Genome-Wide Dissection Of Genetic Basis Of Yield Traits And Heterosis In Brassica Napus

Yield is the most important and complex trait for the genetic improvement of crops, reflecting the culmination of all processes of growth and development and their interaction with the environment.Crop yield is directly determined by yield-component traits.Yield-related traits may also indirectly affect yield by affecting the yield-component traits or by other unknown mechanisms.Although much research has been reported,in each such experiment the genetic architecture and determinants of yield and heterosis has remained ambiguous.Firstly,few QTL have been identified;Secondly,yield,a particularly complex factor,has not been associated with any of the yield-associated traits, relatively simple factors;Thirdly,trials were carried out in a few environments.In this study,we developed two permanent populations of rapeseed(Brassica napus) from a cross,Tapidor(a European winter cultivar)×Ningyou7(a Chinese semi-winter cultivar).One is a double haploid population(named as TNDH) of 202 lines derived by microspore culture of F₁,and the other one is a reconstructed F₂(named as TNRC-F₂) population of 404 lines resulted from the randomly intermating of the 202 lines of TNDH population.The two populations(TNDH and TNRC-F₂) were grown in 10 and 3 year×location combinations(microenvironments),respectively,with a randomized complete block design of 3 replicates.Seed yield and eight yield-associated traits(3 yield-component traits:pod number,seed number and seed weight;5 yield-related traits: branch number,biomass yield,flowering time,maturity time and plant height) were investigated.The genetic architecture and determinants of yield was analyzed thorough identification and meta-analysis of quantitative trait loci(QTL) for seed yield and 8 yield-associated traits from two populations and 10 year×location combinations using a high-density linkage map,comparative mapping and candidate gene in silico mapping. The main results and conclusions are summarized as follows:Construction of linkage map,comparative mapping and in silico mapping of candidate genes:A high-density linkage map of 786 molecular markers was constructed by JoinMap 3.0 software using 1040 molecular markers genotyped with TNDH population. It covered 19 linkage group identified as A1-A10(A genome) and C1-C9(C genome), with a total length of 2117.2 cM and an average distance of 2.7 cM between markers.Based on the 21 syntenic blocks of Arabidopsis identified in previous research, 277 molecular markers with known sequence information were employed as anchored markers for the alignment between the linkage map of TNDH and the genome of Arabidopsis thaliana and more than one hundred syntenic blocks or insertion fragments (islands) were identified.A total of 425 genes of Arabidopsis with known functions relating to flowering time,plant height,branch number,and other traits investigated in this study were collected from the TAIR website and the published papers.A total of 2185 homologous of the 425 Arabidopsis genes were were aligned to the TNDH linkage map by in silico mapping approach.Phenotype and heterosis of two parents and populations:Ningyou7,a semi-winter cultivar,had larger seeds and flowered and matured earlier than the winter cultivar,Tapidor,in all environments.However,for the other six traits,the performance of the two parents in the two macroenvironments(winter and semi-winter) was reversed, such that the means in the two macroenvironments were similar.This reflects the adaptability of the two cultivars to the different macroenvironments and the phenotypic plasticity of flowering time,maturity time and seed weight were smaller than that of other 6 traits.The six traits also showed significant heterosis:seed yield＞pod number and biomass yield＞plant height,seed number and branch number.The midparent heterosis of F₁ was higher than that of the mean of RC-F₂ population,this indicated that heterozygote was generally favorable to heterosis.The midparent heterosis of many cross in RC-F₂ population was greater than that of F₁ cross,this indicated heterozygote are not always favorable to heterosis and homozygote at some loci may further improve heterosis.Genetic correlations and heritability of phenotype and heterosis among different traits:In different microenvironments,pair-wise genetic correlations among the phenotype/midparent heterosis of different traits differed considerably(mostly in degree,a few in direction),which suggested that genetic correlations depended strongly on the environment.In general,seed yield was correlated with all investigated traits(negatively for flowering and maturity times,and positively for