Identification and Validation of Reference Genes for RT-qPCR Analysis in Non-Heading Chinese Cabbage Flowers.
ABSTRACT: Non-heading Chinese cabbage (Brassica rapa ssp. chinensis Makino) is an important vegetable member of Brassica rapa crops. It exhibits a typical sporophytic self-incompatibility (SI) system and is an ideal model plant to explore the mechanism of SI. Gene expression research are frequently used to unravel the complex genetic mechanism and in such studies appropriate reference selection is vital. Validation of reference genes have neither been conducted in Brassica rapa flowers nor in SI trait. In this study, 13 candidate reference genes were selected and examined systematically in 96 non-heading Chinese cabbage flower samples that represent four strategic groups in compatible and self-incompatible lines of non-heading Chinese cabbage. Two RT-qPCR analysis software, geNorm and NormFinder, were used to evaluate the expression stability of these genes systematically. Results revealed that best-ranked references genes should be selected according to specific sample subsets. DNAJ, UKN1, and PP2A were identified as the most stable reference genes among all samples. Moreover, our research further revealed that the widely used reference genes, CYP and ACP, were the least suitable reference genes in most non-heading Chinese cabbage flower sample sets. To further validate the suitability of the reference genes identified in this study, the expression level of SRK and Exo70A1 genes which play important roles in regulating interaction between pollen and stigma were studied. Our study presented the first systematic study of reference gene(s) selection for SI study and provided guidelines to obtain more accurate RT-qPCR results in non-heading Chinese cabbage.
Project description:BACKGROUND: The genomes of non-heading Chinese cabbage (Brassica rapa ssp. chinensis), heading Chinese cabbage (Brassica rapa ssp. pekinensis) and their close relative Arabidopsis thaliana have provided important resources for studying the evolution and genetic improvement of cruciferous plants. Natural growing conditions present these plants with a variety of physiological challenges for which they have a repertoire of genes that ensure adaptability and normal growth. We investigated the differential expressions of genes that control adaptability and development in plants growing in the natural environment to study underlying mechanisms of their expression. RESULTS: Using digital gene expression tag profiling, we constructed an expression profile to identify genes related to important agronomic traits under natural growing conditions. Among three non-heading Chinese cabbage cultivars, we found thousands of genes that exhibited significant differences in expression levels at five developmental stages. Through comparative analysis and previous reports, we identified several candidate genes associated with late flowering, cold tolerance, self-incompatibility, and leaf color. Two genes related to cold tolerance were verified using quantitative real-time PCR. CONCLUSIONS: We identified a large number of genes associated with important agronomic traits of non-heading Chinese cabbage. This analysis will provide a wealth of resources for molecular-assisted breeding of cabbage. The raw data and detailed results of this analysis are available at the website http://nhccdata.njau.edu.cn.
Project description:Leaf heads of cabbage (Brassica oleracea), Chinese cabbage (B. rapa), and lettuce (Lactuca sativa) are important vegetables that supply mineral nutrients, crude fiber and vitamins in the human diet. Head size, head shape, head weight, and heading time contribute to yield and quality. In an attempt to investigate genetic basis of leafy head in Chinese cabbage (B. rapa), we took advantage of recent technical advances of genome resequencing to perform quantitative trait locus (QTL) mapping using 150 recombinant inbred lines (RILs) derived from the cross between heading and non-heading Chinese cabbage. The resequenced genomes of the parents uncovered more than 1 million SNPs. Genotyping of RILs using the high-quality SNPs assisted by Hidden Markov Model (HMM) generated a recombination map. The raw genetic map revealed some physical assembly error and missing fragments in the reference genome that reduced the quality of SNP genotyping. By deletion of the genetic markers in which recombination rates higher than 20%, we have obtained a high-quality genetic map with 2209 markers and detected 18 QTLs for 6 head traits, from which 3 candidate genes were selected. These QTLs provide the foundation for study of genetic basis of leafy heads and the other complex traits.
