Identification and characterization of improved nitrogen efficiency in interspecific hybridized new-type Brassica napus

Background and Aims

Oilseed rape (Brassica napus) is an important oil crop worldwide. The aim of this study was to identify the variation in nitrogen (N) efficiency of new-type B. napus (genome A^rA^rC^cC^c) genotypes, and to characterize some critical physiological and molecular mechanisms in response to N limitation.

Methods

Two genotypes with contrasting N efficiency (D4-15 and D1-1) were identified from 150 new-type B. napus lines, and hydroponic and pot experiments were conducted. Root morphology, plant biomass, N uptake parameters and seed yield of D4-15 and D1-1 were investigated. Two traditional B. napus (genome AⁿAⁿCⁿCⁿ) genotypes, QY10 and NY7, were also cultivated. Introgression of exotic genomic components in D4-15 and D1-1 was evaluated with molecular markers.

Key Results

Large genetic variation existed among traits contributing to the N efficiency of new-type B. napus. Under low N levels at the seedling stage, the N-efficient new-type D4-15 showed higher values than the N-inefficient D1-1 line and the traditional B. napus QY10 and NY7 genotypes with respect to several traits, including root and shoot biomass, root morphology, N accumulation, N utilization efficiency (NutE), N uptake efficiency (NupE), activities of nitrate reductase (NR) and glutamine synthetase (GS), and expression levels of N transporter genes and genes that are involved in N assimilation. Higher yield was produced by the N-efficient D4-15 line compared with the N-inefficient D1-1 at maturity. More exotic genome components were introgressed into the genome of D4-15 (64·97 %) compared with D1-1 (32·23 %).

Conclusions

The N-efficient new-type B. napus identified in this research had higher N efficiency (and tolerance to low-N stress) than traditional B. napus cultivars, and thus could have important potential for use in breeding N-efficient B. napus cultivars in the field.     

Association genetics of the parameters related to nitrogen use efficiency in Brassica juncea L.

Key message

Genome wide association studies allowed prediction of 17 candidate genes for association with nitrogen use efficiency. Novel information obtained may provide better understanding of genomic controls underlying germplasm variations for this trait in Indian mustard.

Abstract

Nitrogen use efficiency (NUE) of Indian mustard (Brassica juncea (L.) Czern & Coss.) is low and most breeding efforts to combine NUE with crop performance have not succeeded. Underlying genetics also remain unexplored. We tested 92 SNP-genotyped inbred lines for yield component traits, N uptake efficiency (NUPEFF), nitrogen utilization efficiency (NUTEFF), nitrogen harvest index (NHI) and NUE for two years at two nitrogen doses (N_o without added N and N₁₀₀ added @100 kg/ha). Genotypes IC-2489-88, M-633, MCP-632, HUJM 1080, GR-325 and DJ-65 recorded high NUE at low N. These also showed improved crop performance under high N. One determinate mustard genotype DJ-113 DT-3 revealed maximum NUTEFF. Genome wide association studies (GWAS) facilitated recognition of 17 quantitative trait loci (QTLs). Environment specificity was high. B-genome chromosomes (B02, B03, B05, B07 and B08) harbored many useful loci. We also used regional association mapping (RAM) to supplement results from GWAS. Annotation of the genomic regions around peak SNPs helped to predict several gene candidates for root architecture, N uptake, assimilation and remobilization. CAT9 (At1g05940) was consistently envisaged for both NUE and NUPEFF. Major N transporter genes, NRT1.8 and NRT3.1 were predicted for explaining variation for NUTEFF and NUPEFF, respectively. Most significant amino acid transporter gene, AAP1 appeared associated with NUE under limited N conditions. All these candidates were predicted in the regions of high linkage disequilibrium. Sequence information of the predicted candidate genes will permit development of molecular markers to aid breeding for high NUE.

