Quantitative trait loci identification reveals zinc finger protein CONSTANS-LIKE 4 as the key candidate gene of stigma color in watermelon(Citrullus lanatus)

Stigma color is a critical agronomic trait in watermelon that plays an important role in pollination.However,there are few reports on the regulation of stigma color in watermelon.In this study,a genetic analysis of the F2 population derived from ZXG1553(P1,with orange stigma)and W1-17(P2,with yellow stigma)indicated that stigma color is a quantitative trait and the orange stigma is recessive compared with the yellow stigma.Bulk segregant analysis sequencing(BSA-seq)revealed a 3.75 Mb segment on chromosome 6 that is related to stigma color.Also,a major stable effective QTL Clqsc6.1(QTL stigma color)was detected in two years between cleaved amplified polymorphic sequencing(CAPS)markers Chr06_8338913 and Chr06_9344593 spanning a~1.01 Mb interval that harbors 51 annotated genes.Cla97C06G117020(annotated as zinc finger protein CONSTANS-LIKE 4)was identified as the best candidate gene for the stigma color trait through RNA-seq,quantitative real-time PCR(qRT-PCR),and gene structure alignment analysis among the natural watermelon panel.The expression level of Cla97C06G117020 in the orange stigma accession was lower than in the yellow stigma accessions with a significant difference.A nonsynonymous SNP site of the Cla97C06G117020 coding region that causes amino acid variation was related to the stigma color variation among nine watermelon accessions according to their re-sequencing data.Stigma color formation is often related to carotenoids,and we also found that the expression trend of ClCHYB(annotated asβ-carotene hydroxylase)in the carotenoid metabolic pathway was consistent with Cla97C06G117020,and it was expressed in low amounts in the orange stigma accession.These data indicated that Cla97C06G117020 and ClCHYB may interact to form the stigma color.This study provides a theoretical basis for gene fine mapping and mechanisms for the regulation of stigma color in watermelon.

Identifying the stability of housekeeping genes to be used for the quantitative real-time PCR normalization in retinal tissue of streptozotocin-induced diabetic rats

AIM:To investigate the stability of the seven housekeeping genes:beta-actin(ActB),glyceraldehyde-3-phosphate dehydrogenase(GAPDH),18s ribosomal unit 5(18s),cyclophilin A(CycA),hypoxanthine-guanine phosphoribosyl transferase(HPRT),ribosomal protein large P0(36B4)and terminal uridylyl transferase 1(U6)in the diabetic retinal tissue of rat model.METHODS:The expression of these seven genes in rat retinal tissues was determined using real-time quantitative reverse transcription polymerase chain reaction(RT-qPCR)in two groups;normal control rats and streptozotocininduced diabetic rats.The stability analysis of gene expression was investigated using geNorm,NormFinder,BestKeeper,and comparative delta-Ct(ΔCt)algorithms.RESULTS:The 36B4 gene was stably expressed in the retinal tissues of normal control animals;however,it was less stable in diabetic retinas.The 18s gene was expressed consistently in both normal control and diabetic rats’retinal tissue.That this gene was the best reference for data normalisation in RT-qPCR studies that used the retinal tissue of streptozotocin-induced diabetic rats.Furthermore,there was no ideal gene stably expressed for use in all experimental settings.CONCLUSION:Identifying relevant genes is a need for achieving RT-qPCR validity and reliability and must be appropriately achieved based on a specific experimental setting.

Detection of Exogenous Gene Copies in Transgenic Soybean by Taqman Quantitative PCR Technique

[Objective] Taqman Quantitative PCR technique was adopted to detect the copies of exogenous nos terminator in transgenic hybrid soybean.[Method] With soybean Lectin as the endogenous reference gene,and gene complex DNA in non-GMO soybeans as the endogenous reference standard,the method of gradient dilution was used for separately calculate Ct value of endogenous reference gene and plasmid DNA and correlation standard curve equation of logarithm of copies,and then to calculate the copies of samples through substituting thus-obtained Ct into the standard curve equation.[Result] The standard curve equation of endogenous reference gene is y=-3.422x＋35.201,R2=0.998;and the standard curve equation of exogenous gene is y=-3.348x＋34.890,R2=0.999.Nos terminator and its lower boundary sequences in transgenic soybean is of single copy.[Conclusion] The study has provided a theoretical basis for determining exogenous gene copies in transgenic soybean.

