A Tourist-like MITE insertion in the upstream region of the BnFLC.A10 gene is associated with vernalization requirement in rapeseed (Brassica napus L.)

  • Jinna Hou1,

    Affiliated with

    • Yan Long1,

      Affiliated with

      • Harsh Raman2,

        Affiliated with

        • Xiaoxiao Zou1,

          Affiliated with

          • Jing Wang1,

            Affiliated with

            • Shutao Dai1,

              Affiliated with

              • Qinqin Xiao1,

                Affiliated with

                • Cong Li1,

                  Affiliated with

                  • Longjiang Fan3,

                    Affiliated with

                    • Bin Liu4, 5 and

                      Affiliated with

                      • Jinling Meng1Email author

                        Affiliated with

                        BMC Plant Biology201212:238

                        DOI: 10.1186/1471-2229-12-238

                        Received: 31 July 2012

                        Accepted: 30 November 2012

                        Published: 15 December 2012

                        Abstract

                        Background

                        Rapeseed (Brassica napus L.) has spring and winter genotypes adapted to different growing seasons. Winter genotypes do not flower before the onset of winter, thus leading to a longer vegetative growth period that promotes the accumulation and allocation of more resources to seed production. The development of winter genotypes enabled the rapeseed to spread rapidly from southern to northern Europe and other temperate regions of the world. The molecular basis underlying the evolutionary transition from spring- to winter- type rapeseed is not known, however, and needs to be elucidated.

                        Results

                        We fine-mapped the spring environment specific quantitative trait locus (QTL) for flowering time, qFT10-4,in a doubled haploid (DH) mapping population of rapeseed derived from a cross between Tapidor (winter-type) and Ningyou7 (semi-winter) and delimited the qFT10-4 to an 80-kb region on chromosome A10 of B. napus. The BnFLC.A10 gene, an ortholog of FLOWERING LOCUS C (FLC) in Arabidopsis, was cloned from the QTL. We identified 12 polymorphic sites between BnFLC.A10 parental alleles of the TN-DH population in the upstream region and in intron 1. Expression of both BnFLC.A10 alleles decreased during vernalization, but decreased more slowly in the winter parent Tapidor. Haplotyping and association analysis showed that one of the polymorphic sites upstream of BnFLC.A10 is strongly associated with the vernalization requirement of rapeseed (r 2 = 0.93, χ 2 = 0.50). This polymorphic site is derived from a Tourist-like miniature inverted-repeat transposable element (MITE) insertion/deletion in the upstream region of BnFLC.A10. The MITE sequence was not present in the BnFLC.A10 gene in spring-type rapeseed, nor in ancestral ‘A’ genome species B. rapa genotypes. Our results suggest that the insertion may have occurred in winter rapeseed after B. napus speciation.

                        Conclusions

                        Our findings strongly suggest that (i) BnFLC.A10 is the gene underlying qFT10-4, the QTL for phenotypic diversity of flowering time in the TN-DH population, (ii) the allelic diversity caused by MITE insertion/deletion upstream of BnFLC.A10 is one of the major causes of differentiation of winter and spring genotypes in rapeseed and (iii) winter rapeseed has evolved from spring genotypes through selection pressure at the BnFLC.A10 locus, enabling expanded cultivation of rapeseed along the route of Brassica domestication.

                        Keywords

                        Rapeseed Flowering time Vernalization Tourist-like MITE FLOWERING LOCUS C Association analysis

                        Background

                        Interaction between various environmental signals and flowering genes is critical for plants to flower and complete their life cycle, and thus important to humans, who rely upon adequate production of fruit and seeds to feed the world’s growing population. Climate change fluctuations accompanying global warming [1, 2] are requiring plant breeders to elucidate the molecular mechanisms underlying flowering, and to develop strategies for manipulating and optimizing the flowering times to maximize crop yields. Four flowering pathways—autonomous, vernalization, photoperiod and gibberellic acid—have been established in Arabidopsis and partially identified in other species [3, 4]. Vernalization is an adaptive trait in which plants acquire the ability to flower following exposure to cold temperatures. A series of genes in the endogenous network involved in this process, and their regulatory relationships, have been identified; genes from different flowering pathways function together with other integrator genes to control flowering [5, 6]. The MADS-box family gene FLOWERING LOCUS C (FLC) represses flowering [7, 8] by suppressing the expression of FLOWERING LOCUS T (FT), a key flowering integrator and confirmed florigen in plants [911], and other floral integrator genes such as FLOWERING DURATION and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS[9, 12, 13]. Expression of FLC is reduced by vernalization [7, 8]. FLC is up-regulated by FRIGIDA (FRI) and repressed by genes in the autonomous pathway [1416]. FLC expression has also been shown to be regulated via histone acetylation and methylation, which alters the expression of a trans-acting regulator common to FLC and members of the related MADS AFFECTING FLOWERING gene [1720].

                        The genus Brassica, which diverged from Arabidopsis 14.5 to 20.4 million years ago [2123] includes more crops of agricultural and horticultural importance than any other genus in the family of Brassicaceae. Comparative analysis has revealed that diploid Brassica genomes are composed of conserved segments triplicated from Arabidopsis[24, 25]. The allopolyploid species B. napus (rapeseed, oilseed rape or canola; genomes AACC, 2n = 4x = 38) is a product of natural hybridization between diploid species B. rapa (2n = 2x = 20, genome AA) and B. oleracea (2n = 2x =18, genome CC). Rapeseed originated in southern Europe along the coastline of the Mediterranean Sea 10,000–100,000 years ago, and was domesticated as an oil crop 400–500 years ago. This crop was originally grown as a spring or semi-winter crop in Mediterranean climates; Its cultivation spread rapidly from southern into northern Europe after the development of winter rapeseed varieties, which do not flower during the long and cold winters. Understanding the evolution of flowering time is critical for domestication and introduction of rapeseed into new agroclimatic regions.

                        Miniature inverted-repeat transposable elements (MITEs) belong to a class of non-autonomous DNA transposable elements known as class II transposons. They are present in high copy number in the genome and contribute to genomic structure variations and intra-species diversity [26, 27]. Differing MITE insertion profiles among varieties of a given species enable tolerance to environmental changes and allow adaptation under selective pressure [26, 28, 29].