Project description:Alterations in leaf adaxial-abaxial (ad-ab) polarity are one of the main factors that influence leaf curvature. In Chinese cabbage, leaf incurvature is an essential prerequisite to the formation of a leafy head. Identifying ad-ab patterning genes and investigating their genetic variation may facilitate elucidation of the mechanisms underlying leaf incurvature during head formation. Comparative genomic analysis of 45 leaf ad-ab patterning genes in Brassica rapa based on 26 homologs of Arabidopsis thaliana indicated that these genes underwent expansion and were retained after whole genome triplication (WGT). We also assessed the nucleotide diversity and selection footprints of these 45 genes in a collection of 94 Brassica rapa accessions that were composed of heading and non-heading morphotypes. Six of the 45 genes showed significant negative Tajima's D indices and nucleotide diversity reduction in heading accessions compared to those in non-heading accessions, indicating that they underwent purifying selection. Further testing of the BrARF3.1 gene, which was one of the selection signals from a larger collection, confirmed that purifying selection did occur. Our results provide genetic evidence that ad-ab patterning genes are involved in leaf incurvature, which is associated with formation of a leafy head, as well as promote an understanding of the genetic mechanism underlying leafy head formation in Chinese cabbage.
Project description:Chinese cabbage (Brassica rapa ssp. chinensis) is an economically and agriculturally significant vegetable crop and is extensively cultivated throughout the world. Heat stress disturbs cellular homeostasis and causes visible growth inhibition of shoots and roots, severe retardation in growth and development, and even death. However, there are few studies on the transcriptome profiling of heat stress in non-heading Chinese cabbage. In this study, we investigated the transcript profiles of non-heading Chinese cabbage from heat-sensitive and heat-tolerant varieties "GHA" and "XK," respectively, in response to high temperature using RNA sequencing (RNA seq). Approximately 625 genes were differentially expressed between the two varieties. The responsive genes can be divided into three phases along with the time of heat treatment: response to stimulus, programmed cell death and ribosome biogenesis. Differentially expressed genes (DEGs) were identified in the two varieties, including transcription factors (TFs), kinases/phosphatases, genes related to photosynthesis and effectors of homeostasis. Many TFs were involved in the heat stress response of Chinese cabbage, including NAC069 TF which was up-regulated at all the heat treatment stages. And their expression levels were also validated by quantitative real-time-PCR (qRT-PCR). These candidate genes will provide genetic resources for further improving the heat-tolerant characteristics in non-heading Chinese cabbage.
Project description:Numerous regulatory genes participate in plant thermotolerance. In Arabidopsis, HEAT-INDUCED TAS1 TARGET2 (HTT2) is an important thermotolerance gene that is silenced by ta-siR255, a trans-acting siRNA. ta-siR255 is absent from heading Chinese cabbage (Brassica rapa ssp. pekinensis). Our previous attempt to overexpress the endogenous BrpHTT2 gene of heading Chinese cabbage (B. rapa ssp. pekinensis) failed because of cosuppression. In theory, heading Chinese cabbage can overexpress Arabidopsis HTT2 to improve thermotolerance in the absence of ta-siR255-mediated gene silencing and the weak potential of coexpression.To test the potential application of HTT2 in improving crop thermotolerance, we transferred p35S::HTT2 to heading Chinese cabbage. We tested the leaf electrical conductivity, hypocotyl elongation, and survival percentage of p35S::HTT2 plants subjected to high-temperature (38 °C) and heat-shock (46 °C) treatment. The leaf electrical conductivity of p35S::HTT2 seedlings under high temperature decreased but did negligibly change under heat shock. The hypocotyl length of p35S::HTT2 seedlings increased under high temperature and heat shock. The survival rate of p35S::HTT2 seedlings increased under heat shock. BrpHsfs, a subset of heat-shock factor genes, were upregulated in p35S::HTT2 plants under high-temperature and heat shock conditions. In the field, transgenic plants with HTT2 appeared greener and formed leafy heads earlier than wild-type plants.Exogenous HTT2 increased the survival rates of heat-shocked heading Chinese cabbage by promoting thermotolerance through decreasing electrical conductivity and extending hypocotyl length. Our work provides a new approach to the genetic manipulation of thermotolerance in crops through the introduction of exogenous thermotolerance genes.
