Identification of quantitative trait loci controlling late bolting in Chinese cabbage

Breeding Science 61: 151–159 (2011)

doi:10.1270/jsbbs.61.151

Identification of quantitative trait loci controlling late bolting in Chinese cabbage (Brassica rapa L.) parental line Nou 6 gou

Tomohiro Kakizaki1), Takeyuki Kato1,2), Nobuko Fukino1), Masahiko Ishida1), Katsunori Hatakeyama1)

and Satoru Matsumoto*1)

1)National Institute of Vegetable and Tea Science, 360 Kusawa, Ano, Tsu, Mie 514-2392, Japan

2)Graduate School of Bioresources, Mie University, 1577 Kurima-machiya, Tsu, Mie 514-8507, Japan

To identify the genes responsible for varietal differences in bolting time in Brassica rapa, we constructed a

linkage map of 220 simple sequence repeat (SSR) markers of B. rapa. To construct the map, we used F2

progeny obtained from a cross between the late-bolting parental line Nou 6 gou (PL6) and the early-bolting

parental line Nou 7 gou (A9709). The linkage map covered 875.6cM and 10 major linkage groups. To in-

vestigate the nature of the vernalization response in the B. rapa parental lines, we examined bolting charac-

teristics under two different conditions i.e., greenhouse and open field. Five quantitative trait loci (QTLs) that

controlled bolting time were detected by experiments in greenhouse and open field. Interestingly, the QTLs

identified in the greenhouse and field experiments did not map to the same loci. Three of five QTLs were

colocalized with the orthologs of well-known flowering genes of Arabidopsis, including FLOWERING

LOCUS C(FLC) and FLOWERING LOCUS T(FT). The QTLs detected in this study may provide useful

information for the selection of bolting traits in Chinese cabbage breeding.

Key Words:simple sequence repeat (SSR) markers, quantitative trait loci (QTL), bolting, Brassica rapa, FLOWERING LOCUS C (FLC), FLOWERING LOCUS T (FT).

Introduction

Vernalization is a process in which plants are exposed to low temperatures for efficient bolting, and this process occurs in many Brassica species. Vernalization is of two types, de-pending on the age of the plant, (1) seed vernalization, in which plants perceive the low temperature signal at the seed germination stage, and (2) plant vernalization, in which plants can perceive the low temperature signal only at a cer-tain developmental stage. B. rapa is among the species that require seed vernalization, including important vegetables such as Chinese cabbages and turnip. Immature bolting, caused by low temperature conditions during spring, reduces yields and compromises the quality of Chinese cabbage. To prevent preharvest bolting, plastic tunnels have been used to maintain a sufficiently high temperature. Although some late-bolting varieties have been developed, unexpected bolt-ing occurs in some cases. Therefore, it is necessary to iden-tify DNA markers that are closely linked to late bolting.

FLOWERING LOCUS C(FLC), which encodes a MADS-box transcription factor, has been identified as a re-pressor of floral transition in Arabidopsis thaliana (Sheldon et al. 1999, 2000). FLC transcription is suppressed at a low temperature, and FLC downregulation promotes flowering (Michaels and Amasino 1999, Sheldon et al. 2000). FLC transcript levels determine the extent of the vernalization re-sponse in flower initiation in a dose-dependent manner (Sheldon et al. 2000). Genetic screening for mutants that re-tain late flowering after extended cold treatment identified two Arabidopsis genes, VERNALIZATION 1(VRN1) and VERNALIZATION 2 (VRN2), that are involved in the vernal-ization response. VRN1 encodes a DNA-binding protein and causes a stable repression of FLC (Levy et al. 2002). Vernal-ization itself epigenetically silences FLC by a process in-volving the polycomb protein VRN2. VRN2 promotes the trimethylation of histone H3K27 (Bastow et al. 2004, Gendall et al. 2001). VERNALIZATION INSENSITIVE 3 (VIN3), which encodes a PHD-domain-containing protein, is involved in the initiation of FLC repression through altera-tion of the structure of FLC chromatin (Sung et al. 2006). In brief, FLC repression during vernalization is accomplished by the cooperative functions of these proteins.

FLOWERING LOCUS T (FT) is also involved in the flo-ral transition pathway. The FT protein moves through the vascular tissues to the shoot apex and induces flowering (Abe et al. 2005, Kardailsky et al. 1999, Kobayashi et al. 1999). The FLC protein negatively regulates FT expression through a direct interaction with the first intron of FT (Helliwell et al. 2006, Lee et al. 2000, Samach et al. 2000).

The Arabidopsis and Brassica clades have been thought to diverge from a common ancestor, 17 to 18 million years ago (Yang et al. 1999, 2006). Because of chromosomal

Communicated by T. Nishio

Received January 12, 2011. Accepted March 8, 2011. *Corresponding author (e-mail: ssmats@affrc.go.jp)

Kakizaki, Kato, Fukino, Ishida, Hatakeyama and Matsumoto 152

rearrangements, including fusions and/or fissions, B. rapa genome has become 4-fold larger than A. thaliana genome (Johnston et al. 2005), and the genome of B. rapa contains four copies of FLC and two copies of FT (Schranz et al. 2002, Yang et al. 2006).