                        Genetic analyses of several mapping populations of Brassica have revealed that both major and minor quantitative trait loci (QTLs) control flowering time. Some of these QTLs have also been shown to collocated with candidate genes for flowering time such as CO, FLC, FT and FRI[3035]. Forty-two QTLs were identified in a doubled haploid (DH) rapeseed mapping population (TN DH) derived from a cross between Tapidor and Ningyou7, but their magnitude and genetic effects varied with growing environment [36]. One major flowering time QTL, qFT10-4, which accounted for more than 50% of phenotypic variation in flowering time in the TN DH populations grown in non-vernalization environments, was colocalized with the ortholog of FLC from Arabidopsis in chromosome A10 and was designated BnFLC.A10[36, 37]. In our study, the candidate gene BnFLC.A10 for qFT10-4 was dissected using a map-based cloning approach, and an association was found between a Tourist-like MITE insertion/deletion in the upstream region of BnFLC.A10 and the stronger vernalization requirement in rapeseed.

                        Results

                        Cloning of BnFLC.A10 from qFT10-4 and allelic divergence

                        To construct a high-resolution map of the qFT10-4 locus, we analyzed a large BC5F2 population (9,000 plants) that was derived from a cross between the DH line TN DH043 (winter-type) and Ningyou7 (semi-winter-type). Four molecular markers developed from the sequence of the Bacterial Artificial Chromosome (BAC) clone JBnB75D10, which contains BnFLC.A10, were used for the analysis (Figure 1A). Eight recombinants were identified and the QTL qFT10-4 was delimited to an 80-kb region that showed collinearity with the top of chromosome 5 of Arabidopsis thaliana (Figure 1B and C). None of the genes in this region except FLC are known to be involved in floral transition.
                        http://static-content.springer.com/image/art%3A10.1186%2F1471-2229-12-238/MediaObjects/12870_2012_1210_Fig1_HTML.jpg
                        Figure 1

                        Cloning of qFT10-4 and detailed structure and allelic divergence of BnFLC.A10 . (A) Positions of markers used to fine-map qFT10-4 are shown in the BAC clone JBnB75D10 of B. napus ‘Tapidor’. Marker IP1IP2 was developed from a specific sequence of BnFLC.A10. (B) Genotypes of recombinants detected among non-flowering plants of the BC5F2 segregation population derived from the TN DH line DH043 (winter-type) and Ningyou7 (semi-winter-type). T and H represent homozygous and heterozygous genotypes, respectively, for the Tapidor allele. (C) Genes identified in the 80-kb region of JBnB75D10 that was delimited with markers T11 and Niab009. Arrows show the relative positions of predicted open reading frames (ORFs). For each ORF, the orthologous gene in A. thaliana is marked and genes that lacked an ortholog are labelled ‘hypothetical’. (D) Schematic diagram of the DNA sequence of BnFLC.A10. The arrow shows the translation start site. Roman numerals indicate the indels (I–IV) between the alleles from Tapidor and Ningyou7. Vertical bars labeled with Arabic numerals represent SNPs (1–8). For the SNPs, the nucleotide found in the Tapidor allele is given first. (E) BnFLC.A10 expression as detected by quantitative PCR during different stages of vernalization (0 to 7 weeks) at 4°C. Expression of the Ningyou7 allele decreased much more rapidly than that of the Tapidor allele during vernalization.

                        To analyze the basis of the vernalization requirement in rapeseed, we cloned and compared BnFLC.A10 sequences (approximately 7 kb) from Tapidor and Ningyou7, the parental lines of the mapping population. No polymorphism was found in the coding sequence (CDS) between the two alleles (BnFLC.A10-T and BnFLC.A10-N). However, there were two insertion/deletions (indels I and II) in the upstream region, together with two indels (indels III and IV) and eight single nucleotide polymorphisms (SNPs 1–8) in intron 1 of BnFLC.A10 (Figure 1D). Expression analysis showed that BnFLC.A10-N was markedly down-regulated upon exposure to cold treatment after 1 week, whereas expression of BnFLC.A10-T decreased gradually over 7 weeks of cold treatment (Figure 1E). This observation provides strong evidence that BnFLC.A10 underlies variation for vernalization requirement and that differences in gene expression establish the basis for allelic variation at the qFT10-4 locus.

                        A 621-bp insertion upstream of BnFLC.A10-T is associated with winter habit in rapeseed

                        To determine whether sequence variations in the two BnFLC.A10 alleles contribute to differences in vernalization requirements or winter growth habit among natural rapeseed populations, we conducted an association analysis using a panel of 79 diverse rapeseed cultivars representing winter, semi-winter and spring genotypes. All of the cultivars were planted in spring environments. Because of lack of vernalization, none of the winter-type cultivars flowered; in contrast all of the spring-type and semi-winter-type cultivars (with one exception) flowered normally (Table 1). The three largest indels (I, II and IV) were analyzed first because the alleles could be easily distinguished by PCR (Figure 2A). For indel I, the 621-bp insertion was absent in all spring and semi-winter cultivars, but was present in all 18 winter cultivars except Coma. The 621-bp insertion showed a highly significant correlation with flowering phenotype (r 2 = 0.93, Table 2; Figure 2B). In contrast, Indel II (r 2 = 0.49) and indel IV (r 2 = 0.56) were only weakly associated with flowering phenotype (Table 2).
                        Table 1

                        Phenotypic and genotypic data for 79 B. napus accessions

                        Accession

                        Origin

                        Type

                        Genotypea

                        Days to flowering

                           

                        InDel I

                        InDel II

                        InDel IV

                        2007

                        2008

                        2009

                        Altex

                        Canada

                        Spring

                        N

                        T

                        T

                        69

                        61

                        64

                        Alto

                        Canada

                         

                        N

                        T

                        T

                        65

                        61

                        61

                        Apomix

                        Unknown

                         

                        N

                        N

                        N

                        65

                        61

                        61

                        Bronowski DH2

                        Poland

                         

                        N

                        N

                        N

                        89

                        85

                        87

                        Bullet

                        Canada

                         

                        N

                        T

                        T

                        67

                        61

                        63

                        Celebra

                        Unknown

                         

                        N

                        N

                        N

                        72

                        72

                        75

                        CENN

                        Unknown

                         

                        N

                        N

                        N

                        78

                        73

                        77

                        Comet

                        Denmark

                         

                        N

                        T

                        T

                        75

                        68

                        68

                        Conzuul

                        Unknown

                         

