmented into a region of interest (ROI) using a simple threshold. Spectra of leaf samples were extracted from ROI. To remove the absolute noises of the spectra, only the date of spectral range 400~1000 nm was used for analysis. Multiplicative scatter correction (MSC) and standard normal variable (SNV) were utilized for data preprocessing. Principal component analysis (PCA) was used to extract the best principal components, and successive projections algorithm (SPA) was used to extract the effective wavelengths. Least squares support vector machine (LS-SVM) was used to obtain the discrimination model of the four different pummelo cultivars. To find out the optimal values of σ2 and γ which were important parameters in LS-SVM modeling, Grid-search technique and Cross-Validation were applied. The first 10 and 11 principal components were extracted by PCA for the hyperspectral data of adaxial surface and abaxial surface, respectively. There were 31 and 21 effective wavelengths selected by SPA based on the hyperspectral data of adaxial surface and abaxial surface, respectively. The best principal components and the effective wavelengths were used as inputs of LS-SVM models, and then the PCA-LS-SVM model and the SPA-LS-SVM model were built. The results showed that 99.46% and 98.44% of identification accuracy was achieved in the calibration set for the PCA-LS-SVM model and the SPA-LS-SVM model, respectively, and a 95.83% of identification accuracy was achieved in the validation set for both the PCA-LS-SVM and the SPA- LS-SVM models, which were built based on the hyperspectral data of adaxial surface. Comparatively, the results of the PCA-LS-SVM and the SPA-LS-SVM models built based on the hyperspectral data of abaxial surface both achieved identification accuracies of 100% for both calibration set and validation set. The overall results demonstrated that use of hyperspectral data of adaxial and abaxial leaf surfaces coupled with the use of PCA-LS-SVM and the SPA-LS-SVM could achieve an accurate identification of pummelo cultivars. It was feasible to use hyperspectral imaging technology to identify different pummelo cultivars, and hyperspectral imaging technology provided an alternate way of rapid identification of pummelo cultivars. Moreover, the results in this paper demonstrated that the data from the abaxial surface of leaf was more sensitive in identifying pummelo cultivars. This study provided a new method for to the fast discrimination of pummelo cultivars.

[Determination of photosynthetic pigments in citrus leaves based on hyperspectral images datas].

The effective region was segmented from the hyperspectral image of citrus leaf by threshold method with the average spectrum extracted and used to describe the corresponding leaf. Based on the different spectral pre-processing methods, the prediction models of three photosynthetic pigments (i. e., chlorophyll a, chlorophyll b, and carotenoid) were calibrated by partial least squares (PLS), BP neural network (BPNN) and least square support vector machine (LS-SVM). The LS-SVM model for chlorophyll a was established based on multiplicative scatter correction (MSC), and the correlation coefficient (Rp) and the root mean square error of prediction (RMSEP) were 0.898 3 and 0.140 4, respectively. The LS-SVM model for chlorophyll b with Rp = 0.912 3 and RMSEP = 0.042 6, was established based on standard normal variable (SNV). The PLS model for carotenoid was established with Rp = 0.712 8 and RMSEP = 0.062 4 based on moving average smoothing (MAS), but the result was no better than the other two. The results illustrated that these three photosynthetic pigments could be nondestructively and real time estimated by hyperspectral image.

[Identification of different Citrus sinensis (L.) Osbeck trees varieties using Fourier transform infrared spectroscopy and hierarchical cluster analysis].

Researched on diversity of the spring leaf samples of seven different Citrus sinensis (L.) Osbeck varieties by Fourier transform infrared (FTIR) spectroscopy technology, the results showed that the Fourier transform infrared spectra of seven varieties leaves was composited by the absorption band of cellulose and polysaccharide mainly, the wave number of characteristics absorption peaks were similar at their FTIR spectra. However, there were some differences in shape of peaks and relatively absorption intensity. The conspicuous difference was presented at the region between 1 500 and 700 cm(-1) by second derivative spectra. Through the hierarchical cluster analysis (HCA) of second derivative spectra between 1 500 and 700 cm(-1), the results showed that the clustering of the different varieties of Citrus sinensis (L.) Osbeck varieties was classification according to genetic relationship. The results showed that FTIR spectroscopy combined with hierarchical cluster analysis could be used to identify and classify of citrus varieties rapidly, it was an extension method to study on early leaves of varieties orange seedlings.

