Comparative Analysis of <em>FLC</em> Homologues in Brassicaceae Provides Insight into Their Role in the Evolution of Oilseed Rape

<div><p>We identified nine <em>FLOWERING LOCUS C</em> homologues (<em>BnFLC</em>) in <em>Brassica napus</em> and found that the coding sequences of all <em>BnFLCs</em> were relatively conserved but the intronic and promoter regions were more divergent. The <em>BnFLC</em> homologues were mapped to six of 19 chromosomes. All of the <em>BnFLC</em> homologues were located in the collinear region of <em>FLC</em> in the <em>Arabidopsis</em> genome except <em>BnFLC.A3b</em> and <em>BnFLC.C3b,</em> which were mapped to noncollinear regions of chromosome A3 and C3, respectively. Four of the homologues were associated significantly with quantitative trait loci for flowering time in two mapping populations. The <em>BnFLC</em> homologues showed distinct expression patterns in vegetative and reproductive organs, and at different developmental stages. <em>BnFLC.A3b</em> was differentially expressed between the winter-type and semi-winter-type cultivars. Microsynteny analysis indicated that <em>BnFLC.A3b</em> might have been translocated to the present segment in a cluster with other flowering-time regulators, such as a homologue of <em>FRIGIDA</em> in <em>Arabidopsis.</em> This cluster of flowering-time genes might have conferred a selective advantage to <em>Brassica</em> species in terms of increased adaptability to diverse environments during their evolution and domestication process.</p> </div

Image2_Genetic architecture for skeletal muscle glycolytic potential in Chinese Erhualian pigs revealed by a genome-wide association study using 1.4M SNP array.tif

Introduction: Muscle glycolytic potential (GP) is a key factor affecting multiple meat quality traits. It is calculated based on the contents of residual glycogen and glucose (RG), glucose-6-phosphate (G6P), and lactate (LAT) contents in muscle. However, the genetic mechanism of glycolytic metabolism in skeletal muscle of pigs remains poorly understood. With a history of more than 400 years and some unique characteristics, the Erhualian pig is called the “giant panda” (very precious) in the world’s pig species by Chinese animal husbandry.Methods: Here, we performed a genome-wide association study (GWAS) using 1.4M single nucleotide polymorphisms (SNPs) chips for longissimus RG, G6P, LAT, and GP levels in 301 purebred Erhualian pigs.Results: We found that the average GP value of Erhualian was unusually low (68.09 μmol/g), but the variation was large (10.4–112.7 μmol/g). The SNP-based heritability estimates for the four traits ranged from 0.16–0.32. In total, our GWAS revealed 31 quantitative trait loci (QTLs), including eight for RG, nine for G6P, nine for LAT, five for GP. Of these loci, eight were genome-wide significant (p −7), and six loci were common to two or three traits. Multiple promising candidate genes such as FTO, MINPP1, RIPOR2, SCL8A3, LIFR and SRGAP1 were identified. The genotype combinations of the five GP-associated SNPs also showed significant effect on other meat quality traits.Discussion: These results not only provide insights into the genetic architecture of GP related traits in Erhualian, but also are useful for pig breeding programs involving this breed.</p

Phylogenetic tree of <i>FLC</i> homologues from <i>Brassica</i>, <i>Arabidopsis</i>, <i>Raphanus</i>, and <i>Sinapis</i> species.

<p><i>BnFLC</i> homologues are highlighted in bold, and <i>AtMAF1</i> (<i>MADS AFFECTING FLOWERING 1</i> of <i>A. thaliana</i>, an <i>AtFLC</i>-like gene) was used as the outgroup. <i>Br</i>, <i>Brassica rapa</i>; <i>Bo</i>, <i>B. oleracea</i>; <i>Rs</i>, <i>Raphanus sativus</i> (radish); <i>Sa</i>, <i>Sinapis alba</i> (white mustard); <i>At</i>, <i>Arabidopsis thaliana</i>; <i>Al</i>, <i>A. lyrata</i>; <i>Ah</i>, <i>A. halleri</i>; <i>Aa</i>, <i>A. arenosa; As, A. suecica</i>. GenBank accession numbers are given in parentheses. Bootstrap support values are shown beside the branches.</p

Quantitative real-time PCR analysis of expression patterns for four <i>BnFLC</i> homologues.

