Jiefu Zhang

High-Density SNP Map Construction and QTL Identification for the Apetalous Character in Brassica napus L.

The apetalous genotype is a morphological ideotype for increasing seed yield and should be of considerable agricultural use; however, only a few studies have focused on the genetic control of this trait in Brassica napus. In the present study, a recombinant inbred line, the AH population, containing 189 individuals was derived from a cross between an apetalous line ‘APL01’ and a normally petalled variety ‘Holly’. The Brassica 60 K Infinium BeadChip Array harboring 52,157 single nucleotide polymorphism (SNP) markers was used to genotype the AH individuals. A high-density genetic linkage map was constructed based on 2,755 bins involving 11,458 SNPs and 57 simple sequence repeats, and was used to identify loci associated with petalous degree (PDgr). The linkage map covered 2,027.53 cM, with an average marker interval of 0.72 cM. The AH map had good collinearity with the B. napus reference genome, indicating its high quality and accuracy. After phenotypic analyses across five different experiments, a total of 19 identified quantitative trait loci (QTLs) distributed across chromosomes A3, A5, A6, A9 and C8 were obtained, and these QTLs were further integrated into nine consensus QTLs by a meta-analysis. Interestingly, the major QTL qPD.C8-2 was consistently detected in all five experiments, and qPD.A9-2 and qPD.C8-3 were stably expressed in four experiments. Comparative mapping between the AH map and the B. napus reference genome suggested that there were 328 genes underlying the confidence intervals of the three steady QTLs. Based on the Gene Ontology assignments of 52 genes to the regulation of floral development in published studies, 146 genes were considered as potential candidate genes for PDgr. The current study carried out a QTL analysis for PDgr using a high-density SNP map in B. napus, providing novel targets for improving seed yield. These results advanced our understanding of the genetic control of PDgr regulation in B. napus.

QTL Identification for Fatty Acid Content in Rapeseed (Brassica napus L.)

Abstract A segregation population BC 1 F 1 of rapeseed ( Brassica napus L.), derived from a cross between a low erucic acid content line APL01 and a high erucic acid content variety M083, was used to construct the genetic map and identify auantitave trait loci (QTLs) linked to main fatty acid composition in rapeseed. The genetic map containing 19 linkage groups was constructed with 251 markers of RAPD, SSR, and SRAP. By means of WinQTLCart scanning, 5 QTLs linked to palmitic acid (C16:0) content were identified, which were located on linkage groups N3, N8, N10, and N13, respectively. The main-effect QTLs qPA8-1 and qPA13 linked to palmatic acid content in rapeseed explained 11.31% and 14.47% of the palmitic acid content variation in the segregation population BC 1 F 1 , respectively. Three QTLs linked to stearic acid (C18:0) content were identified, which were located on the linkage groups N1, N8, and N16. The main-effect QTL qST16 accounted for 12.22% of phenotypic variation. Two QTLs, qOL8 and qOL13 , the main-effect QTLs linked to oleic acid (C18:1) content, were located in the region of m11e37b – A0226Ba267 on linkage group N8 and m18e46 – m20e25a on N13. These QTLs accounted for 11.73% and 27.14% of phenotypic variation, respectively. Three QTLs linked to linoleic acid (C18:2) content were identified, and the main-effect QTL qLI8-1 accounted for 13.25% of phenotypic variation. Three QTLs, qLN1, qLN8 , and qLN11 , linked to linolenic acid (C18:3) content, were identified, but they had small effects on C18:3 and could be regarded as minor-QTLs. Four QTLs were linked to eicosenoic acid (C20:1) content, and three of them, qEI8-1, qEI8-2 , and qEI13 , were main-effect QTLs, accounting for 12.20%, 10.22%, and 11.14% of phenotypic variation, respectively. Two main-effect QTLs linked to erucic acid (C22:1) content, qER8 and qER13 , were located in the region of m11e37b – A0226Ba267 on linkage group N8 and A0301Bb398 – m18e46 on N13, accounted for 16.74% and 31.32% of phenotypic variation, respectively. The main-effect QTLs linked to fatty acid content, which were located in the region near marker m11e27b on linkage group N8 and m18e46 on linkage group N13, could be used in marker-assisted selection in fatty acid improvement in rapeseed.