[Research on zinc content in leaf of Olinda Valencia orange using visible near infrared spectroscopy model].

Olinda valencia orange leaves dry powder-like were taken as sample, and chemical analysis combined with technology of visible near-infrared spectroscopy (Vis/NIRS) was used, through the treatment process of second derivative spectrum of samples of the original spectrum and denoising (Noise). Meanwhile, method of partial least squares (PLS) and cross-validation were used to establish maths model of Zn concentration which applying band combination composited by 400-500 and 1201-1300 nm of characteristic wavelength band. The coefficient of establishing models is 0.9975, while the coefficient of correlation coefficient of prediction is 0.9920. The root mean square error of prediction (RMSEP) of cross-validation is 0.5868. Therefore, the means using visible near-infrared spectroscopy (Vis/NIRS) and the methods of cross-validation and PLS to establish the spectral correction model reflecting the Zn content in leaves and characteristic wavelength bands can detect the Zn content in citrus leaves quantitatively and quickly.

[Study on synchronous correlation between fruit characteristic spectrum and the parameter of internal quality for Hamlin sweet orange fruit].

The purpose of our study is to study the relationship between the reflective spectrum of fruit and the internal quality of the orange fruit and find the suitable mark spectrum that can synchronously measure the several fruit quality index at same time to lay a foundation for the development of a rapid and nondestructive field fruit quality analysis technique by analyzing the visible-near infrared spectrum. Mature Hamlin orange (Citrus sinensis (L)cv. Hamlin sweet orange) fruits were individually mensurated for their reflective spectrum by using FieldSpec-HH spectrometer and for their contents of total soluble solid (TSS), citric acid and vitamin C (Vc) by chemistry analysis. The experiment results showed that the fruit reflectivity values (x) at 988 nm was significantly correlated to both TSS (y)(r = 0.387* *, y = 13. 957x + 5.405), TSS/acid ratio(y)(r = 0. 440* *, y = 75.120x + 37.256), and Vc(r = 0.309*). Both of TSS and Vc contends were positively correlated with the second derivatives of the reflective spectrum at 943 nm, with correlation coefficients of 0.339* and 0.355*. TSS/acid ratio was positively correlated only with the reciprocal log values of the reflective spectrum at 944 nm (r = 0.304*). The results in this study indicated that fruit quality indexes TSS, Vc and TSS/acid can be synchronously, rapidly and nondestructively field measured at the same time by the 988 or 429 nm reflective spectrum test and special regress equation operation.

[Study on the correlation of spectral characteristic and nitrogen content of the soil in citrus orchard of three Gorges Reservoir Area].

The relationship between the spectrum characteristics and nitrogen content of soils in citrus orchard of the Three Gorges Reservoir Area was studied by analyzing the visible-near infrared spectrum. The results showed that the soil reflectivity in creased lineally as the wavelength increases across the visible spectrum and reached a stable plateau in the short wavelength near-infrared region (780-1750 nm)without much fluctuation. In the long wavelength near-infrared region (1750-2400 nm) the reflec tivity of the soils was higher with higher fluctuation. There were three strong absorbance peaks around 1416, 1913 and 2209 nm, respectively, in the long wavelength infrared region. Soil available nitrogen content and total nitrogen content were positively correlated with soil light reflectivity but negatively correlated with catoptric-spectrum values reciprocal logarithm. At 541 nn of visible light region, a high positive correlation was found between the available nitrogen content and the first derivative of the soil reflective spectrum with a correlation coefficient of + 0.605** and the best fitting equation was y = 2E + 09x(2) - 3E + 06x + 890.49, where R2 = 0.5, and x is the first derivative of the soil reflective spectrum. At 1909 nm of the near-infrared long wavelength region, the correlation between the total nitrogen content and the reciprocal-log values of the reflective spectrum of the soils was the best with a correlation coefficient of -0.612**, and the best fitting equation was y = 1.3721x(2) - 2.1075x + 0.8592, where R2 = 0.4, and x is the reciprocal values of the log reflective spectrum of the soils.

