Integrated QTL and eQTL Mapping Provides Insights and Candidate Genes for Fatty Acid Composition, Flowering Time, and Growth Traits in a F2 Population of a Novel Synthetic Allopolyploid Brassica napus.
ABSTRACT: Brassica napus (B. napus, AACC), is an economically important allotetraploid crop species that resulted from hybridization between two diploid species, Brassica rapa (AA) and Brassica olereacea (CC). We have created one new synthetic B. napus genotype Da-Ae (AACC) and one introgression line Da-Ol-1 (AACC), which were used to generate an F2 mapping population. Plants in this F2 mapping population varied in fatty acid content, flowering time, and growth-related traits. Using quantitative trait locus (QTL) mapping, we aimed to determine if Da-Ae and Da-Ol-1 provided novel genetic variation beyond what has already been found in B. napus. Making use of the genotyping information generated from RNA-seq data of these two lines and their F2 mapping population of 166 plants, we constructed a genetic map consisting of 2,021 single nucleotide polymorphism markers that spans 2,929 cM across 19 linkage groups. Besides the known major QTL identified, our high resolution genetic map facilitated the identification of several new QTL contributing to the different fatty acid levels, flowering time, and growth-related trait values. These new QTL probably represent novel genetic variation that existed in our new synthetic B. napus strain. By conducting genome-wide expression variation analysis in our F2 mapping population, genetic regions that potentially regulate many genes across the genome were revealed. A FLOWERING LOCUS C gene homolog, which was identified as a candidate regulating flowering time and multiple growth-related traits, was found underlying one of these regions. Integrated QTL and expression QTL analyses also helped us identified candidate causative genes associated with various biological traits through expression level change and/or possible protein function modification.
Project description:The gene FLOWERING LOCUS T (FT) and its orthologues play a central role in the integration of flowering signals within Arabidopsis and other diverse species. Multiple copies of FT, with different cis-intronic sequence, exist and appear to operate harmoniously within polyploid crop species such as Brassica napus (AACC), a member of the same plant family as Arabidopsis.We have identified six BnFT paralogues from the genome of B. napus and mapped them to six distinct regions, each of which is homologous to a common ancestral block (E) of Arabidopsis chromosome 1. Four of the six regions were present within inverted duplicated regions of chromosomes A7 and C6. The coding sequences of BnFT paralogues showed 92-99% identities to each other and 85-87% identity with that of Arabidopsis. However, two of the paralogues on chromosomes A2 and C2, BnA2.FT and BnC2.FT, were found to lack the distinctive CArG box that is located within intron 1 that has been shown in Arabidopsis to be the binding site for theFLC protein. Three BnFT paralogues (BnA2.FT, BnC6.FT.a and BnC6.FT.b) were associated with two major QTL clusters for flowering time. One of the QTLs encompassing two BnFT paralogues (BnC6.FT.a and BnC6.FT.b) on chromosome C6 was resolved further using near isogenic lines, specific alleles of which were both shown to promote flowering. Association analysis of the three BnFT paralogues across 55 cultivars of B. napus showed that the alleles detected in the original parents of the mapping population used to detect QTL (NY7 and Tapidor) were ubiquitous amongst spring and winter type cultivars of rapeseed. It was inferred that the ancestral FT homologues in Brassica evolved from two distinct copies, one of which was duplicated along with inversion of the associated chromosomal segment prior to the divergence of B. rapa (AA) and B. oleracea (CC). At least ten such inverted duplicated blocks (IDBs) were identified covering a quarter of the whole B. napus genome.Six orthologues of Arabidopsis FT were identified and mapped in the genome of B. napus which sheds new light on the evolution of paralogues in polyploidy species. The allelic variation of BnFT paralogues results in functional differences affecting flowering time between winter and spring type cultivars of oil seed Brassica. The prevalent inverted duplicated blocks, two of which were located by four of the six BnFT paralogues, contributed to gene duplications and might represent predominant pathway of evolution in Brassica.