     

<Emphasis Type="Italic">LMI1</Emphasis>-like genes involved in leaf margin development of <Emphasis Type="Italic">Brassica napus</Emphasis>

In rapeseed (Brassica napus L.), leaf margins are variable and can be entire, serrate, or lobed. In our previous study, the lobed-leaf gene (LOBED-LEAF 1, BnLL1) was mapped to a 32.1 kb section of B. napus A10. Two LMI1-like genes, BnaA10g26320D and BnaA10g26330D, were considered the potential genes that controlled the lobed-leaf trait in rapeseed. In the present study, these two genes and another homologous gene (BnaC04g00850D) were transformed into Arabidopsis thaliana (L.) Heynh. plants to identify their functions. All three LMI1-like genes of B. napus produced serrate leaf margins. The expression analysis indicated that the expression level of BnaA10g26320D determined the difference between lobed- and entire-leaved lines in rapeseed. Therefore, it is likely that BnaA10g26320D corresponds to BnLL1.     

Genetic variation of BnaA3.NIP5;1 expressing in the lateral root cap contributes to boron deficiency tolerance in Brassica napus

Boron (B) is essential for vascular plants. Rapeseed (Brassica napus) is the second leading crop source for vegetable oil worldwide, but its production is critically dependent on B supplies. BnaA3.NIP5;1 was identified as a B-efficient candidate gene in B. napus in our previous QTL fine mapping. However, the molecular mechanism through which this gene improves low-B tolerance remains elusive. Here, we report genetic variation in BnaA3.NIP5;1 gene, which encodes a boric acid channel, is a key determinant of low-B tolerance in B. napus. Transgenic lines with increased BnaA3.NIP5;1 expression exhibited improved low-B tolerance in both the seedling and maturity stages. BnaA3.NIP5;1 is preferentially polar-localized in the distal plasma membrane of lateral root cap (LRC) cells and transports B into the root tips to promote root growth under B-deficiency conditions. Further analysis revealed that a CTTTC tandem repeat in the 5’UTR of BnaA3.NIP5;1 altered the expression level of the gene, which is tightly associated with plant growth and seed yield. Field tests with natural populations and near-isogenic lines (NILs) confirmed that the varieties carried BnaA3.NIP5;1^Q allele significantly improved seed yield. Taken together, our results provide novel insights into the low-B tolerance of B. napus, and the elite allele of BnaA3.NIP5;1 could serve as a direct target for breeding low-B-tolerant cultivars.     

A CACTA‐like transposable element in the upstream region of BnaA9.CYP78A9 acts as an enhancer to increase silique length and seed weight in rapeseed

Rapeseed (Brassica napus L.) is a model plant for polyploid crop research and the second‐leading source of vegetable oil worldwide. Silique length (SL) and seed weight are two important yield‐influencing traits in rapeseed. Using map‐based cloning, we isolated qSLWA9, which encodes a P450 monooxygenase (BnaA9.CYP78A9) and functions as a positive regulator of SL. The expression level of BnaA9.CYP78A9 in silique valves of the long‐silique variety is much higher than that in the regular‐silique variety, which results in elongated cells and a prolonged phase of silique elongation. Plants of the long‐silique variety and transgenic plants with high expression of BnaA9.CYP78A9 had a higher concentration of auxin in the developing silique; this induced a number of auxin‐related genes but no genes in well‐known auxin biosynthesis pathways, suggesting that BnaA9.CYP78A9 may influence auxin concentration by affecting auxin metabolism or an unknown auxin biosynthesis pathway. A 3.7‐kb CACTA‐like transposable element (TE) inserted in the 3.9‐kb upstream regulatory sequence of BnaA9.CYP78A9 elevates the expression level, suggesting that the CACTA‐like TE acts as an enhancer to stimulate high gene expression and silique elongation. Marker and sequence analysis revealed that the TE in B. napus had recently been introgressed from Brassica rapa by interspecific hybridization. The insertion of the TE is consistently associated with long siliques and large seeds in both B. napus and B. rapa collections. However, the frequency of the CACTA‐like TE in rapeseed varieties is still very low, suggesting that this allele has not been widely used in rapeseed breeding programs and would be invaluable for yield improvement in rapeseed breeding.     