                        N

                        T

                        -

                        74

                        68

                        69

                        D.ARoll

                        Unknown

                         

                        N

                        T

                        T

                        72

                        67

                        67

                        Dac-chosen

                        Unknown

                         

                        N

                        N

                        N

                        67

                        67

                        67

                        Dunkeld

                        Australia

                         

                        N

                        N

                        N

                        71

                        71

                        73

                        Erglu

                        Germany

                         

                        N

                        N

                        N

                        76

                        64

                        66

                        Erra

                        Germany

                         

                        N

                        N

                        N

                        69

                        70

                        72

                        Global

                        Canada

                         

                        N

                        T

                        T

                        78

                        64

                        66

                        Granit

                        Sweden

                         

                        N

                        T

                        T

                        79

                        59

                        62

                        Grouse

                        Australia

                         

                        N

                        T

                        -

                        69

                        60

                        57

                        GULLR

                        Sweden

                         

                        N

                        T

                        T

                        78

                        73

                        73

                        Jiayou1

                        Canada

                         

                        N

                        N

                        N

                        76

                        91

                        78

                        Jiayou3

                        Canada

                         

                        N

                        N

                        N

                        75

                        77

                        74

                        Karoo

                        Australia

                         

                        N

                        N

                        N

                        65

                        58

                        66

                        Marnoo

                        Australia

                         

                        N

                        N

                        N

                        69

                        65

                        67

                        Monty

                        Australia

                         

                        N

                        T

                        T

                        69

                        56

                        60

                        Niklas

                        Sweden

                         

                        N

                        T

                        T

                        78

                        67

                        72

                        Nilla

                        Sweden

                         

                        N

                        T

                        T

                        82

                        77

                        73

                        Ning RS-1

                        China

                         

                        N

                        N

                        -

                        83

                        79

                        81

                        Qingyou2

                        China

                         

                        N

                        N

                        N

                        65

                        60

                        63

                        Rioklas

                        Unknown

                         

                        N

                        T

                        T

                        75

                        73

                        74

                        Rucabo

                        Germany

                         

                        N

                        N

                        N

                        74

                        68

                        69

                        Westar

                        Canada

                         

                        N

                        T

                        T

                        50

                        50

                        50

                        Chuanyou11

                        China

                        Semi-winter

                        N

                        N

                        N

                        67

                        71

                        72

                        Fuyou1

                        China

                         

                        N

                        N

                        N

                        66

                        69

                        67

                        Fuyou2

                        China

                         

                        N

                        T

                        H

                        67

                        65

                        65

                        Gànyou14

                        China

                         

                        N

                        T

                        H

                        75

                        77

                        76

                        Gānyou2

                        China

                         

                        N

                        N

                        N

                        67

                        77

                        77

                        Gànyou3

                        China

                         

                        N

                        N

                        N

                        69

                        67

                        68

                        Gānyou5

                        China

                         

                        N

                        N

                        N

                        67

                        62

                        63

                        Huashuang1

                        China

                         

                        N

                        N

                        N

                        72

                        72

                        73

                        Huashuang2

                        China

                         

                        N

                        N

                        T

                        81

                        82

                        78

                        Huashuang3

                        China

                         

                        N

                        T

                        T

                        75

                        72

                        68

                        Huáyou10

                        China

                         

                        N

                        N

                        N

                        69

                        69

                        69

                        Huáyou11

                        China

                         

                        N

                        N

                        N

                        68

                        68

                        68

                        Huáyou12

                        China

                         

                        N

                        N

                        N

                        69

                        69

                        69

                        Huáyou13

                        China

                         

                        N

                        N

                        N

                        72

                        72

                        72

                        Huáyou14

                        China

                         

                        N

                        N

                        N

                        71

                        71

                        71

                        Huáyou16

                        China

                         

                        N

                        N

                        -

                        72

                        76

                        79

                        Huáyou2

                        China

                         

                        N

                        T

                        N

                        67

                        67

                        73

                        Huāyou3

                        China

                         

                        N

                        N

                        N

                        74

                        74

                        74

                        Huāyou4

                        China

                         

                        N

                        N

                        N

                        63

                        63

                        63

                        Huāyou6

                        China

                         

                        N

                        N

                        N

                        67

                        67

                        67

                        Huāyou9

                        China

                         

                        N

                        N

                        N

                        NF

                        NF

                        NF

                        huāyuo5

                        China

                         

                        N

                        T

                        T

                        69

                        68

                        62

                        Huáyuo6

                        China

                         

                        N

                        T

                        -

                        61

                        68

                        72

                        Suyou3

                        China

                         

                        N

                        N

                        N

                        62

                        59

                        59

                        Xiangnongyou2

                        China

                         

                        N

                        N

                        N

                        70

                        70

                        70

                        Xiangnongyou3

                        China

                         

                        N

                        N

                        N

                        67

                        67

                        67

                        Xiangyou13

                        China

                         

                        N

                        N

                        N

                        70

                        73

                        74

                        Youyan2

                        China

                         

                        N

                        N

                        N

                        64

                        60

                        61

                        Zhenyou-1

                        China

                         

                        N

                        T

                        H

                        67

                        78

                        80

                        Zheyou7

                        China

                         

                        N

                        N

                        N

                        72

                        67

                        69

                        Apache

                        UK

                        Winter

                        T

                        T

                        T

                        NF

                        NF

                        NF

                        Bakow

                        Poland

                         

                        T

                        T

                        T

                        NF

                        NF

                        NF

                        Bienvenu

                        France

                         

                        T

                        T

                        T

                        NF

                        NF

                        NF

                        Bolko

                        Poland

                         

                        T

                        T

                        T

                        NF

                        NF

                        NF

                        Brutor

                        France

                         

                        T

                        T

                        T

                        NF

                        NF

                        NF

                        Casino

                        Sweden

                         

                        T

                        T

                        T

                        NF

                        NF

                        NF

                        Ceres

                        Germany

                         

                        T

                        T

                        T

                        NF

                        NF

                        NF

                        Coma

                        Unknown

                         

                        N

                        T

                        H

                        NF

                        NF

                        NF

                        D-083

                        Unknown

                         

                        T

                        T

                        T

                        NF

                        NF

                        NF

                        Diadem

                        Germany

                         