Determining the Copy Number of Exogenous Gene in Transgenic Plant by SYBR Green Real-time Quantitative PCR

[Objective] To explore the feasibility of using SYBR Green real-time quantitative PCR technique to estimate the copy numbers of exogenous gene in a transgenic plant.[Methods] Using SYBR Green real-time quantitative PCR technique,we have determined the copy numbers of the exogenous CYCD3;1 in transgenic Arabidopsis by comparing an endogenous single copy reference gene with CYCD3;1 copy numbers in transgenic plant,meanwhile comparing CYCD3;1 copy numbers between wild plant and transgenic plant.[Results]The exogenous CYCD3;1 copy numbers calculated by this method is identical with results of traditional Southern blot analysis which is highly accurate.[Conclusion]This method is simple,effective and safe for estimating transgene copy numbers.

Validation of housekeeping genes as internal controls for studying the gene expression in Pyropia haitanensis(Bangiales, Rhodophyta) by quantitative real-time PCR

Pyropia haitanensis is an economically important mariculture crop in China and has a high research value for several life phenomena, for example environmental tolerance. To explore the mechanisms underlying these characteristics, gene expression has been investigated at the whole transcriptome level. Gene expression studies using quantitative real-time PCR should start by selecting an appropriate internal control gene; therefore, the absolute expression abundance of six housekeeping genes （18S rRNA （18S）, ubiquitin-conju-ating enzyme （UBC）, actin （ACT）, β-tubulin （TUB）, elongation factors 2 （EF2）, and glyceraldehyde-3-phos- phate dehydrogenase （GAPDH） examined by the quantitative real-time PCR in samples corresponding to different strains, life-cycle stages and abiotic stress treatments. Their expression stabilities were assessed by the comparative cycle threshold （Ct） method and by two different software packages： geNorm and NormFinder. The most stable housekeeping gene is UBC and the least stable housekeeping is GADPH. Thus, it is proposed that the most appropriate internal control gene for expression analyses in P. haitanensis is UBC. The results pave the way for further gene expression analyses of different aspects of P. haitanensis biology including different strains, life-history stages and abiotic stress responses.

Quantitative Trait Loci for Heading Date and Their Relationship with Genetic Control of Yield Traits in Rice (Oryza sativa)

Grain yield and heading date are key factors determining the commercial potential of a rice variety. Mapping of quantitative trait loci （QTLs） in rice has been advanced from primary mapping to gene cloning, and heading date and yield traits have always attracted the greatest attention. In this review, genomic distribution of QTLs for heading date detected in populations derived from intra-specific crosses of Asian cultivated rice （Oryza sativa） was summarized, and their relationship with the genetic control of yield traits was analyzed. The information could be useful in the identification of QTLs for heading date and yield traits that are promising for the improvement of rice varieties.

Evaluation of reference genes for quantitative real-time PCR analysis of gene expression during early development processes of the tongue sole(Cynoglossus semilaevis)

Differential expression of genes is crucial to growth and development of fish. To select the appropriate genes for gene normalization during Cynoglossus semilaevis early developmental process, eight candidate reference genes （ACTB, B2M, EF1A, GADPH, RPL7, TUBA, UBCE and 18S） were tested for their adequacy by using quantitative real-time PCR. The results showed that the expression of all the examined genes exhibited tissue dependent variations in the mature C. semilaevis. EFIA was listed as the most stable reference among the 14 tissues by RefFinder. Furthermore, the recommended comprehensive ranking of the stability determined by RefFinder showed that 18S was the most stable gene during the early developmental stages （from oosphere to 90 days old） in this study. However, when divided the Ct value data of the above mentioned early developmental stages into two separate periods （embryo and post-hatching periods）, TUBA and 18S represented the most stable references of these two developmental periods, respectively. Consequently, the reference gene should be carefully and accurately chosen even for studies of the same species at various developmental processes. The relevant data may help in selecting appropriate reference genes for mRNA expression analysis, and is of great value in the studies of fish growth and development.