Genome-wide transcriptomic analysis uncovers the molecular basis underlying early flowering and apetalous characteristic in Brassica napus L

Floral transition and petal onset, as two main aspects of flower development, are crucial to rapeseed evolutionary success and yield formation. Currently, very little is known regarding the genetic architecture that regulates flowering time and petal morphogenesis in Brassica napus. In the present study, a genome-wide transcriptomic analysis was performed with an absolutely apetalous and early flowering line, APL01, and a normally petalled line, PL01, using high-throughput RNA sequencing. In total, 13,205 differential expressed genes were detected, of which 6111 genes were significantly down-regulated, while 7094 genes were significantly up-regulated in the young inflorescences of APL01 compared with PL01. The expression levels of a vast number of genes involved in protein biosynthesis were altered in response to the early flowering and apetalous character. Based on the putative rapeseed flowering genes, an early flowering network, mainly comprised of vernalization and photoperiod pathways, was built. Additionally, 36 putative upstream genes possibly governing the apetalous character of line APL01 were identified, and six genes potentially regulating petal origination were obtained by combining with three petal-related quantitative trait loci. These findings will facilitate understanding of the molecular mechanisms underlying floral transition and petal initiation in B. napus.

Unconditional and conditional QTL analyses of seed fatty acid composition in Brassica napus L.

BackgroundThe fatty acid composition of B. napus’ seeds determines the oil’s nutritional and industrial values, and affects seed germination. Many studies have reported correlations among C16:0, C18:0, C18:1, C18:2 and C18:3 based on phenotypic data; however, the genetic basis of the fatty acid composition in B. napus is still not well understood.ResultsIn this study, unconditional and conditional quantitative trail locus (QTL) mapping analyses were conducted using a recombinant inbred line in six environments. In total, 21 consensus QTLs each for C16:0, C18:0 and C18:2, 16 for C18:1 and 22 for C18:3 were detected by unconditional mapping. The QTLs with overlapping confidence intervals were integrated into 71 pleiotropically unique QTLs by meta-analysis. Two major QTLs, uuqA5–6 and uuqA5–7, simultaneously affected the fatty acids, except C18:0, in most of environments, with the homologous genes fatty acid desaturase 2 (FAD2) and glycerol-3-phosphate sn-2-acyltransferase 5 (GPAT5) occurring in the confidence interval of uuqA5–6, while phosphatidic acid phosphohydrolase 1 (PAH1) was assigned to uuqA5–7. Moreover, 49, 30, 48, 60 and 45 consensus QTLs were detected for C16:0, C18:0, C18:1, C18:2 and C18:3, respectively, by the conditional mapping analysis. In total, 128 unique QTLs were subsequently integrated from the 232 conditional consensus QTLs. A comparative analysis revealed that 63 unique QTLs could be identified by both mapping methodologies, and 65 additional unique QTLs were only identified in conditional mapping.ConclusionsThus, conditional QTL mapping for fatty acids may uncover numerous additional QTLs that were inhibited by the effects of other traits. These findings provide useful information for better understanding the genetic relationships among fatty acids at the QTL level.

Inheritance and molecular characterization of resistance to AHAS-inhibiting herbicides in rapeseed

Abstract Rapeseed is a very important oil crop in China; however, its production is challenging due to the absence of effective weed management strategies. This is predominantly because of a shortage of herbicide resistance genes. Acetohydroxyacid synthase (AHAS) herbicides inhibit AHAS, a key enzyme involved in branched-chain amino acid synthesis that is required for plant growth. A rapeseed line designated M342 with AHAS herbicide resistance was developed through seed mutagenesis and was studied to assess the level and mode of inheritance of the resistance and to identify the molecular basis of resistance. M342 possessed a high level of cross-resistance to sulfonylureas (SUs) and imidazolinones (IMIs). This resistance was due to AHAS insensitivity to these herbicides and was inherited as a dominant trait conferred by a single nuclear-encoded gene. Molecular analysis revealed the presence of a Trp574Leu mutation in M342, and an allele-specific cleaved amplified polymorphic sequence (AS-CAPS) marker was developed and cosegregated with herbicide resistance in the F2, BC1, and BC2 populations. This mutation altered the transcript levels of BnAHAS1 and BnAHAS3 in M342 compared with those in the wild type, but it did not affect the agronomic or quality traits. The simple genetic inheritance of this mutation and the availability of the cleaved amplified polymorphic sequence (CAPS) marker and herbicide resistance gene should facilitate the development of herbicide-resistant rapeseed cultivars for effective weed control in China.