Project description:Most agronomical traits exhibit quantitative variation, which is controlled by multiple genes and are environmentally dependent. To study the genetic variation of flowering time in Brassica napus, a DH population and its derived reconstructed F(2) population were planted in 11 field environments. The flowering time varied greatly with environments; 60% of the phenotypic variation was attributed to genetic effects. Five to 18 QTL at a statistically significant level (SL-QTL) were detected in each environment and, on average, two new SL-QTL were discovered with each added environment. Another type of QTL, micro-real QTL (MR-QTL), was detected repeatedly from at least 2 of the 11 environments; resulting in a total of 36 SL-QTL and 6 MR-QTL. Sixty-three interacting pairs of loci were found; 50% of them were involved in QTL. Hundreds of floral transition genes in Arabidopsis were aligned with the linkage map of B. napus by in silico mapping; 28% of them aligned with QTL regions and 9% were consistent with interacting loci. One locus, BnFLC10, in N10 and a QTL cluster in N16 were specific to spring- and winter-cropped environments respectively. The number of QTL, interacting loci, and aligned functional genes revealed a complex genetic network controlling flowering time in B. napus.
Project description:Earliness of flowering and maturity are important traits in spring Brassica napus canola-whether grown under long- or short-day condition. By use of a spring B. napus mapping population carrying the genome content of B. oleracea and testing this population under 10 to 18 h photoperiod and 18 to 20 0C (day) temperature conditions, we identified a major QTL on the chromosome C1 affecting flowering time without being influenced by photoperiod and temperature, and a major QTL on C9 affecting flowering time under a short photoperiod (10 h); in both cases, the QTL alleles reducing the number of days to flowering in B. napus were introgressed from the late flowering species B. oleracea. Additive effect of the C1 QTL allele at 14 to18 h photoperiod was 1.1 to 2.9 days; however, the same QTL allele exerted an additive effect of 6.2 days at 10 h photoperiod. Additive effect of the C9 QTL at 10 h photoperiod was 2.8 days. These two QTL also showed significant interaction in the control of flowering only under a short-day (10 h photoperiod) condition with an effect of 2.3 days. A few additional QTL were also detected on the chromosomes C2 and C8; however, none of these QTL could be detected under all photoperiod and temperature conditions. BLASTn search identified several putative flowering time genes on the chromosomes C1 and C9 and located the physical position of the QTL markers in the Brassica genome; however, only a few of these genes were found within the QTL region. Thus, the molecular markers and the genomic regions identified in this research could potentially be used in breeding for the development of early flowering photoinsensitive B. napus canola cultivars, as well as for identification of candidate genes involved in flowering time variation and photosensitivity.
Project description:BACKGROUND: Tocopherols are important antioxidants in vegetable oils; when present as vitamin E, tocopherols are an essential nutrient for humans and livestock. Rapeseed (Brassica napus L, AACC, 2 n = 38) is one of the most important oil crops and a major source of tocopherols. Although the tocopherol biosynthetic pathway has been well elucidated in the model photosynthetic organisms Arabidopsis thaliana and Synechocystis sp. PCC6803, knowledge about the genetic basis of tocopherol biosynthesis in seeds of rapeseed is scant. This project was carried out to dissect the genetic basis of seed tocopherol content and composition in rapeseed through quantitative trait loci (QTL) detection, genome-wide association analysis, and homologous gene mapping. METHODOLOGY/PRINCIPAL FINDINGS: We used a segregating Tapidor × Ningyou7 doubled haploid (TNDH) population, its reconstructed F(2) (RC-F(2)) population, and a panel of 142 rapeseed accessions (association panel). Genetic effects mainly contributed to phenotypic variations in tocopherol content and composition; environmental effects were also identified. Thirty-three unique QTL were detected for tocopherol content and composition in TNDH and RC-F(2) populations. Of these, seven QTL co-localized with candidate sequences associated with tocopherol biosynthesis through in silico and linkage mapping. Several near-isogenic lines carrying introgressions from the parent with higher tocopherol content showed highly increased tocopherol content compared with the recurrent parent. Genome-wide association analysis was performed with 142 B. napus accessions. Sixty-one loci were significantly associated with tocopherol content and composition, 11 of which were localized within the confidence intervals of tocopherol QTL. CONCLUSIONS/SIGNIFICANCE: This joint QTL, candidate gene, and association mapping study sheds light on the genetic basis of seed tocopherol biosynthesis in rapeseed. The sequences presented here may be used for marker-assisted selection of oilseed rape lines with superior tocopherol content and composition.