                        T

                        T

                        T

                        NF

                        NF

                        NF

                        JeT-Neuf

                        France

                         

                        T

                        T

                        T

                        NF

                        NF

                        NF

                        Jupiter

                        Sweden

                         

                        T

                        T

                        T

                        NF

                        NF

                        NF

                        Libritta

                        Germany

                         

                        T

                        T

                        T

                        NF

                        NF

                        NF

                        Liradonna

                        Germany

                         

                        T

                        T

                        T

                        NF

                        NF

                        NF

                        Literavo

                        Germany

                         

                        T

                        T

                        T

                        NF

                        NF

                        NF

                        Matador

                        Sweden

                         

                        T

                        T

                        T

                        NF

                        NF

                        NF

                        Nestor

                        Sweden

                         

                        T

                        T

                        T

                        NF

                        NF

                        NF

                        Panter

                        Sweden

                         

                        T

                        -

                        T

                        NF

                        NF

                        NF

                        Quinta

                        Germany

                         

                        T

                        T

                        T

                        NF

                        NF

                        NF

                        aT, N and H indicate homozygous for Tapidor, homozygous for Ningyou7 and heterozygous genotypes, respectively; - represents an undetectable genotype or a new genotype distinct from the two parents.

                        The gene.

                        http://static-content.springer.com/image/art%3A10.1186%2F1471-2229-12-238/MediaObjects/12870_2012_1210_Fig2_HTML.jpg
                        Figure 2

                        Association of polymorphic sites (indels and SNPs) in BnFLC.A10 and flowering phenotype in rapeseed cultivars that were planted in spring. (A) Genotyping of indels by PCR in some of the analyzed cultivars. Because the polymorphic products for indel III could not be distinguished by PCR, results for this indel are not shown. Lanes 1–19 represent the corresponding PCR products amplified from the genomic DNA of accessions of Apache, Bakow, Bienvenu, Bolko, Brutor, Casino, Ceres, Diadem, JeT-Neuf, Coma, Apomix, Chuanyou11, Dac-chosen, Dunkeld, Erglu, Huashuang2, Jiayou1, Huáyou2 and Karoo. For detailed genotypic information, see Table 1. (B) Association between indel I and flowering phenotype.

                        Table 2

                        Haplotypes detected with the sequence information of BnFLC.A10 from 24 B. napus accessions

                        Haplotype

                        Accession

                        Origin

                        Type

                        InDel

                        SNP

                        Day to flowering

                            

                        I***

                        II*

                        IIIa

                        IV*

                        1*

                        2*

                        3*

                        4

                        5

                        6

                         

                        HapI

                        Tapidor

                        France

                        Winter

                        In

                        DEL

                        6

                        DEL

                        G

                        T

                        A

                        G

                        G

                        A

                        Non-flowering

                         

                        Apache

                        UK

                        Winter

                        In

                        DEL

                        6

                        DEL

                        G

                        T

                        A

                        G

                        G

                        A

                        Non-flowering

                         

                        Bakow

                        Poland

                        Winter

                        In

                        DEL

                        6

                        DEL

                        G

                        T

                        A

                        G

                        G

                        A

                        Non-flowering

                         

                        Bienvenu

                        France

                        Winter

                        In

                        DEL

                        6

                        DEL

                        G

                        T

                        A

                        G

                        G

                        A

                        Non-flowering

                         

                        Brutor

                        France

                        Winter

                        In

                        DEL

                        6

                        DEL

                        G

                        T

                        A

                        G

                        G

                        A

                        Non-flowering

                         

                        Casino

                        Sweden

                        Winter

                        In

                        DEL

                        6

                        DEL

                        G

                        T

                        A

                        G

                        G

                        A

                        Non-flowering

                         

                        Quinta

                        Germany

                        Winter

                        In

                        DEL

                        6

                        DEL

                        G

                        T

                        A

                        G

                        G

                        A

                        Non-flowering

                        HapII

                        Alto

                        Canada

                        Spring

                        DEL

                        DEL

                        6

                        DEL

                        G

                        T

                        A

                        G

                        G

                        A

                        62

                         

                        Bullet

                        Canada

                        Spring

                        DEL

                        DEL

                        6

                        DEL

                        G

                        T

                        A

                        G

                        G

                        A

                        64

                         

                        Comet

                        Denmark

                        Spring

                        DEL

                        DEL

                        6

                        DEL

                        G

                        T

                        A

                        G

                        G

                        A

                        70

                         

                        GULLR

                        Sweden

                        Spring

                        DEL

                        DEL

                        6

                        DEL

                        G

                        T

                        A

                        G

                        G

                        A

                        75

                         

                        Westar

                        Canada

                        Spring

                        DEL

                        DEL

                        6

                        DEL

                        G

                        T

                        A

                        G

                        G

                        A

                        50

                        HapIII

                        Qingyou2

                        China

                        Spring

                        DEL

                        In

                        6

                        DEL

                        G

                        T

                        A

                        G

                        G

                        A

                        63

                        HapIV

                        Erglu

                        Australia

                        Spring

                        DEL

                        In

                        7

                        In

                        A

                        C

                        T

                        G

                        G

                        A

                        69

                        HapV

                        Gānyou5

                        China

                        Semi-winter

                        DEL

                        In

                        7

                        In

                        A

                        C

                        T

                        A

                        C

                        G

                        64

                         

                        Huashuang1

                        China

                        Semi-winter

                        DEL

                        In

                        7

                        In

                        A

                        C

                        T

                        A

                        C

                        G

                        72

                         

                        Huāyou4

                        China

                        Semi-winter

                        DEL

                        In

                        7

                        In

                        A

                        C

                        T

                        A

                        C

                        G

                        63

                         

                        Karoo

                        Australia

                        Spring

                        DEL

                        In

                        7

                        In

                        A

                        C

                        T

                        A

                        C

                        G

                        63

                         

                        Suyou3

                        China

                        Semi-winter

                        DEL

                        In

                        7

                        In

                        A

                        C

                        T

                        A

                        C

                        G

                        60

                         

                        Xiangyou13

                        China

                        Semi-winter

                        DEL

                        In

                        7

                        In

                        A

                        C

                        T

                        A

                        C

                        G

                        72

                        HapVI

                        Erra

                        Germany

                        Spring

                        DEL

                        In

                        10

                        In

                        A

                        C

                        T

                        A

                        C

                        G

                        70

                         

                        Jiayou1

                        China

                        Spring

                        DEL

                        In

                        10

                        In

                        A

                        C

                        T

                        A

                        C

                        G

                        82

                         

                        NingRS-1

                        China

                        Semi-winter

                        DEL

                        In

                        10

                        In

                        A

                        C

                        T

                        A

                        C

                        G

                        81

                         

                        Ningyou7

                        China

                        Semi-winter

                        DEL

                        In

                        10

                        In

                        A

                        C

                        T

                        A

                        C

                        G

                        72

                        Symbols * and *** represent p = 0.05 and 0.001 levels of significance of each site’s influence on flowering time.