Development and Application of Allele-Specific PCR Markers for Imidazolinone-Resistant Gene BnALS1R in Brassica napus

A BnALS1R gene conferring resistance to the imidazolinone herbicides was previously isolated from an ALS(acetolactate synthase)inhabiting herbicide-resistant mutant line M9 in Brassica napus.A single nucleotide polymorphism leading to an amino acid substitution from serine to asparagine at site 638 of ALS was found between the BnALS1R in the mutant and BnALS1 in the wild type.We reported here the development of polymorphic molecular marker for allele-specific PCR(AS-PCR)assays to distinguish herbicide-susceptible and resistant ALS alleles in either homozygous or heterozygous genotypes.Thirty primers were designed according to the SNP(site 638,Ser was replaced by Asn)and the difference in DNA sequence between the BnALS1 and BnALS3 cloned from M9 and other susceptible cultivars.Three primers of them with polymorphism were obtained,which can differentiate the homozygous resistant M9 from susceptible rapeseed.Meanwhile,these PCR markers can effectively discriminate among three genotypes using polymorphic primers and were evaluated in F2,BC1,and BC2 populations.Observed segregation ratios fitted the expected 1 2 1,1 1,and 1 1 ratios,respectively,which confirms the single-locus Mendel model.Furthermore,we used the markers for detecting the resistant gene and evaluating the size of DNA segments introgressed from the M9 donor in the resistant restoring lines containing BnALS1R.All the resistant restoring lines had the DNA banding pattern of BnALS1R.Therefore,PCR-based assays using the markers could be used for the detection and characterization of the herbicide-resistant gene BnALS1R in rapeseed.The validated AS-PCR markers will facilitate the breeding of herbicide-resistant rapeseed by using BnALS1R in marker-assisted selection.

Quantitative Trait Transcripts Mapping Coupled with Expression Quantitative Trait Loci Mapping Reveal the Molecular Network Regulating the Apetalous Characteristic in Brassica napus L.

The apetalous trait of rapeseed (Brassica napus, AACC, 2n = 38) is important for breeding an ideal high-yield rapeseed with superior klendusity to Sclerotinia sclerotiorum. Currently, the molecular mechanism underlying the apetalous trait of rapeseed is unclear. In this study, 14 petal regulators genes were chosen as target genes (TGs), and the expression patterns of the 14 TGs in the AH population, containing 189 recombinant inbred lines derived from a cross between apetalous “APL01” and normal “Holly,” were analyzed in two environments using qRT-PCR. Phenotypic data of petalous degree (PDgr) in the AH population were obtained from the two environments. Both quantitative trait transcript (QTT)-association mapping and expression QTL (eQTL) analyses of TGs expression levels were performed to reveal regulatory relationships among TGs and PDgr. QTT mapping for PDgr determined that PLURIPETALA (PLP) was the major negative QTT associated with PDgr in both environments, suggesting that PLP negatively regulates the petal development of line “APL01.” The QTT mapping of PLP expression levels showed that CHROMATIN-REMODELING PROTEIN 11 (CHR11) was positively associated with PLP expression, indicating that CHR11 acts as a positive regulator of PLP expression. Similarly, QTT mapping for the remaining TGs identified 38 QTTs, associated with 13 TGs, and 31 QTTs, associated with 10 TGs, respectively, in the first and second environments. Additionally, eQTL analyses of TG expression levels showed that 12 and 11 unconditional eQTLs were detected in the first and second environment, respectively. Based on the QTTs and unconditional eQTLs detected, we presented a hypothetical molecular regulatory network in which 14 petal regulators potentially regulated the apetalous trait in “APL01” through the CHR11-PLP pathway. PLP acts directly as the terminal signal integrator negatively regulating petal development in the CHR11-PLP pathway. These findings will aid in the understanding the molecular mechanism underlying the apetalous trait of rapeseed.