Project description:Brassica napus is an important oilseed crop. Dissection of the genetic architecture underlying oil-related biological processes will greatly facilitates the genetic improvement of rapeseed. The differential gene expression during pod development offers a snapshot on the genes responsible for oil accumulation in. To identify candidate genes in the linkage peaks reported previously, we used RNA sequencing (RNA-Seq) technology to analyze the pod transcriptomes of German cultivar Sollux and Chinese inbred line Gaoyou.The RNA samples were collected for RNA-Seq at 5-7, 15-17 and 25-27 days after flowering (DAF). Bioinformatics analysis was performed to investigate differentially expressed genes (DEGs). Gene annotation analysis was integrated with QTL mapping and Brassica napus pod transcriptome profiling to detect potential candidate genes in oilseed.Four hundred sixty five and two thousand, one hundred fourteen candidate DEGs were identified, respectively, between two varieties at the same stages and across different periods of each variety. Then, 33 DEGs between Sollux and Gaoyou were identified as the candidate genes affecting seed oil content by combining those DEGs with the quantitative trait locus (QTL) mapping results, of which, one was found to be homologous to Arabidopsis thaliana lipid-related genes.Intervarietal DEGs of lipid pathways in QTL regions represent important candidate genes for oil-related traits. Integrated analysis of transcriptome profiling, QTL mapping and comparative genomics with other relative species leads to efficient identification of most plausible functional genes underlying oil-content related characters, offering valuable resources for bettering breeding program of Brassica napus.This study provided a comprehensive overview on the pod transcriptomes of two varieties with different oil-contents at the three developmental stages.
Project description:BACKGROUND:Optimum flowering time is a key agronomic trait in Brassica napus. To investigate the genetic architecture and genetic regulation of flowering time in this important crop, we conducted quantitative trait loci (QTL) analysis of flowering time in a recombinant inbred line (RIL) population, including lines with extreme differences in flowering time, in six environments, along with RNA-Seq analysis. RESULTS:We detected 27 QTLs distributed on eight chromosomes among six environments, including one major QTL on chromosome C02 that explained 11-25% of the phenotypic variation and was stably detected in all six environments. RNA-Seq analysis revealed 105 flowering time-related differentially expressed genes (DEGs) that play roles in the circadian clock/photoperiod, autonomous pathway, and hormone and vernalization pathways. We focused on DEGs related to the regulation of flowering time, especially DEGs in QTL regions. CONCLUSIONS:We identified 45 flowering time-related genes in these QTL regions, eight of which are DEGs, including key flowering time genes PSEUDO RESPONSE REGULATOR 7 (PRR7) and FY (located in a major QTL region on C02). These findings provide insights into the genetic architecture of flowering time in B. napus.
Project description:Flowering time adaptation is a major breeding goal in the allopolyploid species Brassica napus. To investigate the genetic architecture of flowering time, a genome-wide association study (GWAS) of flowering time was conducted with a diversity panel comprising 523 B. napus cultivars and inbred lines grown in eight different environments. Genotyping was performed with a Brassica 60K Illumina Infinium SNP array. A total of 41 single-nucleotide polymorphisms (SNPs) distributed on 14 chromosomes were found to be associated with flowering time, and 12 SNPs located in the confidence intervals of quantitative trait loci (QTL) identified in previous researches based on linkage analyses. Twenty-five candidate genes were orthologous to Arabidopsis thaliana flowering genes. To further our understanding of the genetic factors influencing flowering time in different environments, GWAS was performed on two derived traits, environment sensitivity and temperature sensitivity. The most significant SNPs were found near Bn-scaff_16362_1-p380982, just 13 kb away from BnaC09g41990D, which is orthologous to A. thaliana CONSTANS (CO), an important gene in the photoperiod flowering pathway. These results provide new insights into the genetic control of flowering time in B. napus and indicate that GWAS is an effective method by which to reveal natural variations of complex traits in B. napus.
