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

Abstract

Background: Sucrose is the primary photosynthesis product and the principal translocating form within higher plants. Sucrose transporters (SUC/SUT) play a critical role in phloem loading and unloading. Photoassimilate transport is a major limiting factor for seed yield. Our previous research demonstrated that SUT co-localizes with yield-related quantitative trait loci. This paper reports the isolation of BnA7.SUT1 alleles and their promoters and their association with yield-related traits.

Results: Two novel BnA7.SUT1 genes were isolated from B. napus lines 'Eagle' and 'S-1300' and designated as BnA7.SUT1.a and BnA7.SUT1.b, respectively. The BnA7.SUT1 protein exhibited typical SUT features and showed high amino acid homology with related species. Promoters of BnA7.SUT1.a and BnA7.SUT1.b were also isolated and classified as pBnA7.SUT1.a and pBnA7.SUT1.b, respectively. Four dominant sequence-characterized amplified region markers were developed to distinguish BnA7.SUT1.a and BnA7.SUT1.b. The two genes were estimated as alleles with two segregating populations (F2 and BC1) obtained by crossing '3715'×'3769'. BnA7.SUT1 was mapped to the A7 linkage group of the TN doubled haploid population. In silico analysis of 55 segmental BnA7.SUT1 alleles resulted three BnA7.SUT1 clusters: pBnA7.SUT1.a- BnA7.SUT1.a (type I), pBnA7.SUT1.b- BnA7.SUT1.a (type II), and pBnA7.SUT1.b- BnA7.SUT1.b (type III). Association analysis with a diverse panel of 55 rapeseed lines identified single nucleotide polymorphisms (SNPs) in promoter and coding domain sequences of BnA7.SUT1 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 BnA7.SUT1 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 BnA7.SUT1 expression positively correlated with EFB and TSW.

Conclusions: Sequence, mapping, association, and expression analyses collectively showed significant diversity between the two BnA7.SUT1 alleles, which control some of the phenotypic variation for branch number and seed weight in B. napus consistent with expression levels. The associations between allelic variation and yield-related traits may facilitate selection of better genotypes in breeding.

Figure 1

Comparison of the nucleotide sequences of BnA7.SUT1.a and BnA7.SUT1.b. The gray bars and black lines denote exons and introns in the CDS, while thick black lines indicate the promoter of BnA7.SUT1. The black rhombus and arrowhead with names distinguish amino acid sites and the locations of primers, respectively.

Figure 2

LD across the 51 BnA7.SUT1 loci. Positions of polymorphisms in the alignment are given. Positions 138, 357, 999, and 1085 are indel polymorphisms, and the remaining polymorphisms are SNPs.

Figure 3

Sequence comparison of three genotypes in the promoter and 5'UTR of BnA7.SUT1. ^a G, genotype; aa, ba, bb represent the 5'-ends of pBnA7.SUT1.a- BnA7.SUT1.a, pBnA7.SUT1.b- BnA7.SUT1.a, and pBnA7.SUT1.b- BnA7.SUT1.b, respectively.

Figure 4

Mapping of BnA7.SUT1 in the TN DH linkage map. The marker sRsut1 was derived from the BAC containing BnA7.SUT1.

Figure 5

Estimation of the most appropriate group K values, calculation SD over 10 independent runs. Mean L(K) over 10 runs for each K value. (B) Change rate of likelihood value calculated as L'(K) = L(K) -L(K-1). We followed the method of Evanno et al. [64].

Figure 6

Box-plots showing distributions of EFB, SP, BY, and TSW within types I, II, and III. (A) (B) (C) (D) are phenotypic details for EFB, SP, BY, and TSW, respectively. 08WH, year 2008 Wuhan; 09WH, year 2009 Wuhan; 09YC, year 2009 Yichang; 09HG, year 2009 Huanggang. aa represents genotype pBnA7.SUT1.a- BnA7.SUT1.a; ba represents genotype pBnA7.SUT1.b- BnA7.SUT1.a; and bb represents genotype pBnA7.SUT1.b- BnA7.SUT1.b. * t-test between ba genotype and aa genotype, between bb genotype and aa genotype significant at P = 0.05, ** significant at P = 0.01; ※ t-test between bb genotype and ba genotype significant at P = 0.05, ※ ※ significant at P = 0.01.

Figure 7

Real-time PCR analysis of BnA7.SUT1 expression. (A) Expression of BnA7.SUT1 in different organs, including source leaf and stem, bud, pod 25 DAF, pericarp of pod, and young seeds among diverse genotypes. (B) Expression in leaf including various developmental stages. 70 DAS is the reproductive stage of winter B. napus. (C) Expression in the pod during the developmental period. Pistil was dissected from the bud.