Identification and Functional Analysis of Two New Mutant BnFAD2 Alleles That Confer Elevated Oleic Acid Content in Rapeseed

Rapeseed (Brassica napus L.) is a vital oil crop worldwide. High oleic acid content is a desirable quality trait for rapeseed oil, which makes it more beneficial to human health. However, many germplasm resources with high oleic acid content in rapeseed have not been evaluated with regard to their genotypes, making it difficult to select the best strains with this trait for the breeding of high oleic acid rapeseed variety. This work was to explore the gene-regulation mechanism of this trait using a new super-high oleic acid content (∼85%) line N1379T as genetic material. In this study, the sequences of four homologous fatty acid desaturase (BnFAD2) genes were compared between super-high (∼85%, N1379T) and normal (∼63%) oleic acid content lines. Results showed that there were two single-nucleotide polymorphisms (SNPs) in BnFAD2-1 and BnFAD2-2, respectively, which led to the amino acid changes (E106K and G303E) in the corresponding proteins. Functional analysis of both genes in yeast confirmed that these SNPs were loss-of-function mutations, thus limiting the conversion of oleic acid to linoleic acid and resulting in the considerable accumulation of oleic acid. Moreover, two specific cleaved amplified polymorphic sequences (CAPS) markers for the two SNPs were developed to identify genotypes of each line in the F2 and BC1 populations. Furthermore, these two mutant loci of BnFAD2-1 and BnFAD2-2 genes were positively associated with elevated oleic acid levels and had a similar effect with regard to the increase of oleic acid content. Taken together, these two novel SNPs in two different BnFAD2 genes jointly regulated the high oleic acid trait in this special germplasm. The study provided insight into the genetic regulation involved in oleic acid accumulation and highlighted the use of new alleles of BnFAD2-1 and BnFAD2-2 in breeding high oleic acid rapeseed varieties.

Comparative analysis of miRNAs of two rapeseed genotypes in response to acetohydroxyacid synthase-inhibiting herbicides by high-throughput sequencing

Acetohydroxyacid synthase (AHAS), also called acetolactate synthase, is a key enzyme involved in the first step of the biosynthesis of the branched-chain amino acids valine, isoleucine and leucine. Acetohydroxyacid synthase-inhibiting herbicides (AHAS herbicides) are five chemical families of herbicides that inhibit AHAS enzymes, including imidazolinones (IMI), sulfonylureas (SU), pyrimidinylthiobenzoates, triazolinones and triazolopyrimidines. Five AHAS genes have been identified in rapeseed, but little information is available regarding the role of miRNAs in response to AHAS herbicides. In this study, an AHAS herbicides tolerant genotype and a sensitive genotype were used for miRNA comparative analysis. A total of 20 small RNA libraries were obtained of these two genotypes at three time points (0h, 24 h and 48 h) after spraying SU and IMI herbicides with two replicates. We identified 940 conserved miRNAs and 1515 novel candidate miRNAs in Brassica napus using high-throughput sequencing methods combined with computing analysis. A total of 3284 genes were predicted to be targets of these miRNAs, and their functions were shown using GO, KOG and KEGG annotations. The differentiation expression results of miRNAs showed almost twice as many differentiated miRNAs were found in tolerant genotype M342 (309 miRNAs) after SU herbicide application than in sensitive genotype N131 (164 miRNAs). In additiond 177 and 296 miRNAs defined as differentiated in sensitive genotype and tolerant genotype in response to SU herbicides. The miR398 family was observed to be associated with AHAS herbicide tolerance because their expression increased in the tolerant genotype but decreased in the sensitive genotype. Moreover, 50 novel miRNAs from 39 precursors were predicted. There were 8 conserved miRNAs, 4 novel miRNAs and 3 target genes were validated by quantitative real-time PCR experiment. This study not only provides novel insights into the miRNA content of AHAS herbicides tolerant rapeseed in response to AHAS herbicides, but also demonstrates that miRNAs may be involved in AHAS herbicides tolerance.