Joint Analysis Method for Major Genes Controlling Multiple Correlated Quantitative Traits

Based on the major gene and polygene mixed inheritance model for multiple correlated quantitative traits, the authors proposed a new joint segregation analysis method of major gene controlling multiple correlated quantitative traits, which include major gene detection and its effect and variation estimation. The effect and variation of major gene are estimated by the maximum likelihood method implemented via expectation-maximization （EM） algorithm. Major gene is tested with the likelihood ratio （LR） test statistic. Extensive simulation studies showed that joint analysis not only increases the statistical power of major gene detection but also improves the precision and accuracy of major gene effect estimates. An example of the plant height and the number of tiller of F2 population in rice cross Duonieai x Zhonghua 11 was used in the illustration. The results indicated that the genetic difference of these two traits in this cross refers to only one pleiotropic major gene. The additive effect and dominance effect of the major gene are estimated as -21.3 and 40.6 cm on plant height, and 22.7 and -25.3 on number of tiller, respectively. The major gene shows overdominance for plant height and close to complete dominance for number of tillers.

Selection of Reference Genes for Gene Expression Analysis in Nilaparvata lugens with Different Levels of Virulence on Rice by Quantitative Real-Time PCR

The brown planthopper Nilaparvata lugens Stal （Homoptera： Delphacidae） can cause hopperburn by feeding on rice and also can transmit the grassy stunt disease. Resistant rice varieties have been developed, but several N. lugens strains can recover their virulence to these resistant rice varieties. In the present study, reference genes with stable expression levels in N. lugens populations showed different levels of virulence to susceptible and resistant rice varieties. The expression of six candidate reference genes in N. lugens feeding on susceptible and resistant rice varieties was analyzed. These genes were evaluated for their potential use in the analysis of differential gene expression. Polymerase chain reaction data was generated from N. lugens, including two different treatments （resistant or susceptible rice） and three virulent N. lugens populations. Three software programs （BestKeeper, Normfinder and geNorm） were used to assess the candidate reference genes. Both geNorm and Normfinder identified the genes 18S, E-ACT, E-TUB and a-TUB as the most stable reference genes. BestKeeper identified ETIF1 as the optimal reference gene with the least overall variation, whereas 18S and a-TUB were the second and third most stably expressed genes, respectively. Therefore, we concluded that the genes 18S and a-TUB were the most suitable reference genes in N. lugens. These results will facilitate future transcript profiling studies on N. lugens populations that show variation in virulence levels on different rice varieties.

Incorporating empirical knowledge into data-driven variable selection for quantitative analysis of coal ash content by laser-induced breakdown spectroscopy

Laser-induced breakdown spectroscopy(LIBS)has become a widely used atomic spectroscopic technique for rapid coal analysis.However,the vast amount of spectral information in LIBS contains signal uncertainty,which can affect its quantification performance.In this work,we propose a hybrid variable selection method to improve the performance of LIBS quantification.Important variables are first identified using Pearson's correlation coefficient,mutual information,least absolute shrinkage and selection operator(LASSO)and random forest,and then filtered and combined with empirical variables related to fingerprint elements of coal ash content.Subsequently,these variables are fed into a partial least squares regression(PLSR).Additionally,in some models,certain variables unrelated to ash content are removed manually to study the impact of variable deselection on model performance.The proposed hybrid strategy was tested on three LIBS datasets for quantitative analysis of coal ash content and compared with the corresponding data-driven baseline method.It is significantly better than the variable selection only method based on empirical knowledge and in most cases outperforms the baseline method.The results showed that on all three datasets the hybrid strategy for variable selection combining empirical knowledge and data-driven algorithms achieved the lowest root mean square error of prediction(RMSEP)values of 1.605,3.478 and 1.647,respectively,which were significantly lower than those obtained from multiple linear regression using only 12 empirical variables,which are 1.959,3.718 and 2.181,respectively.The LASSO-PLSR model with empirical support and 20 selected variables exhibited a significantly improved performance after variable deselection,with RMSEP values dropping from 1.635,3.962 and 1.647 to 1.483,3.086 and 1.567,respectively.Such results demonstrate that using empirical knowledge as a support for datadriven variable selection can be a viable approach to improve the accuracy and reliability of LIBS quantification.