甘蓝型油菜抗茎倒材料的筛选与候选基因挖掘

该研究采用茎秆抗折力指标评价了200份甘蓝型油菜种质资源抗茎倒伏能力,据此筛选出极端抗茎倒材料和不抗茎倒材料各1份,随后测定了2份材料成熟期茎秆的理化组分含量,并对2份极端抗茎倒差异材料蕾苔期、盛花期茎秆进行转录组测序分析,为甘蓝型油菜抗茎倒伏的遗传改良奠定基础。结果表明:(1)200份甘蓝型油菜种质资源的茎秆抗折力和茎粗均为正态分布,均属于数量遗传性状,并依据茎秆抗折力和农艺性状筛选出生育期相近,株型相似,茎秆粗度差异不显著,但茎秆抗折力差异显著的极端不抗茎倒材料GY172和抗茎倒材料GY199。(2)GY199的韧皮部比GY172更加致密,而GY172成熟期茎秆中的半纤维素、木质素、中性洗涤纤维及总可溶性糖含量均显著高于GY199,而其纤维素含量极显著低于GY199,即成熟期茎秆纤维素含量与这2个材料的茎秆抗折力呈正相关。(3)蕾苔期、盛花期茎秆转录组测序发现,碳代谢、碳固定、磷酸戊糖途径、氨基酸的生物合成、糖酵解/糖异生等途径的14个基因(BnaA10G0056100ZS、BnaC08G0455100ZS、BnaA08G0262400ZS、BnaC08G0239700ZS、BnaA07G0362300ZS、BnaC02G0081300ZS、BnaC04G0273000ZS等)以及纤维素合成相关的9个基因(BnaA05G0152200ZS、BnaA01G0411100ZS、BnaA03G0018900ZS、BnaA03G0037800ZS等)在抗茎倒材料GY199中显著上调表达,这些基因可能参与调控了茎秆强度性状,可作为油菜抗茎倒候选基因。     

Genetic mapping,cloning, and functional characterization of the <Emphasis Type="Italic">BnaX.VTE4</Emphasis> gene encoding a γ-tocopherol methyltransferase from oilseed rape

Rapeseed (Brassica napus L.) is one of the major oilseed crops and an important source for tocopherols known as vitamin E in human nutrition. Increasing the tocopherol content and altering the tocopherol composition is a major goal in rapeseed breeding. The genes encoding enzymes from the tocopherol pathway have been cloned from model species. However, only scant data about tocopherol genes from crop species have been available. We have cloned four sequences of a gene family from B. napus with homology to the Arabidopsis thaliana VTE4 gene. The sequences were amplified by PCR with primers derived from the A. thaliana gene. BAC-clones were isolated to analyze the genomic structure of the BnaX.VTE4-loci. In contrast to the A. thaliana gene all B. napus sequences have two additional introns. For functional analysis, the BnaA.VTE4.a1 sequence was transformed into A. thaliana. Seeds from transgenic offspring showed a 50-fold increase of the α-tocopherol fraction which is in accordance with the predicted function of the gene. A marker assay was established and the BnaA.VTE4.a1 sequence was mapped to the end of chromosome A02 of the Tapidor × Ningyou7 genetic map, where also two QTL for α-tocopherol content had been mapped. Thus, the BnaA.VTE4.a1 gene is a promising candidate for these QTL and can be used for marker assisted selection for α-tocopherol content in rapeseed. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. The nucleotide sequences data reported in this paper have been submitted to GenBank nucleotide sequence database with the accession numbers EU637012 to EU637015 and FJ435091 to FJ435093.     

Genome-wide association study dissects the genetic control of plant height and branch number in response to low-phosphorus stress in Brassica napus

Background and AimsOilseed rape (Brassica napus) is one of the most important oil crops worldwide. Phosphorus (P) deficiency severely decreases the plant height and branch number of B. napus. However, the genetic bases controlling plant height and branch number in B. napus under P deficiency remain largely unknown. This study aims to mine candidate genes for plant height and branch number by genome-wide association study (GWAS) and determine low-P-tolerance haplotypes.MethodsAn association panel of B. napus was grown in the field with a low P supply (P, 0 kg ha⁻¹) and a sufficient P supply (P, 40 kg ha⁻¹) across 2 years and plant height and branch number were investigated. More than five million single-nucleotide polymorphisms (SNPs) were used to conduct GWAS of plant height and branch number at two contrasting P supplies.Key ResultsA total of 2127 SNPs were strongly associated (P < 6·25 × 10⁻⁰⁷) with plant height and branch number at two P supplies. There was significant correlation between phenotypic variation and the number of favourable alleles of associated loci on chromosomes A10 (chrA10_821671) and C08 (chrC08_27999846), which will contribute to breeding improvement by aggregating these SNPs. BnaA10g09290D and BnaC08g26640D were identified to be associated with chrA10_821671 and chrC08_27999846, respectively. Candidate gene association analysis and haplotype analysis showed that the inbred lines carrying ATT at BnaA10g09290Hap1 and AAT at BnaC08g26640Hap1 had greater plant height than lines carrying other haplotype alleles at low P supply.ConclusionOur results demonstrate the power of GWAS in identifying genes of interest in B. napus and provided insights into the genetic basis of plant height and branch number at low P supply in B. napus. Candidate genes and favourable haplotypes may facilitate marker-based breeding efforts aimed at improving P use efficiency in B. napus.     