                        aThe repeats of the CTT motif in indel III; In = insertion; DEL = deletion.

                        Haplotyping of BnFLC.A10 specific markers for indels I–IV and SNPs 1–6 (Figure 1D) confirmed that most winter rapeseeds had a 621-bp insertion in the upstream region of BnFLC.A10 (haplotype I), whereas the 621-bp fragment was absent in the spring types (Table 2). These results suggest that indel I (with the 621-bp fragment present or absent) in the upstream region of BnFLC.A10 plays a very important role in modulating flowering time in natural rapeseed germplasm and potential development of a winter growth habit.

                        The 621-bp insertion in the upstream region of BnFLC.A10 is a Tourist-like MITE

                        To further characterize the 621-bp insertion sequence in winter-type rapeseed accessions, BnFLC.A10 alleles from the eight cultivars that representing haplotype I (Table 2) were sequenced and aligned. All the sequenced genotypes showed 100% identity. The inserted sequence possessed typical characteristics of a Tourist-like MITE [3840], with 14-bp terminal inverted repeat (TIR) sequences flanked by target sequence duplications (TSDs) of TAA (Figure 3A). Between the TIR sequences, an AT-rich (67%) core that contained 12 classes of important motifs (such as the TATA box and CAAT box) was identified (Additional file 1). These motifs might function in transcriptional initiation or promotion, or in response to different stimuli and signals (Additional file 1). At least four homologs of the MITE insertion (BLASTN expected value < 1e-10) were identified in the genomic sequence of B. napus from public databases (http://​www.​ncbi.​nlm.​nih.​gov) and up to 200 copies (E-value < 1e-10) were identified in the sequenced genome of B. rapa, the ancestral source of the A genome in B. napus. These homologs defined a new family of MITEs, which we named Monkey King (Figure 3B) after the subject of a Chinese myth. (In “Journey to the west”, Monkey King is capable of 72 methods of transformation and can transform hundreds of monkeys with one of his hairs. He also jumps long distances with a cloud somersault).
                        http://static-content.springer.com/image/art%3A10.1186%2F1471-2229-12-238/MediaObjects/12870_2012_1210_Fig3_HTML.jpg
                        Figure 3

                        Structure of the 621-bp MITE and its family. (A) Basic structure of the 621-bp MITE and elements in the Monkey King family. The consensus sequences of the TIRs and TSDs are shown. The length and content of the core AT-rich sequence varied among homologs of Monkey King. The numbers marked in the frame of core sequences represent the length (without TAAs and TSDs) of Monkey King upstream of BnFLC.A10 and its homologs in the B. rapa genome. W=(A/T), Y=(C/T), M=(A/C), R=(A/G), and S=(C/G). (B) Flanking sequence of Monkey King at the 5’ upstream end of BnFLC.A10-T and the corresponding Related Empty Sites (RESites) in B.rapa. (C) Three replicates of the GACTGGTT motif scattered near the 5’ end region of Monkey King. The sequence of Monkey King is shown, with dots representing omitted portions. TSDs are underlined and TIRs are marked with arrows. GACTGGTT motifs are framed in red; all of the three duplications are located near the 5’ end region.

                        Origin and transmission of the BnFLC.A10 621-bp insertion in B. napus and its A genome ancestral species B. rapa

                        To understand the evolutionary process behind the adaptation associated with the insertion of Monkey King into the upstream region of BnFLC.A10 and to trace its origin and transmission, we investigated an additional 154 spring cultivars of B. napus and 103 cultivars (including the genome sequenced cultivar, Chiifu [41]) belonging to nine subspecies of B. rapa (oilseed, swede and fodder types, Additional file 2). No Monkey King insertion was detected in the upstream region of BnFLC.A10 in any of the accessions, even though the empty site of insertion was almost 100% identical to the sequences that flanking the Monkey King insertion in BnFLC.A10 in winter rapeseed (Figure 3B and Additional file 2). On the other hand, hundreds of copies of Monkey King were detected in the whole genome, but not in the BrFLC.A10 upstream region of B. rapa ‘Chiifu’. This suggests that Monkey King may have pre-existed in the B. rapa genome but was inactive, after the generation of B. napus, it was activated and inserted into the upstream region of BnFLC.A10, giving rise to winter rapeseed.

                        Discussion

                        In this study, we used positional cloning to dissect the major flowering time QTL, qFT10-4, which was detected only in the spring-cropped TN DH population. The QTL qFT10-4 on chromosome A10 was delimited in a narrow 80-kb genomic region and annotation of different genes allowed us to identify BnFLC.A10, an ortholog of FLC, as the candidate gene. We demonstrated for the first time that flowering time variation at the qFT.10-4 locus is conditioned by the major vernalization response gene, BnFLC.A10; the MITE insertion upstream of BnFLC.A10 show significant association with the flowering time variation between winter and spring rapeseed.