the other 6 traits),notably with the highest average coefficients of determination.This indicated seed yield strongly depended on other traits investigated in this research,reflecting the synthesis and complexity of seed yield.Despite strong genotypic and environmental effects for all investigated traits,there were significant genotype×environment interactions.The broad-sense heritability of trait performance was higher than that of midparent heterosis:flowering time＞seed weight and maturity time＞plant height and seed number＞biomass yield,branch number and pod number＞seed yield.Correlations of general heterozygosity/special heterozygosity and heterosis and hybrid performance:In general,the correlation of specail heterozygosity and hybrid performance and midparent heterosis was higher than that of general heterozygosity and hybrid performance and heterosis.But the correlation of hybrid performance and heterozugosity were not significantly different with that of heterosis and heterozygosity.In different microenvironments,the correlation coefficients showed significant difference: the highest for S5,S6 and N6 were not significantly different with each other.For different traits,the correlation coeffecients showed great variations:seed yield＞pod number,biomass yield and seed weight＞seed number per pod and branch number＞flowering time and maturity time.This result was well accordant with the heterosis level of these traits.In general,these correlation coefficients were all low,this indicated that heterosis could not be exactly predicted by molecular marker heterozygosity.Detection of QTL and mode of inheritance:A total of 1022 QTL(614 and 408 respectively at P=0.05 and 0.5) were detected for the nine traits from two populations and 10 microenvironments using WinQTLCart 2.5 software.After deleting 152 non-overlapping QTL at P=0.5,a total of 870 QTL were identified for the nine traits(then named as "identified-QTL"),of which nearly 10%(85) were for seed yield.The identified-QTL alleles that increased flowering and maturity times but decreased seed weight came mostly from Tapidor,and that increased branches,bioamass yield,plant height,pod number,seed number and seed yield came basically equally from the two parents,which accorded well with the performance of the two parents for these traits.For RC-F₂ population,the proportion of partial-dominant QTL was the highest,followed by additive QTL,the least was dominant and over-dominant QTL.For seed yield,more QTL showed dominant and over-dominant mode,then followed by pod number and biomass yield,followed by seed number,seed weight,branch number and plant height,the least was flowering time and maturity time:no over-dominant QTL was found.Meta-analysis of identified-QTL in different experiments and expression response of consensus-QTL in natural environments:Where the confidence intervals of identified-QTL for each trait in different experiments overlapped,the 694 overlapping identified-QTL were integrated into 225 reproducible "consensus-QTL" by meta-analysis using BioMercator 2.1 software,with reduced confidence intervals from an average of 6.5 to 3.7 cM.The other 176 non-overlapping identified-QTL were not involoved in the "actual" integration.The total 401 consensus-QTL were classified into two types:15 major QTL(those occurring at least once with R²â‰¥20%or at least twice with R²â‰¥10%) and 386 minor QTL(the remainder with relatively small effect).Nearly half consensus-QTL,particularly those of seed yield,was specifically detected in one of the 10 microenvironments and few were detected in more than half microenvironments.Two thirds of the consensus-QTL was detected in either the winter or semi-winter macroenvironment,and only one third appeared in both.The high proportion of environment-specific QTL indicates the large impact the natural environment has on the genes underlying seed yield and yield-associated traits.Meta-analysis of consensus-QTL for different traits and the identification of indicator-QTL:Most of the consensus-QTL determined for each trait overlapped with those determined for other traits,the 329 overlapping consensus-QTL were integrated into 111 pleiotropic "unique-QTL" by meta-analysis again,with reduced average putative confidence interval of 2.5 cM.Another 72 consensus-QTL were not invooved into the "actual" integration.The 55 consensus-QTL for seed yield were classified into two types: (â…°) 8 non-overlapping QTL,and(â…±) 47 overlapping QTL,which were integrated into pleiotropic unique-QTL with shorter average confidence interval of 2.5 cM.Additionally, six of eight "independent" QTL for seed yield overlapped other QTL for traits not published in this paper.Seed yield showed the highest