<p>Different letters above a bar indicate a significant difference (<i>P</i><0.05). Expression levels at the cotyledon stage were considered to be the control. Relative expression values of each <i>BnFLC</i> homologue were normalized with the reference gene <i>β-Actin.</i> (A) Comparison of the relative expression levels of <i>BnFLC</i> homologues in different tissues of the winter cultivar Tapidor. Samples underlined with a solid or dashed line were collected from nonvernalized and vernalized plants, respectively. (B) Relative expression levels of <i>BnFLCs</i> in leaves and cotyledons at different developmental stages in the semi-winter cultivar Ningyou7. No cold treatment was applied throughout all of the developmental stages. (C) Vernalization responsiveness of <i>BnFLC</i> homologues in Tapidor and Ningyou7. The relative fold change between four-week-old leaves (without vernalization) and seven-week-old leaves (four-week-old plants followed by three weeks of cold treatment) was measured.</p

Location of <i>BnFLC</i> homologues and flowering-time QTL on the partial linkage map for TN-DH population.

<p>Genome blocks related to <i>BnFLC</i> homologues (underlined) were defined by comparative mapping of <i>B. napus</i> and <i>A. thaliana</i> and using the locus boundaries of the blocks reported by Schranz <i>et al. </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045751#pone.0045751-Schranz1" target="_blank">[19]</a>. Arrows at left indicate an opposite orientation of the blocks, and a dashed line represents undefined boundaries. Solid and dashed vertical bars on the right represent the position of flowering-time QTL colocalized with <i>BnFLC</i> homologues under winter- and spring-cropping environments, respectively. Triangles indicate the approximate position of flowering-time QTL associated with <i>BnFLC</i> genes, which were detected from the Skipton/Ag-Spectrum population.</p

Microsynteny analysis of homologous regions related to <i>BnFLC.A3b</i>, <i>BnFRI.A3</i>, and <i>BnCBF.A3</i>.

<p>Collinear gene models are connected by dotted lines. (A) The colored bar in the center represents the portion of chromosome 3 of <i>B. napus</i> that contains the three genes. The blue arrow indicates the opposite orientation of the ‘W’ block. The left-hand box represents a region of the ‘J’ block (BnA3-J) in which <i>BnFLC.A3b</i> and <i>BnCBF.A3</i> are located, and alignment of conserved gene models of BnC3-J of <i>B. napus,</i> the triplicated regions (BrA3-J, BrA4-J and BrA5-J) of <i>B. rapa</i>, and the homologous regions in <i>A. thaliana</i> and <i>A. lyrata</i>. Red lines connect the collinear gene models in the ‘J’ block next to <i>FLC</i> and <i>CBF</i> homologues. The right-hand box represents a region of the ‘W’ block in which <i>BnFRI.A3</i> is located, and alignment of conserved gene models in the triplicated regions (BrA2-W, BrA3-W and BrA4-W) of <i>B. rapa,</i> and the homologous regions in <i>A. thaliana</i> and <i>A. lyrata</i>. Red lines connect the <i>FRI</i> homologues and the <i>FRI</i> homologous fragments. (B) Detailed structure of a region in BrA3-J (indicated by a green box in the left-hand box of (a) from the BAC clone KBrH038M21; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045751#pone.0045751-Mun1" target="_blank">[35]</a>). Repeat sequences were identified between the homologues of <i>At2g30000</i> and <i>At2g30020</i>. <i>GF-1</i> and <i>GF-2</i> represent two gene fragments homologous to the ‘R’ block genes, <i>At5G10200</i> and <i>At5G10150</i>, respectively.</p

Image1_Genetic architecture for skeletal muscle glycolytic potential in Chinese Erhualian pigs revealed by a genome-wide association study using 1.4M SNP array.tif

Introduction: Muscle glycolytic potential (GP) is a key factor affecting multiple meat quality traits. It is calculated based on the contents of residual glycogen and glucose (RG), glucose-6-phosphate (G6P), and lactate (LAT) contents in muscle. However, the genetic mechanism of glycolytic metabolism in skeletal muscle of pigs remains poorly understood. With a history of more than 400 years and some unique characteristics, the Erhualian pig is called the “giant panda” (very precious) in the world’s pig species by Chinese animal husbandry.Methods: Here, we performed a genome-wide association study (GWAS) using 1.4M single nucleotide polymorphisms (SNPs) chips for longissimus RG, G6P, LAT, and GP levels in 301 purebred Erhualian pigs.Results: We found that the average GP value of Erhualian was unusually low (68.09 μmol/g), but the variation was large (10.4–112.7 μmol/g). The SNP-based heritability estimates for the four traits ranged from 0.16–0.32. In total, our GWAS revealed 31 quantitative trait loci (QTLs), including eight for RG, nine for G6P, nine for LAT, five for GP. Of these loci, eight were genome-wide significant (p −7), and six loci were common to two or three traits. Multiple promising candidate genes such as FTO, MINPP1, RIPOR2, SCL8A3, LIFR and SRGAP1 were identified. The genotype combinations of the five GP-associated SNPs also showed significant effect on other meat quality traits.Discussion: These results not only provide insights into the genetic architecture of GP related traits in Erhualian, but also are useful for pig breeding programs involving this breed.</p