A Comparison Between Northern Blotting and Quantitative Real-Time PCR as a Means of Detecting the Nutritional Regulation of Genes Expressed in Roots of Arabidopsis thaliana

Quantitative real-time PCR （qRT-PCR） has become a routine and robust technique for measuring the expression of genes of interest, validating microarray experiments and monitoring biomarkers. However, concerns have been raised over the accuracy of qRT-PCR in China as well as in the rest of the world. We have previously used qRT-PCR to study the response of ANR1 and other root-expressed MADS-box genes to fluctuations in the supply of nitrate, phosphate and sulphate under hydroponic growth conditions. In this study, we have used both Northern blotting and qRT-PCR analyses to confirm the nutritional regulation of MADS-box genes in Arabidopsis thaliana and test whether both technologies produce the same results. The information obtained indicated that the qRT-PCR results are consistent with those obtained by Northern blotting hybridization for all the tested root-expressed MADS-box genes, in response to different nitrate, phosphate and sulphate growth conditions. Furthermore, our novel results showed that the expressions of AGL12, AGL18, and AGL19 were all down regulated in response to S and P re-supply in both qRT-PCR and Northern blotting analyses.

Reference genes for quantitative real-time PCR analysis and quantitative expression of P5CS in Agropyron mongolicum under drought stress

Reference genes, stably expressing in different tissues and cells, are commonly used as the references in expression analysis. Selecting the optimum reference gene is crucial to the success of experiments. In this study, the expression stabilities of nine common reference genes, including ACT2, 18 S r RNA, APRT, EF-1α, RNA POL II, TUBα, TUBβ, GAPDH and TLF of Agropyron mongolicum, were studied under drought condition. Among them, 18 S r RNA was found to be the most optimum reference gene under drought stress by the analyzing of ge Norm and Norm Finder software. Quantitative expression levels of P5 CS using 18 S r RNA as the reference gene, and proline contents under drought stress in A. mongolicum were further operated, and we found the expression level of P5 CS gene and proline content had a significantly positive relationship（R^2=0.7763, P〈0.05）. This study established and validated 18 S r RNA as the reference genes in A. mongolicum under drought stress, providing a powerful tool for the quantitative expression analysis of drought genes in A. mongolicum.

Major Gene Identification and Quantitative Trait Locus Mapping for YieldRelated Traits in Upland Cotton(Gossypium hirsutum L.)

Segregation analysis of the mixed genetic model of major gene plus polygene was used to identify the major genes for cotton yield-related traits using six generations P1, P2, F1, B1, B2, and F2 generated from the cross of Baimian 1 x TM-1. In addition to boll size and seed index, the major genes for the other five traits were detected： one each for seed yield, lint percentage, boll number, lint index; and two for lint yield. Quantitative trait locus/loci （QTL） mapping was performed in the F2 and F2：3 populations of above cross through molecular marker technology, and a total of 50 QTL （26 suggestive and 24 significant） for yield-related traits were detected. Four common QTL were discovered： qLP-3b（F2）/qLP-3（F2：3） and qLP-19b （F2）/qLP-19（F2：3） for lint percentage, qBN-17（F2）/qBN-17（F2：3） for boll number, and qBS-26b（F2）/qBS-26（F2：3） for boll size. Especially, qLP- 3b（Fz）/qLP-3（F2：3）, not only had LOD scores 〉3 but also exceeded the permutation threshold （5.13 and 5.29, respectively）, correspondingly explaining 23.47 and 29.55% of phenotypic variation. This QTL should be considered preferentially in marker assisted selection （MAS）. Segregation analysis and QTL mapping could mutually complement and verify, which provides a theoretical basis for genetic improvement of cotton yield-related traits by using major genes （QTL）.