Combined analysis of mRNA and miRNA identifies dehydration and salinity responsive key molecular players in citrus roots.

Citrus is one of the most economically important fruit crops around world. Drought and salinity stresses adversely affected its productivity and fruit quality. However, the genetic regulatory networks and signaling pathways involved in drought and salinity remain to be elucidated. With RNA-seq and sRNA-seq, an integrative analysis of miRNA and mRNA expression profiling and their regulatory networks were conducted using citrus roots subjected to dehydration and salt treatment. Differentially expressed (DE) mRNA and miRNA profiles were obtained according to fold change analysis and the relationships between miRNAs and target mRNAs were found to be coherent and incoherent in the regulatory networks. GO enrichment analysis revealed that some crucial biological processes related to signal transduction (e.g. 'MAPK cascade'), hormone-mediated signaling pathways (e.g. abscisic acid- activated signaling pathway'), reactive oxygen species (ROS) metabolic process (e.g. 'hydrogen peroxide catabolic process') and transcription factors (e.g., 'MYB, ZFP and bZIP') were involved in dehydration and/or salt treatment. The molecular players in response to dehydration and salt treatment were partially overlapping. Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) analysis further confirmed the results from RNA-seq and sRNA-seq analysis. This study provides new insights into the molecular mechanisms how citrus roots respond to dehydration and salt treatment.

Genome-wide analysis of citrus R2R3MYB genes and their spatiotemporal expression under stresses and hormone treatments.

The R2R3MYB proteins represent one of the largest families of transcription factors, which play important roles in plant growth and development. Although genome-wide analysis of this family has been conducted in many species, little is known about R2R3MYB genes in citrus, In this study, 101 R2R3MYB genes has been identified in the citrus (Citrus sinesis and Citrus clementina) genomes, which are almost equal to the number of rice. Phylogenetic analysis revealed that they could be subdivided into 21 subgroups. The evolutionary relationships and the intro-exon organizations were also analyzed, revealing strong gene conservation but also the expansions of particular functional genes during the plant evolution. Tissue-specific expression profiles showed that 95 citrus R2R3MYB genes were expressed in at least one tissue and the other 6 genes showed very low expression in all tissues tested, suggesting that citrus R2R3MYB genes play important roles in the development of all citrus organs. The transcript abundance level analysis during abiotic conditions (NaCl, abscisic acid, jasmonic acid, drought and low temperature) identified a group of R2R3MYB genes that responded to one or multiple treatments, which showed a promising for improving citrus adaptation to stresses. Our results provided an essential foundation for the future selection of the citrus R2R3MYB genes for cloning and functional dissection with an aim of uncovering their roles in citrus growth and development.

[Study on the changes of incidence rates on birth defects through hospital based surveillance program in Guangdong province during 1997-2007].

OBJECTIVE: To analyze the tendency of hospital based surveillance incidence rates of birth defects in Guangdong province to provide reference evidence for related intervention program. METHODS: Data from hospital based birth defects surveillance of Guangdong province during 1997 - 2007 were analyzed retrospectively. RESULTS: The overall surveillance rates of birth defects in Guangdong province showed an increasing trend form 98.9 per 10 000 birth in 1997 to 244.2 per 10 000 birth in 2007. Among the frequently seen birth defects, the rate of congenital heart diseases increased sharply, but the rates of congenital talipes equinovarus, polydactyly, cleft lip with/without cleft palate showed a slight increasing trend. It seemed that there was a significant difference between different maternal age interval and gender of the infants. Mother's illness, drug use, and exposure to environment harmful factors in early pregnancy stage, high maternal age, negative bearing history were the suspect risk factors for birth defects. CONCLUSION: The increasing trend of surveillance rate of birth defects seemed to be comprehensively affected by the factors as: method being adopted, target and extension of birth defects surveillance, technology used for diagnosis, kinds of risk factor, and quality control on surveillance programs.