correlation with the other 8 traits, and a high percentage(85%) of QTL for seed yield colocalized with QTL for other yield-associated traits,which indicated that the QTL of yield-associated traits were potential contributors to the colocalized QTL for seed yield.To estimate which QTL of yield-associated trait(s) were more likely to have pleiotropic effect on seed yield at a particular locus,the idea of indicator-QTL was proposed to identify the probable genetic determinant of the colocalized QTL for seed yield.The indicator-QTL are determined by their larger LOD scores,better reproducibility,overlapping confidence intervals and presence in common environment(s) with the QTL for seed yield.Twenty-nine of 47 pleiotropic QTL for seed yield colocalized with more than two consensus-QTL,which indicated that,in addition to pleiotropy,the effect of the QTL for seed yield could be a synthetic effect of several underlying tightly linked QTL of different yield-associated traits.Multiple indicator-QTL were chosen for 14 pleiotropic QTL for seed yield and a total of 63 indicator-QTL were determined.Multiple indicator-QTL came from the same or different environments,indicating the synthesis,variability and plasticity of QTL for yield which was strongly dependent on the environmental conditions.Indicator-QTL, from easily measured traits,usually had more stable expression,higher LOD scores,larger R² values,and identifiable candidate genes than the colocalized QTL for seed yield and thus will facilitate the cloning of yield QTL,assuming that pleiotropy and not linkage is the cause of the colocalization of the indicator-QTL with the corresponding QTL for seed yield.There is indirect evidence that supports the idea that pleiotropy is likely to be the genetic cause of the co-localization of indicator-QTL and seed yield QTL:(1) the peak positions of the two kinds of QTL are very close to each other;(2) the proportion of the overlapping confidence intervals of the two kinds of QTL is very high;(3) the additive-effect directions of the seed yield QTL are generally opposite(-) to that of indicator-QTL for two traits(flowering time and maturity time) while mostly the same(+) for the other six traits,which accorded well with the signs of the genetic-correlation coefficients of seed yield with the above eight yield-associated traits;(4) in many cases, the additive effect of the seed yield QTL was very near to the putative change in the value of seed yield that was estimated from the additive effect of the indicator-QTL.Detection,distribution,type,effect and environmental response of epistatic interactions:A total of 538 E-QTL were found in two populations in all microenvironments for nine traits,most of them did not show significant main-effect.The loci involved into E-QTL showed uneven distribution in the genome with significant hotspot that was identified in different microenvironments.Only 1%of the E-QTL was reproducible and this proportion was significantly lower than that of maize and rice(10%). This reflected the high variability and plasticity of the epistatic interactions in polyploid. For the E-QTL identified in RC-F₂ population,the total number and R² of AA interaction was the largest,followed by AD/DA interaction,the least was DD interaction.For the M-QTL,dominant effect was responsibole for a minor part of variance explained.For seed yield,the variance explained by E-QTL was higher than that of M-QTL.This strongly suggested that epistasis,especially AA epistasis,was the major genetic basis of heterosis in Brassica napus.Double homozygote was most likely to be the best and worst genotype.Double heterozygote was most unlikely be the best and worst genotype.Our research has great significance for science and application:(1) We identified hundreds of QTL for yield traits and their heterosis,more than half of them were confirmed by different experiments and provided an important genetic resource for marker-assisted selection,QTL cloning and gene function analysis.(2) The mode of expression of QTL for yield traits and heterosis in natural environments was revealed by meta-analysis and this may have significant implications for crop genetics and breeding. (3) The complexity of the genetic architecture of yield and heterosis was demonstrated by meta-analysis,illustrating the pleiotropy,synthesis,variability,and plasticity of yield QTL and the importance,high variability and plasticity of epistasis.(4)The idea of estimating indicator-QTL for yield QTL and identifying potential candidate genes for yield not only deepens our understanding of the genetic determinants of yield and facilitates the cloning of yield QTL but also provides an advance in methodology for complex traits.