Two quantitative trait loci (QTLs), VFR1 and VFR2, that control days to flowering have been identified in B. rapa by non-vernalized experiments (Osborn et al. 1997). VFR2 was found to contain BrFLC1 (Schranz et al. 2002). Osborn et al. (1997) also reported two additional QTLs, FR1 and FR2, which control vernalization-independent flowering. Schranz et al. (2002) mapped BrFLC2 and BrFLC5 to FR1 and FR2, respectively. Li et al. (2009) reported that BrFLC1 and BrFLC2 were linked to QTLs that control bolting, budding, and flowering time. They used F2 progeny derived from Yellow Sarson and the Japanese commercial variety “Osome.”The natural variation of the splicing site in BrFLC1 was thought to contribute to flowering time in a study using 121 B. rapa accessions (Yuan et al. 2009). Colocalization of the flowering-time QTL with flowering-related genes, including BrFLC1 and BrFLC2 has been detected in multiple segre-gating populations of B. rapa (Lou et al. 2007). These studies strongly suggest that the BrFLC genes have retained redundant functions in the vernalization response in B. rapa. In B. napus, three FT orthologs are associated with two QTLs for flowering time (Wang et al. 2009). Allelic varia-tion in FT orthologs in B. napus is may be responsible for the variation in flowering time in winter and spring cultivars.

Although several studies on QTLs for flowering time in B. rapa are available, progenies of hybrids between distantly related lines have been used in these studies. In the breeding of Chinese cabbage cultivars, a cross between different Chinese cabbage lines is usually performed. For development of DNA markers useful in Chinese cabbage breeding, QTLs should be identified in progenies resulting from a cross be-tween different Chinese cabbage lines. In this study, we used an extremely late-bolting parental line (PL6) and an early-bolting parental line (A9709) of Chinese cabbage, and per-formed QTL analysis to identify genetic markers associated with the late-bolting trait in PL6.

Materials and Methods

Plant materials and investigation of bolting time Two parental lines of Chinese cabbage (Brassica rapa L.subsp. pekinensis), Chukanbohon Nou 6 gou (PL6) and Chukanbohon Nou 7 gou (A9709), were used. An F2 popula-tion (n=478) was produced by self-pollinating an F1 plant de-rived from a cross between PL6 (female) and A9709 (male). Temperature is the most important stimulus for bolting, and therefore, we divided the F2 population into five subsets, and cultivated them under different conditions or during various periods (Table1). One subset was cultivated in pots (diame-ter, 9cm) in a heated greenhouse, and the other subsets were grown in the field at the National Institute of Vegetable and Tea Science from January to May in 2009 and 2010 (Tsu, Mie, Japan; 34°46′N, 136°25′E). In the pot-cultivation trials, the number of days required for the top of the inflorescence to reach 10cm above the soil was recorded from the day of transfer to the greenhouse as the starting date. In the case of field cultivation, the length of the stem was recorded. Linkage and QTL analysis

We constructed a linkage map by using genotyping data of 96 F2 progeny derived from a cross between PL6 and A9709 in pot cultivation (09-pot population). The DNA markers used in the study were 43 simple sequence repeat (SSR) markers (prefix BRMS) reported by Suwabe et al. (2002, 2004, 2006), 30 SSR markers (prefixes Na, Ni, Ol and Ra) reported by Lowe et al. (2004), 99 SSR markers (prefix KBr) reported by Hatakeyama et al. (2010), 14 EST-based SSR markers (prefix BRE) developed by Abe et al. (unpublished data), and 7 SSR markers (prefix ENA or EJU) reported by Choi et al. (2007). For genotyping the S locus, we amplified S locus glycoprotein by using primer pairs re-ported by Nishio et al. (1996). To increase the number of SSR makers, we used the read2Marker program and devel-oped 19 additional SSR markers (Fukuoka et al. 2005) from BAC sequences of the B. rapa KBr (H, B and S) libraries from the Brassica Genome Gateway (http://brassica.bbsrc. https://www.sodocs.net/doc/2410983350.html,/). The primers used for linkage map construction are listed in Supplemental Table1. PCR reactions and detection of polymorphisms were performed as described previously (Hatakeyama et al. 2010). Linkage analysis was performed and genetic map was constructed using the J oinmap 4.0 software (van Ooijen 2006). The QTL analysis was per-formed using the MapQTL 5.0 software (van Ooijen 2004) under the “interval mapping” option. A second map was constructed using 96 F2 plants in the field experiment that were transplanted in February 2009 (09-fieldA population).