                        Control of flowering time by vernalization has previously been shown to depend on a complex regulatory network, especially in amphidiploid rapeseed. In one study of the relationship between flowering time and FLC orthologs, five BnFLC sequences were isolated from B. napus cDNA library and in another study six FLC paralogs have been identified in B. napus by comparative analysis of B. napus and Arabidopsis genomes [36, 42]. The fact that indel I in the upstream region of BnFLC.A10 cosegregated with flowering phenotype in the TN DH population but in only some of the diverse cultivars might be due to the contribution of other flowering time QTLs, including other BnFLCs, with very small genetic effects. For examples, one of the BnFLC paralogs, which was located in linkage group A3 (BnFLC.A3b), colocalized with the flowering time QTL and thus might contribute to the vernalization response in certain cultivars [43]. In fact, at least nine copies of Bn.FLC genes exist in rapeseed [43]. Other genes, such as FRIGIDA, also regulate FLC expression in rapeseed; BnaA.FRI.a, one of orthologs of FRIGIDA in Arabidopsis, contributes to flowering time variation in rapeseed, which may partly explain why indel I did not totally cosegregated with flowering time in the association analysis [33]. In our research, the cultivar “Coma” that lacked the BnFLC.A10 upstream MITE insertion still exhibited the winter characteristic. It is thus possible that other copies of BnFLCs or related genes from the vernalization pathway may contribute to vernalization response in B. napus. The expression of one of the other BnFLC copies, or of all BnFLC copies acting in concert in the Coma genome, may be sufficient to inhibit flowering transition under spring environmental conditions, therefore enabling the cultivar ‘Coma’ to function as a winter rapeseed. Genetic diversity with respect to BnFLCs and other Arabidopsis vernalization pathway gene homologs has been associated with vernalization in B. napus, but no gene or polymorphic site as strongly associated with vernalization requirement of rapeseed as the BnFLC.A10 upstream MITE insertion has been dissected previously. The MITE insertion into BnFLC.A10 appears to be one of the most important causative factors of vernalization requirement in winter rapeseed cultivars.

                        It is believed that rapeseed originated from a natural hybridization between B. rapa and B. oleracea that occurred in southern Europe along the Mediterranean coastline approximately 10,000–100,000 years ago. Given the warm climate in this region year-round, naturally occurring rapeseed genotypes and their ancestors may not have needed to develop an adaptation requiring prolonged vernalization. The activation of Monkey King in B. napus genome would have introduced diversity into the germplasm upon which selective pressure could act. The insertion of Monkey King in the upstream region of BnFLC.A10 resulted in strong dependence on vernalization for flowering; this characteristic was then selected by plant breeders during the development of winter-type rapeseed cultivars of rapeseed that could be grown in northern Europe and other temperate regions of the world.

                        Several studies have examined the effect of MITEs on neighboring gene expression. For example, the DNA methylation level of a MITE can influence expression of neighboring genes. An assay of transient and stably-transformed rice revealed that the MITE Kiddo, when present in the promoter of the rice ubiquitin2 (rubq2) gene, was responsible for up to 20% of neighboring gene expression; most notably, when DNA methylation of Kiddo was blocked, transcript levels of endogenous rubq2 increased threefold [44]. An association has also been reported between a MITE inserted in the upstream regulator region of the gene Vgt1 (Vegetative to generative transition) and early flowering in Northern maize genotypes [45, 46]. In our study, the MITE upstream of BnFLC.A10 was positively associated with gene expression and induced BnFLC.A10 expression during vernalization. The inserted MITE seems to attenuate cold-induced BnFLC.A10 repression rather than increase its expression, in winter rapeseed. This result is very similar to that observed in Arabidopsis, where FLC expression was correlated with flowering time and vernalization requirement in unvernalized or long days, but not as strongly as anticipated [47, 48]. We thus conclude that either decreased rate of FLC expression during vernalization or additional epistatic interaction with other genes is more important for control of flowering time and vernalization requirement than variation of FLC expression under unvernalized conditions. Using motif prediction, motifs associated with gene regulation were found to exist in the Monkey King sequence (Additional file 1). Most of these motifs were located in gene promoter and enhancer regions (TATA box and CAAT box) or were light responsive elements (Sp1) (Additional file 1) associated with response to environmental signals in different organisms. Certain transcriptional factors presumably bind to this region to more efficiently initiate or enhance the expression of neighbouring genes. The actual protein binding ability of the 621-bp insertion was evaluated using electrophoretic mobility shift assays (EMSAs). Nuclear protein(s) extracted from Tapidor before vernalization were able to bind to some fragments from the middle of the 621-bp Monkey King region that contained TATA box motifs (unpublished data). These results suggest that Monkey King can bind to specific transcription factors that may initiate or enhance BnFLC.A10 expression in winter rapeseed cultivars, giving rise to their stronger vernalization requirement.

                        Our analysis also indicated that Monkey King is involved in gene regulation in many different settings in the genome. For example, we found three copies of the sequence GACTGGTT near the 5’ end of Monkey King (Figure 3C); this motif is conserved in the upstream region of Dsg1 (desmoglein1, which encodes desmosomal cadherin) in mice. The motif in Dsg1 is recognized by GRHL1 (grainyhead-like 1, a homolog of the Drosophila gene grainyhead) and increases Dsg1 expression [49]. Part of the Monkey King sequence is transcribed in Brassica genomes (http://​www.​ncbi.​nlm.​nih.​gov, Table 3), and has been identified in the 3' untranslated region of the WRKY21-1 gene (EU912394). Other transcripts that share high similarity with portions of the Monkey King sequence have been found in the expressed sequence tag library of rapeseed (http://​www.​ncbi.​nlm.​nih.​gov). The presence of these transcripts suggests the existence of a novel gene regulatory mechanism that is similar to the method by which exon shuffling generates new genes [50, 51] or overlapping transcripts generate siRNAs to regulate gene expression [52, 53]. It is possible that transcripts derived from Monkey King might regulate native gene expression through siRNA-induced DNA methylation. MITE activities within BnFLC.Al0 may have shaped phenotypic diversity and influenced mechanisms of adaptation to diverse climates during the evolutionary process.
                        Table 3

                        20 sequences that show high similarity with Monkey King in the B. napus EST library

                        Accession

                        Aligned position in Monkey King (bp)

                        Query coverage

                        E value

                        Max identity

                        Description

                        CD826040.1

                        29-586

                        90%

                        0

                        89%

                        BN25.062J15F011130 BN25 Brassica napus cDNA clone BN25062J15, mRNA sequence

                        EV022063.1

                        1-416

                        67%

                        3.00E-164

                        91%

                        BNSCS2CT_UP_086_C03_19APR2007_027 Brassica napus BNSCS2CT Brassica napus cDNA 5', mRNA sequence

                        EE567409.1

                        29-437

                        66%

                        5.00E-157

                        91%

                        BNZB_UP_149_C07_29SEP2005_059 Brassica napus BNZB Brassica napus cDNA 5', mRNA sequence

                        ES968675.1

                        5-416

                        66%

                        2.00E-151

                        90%

                        BNZB_UP_208_D10_15MAR2006_074 Brassica napus BNZB Brassica napus cDNA 5', mRNA sequence