Project description:Long non-coding RNAs (lncRNAs) mediate important epigenetic regulation in various biological processes related to the stress response in plants. However, the systematic analysis of the lncRNAs expressed in Brassica rapa under heat stress has been elusive. In this study, we performed a genome-wide analysis of the lncRNA expression profiles in non-heading Chinese cabbage leaves using strand-specific RNA-sequencing. A total of 4594 putative lncRNAs were identified with a comprehensive landscape of dynamic lncRNA expression networks under heat stress. Co-expression networks of the interactions among the differentially expressed lncRNAs, mRNAs and microRNAs revealed that several phytohormones were associated with heat tolerance, including salicylic acid (SA) and brassinosteroid (BR) pathways. Of particular importance is the discovery of 25 lncRNAs that were highly co-expressed with 10 heat responsive genes. Thirty-nine lncRNAs were predicted as endogenous target mimics (eTMs) for 35 miRNAs, and five of them were validated to be involved in the heat tolerance of Chinese cabbage. Heat responsive lncRNA (TCONS_00048391) is an eTM for bra-miR164a, that could be a sponge for miRNA binding and may be a competing endogenous RNA (ceRNA) for the target gene NAC1 (Bra030820), affecting the expression of bra-miR164a in Chinese cabbage. Thus, these findings provide new insights into the functions of lncRNAs in heat tolerance and highlight a set of candidate lncRNAs for further studies in non-heading Chinese cabbage.
Project description:BACKGROUND: Brassica rapa is an economically important crop species. During its long breeding history, a large number of morphotypes have been generated, including leafy vegetables such as Chinese cabbage and pakchoi, turnip tuber crops and oil crops. RESULTS: To investigate the genetic variation underlying this morphological variation, we re-sequenced, assembled and annotated the genomes of two B. rapa subspecies, turnip crops (turnip) and a rapid cycling. We then analysed the two resulting genomes together with the Chinese cabbage Chiifu reference genome to obtain an impression of the B. rapa pan-genome. The number of genes with protein-coding changes between the three genotypes was lower than that among different accessions of Arabidopsis thaliana, which can be explained by the smaller effective population size of B. rapa due to its domestication. Based on orthology to a number of non-brassica species, we estimated the date of divergence among the three B. rapa morphotypes at approximately 250,000 YA, far predating Brassica domestication (5,000-10,000 YA). CONCLUSIONS: By analysing genes unique to turnip we found evidence for copy number differences in peroxidases, pointing to a role for the phenylpropanoid biosynthesis pathway in the generation of morphological variation. The estimated date of divergence among three B. rapa morphotypes implies that prior to domestication there was already considerably divergence among B. rapa genotypes. Our study thus provides two new B. rapa reference genomes, delivers a set of computer tools to analyse the resulting pan-genome and uses these to shed light on genetic drivers behind the rich morphological variation found in B. rapa.
Project description:Self-incompatibility (SI) is a genetic mechanism flowering plants adopted to reject self-pollen and promote outcrossing. In the Brassicaceae family plants, the stigma tissue plays a key role in self-pollen recognition and rejection. We reported earlier in Chinese cabbage (<i>Brassica rapa</i>) that stigma tissue showed upregulated ethylene responses and programmed cell death (PCD) upon compatible pollination, but not in SI responses. Here, we show that SI is significantly compromised or completely lost in senescent flowers and young flowers of senescent plants. Senescence upregulates senescence-associated genes in <i>B. rapa</i>. Suppressing their expression in young stigmas by antisense oligodeoxyribonucleotide abolishes compatible pollination-triggered PCD and inhibits the growth of compatible pollen tubes. Furthermore, ethylene biosynthesis genes and response genes are upregulated in senescent stigmas, and increasing the level of ethylene or inhibiting its response increases or decreases the expression of senescence-associated genes, respectively. Our results show that senescence causes PCD in stigmatic papilla cells and is associated with the breakdown of SI in Chinese cabbage and in radish.
