Map-based Cloning Of Boron Efficient QTL And Analysis Of Expression Profiling In Responses To Boron Stresses In Brassica Napus

Being an essential micronutrient element for the growth and development of plants,boron(B)is widely involved in diverse physiological and biochemical processes.Oil seedrape(Brassica napus L.)is extremely susceptible to B stresses(especially B deficiency);which seriously inhibit the growth of B.napus and further lead to a severe decline in the seed yield and quality.In the agricultural production,application of the chemical B fertilizers is the main step to address the B-deficiency problems in soils.However,borate rock belong to a kind of non-renewable resources,whose reserves are continually decreasing.Among the 14 essential mineral nutrient elements,the narrowest range occurs between B deficiency and B toxicity in soils;high concentrations of B easily pose a serious toxic threat to the tissues and organs of plants.Therefore,identification of the germ plasm resistant to B stresses,cloning of the corresponding genes and further revelation of the genetic and molecular mechanisms underlying the tolerance to B stresses are of great importance for sustainable development of B.napus.Based on the research background and aims,first,the present study analyzed the morphological,physiological,transcriptomic differences in responses to B deficiency and genome-wide genetic variations between the B-efficient(B-deficiency-resistant)cultivar QY10 and the B-inefficient(B-deficiency-sensitive)cultivar W10;then,based on the identification of the major B-efficiency quantitative trait locus(QTL)q BEC-A3 a and its corresponding near-isogenic lines previously established,we fine mapped q BEC-A3 a and cloned the gene mainly responsible for the resistance to B deficiency;eventually,we analyzed the differential transcriptional responses of oilseed rape to B deficiency and B toxicity on a genome-wide scale.The main results obtained in the present research were as below:(1)Morphological,physiological and transcriptional differences in responses to B deficiency and genomic variations between the rapeseed genotypes with contrasting B efficieniesUnder hydroponic culture systems,B deficiency resulted in the severe inhibition of the leaves and roots,a dramatic decline in the total dry weight and B accumulations of the B-efficient cultivar W10 compared with the B-efficient cultivar QY10.Triggered by B deficiency,W10 presented large amount of reactive oxygen species(ROS)in the root tips and a shorter non-root-hair zone,and the leaves and roots of W10 suffered from lipid peroxidation.The ultrastructure of the mesophyll cells revealed that under B deficiency,W10 presented a series of physiological defects,such as abnormal cell morphology,plasmolysis,disorderly arrayed chloroplasts and thicker and swollen cell walls.At the maturity stage,W10 showed abnormal floral organs,of which the seed yield were significantly reduced.Through the whole-genome re-sequencing,we identified a total of 1,605,747 SNP and 218,755 In Del polymorphic sites on a genome-wide scale,which were non-evenly distributed along the 19 chromosomes of B.napus,and the DNA polymorphisms in the An sub-genome were higher than those of the Cn sub-genome.Through the digital gene expression(DGE)profiling sequencing,we identified a sum of 21,743 and 14,343 differentially expressed genes(DEGs)in the leaves and roots of QY10 and W10,which were mainly involved in the uptake,transport and distribution of B,scavenging ROS and the maintenance of cell wall structure and plasma membrane integrity.(2)Fine mapping and cloning of the major-effect B-efficiency QTL q BEC-A3aBased on the identification of the major B-efficiency QTL q BEC-A3 a and its corresponding near-isogenic lines(BC4F2)previously established,q BEC-A3 a was demonstrated to improve the tolerance of oilseed rape to B deficiency under hydroponic culture,pot culture and suspension cell systems,and we employed the QTL-seq method to validate the unique presence of q BEC-A3 a.Subsequently,we combined analysis of genotypes,screening of the BC4F3 recombinants and identification of the B efficiencies of the BC4F3:4 family lines derived from the corresponding recombinants,and delimited the region of q BEC-A3 a from 6.6 c M to 1.74 c M;similarly,we used the BC4F4/BC4F4:5 families,further refining q BEC-A3 a to a genomic region spanning 119 kb.Within this region,there were 21 annotated genes;among them,the ORF16(Bna A03g24370D)was highly homologous(92.4%)to At NIP5;1,an influx boric acid channel in A.thaliana.The graft experiment by our team found that B efficiency of oilseed rape is governed by the roots,therefore,the candidate genes underlying B efficiency were defined as the DEGs in the roots between the B-efficient and-inefficient genotypes.The DGE profiling revealed the expression levels of Bna A03g24370 D in the roots of the B-efficient parent QY10 and the B-efficient near-isogenic line(NIL)NILQ were significantly higher than that of the B-ineffficient parent W10;thus,it was considered as the candidate gene underlying B efficiency and designated as Bna A3.NIP5;1.Comparative sequencing revealed that the sequence amino acid sequences of the proteins encoded by Bna A3.NIP5;1 were identical between QY10 and W10;an In Del and 11 SNP sites were identified in the 5'UTR region,which were potentially involved in the regulation of the differential expression of Bna A3.NIP5;1 between the rapeseed cultivars with contrasting B efficiencies.Phylogenetic analysis revealed that Bna A3.NIP5;1 belonged to NIP II subgroup in the MIP superfamily,which originated from Bra A3.NIP5;1 of the diploid ancestor B.rapa.Evolution selective pressure revealed the nonsynonymous(Ka)and synonymous(Ks)nucleotide substitution rate of Bna A3.NIP5;1 was less than one compared with At NIP5;1,which indicated that Bna A3.NIP5;1 was under positive selection.Co-expression analysis of the genes related to B uptake,transport and distribution in Brassica napus showed that Bna A3.NIP5;1 played a core role in the network involving efficient B uptake in the roots.Additionally,we performed an analysis of QTL-seq using a doubled haploid comprising 190 lines derived from QY10 and W10,and identified two new B-efficient QTLs q BEC-C2 a and q BEC-C2 b on the chromosome C2;subsequently,based on the results of QTL-seq and differential expression analysis of the annotated genes,Bna C2.NIP5;1,a homologue of At NIP5;1,and an ABC transporter gene Bna C2.ABCG21 were assumed to be the candidates under B efficiency.In the present study,an integrated analysis of QTL-seq and RNA-seq that were assisted by high-throughput sequencing provided validated insights into the high-resolution detection of QTLs and rapid cloning of quantitative trait genes(QTGs)in plant species with complex genomes.(3)Differential transcriptomic responses of oilseed rape to B deficiency and B toxicityWe analyzed the transcriptome differences under B deficiency and B toxicity using the B-deficiency-sensitive cultivar W10.Under both B deficiency and B toxicity,the biomasses,root length,B contents and photosynthetic pigments of the rapeseed plants were markedly decreased whereas the anthocyanin concentrations in the leaves were significantly elevated.In the leaves,there were 2,950 and 2,439 genes differentially expressed under B deficiency and B toxicity,respectively,which were more than the number of the DEGs in the roots.The transcriptomic analysis indicated that efficient uptake,transport,distribution and compartmentation of B were modulated through the transcriptional regulation of the B transporter genes,including Bna BOR1 s,Bna BOR2 s and Bna BOR4 s,and the B channel genes,including Bna NIPs,Bna TIPs and Bna PIPs,which contributed to the maintenance of B homeostasis in B.napus and the enhanced tolerance of oidseed rape to B stresses.