                        EE558281.1

                        1-400

                        64%

                        2.00E-151

                        90%

                        BNZB_RP_027_G10_28APR2004_068 Brassica napus BNZB Brassica napus cDNA 5', mRNA sequence

                        EE564397.1

                        30-613

                        94%

                        2.00E-151

                        84%

                        BNZB_UP_107_G11_23AUG2004_083 Brassica napus BNZB Brassica napus cDNA 5', mRNA sequence

                        GT085003.1

                        140-613

                        76%

                        1.00E-143

                        86%

                        c08_20na_1j.s 20na Brassica napus cDNA clone c08_20na_1j 5, mRNA sequence

                        ES956896.1

                        87-416

                        53%

                        8.00E-125

                        91%

                        9RDBNGA_UP_157_G11_10MAR2006_083 Brassica napus 9RDBNGA Brassica napus cDNA 5', mRNA sequence

                        EE567134.1

                        379-618

                        38%

                        4.00E-108

                        96%

                        BNZB_UP_144_H11_27SEP2005_081 Brassica napus BNZB Brassica napus cDNA 5', mRNA sequence

                        EE567253.1

                        373-618

                        39%

                        1.00E-107

                        95%

                        BNZB_UP_147_A07_27SEP2005_063 Brassica napus BNZB Brassica napus cDNA 5', mRNA sequence

                        EE559708.1

                        379-618

                        38%

                        1.00E-107

                        96%

                        BNZB_UP_048_D04_11MAY2004_026 Brassica napus BNZB Brassica napus cDNA 5', mRNA sequence

                        EE566332.1

                        315-552

                        38%

                        2.00E-091

                        92%

                        BNZB_UP_133_A11_27SEP2005_095 Brassica napus BNZB Brassica napus cDNA 5', mRNA sequence

                        ES903789.1

                        5-203

                        32%

                        3.00E-079

                        93%

                        BNARO4GH_T3_002_A02_24NOV2006_016 Brassica napus BNARO4GH Brassica napus cDNA 5', mRNA sequence

                        EE567417.1

                        390-613

                        36%

                        9.00E-055

                        84%

                        BNZB_UP_149_D08_29SEP2005_058 Brassica napus BNZB Brassica napus cDNA 5', mRNA sequence

                        FG554276.1

                        441-613

                        27%

                        7.00E-051

                        88%

                        BN18DYSC_UP_016_A09_18FEB2008_079 BN18DYSC Brassica napus cDNA 5', mRNA sequence

                        EE568964.1

                        438-613

                        28%

                        1.00E-048

                        86%

                        BNZB_UP_170_G02_30SEP2005_004 Brassica napus BNZB Brassica napus cDNA 5', mRNA sequence

                        EV193796.1

                        438-613

                        28%

                        1.00E-043

                        85%

                        0091281 Brassica napus Cold acclimation - dark Brassica napus cDNA, mRNA sequence

                        FG577502.1

                        539-613

                        12%

                        2.00E-021

                        92%

                        BN24DYSC_UP_080_D10_8FEB2008_074 BN24DYSC Brassica napus cDNA 5', mRNA sequence

                        Conclusions

                        This study demonstrated that BnFLC.A10 is the highly likely causative gene underlying qFT10-4, which accounted for most flowering time variation in the TN DH population under spring environmental conditions. Comparision of allelic sequences from Tapidor and Ningyou7 revealed the presence of a Tourist-like MITE insertion in winter-type cultivar Tapidor. Association analysis among winter- and spring-type rapeseeds revealed that the presence of the Tourist-like MITE insertion is very strongly associated with vernalization requirement, and suggested that it appeared after B. napus was generated as a product of natural hybridization between B. rapa and B. oleracea. MITE activity led to genetic and phenotypic diversities among varieties and provided the fuel for evolutionary selection. As a result, winter genotypes may have evolved from spring genotypes; this useful variation has subsequently been used as a genetic resource for the development of winter cultivars enabling worldwide production of rapeseed.

                        Methods

                        Plant materials

                        For fine mapping of the BnFLC.A10 locus, we used 9,000 plants derived from four BC5F1 individuals: 8y085-1, 8y086-1, 8y086-2 and 8y086-4. TN DH043 (the 43rd line of the TN DH population) was crossed wiht Ningyou7 (semi-winter recurrent parent) and seeds were collected from the F1 generation. Plants were then backcrossed with Ningyou7 over five successive generations (BC1 to BC5). Molecular markers were used to track the Tapidor allele at the BnFLC.A10 locus in the F1 backcross. The BC5F2 near-isogenic lines were planted in the spring of 2009 for phenotyping with respect to flowering time. A panel of 79 diverse rapeseed cultivars (Table 1) was used for the association analysis. These cultivars were planted in spring during three successive years (2007–2009) for phenotyping. Climatic conditions during the planting season and geographic features of the planting site were as described previously [36]. The spring rapeseed and B. rapa accessions representing nine subspecies that were used to detect the presence of Monkey King upstream of BnFLC.A10 and BrFLC.A10 are listed in Additional file 2. These accessions were obtained from the National Brassica Germplasm Improvement Program (Wagga Wagga, Australia), the Australian Temperate Field Crops Collection (Horsham, Australia), and from the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences (Beijing, China).

                        Phenotypic evaluation

                        Flowering times of the different cultivars used for the association analysis were recorded as the number of days from the day of sowing to the day when 50% of plants in the plot flowered. In the BC5F2 populations, days to flowering (DTF) were recorded as the number of days from the day of sowing to the day when the first flower opened. The phenotype ‘non-flowering’ was assigned when plants showed no visible buds at autumn harvest in middle-October. Phenotypes of the 79 cultivars used for the association analysis are listed in Table 1.

