Characterization of Sucrose transporter alleles and their association with seed yield-related traits in Brassica napus L

<p>Abstract</p> <p>Background</p> <p>Sucrose is the primary photosynthesis product and the principal translocating form within higher plants. <it>Sucrose transporters </it>(<it>SUC/SUT</it>) play a critical role in phloem loading and unloading. Photoassimilate transport is a major limiting factor for seed yield. Our previous research demonstrated that <it>SUT </it>co-localizes with yield-related quantitative trait loci. This paper reports the isolation of <it>BnA7.SUT1 </it>alleles and their promoters and their association with yield-related traits.</p> <p>Results</p> <p>Two novel <it>BnA7.SUT1 </it>genes were isolated from <it>B. napus </it>lines 'Eagle' and 'S-1300' and designated as <it>BnA7.SUT1.a </it>and <it>BnA7.SUT1.b</it>, respectively. The BnA7.SUT1 protein exhibited typical SUT features and showed high amino acid homology with related species. Promoters of <it>BnA7.SUT1.a </it>and <it>BnA7.SUT1.b </it>were also isolated and classified as <it>pBnA7.SUT1.a </it>and <it>pBnA7.SUT1.b</it>, respectively. Four dominant sequence-characterized amplified region markers were developed to distinguish <it>BnA7.SUT1.a </it>and <it>BnA7.SUT1.b</it>. The two genes were estimated as alleles with two segregating populations (F₂and BC₁) obtained by crossing '3715'×'3769'. <it>BnA7.SUT1 </it>was mapped to the A7 linkage group of the TN doubled haploid population. <it>In silico </it>analysis of 55 segmental <it>BnA7.SUT1 </it>alleles resulted three <it>BnA7.SUT1 </it>clusters: <it>pBnA7.SUT1.a- BnA7.SUT1.a </it>(type I), <it>pBnA7.SUT1.b- BnA7.SUT1.a </it>(type II), and <it>pBnA7.SUT1.b- BnA7.SUT1.b </it>(type III). Association analysis with a diverse panel of 55 rapeseed lines identified single nucleotide polymorphisms (SNPs) in promoter and coding domain sequences of <it>BnA7.SUT1 </it>that were significantly associated with one of three yield-related traits: number of effective first branches (EFB), siliques per plant (SP), and seed weight (n = 1000) (TSW) across all four environments examined. SNPs at other <it>BnA7.SUT1 </it>sites were also significantly associated with at least one of six yield-related traits: EFB, SP, number of seeds per silique, seed yield per plant, block yield, and TSW. Expression levels varied over various tissue/organs at the seed-filling stage, and <it>BnA7.SUT1 </it>expression positively correlated with EFB and TSW.</p> <p>Conclusions</p> <p>Sequence, mapping, association, and expression analyses collectively showed significant diversity between the two <it>BnA7.SUT1 </it>alleles, which control some of the phenotypic variation for branch number and seed weight in <it>B. napus </it>consistent with expression levels. The associations between allelic variation and yield-related traits may facilitate selection of better genotypes in breeding.</p

A Major and Stable QTL for Bacterial Wilt Resistance on Chromosome B02 Identified Using a High-Density SNP-Based Genetic Linkage Map in Cultivated Peanut Yuanza 9102 Derived Population

Bacterial wilt (BW) is one of the important diseases limiting the production of peanut (Arachis hypogaea L.) worldwide. The sufficient precise information on the quantitative trait loci (QTL) for BW resistance is essential for facilitating gene mining and applying in molecular breeding. Cultivar Yuanza 9102 is BW resistant, bred from wide cross between cultivated peanut Baisha 1016 and a wild diploid peanut species A. chacoense with BW resistance. In this study, we aim to map the major QTLs related to BW-resistance in Yuanza 9102. A high density SNP-based genetic linkage map was constructed through double-digest restriction-site-associated DNA sequencing (ddRADseq) technique based on Yuanza 9102 derived recombinant inbred lines (RILs) population. The map contained 2,187 SNP markers distributed on 20 linkage groups (LGs) spanning 1566.10 cM, and showed good synteny with AA genome from A. duranensis and BB genome from A. ipaensis. Phenotypic frequencies of BW resistance among RIL population showed two-peak distribution in four environments. Four QTLs explaining 5.49 to 23.22% phenotypic variance were identified to be all located on chromosome B02. The major QTL, qBWB02.1 (12.17–23.33% phenotypic variation explained), was detected in three environments showing consistent and stable expression. Furthermore, there was positive additive effect among these major and minor QTLs. The major QTL region was mapped to a region covering 2.3 Mb of the pseudomolecule B02 of A. ipaensis which resides in 21 nucleotide-binding site -leucine-rich repeat (NBS-LRR) encoding genes. The result of the major stable QTL (qBWB02.1) not only offers good foundation for discovery of BW resistant gene but also provide opportunity for deployment of the QTL in marker-assisted breeding in peanut