The vegetative nitrogen response of sorghum lines containing different alleles for nitrate reductase and glutamate synthase

Improving the nitrogen (N) responsiveness of crops is crucial for food security and environmental sustainability, and breeding N use efficient (NUE) crops has to exploit genetic variation for this complex trait. We used reverse genetics to examine allelic variation in two N metabolism genes. In silico analysis of the genomes of 44 genetically diverse sorghum genotypes identified a nitrate reductase and a glutamate synthase gene that were under balancing selection in improved sorghum cultivars. We hypothesised that these genes are a potential source of differences in NUE, and selected parents and progeny of nested association mapping populations with different allelic combinations for these genes. Allelic variation was sourced from African (Macia) and Indian (ICSV754) genotypes that had been incorporated into the Australian elite parent R931945-2-2. Nine genotypes were grown for 30 days in a glasshouse and supplied with continuous limiting or replete N, or replete N for 27 days followed by 3 days of N starvation. Biomass, total N and nitrate contents were quantified together with gene expressions in leaves, stems and roots. Limiting N supply universally resulted in less shoot and root growth, increased root weight ratio and reduced tissue nitrate and total N concentrations. None of the tested genotypes exceeded growth or NUE of the elite parent R931945-2-2 indicating that the allelic combinations did not confer an advantage during early vegetative growth. Thus, the next steps for ascertaining potential effects on NUE include growing plants to maturity. We conclude that reverse genetics that take advantage of rapidly expanding genomic databases enable a systematic approach for developing N-efficient crops.     

Influence of Biochar on Nitrogen Use Efficiency and Root Morphology of Rice-Seedling in Two Contrasting Paddy Soils

Biochar may affect the root morphology and nitrogen (N) use efficiency (NUE) of rice at seedling stage, which has not been clearly verified until now. To clarify it, we conducted a pot experiment regarding to two soil types (Hydragric Anthrosol and Haplic Acrisol), two biochar application rates (0.5 wt% and 1.5 wt %) and two rice varieties (common rice var. Xiushui134 and hybrid super rice var. Zhongkejiayou12-6) meanwhile. Seedling NUE of common rice Xiuhui134 was significantly increased (p < 0.05) by 78.2% in Hydragric Anthrosol and by 91.4% in Haplic Acrisol following biochar addition with 1.5 wt%. However, biochar addition exerted no influence on seedling NUE of super rice Zhongkejiayou12-6 in both soils. Overall, 0.09–0.10 units higher soil pH and 105– 116% higher soil NH₄⁺ -N were observed in Xiushui134 growing two soils with 1.5 wt% biochar. In addition, improved root morphology (including longer root length, larger root surface area, bigger root volume, and more root tips) contributed to the higher seedling NUE of Xiushui134 in two soils. The soil pH and NH₄⁺ -N content, also the root morphology were influenced by biochar, which though could not thoroughly explained the NUE of Zhongkejiayou12-6. In conclusion, biochar application to paddy soil changed soil pH and NH₄⁺ -N content, root growth, and the consequent seedling NUE of rice, which effects are relative with rice cultivar, biochar addition rate, and soil type.     