High-density SNP genetic linkage map construction and quantitative trait locus mapping for resistance to cucumber mosaic virus in tobacco(Nicotiana tabacum L.)

Genetic linkage maps are essential for studies of genetics, genomic structure, and genomic evolution, and for mapping quantitative trait loci (QTL). Identification of molecular markers and construction of genetic linkage maps in tobacco (Nicotiana tabacum L.), a classical model plant and important economic crop, have remained limited. In the present study we identified a large number of single nucleotide polymorphism (SNP) markers and constructed a high-density SNP genetic map for tobacco using restriction site-associated DNA sequencing. In 1216.30 Gb of clean sequence obtained using the Illumina HiSeq 2000 sequencing platform, 99,647,735 SNPs were identified that differed between 203 sequenced plant genomes and the tobacco reference genome. Finally, 13,273 SNP markers were mapped on 24 high-density tobacco genetic linkage groups. The entire linkage map spanned 3421.80 cM, with a mean distance of 0.26 cM between adjacent markers. Compared with genetic linkage maps published previously, this version represents a considerable improvement in the number and density of markers. Seven QTL for resistance to cucumber mosaic virus (CMV) in tobacco were mapped to groups 5 and 8. This high-density genetic map is a promising tool for elucidation of the genetic bases of QTL and for molecular breeding in tobacco.

Validation of Reference Genes for Quantitative Real-Time PCR in Laodelphax striatellus

The normalization of quantitative real-time PCR （qPCR） is important to obtain accurate gene expression data, and the most common method for qPCR normalization is to use reference genes. However, reference genes can be regulated under different conditions, qPCR has recently been used for gene expression study in Laodelphax striatellus, but there is no study on validation of the reference genes. In this study, five new housekeeping genes （LstrTUB1, LstrTUB2, LstrTUB3, LstrARF and LstrRPL9） in L. striatellus were cloned and deposited in the GenBank with accession numbers of JF728809, JF728810, JF728811, JF728807 and JF728806, respectively. Furthermore, mRNA expressions of the five genes and β-actin were measured by qPCR with insect samples of different instar at nymph stage, and the expression stabilities were determined by the software geNorm and NormFinder. As a result, ARF and RPL9 were consistently more stable than β-actin, while three TUB genes were less stable than β-actin. To determine the optimal number of reference genes used in qPCR, a pairwise variations analysis by geNorm indicated that two references ARF and RPL9 were required to obtain the accurate quantification. These results were fiarther confirmed by the validation qPCR experiment with chitinase gene as the target gene, in which the standard error of the mRNA quantification by using binary reference ARF-RPL9 was much lower than those by ARF, RPL9 or β-actin alone. Taken together, our study suggested that the combination of ARF-RPL9 could replace β-actin as the reference genes for qPCR in L. striatellus.

Identification of normalization factors for quantitative realtime RT-PCR analysis of gene expression in Pacific abalone Haliotis discus hannai

Quantitative real-time reverse transcription-polymerase chain reaction （qRT-PCR） is widely used in studies of gene expression. In most of these studies, housekeeping genes are used as internal references without validation. To identify appropriate reference genes for qRT-PCR in Pacific abalone Haliotis discus hannai, we examined the transcription stability of six housekeeping genes in abalone tissues in the presence and absence of bacterial infection. For this purpose, abalone were infected with the bacterial pathogen Fibrio anguillarum for 12 h and 48 h. The mRNA levels of the housekeeping genes in five tissues （digestive glands, foot muscle, gill, hemocyte, and mantle） were determined by qRT-PCR. The PCR data was subsequently analyzed with the geNorm and NormFinder algorithms. The results show that in the absence of bacterial infection, elongation factor-l-alpha and beta-actin were the most stably expressed genes in all tissues, and thus are suitable as cross-tissue type normalization factors. However, we did not identify any universal reference genes post infection because the most stable genes varied between tissue types. Furthermore, for most tissues, the optimal reference genes identified by both algorithms at 12 h and 48 h post-infection differed. These results indicate that bacterial infection induced significant changes in the expression of abalone housekeeping genes in a manner that is dependent on tissue type and duration of infection. As a result, different normalization factors must be used for different tissues at different infection points.