                        Sequencing of BnFLC.A10 alleles from Tapidor and Ningyou7

                        A fragment amplified from the Tapidor genome with primer pair “Exon 4-7” (Table 4) was used as a probe to screen the Tapidor BAC library [54]. From 12 BAC clones that contained BnFLC.A10, one clone with the code JBnB75D10 was selected and sequenced to obtain the BnFLC.A10-T allele. Primers (P4, P5, exon1-2,exon2-4, exon4-7, Table 4)were designed based on the basis of the BnFLC.A10-T sequence and used to obtain the sequence of BnFLC.A10-N. The amplicons were cloned into a pGEM-T Easy vector (Promega, Madison, WI, USA) and sequenced to determine the BnFLC.A10-N sequence. Information on primers and amplified gene regions is provided in Table 4. BnFLC.A10-N and BnFLC.A10-T sequences were obtained, accession numbers [GenBank: JX901141 and JX901142].
                        Table 4

                        Sequence information for primers used for polymorphism and BnFLC.A10 gene expression analysis

                        Primer name

                        Sequence(5'-3')

                        BnFLC.A10 specific primers

                        Exon1-2 f

                        CATCCGTCGCTCTTCTTGTC

                        Exon1-2 r

                        GTTGCTTTCCATATCGATCAAG

                        Exon2-4 f

                        AACATGATGATGATCTTAAAGCCT

                        Exon2-4 r

                        CTCCAGCTGAACCAGGGAAC

                        Exon4-7 f

                        CTTGAGGAATCAAATGTCGATAA

                        Exon4-7 r

                        CGGAGATTTGTCCTGGTGAG

                        InDel1 f (P4 f)

                        GGTTCCTTTTCTTTTCGTTTGGG

                        InDel1 r (P4 r)

                        GAAGTAAAGTCGGACAAGAAGG

                        InDel2 f (P5 f)

                        CCTTCTTGTCCGACTTTACTTC

                        InDel2 r (P5 r)

                        CGTTGCTCCTACTTTGTCTATC

                        InDel3 (IP1IP2) f

                        CGTCGCTCTTCTTGTCGTCTC

                        InDel3 (IP1IP2) r

                        TATGCATCACAGCGTGTCAAA

                        InDel4 f

                        GTGTTCAGCTGTCGCTTCTAT

                        InDel4 r

                        CTAACGCTGGCTTTGATCTT

                        Itr1f

                        AATACTTCCTGCGAATCTTGTG

                        Itr1r

                        AGTTTGCTTCTAAGTCCCCAAT

                        SSR primers developed from JBnB75D10

                        25GTTA f

                        ACTTTCATCACCATTGCAGACA

                        25GTTA r

                        AAGAGCAGCCATTGTATCAGGT

                        T11 f

                        TTCCCAAGCTTGCTGGTACT

                        T11 r

                        GAGATTTCCCTCGCTTGATG

                        NIAB009 f

                        TACGCTAGTGAGAACACCTCCA

                        NIAB009 r

                        GCTTTAGCAAGAAAACTCGGAA

                        q-RT PCR primers

                        Prt f

                        TCCGTCGCTCTTCTTGTCGT

                        Prt r

                        GCTGAACCAGGGAACCCACA

                        actin2 F

                        CTGTGCCAATCTACGAGGGTTTC

                        actin2 R

                        CTTACAATTTCCCGCTCTGCTGT

                        18S f

                        GAGTATGGTCGCAAGGCTGAAA

                        18S r

                        CGCTCCACCAACTAAGAACGG

                        Gene annotation for the BAC sequence

                        Gene annotation was carried out using the FGENESH program by selection of the organism category “Dicot plants (A. thaliana)” and alignment with A. thaliana genes. Simple sequences and transposons were identified using RepeatMasker (http://​www.​repeatmasker.​org/​, validated 19th September, 2011) followed by manual inspection. We predicted the function of genes that were not aligned with A. thaliana orthologs from their conserved domains.

                        RNA extraction and q-RT PCR

                        Plants were grown under long-day conditions (16 h light/8 h dark) at 23°C until they had developed to the six-leaf stage, at which point they were transferred to 4°C for vernalization. BnFLC.A10 expression was analyzed in plants that had been subjected to a (control), 1, 4 and 7 weeks of vernalization. Total RNA was extracted from plant leaves using TRIzol® reagent (Invitrogen, Carlsbad, California, USA). Total RNA (2 μg) was reverse-transcribed using M-MLV Reverse Transcriptase (Promega). An iQ5 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) was used for quantitative RT-PCR to detect levels of BnFLC.A10 expression in the two parents. BnFLC.A10 primers (Prt f/Prt r, Table 4) amplified a 235-bp fragment of the BnFLC.A10 CDS. Two genes, actin2 and 18S rRNA (Table 4), were used to normalize expression levels. Three biological and technical replicates were analyzed.

                        Natural variation in BnFLC.A10

                        Allele-specific primers “Itr1f/Itr1r” (Table 4) were used to distinguish the BnFLC.A10 Tapidor allele from the Ningyou7 BnFLC.A10 variant in 24 rapeseed cultivars. The PCR products were cloned into a pGEM-T Easy vector (Promega) for sequencing. Plasmid prepared from two to four colonies from each PCR product was sequenced separately to minimize the contribution of polymerase errors to sequence variation.

                        Screening of homologous sequences of Monkey King in the B. rapa genome

                        To identify homologous sequences, the full length MITE sequence was queried against the B. rapa genome in the brassicadb database (http://​brassicadb.​org/​) using BLAST. Results were filtered using an E value<1e-10 as the cutoff.

                        Declarations

                        Acknowledgements

                        We thank Drs. Caroline Dean (Department of Cell and Developmental Biology, John Innes Centre, UK), Christian Jung (Plant Breeding Institute, Christian-Albrechts-University of Kiel, Germany) and Qifa Zhang (National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, China) for their invaluable comments; Drs Xiaowu Wang (The Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing, China) and Bob Redden (Horsham) for providing seeds of the Brassica rapa accessions; Rosy Raman and Ms Ekaterina Simova-Samuelian for indel analysis; and Huazhong Agricultural University (Wuhan, China), and NSW DPI (Wagga Wagga, Australia) for supporting this research. Financial support for this work was provided by the National Basic Research and Development Programme (2006CB101600) and National Natural Science Foundation of China (30900788).

                        Authors’ Affiliations

                        (1)
                        National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University
                        (2)
                        EH Graham Centre for Agricultural Innovation (an alliance between the Charles Sturt University and NSW Department of Primary Industries), Wagga Wagga Agricultural Institute
                        (3)
                        Department of Agronomy & James D. Watson Institute of Genome Sciences, Zhejiang University
                        (4)
                        Center of Systematic Genomics, Xinjiang Institute of Ecology and Geography, Chinese Academy of Sciences
                        (5)
                        Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences

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                        This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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