Sequence,expression divergence,and complementation of homologous <Emphasis Type="Italic">ALCATRAZ</Emphasis> loci in <Emphasis Type="Italic">Brassica napus</Emphasis>

The genomic era provides new perspectives in understanding polyploidy evolution, mostly on the genome-wide scale. In this paper, we show the sequence and expression divergence between the homologous ALCATRAZ (ALC) loci in Brassica napus, responsible for silique dehiscence. We cloned two homologous ALC loci, namely BnaC.ALC.a and BnaA.ALC.a in B. napus. Driven by the 35S promoter, both the loci complemented to the alc mutation of Arabidopsis thaliana, yet only the expression of BnaC.ALC.a was detectable in the siliques of B. napus. Sequence alignment indicated that BnaC.ALC.a and BolC.ALC.a, or BnaA.ALC.a and BraA.ALC.a, possess a high level of similarity. The understanding of the sequence and expression divergence among homologous loci of a gene is of due importance for an effective gene manipulation and TILLING (or ECOTILLING) analysis for the allelic DNA variation at a given locus. S. Hua and I. H. Shamsi contributed equally to this work.     

Proteomic alterations of <Emphasis Type="Italic">Brassica napus</Emphasis> root in response to boron deficiency

Boron (B) deficiency is a worldwide problem, and Brassica napus is one of the most sensitive crops to B deficiency. To better understand the B starvation response of Brassica napus, we conducted a comparative proteomic analysis of seedling stage Brassica napus root between B-sufficient and B-limited conditions: 45 differentially expressed proteins were successfully identified by 2-DE coupled with MALDI-TOF/TOF-MS and LTQ-ESI-MS/MS analysis. Among these proteins, 10 were down-regulated and 35 were up-regulated under B-limited condition. Combining GO and KEGG analyses with data from previous reports, proteins were categorized into several functional groups, including antioxidant and detoxification, defense-related proteins, signaling and regulation, carbohydrate and energy metabolism, amino acid and fatty acid metabolism, protein translation and degradation, cell wall structure, and transporter. The genes of selected proteins were analyzed by quantitative RT-PCR. Our results provide novel information for better understanding the physiological and biochemical responses to B deficiency in plants.     

Dissection of genetic architecture for glucosinolate accumulations in leaves and seeds of Brassica napus by genome‐wide association study

Glucosinolates (GSLs), whose degradation products have been shown to be increasingly important for human health and plant defence, compose important secondary metabolites found in the order Brassicales. It is highly desired to enhance pest and disease resistance by increasing the leaf GSL content while keeping the content low in seeds of Brassica napus, one of the most important oil crops worldwide. Little is known about the regulation of GSL accumulation in the leaves. We quantified the levels of 9 different GSLs and 15 related traits in the leaves of 366 accessions and found that the seed and leaf GSL content were highly correlated (r = 0.79). A total of 78 loci were associated with GSL traits, and five common and eleven tissue‐specific associated loci were related to total leaf and seed GSL content. Thirty‐six candidate genes were inferred to be involved in GSL biosynthesis. The candidate gene BnaA03g40190D (BnaA3.MYB28) was validated by DNA polymorphisms and gene expression analysis. This gene was responsible for high leaf/low seed GSL content and could explain 30.62% of the total leaf GSL variation in the low seed GSL panel and was not fixed during double‐low rapeseed breeding. Our results provide new insights into the genetic basis of GSL variation in leaves and seeds and may facilitate the metabolic engineering of GSLs and the breeding of high leaf/low seed GSL content in B. napus.     

The combined effect of constant water deficit and nitrogen supply on WUE, NUE and Δ13C in durum wheat potted plants