Evaluation of Reference Genes for Quantitative Real-Time PCR Analysis in Manila Clam Ruditapes philippinarum Under Hypoxic Stress

Quantitative real-time PCR(qRT-PCR)has been widely used for gene expression analysis,and selection of reference genes is a key point to obtain accurate results.To find out optimal reference genes for qRT-PCR in Manila clam Ruditapes philippinarum in response to hypoxia,different tissues were used and compared to evaluate the stability of candidate reference genes under low oxygen stress(DO 0.5mgL^(−1) and DO 2.0mgL^(−1))and normal condition(DO 7.5mgL^(−1)).Seven candidate reference genes were selected to evaluate the stability of their expression levels.The reference genes were evaluated by Delta Ct,BestKeeper,NormFinder and geNorm,and then screened by RefFinder calculation.Under hypoxic stress of 0.5mgL^(−1),the most suitable reference gene for gill and hepatopancreas was RPL31,and the optimal reference genes for axe foot and adductor muscle were TUB and HIS,respectively.For hypoxic stress of 2.0mgL^(−1),the most stable reference gene for gill and hepatopancreas was RPL31,and the optimal reference genes for axe foot and adductor muscle were RPS23 and EF1A,respectively.At the normal condition,HIS and EF1A were identified as the optimal internal reference genes in gill and hepatopancreas respectively,and GFRP2 was the best internal reference gene for axe foot and adductor muscle.The present findings will provide important basis for the selection of reference genes for qRT-PCR analysis of gene expression level in bivalves under hypoxic stress,which might be helpful for the analysis of other molluscs too.

Establishment of Fluorescence Quantitative RT-PCR Assay for Detection of Equine Arteritis Virus

[Objective]The paper was to establish a fluorescence quantitative RT-PCR method for detection of equine arteritis virus(EAV).[Method]Primers and probes were developed for the EAV ORF7 gene sequence,and the reaction system was optimized.Standard curves were established,leading to the initial development of the EAV fluorescence quantitative RT-PCR assay.The accuracy,specificity,and sensitivity of this method were subsequently evaluated.[Result]The EAV fluorescence quantitative RT-PCR assay demonstrated optimal performance at an annealing temperature of 61 C,with a final concentration of primer and probe set at 0.6μmol/L.The plasmid standard demonstrated a strong linear correlation with Ct values within the range of 1.6×10^(7)-1.6×10^(2)copies/μL.The equation of the standard curve was determined to be y=-2.68x+32.88,with an R^(2) value of 0.9927.Consequently,the EAV fluorescence quantitative RT-PCR assay was successfully established.The methodology employed was effective in detecting EAV,Theileria equi,equine herpesvirus-1(EHV-1),equine herpesvirus-4(EHV-4),and equine influenza virus(EIV).The findings indicated that the method was specifically capable of detecting EAV,while the other pathogens tested yielded negative results.The method demonstrated a high degree of specificity.It was employed to detect the standard plasmid cRNA synthesized through in vitro transcription following a 10-fold dilution.The results indicated that the minimum detection limit of the method was 1.6×10^(2) copies/μL,and it exhibited high sensitivity.The coefficient of variation,both within and between groups,was maintained at 1.8%,indicating good reproducibility.In this study,the fluorescence quantitative RT-PCR assay developed was utilized alongside the EAV fluorescence quantitative RT-PCR assay established by previous researchers to analyze a total of 234 clinical samples.Both methods yielded a positive detection rate of 14.1%,and the coincidence rate between the two techniques was found to be 100%.[Conclusion]The fluorescence quantitative RT-PCR assay developed in this study offers a novel approach and concept for the prevention and control of equine viral arteritis(EVA).