Project description:In Chinese cabbage (Brassica rapa L. ssp. pekinensis), leaf adaxial-abaxial (ad-ab) polarity is tightly related to leaf incurvature, an essential factor for the formation of leafy heads. Therefore, identification of the genes responsible for leaf ad-ab polarity and studying their genetic variation may clarify the mechanism of leafy head formation. By comparing the sequences of the genes regulating leaf ad-ab polarity development in Arabidopsis thaliana (A. thaliana), 41 candidate genes distributed on 10 chromosomes were found to be responsible for the establishment of ad-ab polarity in Chinese cabbage. Orthologous genes, including 10 single copies, 14 double copies, and one triple copies, were detected in the Chinese cabbage. The gene structure and conserved domain analyses showed that the number of exons of the 41 candidate genes range from one to 25, and that most genes share the conserved motifs 1, 6, and 10. Based on the 41 candidate genes, 341 simple sequence repeats (SSRs) were detected, including five replicated types: single, double, triple, quintuple, and sextuple nucleotide replications. Among these sequence repeat (SSR) loci, 323 loci were used to design 969 specific primers, and 362 primer pairs were selected randomly and evaluated using 12 Chinese cabbage accessions with different heading types. 23 primer pairs resulting with clear, polymorphic bands, combined with other 127 markers, was used to construct a linkage map by using an F2 population containing 214 lines derived from the hybrid of the overlapping heading Chinese cabbage "14Q-141" and the outward curling heading Chinese cabbage "14Q-279." The result showed that the sequences of markers in the genetic linkage map and the physical map was consistent in general. Our study could help to accelerate the breeding process of leafy head quality in Chinese cabbage.
Project description:BACKGROUND:Members of the MtN3/saliva/SWEET gene family are present in various organisms and are highly conserved. Their precise biochemical functions remain unclear, especially in Chinese cabbage. Based on the whole genome sequence, this study aims to identify the MtN3/saliva/SWEETs family members in Chinese cabbage and to analyze their classification, gene structure, chromosome distribution, phylogenetic relationship, expression pattern, and biological functions. RESULTS:We identified 34 SWEET genes in Chinese cabbage and analyzed their localization on chromosomes and transmembrane regions of their corresponding proteins. Analysis of a phylogenetic tree indicated that there were at least 17 supposed ancestor genes before the separation in Brassica rapa and Arabidopsis. The expression patterns of these genes in different tissues and flower developmental stages of Chinese cabbage showed that they are mainly involved in reproductive development. The Ka/Ks ratio between paralogous SWEET gene pairs of B. rapa were far less than 1. In our previous study, At2g39060 homologous gene Bra000116 (BraSWEET9, also named BcNS, Brassica Nectary and Stamen) played an important role during flower development in Chinese cabbage. Instantaneous expression experiments in onion epidermal cells showed that the gene encoding this protein is localized to the plasma membrane. A basal nectary split is the phenotype of transgenic plants transformed with the antisense expression vector. CONCLUSION:This study is the first to perform a sequence analysis, structures analysis, physiological and biochemical characteristics analysis of the MtN3/saliva/SWEETs gene in Chinese cabbage and to verify the function of BcNS. A total of 34 SWEET genes were identified and they are distributed among ten chromosomes and one scaffold. The Ka/Ks ratio implies that the duplication genes suffered strong purifying selection for retention. These genes were differentially expressed in different floral organs. The phenotypes of the transgenic plants indicated that BcNs participates in the development of the floral nectary. This study provides a basis for further functional analysis of the MtN3/saliva/SWEETs gene family.