Table1_Genetic architecture for skeletal muscle glycolytic potential in Chinese Erhualian pigs revealed by a genome-wide association study using 1.4M SNP array.DOCX

Introduction: Muscle glycolytic potential (GP) is a key factor affecting multiple meat quality traits. It is calculated based on the contents of residual glycogen and glucose (RG), glucose-6-phosphate (G6P), and lactate (LAT) contents in muscle. However, the genetic mechanism of glycolytic metabolism in skeletal muscle of pigs remains poorly understood. With a history of more than 400 years and some unique characteristics, the Erhualian pig is called the “giant panda” (very precious) in the world’s pig species by Chinese animal husbandry.Methods: Here, we performed a genome-wide association study (GWAS) using 1.4M single nucleotide polymorphisms (SNPs) chips for longissimus RG, G6P, LAT, and GP levels in 301 purebred Erhualian pigs.Results: We found that the average GP value of Erhualian was unusually low (68.09 μmol/g), but the variation was large (10.4–112.7 μmol/g). The SNP-based heritability estimates for the four traits ranged from 0.16–0.32. In total, our GWAS revealed 31 quantitative trait loci (QTLs), including eight for RG, nine for G6P, nine for LAT, five for GP. Of these loci, eight were genome-wide significant (p −7), and six loci were common to two or three traits. Multiple promising candidate genes such as FTO, MINPP1, RIPOR2, SCL8A3, LIFR and SRGAP1 were identified. The genotype combinations of the five GP-associated SNPs also showed significant effect on other meat quality traits.Discussion: These results not only provide insights into the genetic architecture of GP related traits in Erhualian, but also are useful for pig breeding programs involving this breed.</p

Image3_Genetic architecture for skeletal muscle glycolytic potential in Chinese Erhualian pigs revealed by a genome-wide association study using 1.4M SNP array.tif

Introduction: Muscle glycolytic potential (GP) is a key factor affecting multiple meat quality traits. It is calculated based on the contents of residual glycogen and glucose (RG), glucose-6-phosphate (G6P), and lactate (LAT) contents in muscle. However, the genetic mechanism of glycolytic metabolism in skeletal muscle of pigs remains poorly understood. With a history of more than 400 years and some unique characteristics, the Erhualian pig is called the “giant panda” (very precious) in the world’s pig species by Chinese animal husbandry.Methods: Here, we performed a genome-wide association study (GWAS) using 1.4M single nucleotide polymorphisms (SNPs) chips for longissimus RG, G6P, LAT, and GP levels in 301 purebred Erhualian pigs.Results: We found that the average GP value of Erhualian was unusually low (68.09 μmol/g), but the variation was large (10.4–112.7 μmol/g). The SNP-based heritability estimates for the four traits ranged from 0.16–0.32. In total, our GWAS revealed 31 quantitative trait loci (QTLs), including eight for RG, nine for G6P, nine for LAT, five for GP. Of these loci, eight were genome-wide significant (p −7), and six loci were common to two or three traits. Multiple promising candidate genes such as FTO, MINPP1, RIPOR2, SCL8A3, LIFR and SRGAP1 were identified. The genotype combinations of the five GP-associated SNPs also showed significant effect on other meat quality traits.Discussion: These results not only provide insights into the genetic architecture of GP related traits in Erhualian, but also are useful for pig breeding programs involving this breed.</p

Analysis of the upstream regions of <i>Brassica FLC</i> homologues and <i>AtFLC.</i>

<p>(A) Comparison of the potential conserved <i>cis</i>-blocks upstream of <i>AtFLC</i> and <i>Brassica FLC</i> (0 to −2880 bp) homologues. Different conserved segments (more than 75% sequence identity, and referred to as <i>cis</i>-blocks in the text) are shown as boxes with different shading and are numbered for comparison. Arrows at the 5′ end indicate the approximate position of the neighbour gene (<i>At5g10150</i> and its homologues; unfilled) or the gene fragment (grey) upstream of <i>FLC.</i> Dashed lines represent upstream sequences that were not determined. (B) Alignment of 30 bp sequences that contain putative <i>cis</i>-regulatory elements (G-box and CAAT-box) in <i>cis</i>-block 4 among <i>FLC</i> homologues. (C) Alignment for the sequences in <i>cis</i>-block 5 among <i>FLC</i> homologues. The position of the putative CAAT-box (in the minus strand) is shown.</p