Analyses and identifications of quantitative trait loci and candidate genes controlling mesocotyl elongation in rice

Rice direct seeding has the significant potential to save labor and water,conserve environmental resources,and reduce greenhouse gas emissions tremendously.Therefore,rice direct seeding is becoming the major cultivation technology applied to rice production in many countries.Identifying and utilizing genes controlling mesocotyl elongation is an effective approach to accelerate breeding procedures and meet the requirements for direct-seeded rice(DSR) production.This study used a permanent mapping population with 144 recombinant inbred lines(RILs) and 2 828 bin-markers to detect quantitative trait loci(QTLs) associated with mesocotyl length in 2019 and 2020.The mesocotyl lengths of the rice RILs and their parents,Lijiangxintuanheigu(LTH) and Shennong 265(SN265),were measured in a growth chamber at 30°C in a dark environment.A total of 16 QTLs for mesocotyl length were identified on chromosomes 1(2),2(4),3(2),4,5,6,7,9,11(2),and 12.Seven of these QTLs,including qML1a,qML1b,qML2d,qML3a,qML3b,qML5,and qML11b,were reproducibly detected in both years via the interval mapping method.The major QTL,qML3a,was reidentified in two years via the composite interval mapping method.A total of 10 to 413 annotated genes for each QTL were identified in their smallest genetic intervals of 37.69 kb to 2.78 Mb,respectively.Thirteen predicted genes within a relatively small genetic interval(88.18 kb) of the major mesocotyl elongation QTL,qML3a,were more thoroughly analyzed.Finally,the coding DNA sequence variations among SN265,LTH,and Nipponbare indicated that the LOC_Os03g50550 gene was the strongest candidate gene for the qML3a QTL controlling the mesocotyl elongation.This LOC_Os03g50550 gene encodes a mitogen-activated protein kinase.Relative gene expression analysis using qRT-RCR further revealed that the expression levels of the LOC_Os03g50550 gene in the mesocotyl of LTH were significantly lower than in the mesocotyl of SN265.In conclusion,these results further strengthen our knowledge about rice’s genetic mechanisms of mesocotyl elongation.This investigation’s discoveries will help to accelerate breeding programs for new DSR variety development.

Assessment of internal controls for data normalization of gene expression after different bacterial stimulation by quantitative real-time PCR in golden pompano Trachinotus blochii

Trachinotus blochii is one of the important commercial fish species.In this study,we aim to confirm the reliability reference genes in T.blochii during different bacterial challenge through quantitative real-time PCR(qRT-PCR).The expression of the seven selected genes in four immune organs(i.e.,spleen,kidney,intestine,and gill)stimulated with Vibrio harveyi,Edwardsiella tarda,and Streptococcus agalactiae were determined by qRT-PCR.The PCR data was analyzed using the geNorm and NormFinder algorithms.The results showed the selection of the internal controls should be tissue specific when studying gene expression in response to bacterial stimulation.After 48 h of stimulation with V.harveyi,geNorm ranked EF1 A/Actin,18 S rRNA/B2M,UBCE/B2M,and 18 S rRNA/B2M,as the most stably expressed genes in spleen,kidney,intestine,and gill,respectively.After 48 h of stimulation with E.tarda,geNorm ranked 18 S rRNA/EF1 A,18 S rRNA/B2M,B2M/RPL13,and 18 S rRNA/EF1 A,as the most stably expressed genes in spleen,kidney,intestine,and gill,respectively.After 48 h of stimulation with S.agalactiae,18 S rRNA/EF1 A,18 S rRNA/B2 M,B2 M/Actin,and 18 S rRNA/B2M were ranked as the most stably expressed genes in spleen,kidney,intestine,and gill,respectively.Compared to the results analyzed by geNorm,reference genes received similar rankings when using NormFinder software.The results showed that the reference genes appeared to be not only tissue specific,but also specific to the infecting species of bacteria.If one gene is preferred when T.blochii were infected by bacteria,18 S rRNA,B2M,B2M,18 S rRNA may be used in spleen,kidney,intestine,and gill,respectively.