Project description:Flowering, the transition from the vegetative to the generative phase, is a decisive time point in the lifecycle of a plant. Flowering is controlled by a complex network of transcription factors, photoreceptors, enzymes and miRNAs. In recent years, several studies gave rise to the hypothesis that this network is also strongly involved in the regulation of other important lifecycle processes ranging from germination and seed development through to fundamental developmental and yield-related traits. In the allopolyploid crop species Brassica napus, (genome AACC), homoeologous copies of flowering time regulatory genes are implicated in major phenological variation within the species, however the extent and control of intraspecific and intergenomic variation among flowering-time regulators is still unclear. To investigate differences among B. napus morphotypes in relation to flowering-time gene variation, we performed targeted deep sequencing of 29 regulatory flowering-time genes in four genetically and phenologically diverse B. napus accessions. The genotype panel included a winter-type oilseed rape, a winter fodder rape, a spring-type oilseed rape (all B. napus ssp. napus) and a swede (B. napus ssp. napobrassica), which show extreme differences in winter-hardiness, vernalization requirement and flowering behavior. A broad range of genetic variation was detected in the targeted genes for the different morphotypes, including non-synonymous SNPs, copy number variation and presence-absence variation. The results suggest that this broad variation in vernalization, clock and signaling genes could be a key driver of morphological differentiation for flowering-related traits in this recent allopolyploid crop species.
Project description:Background:Brassica napus is one of the most important oilseed crops, and also an important biofuel plant due to its low air pollution and renewability. Growth period are important traits that affect yield and are crucial for its adaptation to different environments in B. napus. Results:To elucidate the genetic basis of growth period traits, genome-wide association analysis (GWAS) and linkage mapping were employed to detect the quantitative trait loci (QTL) for days to initial flowering (DIF), days to final flowering (DFF), flowering period (FP), maturity time (MT), and whole growth period (GP). A total of 146 SNPs were identified by association mapping, and 83 QTLs were identified by linkage mapping using the RIL population. Among these QTLs, 19 were pleiotropic SNPs related to multiple traits, and six (q18DFF.A03-2, q18MT.A03-2, q17DFF.A05-1, q18FP.C04, q17DIF.C05 and q17GP.C09) were consistently detected using both mapping methods. Additionally, we performed RNA sequencing to analyze the differential expression of gene (DEG) transcripts between early- and late-flowering lines selected from the RIL population, and the DEGs were integrated with association mapping and linkage analysis to confirm their roles in the growth period. Consequently, 12 candidate genes associated with growth period traits were identified in B. napus. Among these genes, seven have polymorphic sites in the coding sequence and the upstream 2-kb sequence based on the resequencing data. The haplotype BnaSOC1.A05-Haplb and BnaLNK2.C06-Hapla showed more favorable phenotypic traits. Conclusions:The candidate genes identified in this study will contribute to our genetic understanding of growth period traits and can be used as targets for target mutations or marker-assisted breeding for rapeseed adapted to different environments.
Project description:Silique length (SL) and seed weight (SW) are important yield-associated traits in rapeseed (Brassica napus). Although many quantitative trait loci (QTL) for SL and SW have been identified in B. napus, comparative analysis for those QTL is seldom performed. In the present study, 20 and 21 QTL for SL and SW were identified in doubled haploid (DH) and DH-derived reconstructed F2 populations in rapeseed, explaining 55.1-74.3% and 24.4-62.9% of the phenotypic variation across three years, respectively. Of which, 17 QTL with partially or completely overlapped confidence interval on chromosome A09, were homologous with two overlapped QTL on chromosome C08 by aligning QTL confidence intervals with the reference genomes of Brassica crops. By high density selective genotyping of DH lines with extreme phenotypes, using a Brassica single-nucleotide polymorphism (SNP) array, the QTL on chromosome A09 was narrowed, and aligned into 1.14-Mb region from 30.84 to 31.98?Mb on chromosome R09 of B. rapa and 1.05-Mb region from 27.21 to 28.26?Mb on chromosome A09 of B. napus. The alignment of QTL with Brassica reference genomes revealed homologous QTL on A09 and C08 for SL. The narrowed QTL region provides clues for gene cloning and breeding cultivars by marker-assisted selection.