Development Of Boron Efficient Near Isogenic Lines And Mapping Of A Boron Efficient Locus BnBE2 Of Brassica Napus

Boron(B) is an essential mineral nutrient for the growth and development in higher plants.Rapeseed(Brassica napus) is acutely sensitive to B deficiency.The main production area of rapeseed in China is deficient in soil available B.Boron deficiency causes the symptom of "flowering without seed setting",or even no seed harvest.At present,applying B fertilizer is the main method to settle this problem.However,B isn't a regenerated mineral resource.Considerable genotypic variations in response to B deficiency exist among rapeseed varieties.This raises the possibility of genetic improvement for the trait tolerant to low B stress.Based on the identification of B efficiency for some rapeseed cultivars with different genotype and QTL mapping for B efficiency,the B efficiency was proved to be controlled by quantitative trait loci in Brassica napus.To free mapping and clone these genes,this study was involved in developing a stable and available solution culture method to validate the B efficiency of Brassica napus at the seedling stage,constructing B-efficient near isogenic lines with random markers assisted background selection,mapping a B-efficient locus,named BnBE2.The main results were as follows:1.Development a system for evaluating B efficiency at the seedling stageThe growth of several genotypes was investigated under different B levels with solution culture.The results showed that significant difference in the phenotype growth among the different genotypes was found at the B level of 0.25μM.Thus,the B concentration is defined as a critical B stress level at which B-efficient cultivars had normal growth without B-deficient symptom while B-inefficient cultivars showed typical signs of B deficiency.The system is suitable for B efficiency evaluation not only for different rapeseed cukivars but also for a genetic segregating population.Under the optimum experiment conditions the method has good repeatability.2.Constructing B efficient near isogenic linesBased on the identification for B efficiency of each BC generation at the critical B level(0.25μM B),B efficient NIL were developed with random marker assisted background selection.It was proved that random molecular marker assisted background selection is effective and necessary in an early backcrossing generation to accelerate the construction of NILs.3.Mapping B-efficient gene,BnBE23.1 Genetic analysis for B efficiency in BC populationsAccording to the result of phenotypic investigation in three BC populations(BC₄S₁, BC₅,BC₆) under the low B level at the seedling stage,the ratio of B-efficient individuals to B-inefficient individuals was 1:1 in the BC₅ and BC₆ population and 3:1 in the BC₄S₁ population,respectively.These results indicated that the B efficiency in the BC line was considered as Mendel factor,controlled by a single locus.Thus the homozygous of the B-efficient BC plants and the recurrent parent would be considered to be NILs for B efficiency,which should be useful for further free mapping and cloning the B-efficient gene.3.2 Identification of linked markers and mapping the B efficient gene Based on the B efficiency identification of BC₅ population,DNA bulks were constructed from ten extreme B-efficient and ten extreme B-inefficient plants, respectively.Six markers were identified linked to BnBE2 using bulked segregant analysis(BSA) in combination with molecular markers AFLP and SRAP techniques.The six markers were all mapped in a region of C4.All of the most homologous sequences were from the Brassica oleracea genome based on the analysis of sequence BLAST against the Brassica database,which was an indication of the correctness of the mapping.3.3 Conversion of the linked AFLP marker into an SSCP markerThe sequence of the linked marker $6M32 was performed a BLAST against the Brassica database and found to be high homologous to a BAC clone BOHZW41 of Brassica oleracea(CC).Primer was designed based on the flanking sequence and the PCR product was resolved on an 8%nondenaturing polyacrylamide gel and an SSCP marker was successfully detected.The AFLP marker was converted into an SSCP marker successfully,which proved the feasibility of this method in converting markers.The secondary structure was predicted for the sequences of the two parents using the software of RNA structure 3.2.The sequence difference,including 3 base pairs difference between the two parents,revealed the reason producing the difference of conformation. 3.4 Development of linked markers by comparative mappingThe genome of Brassica napus(AACC) includes A and C genome,which originated from Brassica rapa and Brassica oleracea.A genome and C genome share orthologous regions in the genus Brassica.Therefore three SSR markers within A4 on other Brassica napus genetic map were mapped on the target region of BriBE2 on C4.4.Physiological mechanism for the B efficient locus,BnBE2The result obtained from study on uptake,accumulation and utilization of B showed that the B-efficient NIL has lower B content and higher B utilization efficiency under the low B condition as compared with the B-inefficient recurrent parent,and the B efficiency is attributed to the higher B utilization efficiency or less demand for B,and this locus is different from BE1 and thus named as BnBE2.5.Association analysis of molecular markers flanked to BnBE2 for several lines of Brassica napus with different B efficiencyAn analysis of the flanking markers linked to BnBE2 in different B efficiency rapeseed cultivars was illustrated and the result showed that there is poor correlation between the flanking markers polymorphism bands and B efficiency,which indicates that the B efficiency is a complex trait controlled by multiple loci in Brassica napus.