Water scarcity and nitrogen shortage are the main constraints on durum wheat productivity. This paper examines the combined effects of a constant water deficit and nitrogen supply (NS) on growth, photosynthesis, stomatal conductance (g_s) and transpiration, instantaneous and time‐integrated water use efficiency (WUE) and nitrogen use efficiency (NUE) and carbon isotope discrimination (Δ¹³C) in durum wheat genotypes grown in pots under greenhouse conditions. Three water levels (40%, 70% and 100% container capacity), two nitrogen doses (high and low N) and four genotypes were assayed in a total of 24 experimental treatments. Water and nitrogen treatments were imposed 2 weeks after plant emergence. The growth, nitrogen content and Δ¹³C of the shoot and the gas exchange in the flag leaf were determined about 2 weeks after anthesis. As expected, both water and NS had a strong positive effect on growth. However, a reduction in water supply had low effect decreasing photosynthesis and transpiration, Δ¹³C and NUE and increasing WUE. On the contrary, increasing the level of nitrogen supplied had a significant negative effect on g_s, which decreased significantly the ratio of intercellular to ambient CO₂ concentrations and Δ¹³C, and increased both instantaneous and time‐integrated WUE. In addition, a higher N level also negatively affected the instantaneous and time‐integrated NUE. The Δ¹³C of shoots correlated significantly and negatively with either instantaneous or time‐integrated measurements of WUE. Moreover, within each NS, Δ¹³C also correlated negatively with the integrated NUE. We concluded that under our experimental conditions, Δ¹³C gives information about the efficiency with which not just water but also nitrogen are used by the plant. In addition, this study illustrates that a steady water limitation may strongly affect biomass without consistent changes in WUE. The lack of effect of the different water regimes on gas exchange, WUE and Δ¹³C illustrate the importance of how stress is imposed during growth.     

Genetics of Clubroot Resistance in <Emphasis Type="Italic">Brassica</Emphasis> Species

Clubroot disease, caused by the obligate plant pathogen Plasmodiophora brassicae Wor., is one of the most economically important diseases affecting Brassica crops in the world. The genetic basis of clubroot resistance (CR) has been well studied in three economically important Brassica species: B. rapa, B. oleracea, and B. napus. In B. rapa, mainly in Chinese cabbage, one minor and seven major CR genes introduced from European fodder turnips have been identified. Mapping of these CR genes localized Crr1 on R8, Crr2 on R1, CRc on R2, and Crr4 on R6 linkage groups of Chinese cabbage. Genes Crr3, CRa, CRb, and CRk mapped to R3, but at two separate loci, CRa and CRb are independent of Crr3 and CRk, which are closely linked. Further analysis suggested that Crr1, Crr2, and CRb have similar origins in the ancestral genome as in chromosome 4 of Arabidopsis thaliana. Genetic analysis of clubroot resistance genes in B. oleracea suggests that they are quantitative traits. Twenty-two quantitative trait loci (QTLs) were mapped in different linkage groups of B. oleracea. In B. napus, genetic analysis of clubroot resistance was found to be governed by one or two dominant genes, whereas resistance conferred by two recessive genes is reported. The quantitative analysis approach, however, proved that they are polygenic. In total, at least 16 QTLs have been detected on eight chromosomes of B. napus, N02, N03, N08, N09, N13, N15, N16, and N19. The chromosomal location of the other six QTLs is not clear. Cloning of any of these QTLs or resistance loci was not, however, possible until recently. Progress in genomics, particularly the techniques of comparative mapping and genome sequencing, supplements cloning and allows improved characterization of CR genes. Further development of DNA markers linked to CR genes will in turn hasten the breeding of clubroot-resistant Brassica cultivars.     

Genetic variation in eggplant for Nitrogen Use Efficiency under contrasting NO3‐ supply

Eggplant (Solanum melongena L.) yield is highly sensitive to N fertilization, the excessive use of which is responsible for environmental and human health damage. Lowering N input together with the selection of improved Nitrogen‐Use‐Efficiency (NUE) genotypes, more able to uptake, utilize, and remobilize N available in soils, can be challenging to maintain high crop yields in a sustainable agriculture. The aim of this study was to explore the natural variation among eggplant accessions from different origins, in response to Low (LN) and High (HN) Nitrate (NO₃^‐) supply, to identify NUE‐contrasting genotypes and their NUE‐related traits, in hydroponic and greenhouse pot experiments. Two eggplants, AM222 and AM22, were identified as N‐use efficient and inefficient, respectively, in hydroponic, and these results were confirmed in a pot experiment, when crop yield was also evaluated. Overall, our results indicated the key role of N‐utilization component (NUtE) to confer high NUE. The remobilization of N from leaves to fruits may be a strategy to enhance NUtE, suggesting glutamate synthase as a key enzyme. Further, omics technologies will be used for focusing on C‐N metabolism interacting networks. The availability of RILs from two other selected NUE‐contrasting genotypes will allow us to detect major genes/quantitative trait loci related to NUE.