A male sterility-associated cytotoxic protein ORF288 in Brassica juncea causes aborted pollen development

Cytoplasmic male sterility (CMS) is a widespread phenomenon in higher plants, and several studies have established that this maternally inherited defect is often associated with a mitochondrial mutant. Approximately 10 chimeric genes have been identified as being associated with corresponding CMS systems in the family Brassicaceae, but there is little direct evidence that these genes cause male sterility. In this study, a novel chimeric gene (named orf288) was found to be located downstream of the atp6 gene and co-transcribed with this gene in the hau CMS sterile line. Western blotting analysis showed that this predicted open reading frame (ORF) was translated in the mitochondria of male-sterile plants. Furthermore, the growth of Escherichia coli was significantly repressed in the presence of ORF288, which indicated that this protein is toxic to the E. coli host cells. To confirm further the function of orf288 in male sterility, the gene was fused to a mitochondrial-targeting pre-sequence under the control of the Arabidopsis APETALA3 promoter and introduced into Arabidopsis thaliana. Almost 80% of transgenic plants with orf288 failed to develop anthers. It was also found that the independent expression of orf288 caused male sterility in transgenic plants, even without the transit pre-sequence. Furthermore, transient expression of orf288 and green fluorescent protein (GFP) as a fused protein in A. thaliana protoplasts showed that ORF288 was able to anchor to mitochondria even without the external mitochondrial-targeting peptide. These observations provide important evidence that orf288 is responsible for the male sterility of hau CMS in Brassica juncea

BnMs3 is required for tapetal differentiation and degradation, microspore separation, and pollen-wall biosynthesis in Brassica napus

7365AB, a recessive genetic male sterility system, is controlled by BnMs3 in Brassica napus, which encodes a Tic40 protein required for tapetum development. However, the role of BnMs3 in rapeseed anther development is still largely unclear. In this research, cytological analysis revealed that anther development of a Bnms3 mutant has defects in the transition of the tapetum to the secretory type, callose degradation, and pollen-wall formation. A total of 76 down-regulated unigenes in the Bnms3 mutant, several of which are associated with tapetum development, callose degeneration, and pollen development, were isolated by suppression subtractive hybridization combined with a macroarray analysis. Reverse genetics was applied by means of Arabidopsis insertional mutant lines to characterize the function of these unigenes and revealed that MSR02 is only required for transport of sporopollenin precursors through the plasma membrane of the tapetum. The real-time PCR data have further verified that BnMs3 plays a primary role in tapetal differentiation by affecting the expression of a few key transcription factors, participates in tapetal degradation by modulating the expression of cysteine protease genes, and influences microspore separation by manipulating the expression of BnA6 and BnMSR66 related to callose degradation and of BnQRT1 and BnQRT3 required for the primary cell-wall degradation of the pollen mother cell. Moreover, BnMs3 takes part in pollen-wall formation by affecting the expression of a series of genes involved in biosynthesis and transport of sporopollenin precursors. All of the above results suggest that BnMs3 participates in tapetum development, microspore release, and pollen-wall formation in B. napus

CRISPR/Cas9-Mediated Targeted Mutagenesis of BnaCOL9 Advances the Flowering Time of Brassica napus L.

Rapeseed (Brassica napus L.) is one of the most important oil crops in the world. The planting area and output of rapeseed are affected by the flowering time, which is a critical agronomic feature. COL9 controls growth and development in many different plant species as a member of the zinc finger transcription factor family. However, BnaCOL9 in rapeseed has not been documented. The aim of this study was to apply CRISPR/Cas9 technology to create an early-flowering germplasm resource to provide useful material for improving the early-maturing breeding of rapeseed. We identified four COL9 homologs in rapeseed that were distributed on chromosomes A05, C05, A03, and C03. We successfully created quadruple BnaCOL9 mutations in rapeseed using the CRISPR/Cas9 platform. The quadruple mutants of BnaCOL9 flowered earlier than the wild-type. On the other hand, the flowering time of the BnaCOL9 overexpression lines was delayed. An analysis of the expression patterns revealed that these genes were substantially expressed in the leaves and flowers. A subcellular localization experiment demonstrated that BnaCOL9 was in the nucleus. Furthermore, we discovered that two key flowering-related genes, BnaCO and BnaFT, were highly elevated in the BnaCOL9 mutants, but dramatically downregulated in the BnaCOL9 overexpression lines. Our findings demonstrate that BnaCOL9 is a significant flowering inhibitor in rapeseed and may be employed as a crucial gene for early-maturing breeding

BnaA.bZIP1 Negatively Regulates a Novel Small Peptide Gene, BnaC.SP6, Involved in Pollen Activity

Small peptides secreted to the extracellular matrix control many aspects of the plant’s physiological activities which were identified in Arabidopsis thaliana, called ATSPs. Here, we isolated and characterized the small peptide gene Bna.SP6 from Brassica napus. The BnaC.SP6 promoter was cloned and identified. Promoter deletion analysis suggested that the -447 to -375 and -210 to -135 regions are crucial for the silique septum and pollen expression of BnaC.SP6, respectively. Furthermore, the minimal promoter region of p158 (-210 to -52) was sufficient for driving gene expression specifically in pollen and highly conserved in Brassica species. In addition, BnaA.bZIP1 was predominantly expressed in anthers where BnaC.SP6 was also expressed, and was localized to the nuclei. BnaA.bZIP1 possessed transcriptional activation activity in yeast and protoplast system. It could specifically bind to the C-box in p158 in vitro, and negatively regulate p158 activity in vivo. BnaA.bZIP1 functions as a transcriptional repressor of BnaC.SP6 in pollen activity. These results provide novel insight into the transcriptional regulation of BnaC.SP6 in pollen activity and the pollen/anther-specific promoter regions of BnaC.SP6 may have their potential agricultural application for new male sterility line generation

Genome-Wide DNA Methylation Comparison between Brassica napus Genic Male Sterile Line and Restorer Line

DNA methylation is an essential epigenetic modification that dynamically regulates gene expression during plant development. However, few studies have determined the DNA methylation profiles of male-sterile rapeseed. Here, we conducted a global comparison of DNA methylation patterns between the rapeseed genic male sterile line 7365A and its near-isogenic fertile line 7365B by whole-genome bisulfite sequencing (WGBS). Profiling of the genome-wide DNA methylation showed that the methylation level in floral buds was lower than that in leaves and roots. Besides, a total of 410 differentially methylated region-associated genes (DMGs) were identified in 7365A relative to 7365B. Traditional bisulfite sequencing polymerase chain reaction (PCR) was performed to validate the WGBS data. Eleven DMGs were found to be involved in anther and pollen development, which were analyzed by quantitative PCR. In particular, Bnams4 was hypo-methylated in 7365A, and its expression was up-regulated, which might affect other DMGs and thus control the male sterility. This study provided genome-wide DNA methylation profiles of floral buds and important clues for revealing the molecular mechanism of genic male sterility in rapeseed

Genome-Wide Association Study Reveals the Genetic Architecture Underlying Salt Tolerance-Related Traits in Rapeseed (Brassica napus L.)

Soil salinity is a serious threat to agriculture sustainability worldwide. Salt tolerance at the seedling stage is crucial for plant establishment and high yield in saline soils; however, little information is available on rapeseed (Brassica napus L.) salt tolerance. We evaluated salt tolerance in different rapeseed accessions and conducted a genome-wide association study (GWAS) to identify salt tolerance-related quantitative trait loci (QTL). A natural population comprising 368 B. napus cultivars and inbred lines was genotyped with a Brassica 60K Illumina Infinium SNP array. The results revealed that 75 single-nucleotide polymorphisms (SNPs) distributed across 14 chromosomes were associated with four salt tolerance-related traits. These SNPs integrated into 25 QTLs that explained 4.21–9.23% of the phenotypic variation in the cultivars. Additionally, 38 possible candidate genes were identified in genomic regions associated with salt tolerance indices. These genes fell into several functional groups that are associated with plant salt tolerance, including transcription factors, aquaporins, transporters, and enzymes. Thus, salt tolerance in rapeseed involves complex molecular mechanisms. Our results provide valuable information for studying the genetic control of salt tolerance in B. napus seedlings and may facilitate marker-based breeding for rapeseed salt tolerance