Table1.Five environmental conditions for bolting time evaluation

Trial Sowing date Date of transplanting Date of recording Conditions Number of F2 plants 09-pot2008.11.135°C/24h light for 49days a heated greenhouse b96

09-fieldA2009.1.82009.2.92009.4.23Open field96

09-fieldB2009.1.272009.2.262009.5.12Open field96

10-fieldA2010.1.82010.2.102010.4.28Open field109

10-fieldB2010.2.162010.3.192010.5.27Open field81

a25-days-old plants were treated with low temperature for 49days under continuous light.

b The minimum temperarute in the greenhouse was 20°C.

QTLs for bolting time in Chinese cabbage153

The logarithm of odds (LOD) thresholds for QTLs (α=0.05) were estimated to be 3.5 and 4.5 in the 09-pot and 09-fieldA populations, respectively, by 1000 permutations. RNA isolation and real-time RT-PCR analysis of BrFTa and BrFLC2

Two parental lines, A9709 and PL6, of 15-day-old plants were vernalized at 5°C for up to 49days under a 12-h light cycle. Total RNA was extracted from leaf tissues at 7-day intervals for 49days by using an RNeasy Plant Minikit (QIAGEN), according to the manufacturer’s instructions. cDNA was synthesized using the PrimeScript? RT reagent kit (TaKaRa) with random hexamer and oligo d(T) primers. Real-time RT-PCR was performed on a Thermal Cycler Dice Real-Time System (TaKaRa) using SYBR Premix Ex Taq II (TaKaRa). The real-time PCR primers for BrFTa and BrFLC2 are listed in Supplemental Table2. The transcript level of each gene was normalized to that of BrACTIN (Li et al. 2009).

Sequence and expression analysis of the BrFLC genes To determine polymorphisms in BrFLC1 and BrFLC5, genomic fragments were amplified using the specific prim-ers listed in Supplemental Table2 and a program consisting of 35 cycles (94°C for 30s, 56°C for 30s and 72°C for 2min). PCR products were directly sequenced using an ABI3730 DNA Analyzer (Applied Biosystems) and ana-lyzed using the BioEdit program (http://www.mbio.ncsu. edu/bioedit/bioedit.html). For expression analysis of BrFLC1, young leaves were collected from 15-day-old plants in the greenhouse at 20°C, total RNA was extracted using an RNeasy Plant Minikit (QIAGEN), and RT-PCR was performed according to the method described by Yuan et al. (2009). To analyze the expression of 4 BrFLC genes during cold treatment, RT-PCR was performed using the specific primers listed in Supplemental Table2 and a pro-gram consisting of 35 cycles (94°C for 30s, 58°C for 30s and 72°C for 30s).

Results

Phenotypic variation

The bolting characteristics of the PL6 and A9709 parents, the F1 hybrids, and the F2 population were evaluated under five different environmental conditions (Table1). To ensure that the plants in the pot experiment received an appropriate cold treatment, we incubated 25-day-old plants at 5°C for 49days under continuous light. After the cold treatment, the plants were transplanted into pots (diameter, 9cm) and placed in a heated greenhouse (February to March 2009). In the field experiment, seeds were germinated in pots placed in the greenhouse and transplanted into a field without sub-jecting them to a cold treatment. To examine bolting charac-teristics, we determined the number of days needed for the stem length to reach 10cm (pot experiment) or the stem length per se (field experiment). The distribution of stem length and bolting time in the F2 population is shown in Fig.1. The bolting time significantly differed between A9709 and PL6, both in greenhouse and field cultivation (Fig.1A, 1B). The F2 populations did not show a typical dis-crete segregation, and this finding indicated that bolting is controlled by multiple genes. In the pot experiment (desig-nated 09-pot), the number of days needed for the stem length to reach 10cm was 10±0, 27.5±1.3 and 19.3±1.0 (mean±SD), in A9709, PL6, and F1, respectively (Fig.1A). In the field experiment in 2009 (designated 09-fieldA), the mean±SD of the stem length was 33.7±4.8, 5.7±1.7 and 10.1±1.7cm in A9709, PL6, and F1, respectively (Fig.1B). QTL for late bolting in PL6

QTL analysis in the 09-pot experiment identified three QTLs on two different linking groups (LGs), and that in the 09-fieldA experiment identified two QTLs on two LGs (Fig.2). The QTL that had the greatest effect on bolting in pot cultivation, pot-QTL1, was detected on R07 located in close proximity to the KBrB092C03 marker and accounted for 25.3% of the observed variation (Table

2). The flowering Fig.1.Frequency distributions of days to bolting and stem length of 96 F2 plants derived from a cross between the parental lines PL6 and A9709 in pot cultivation (A) and field cultivation (B).

Kakizaki, Kato, Fukino, Ishida, Hatakeyama and Matsumoto

154promoter BrFTa was present in a BAC sequence containing KBrB092C03. Two other QTLs, pot-QTL2 and pot-QTL3,were mapped on R07 and R02, respectively (Fig.2). These three pot QTLs accounted for 64.2% of the observed varia-tion (Table 2). No FLC or FT homolog was mapped near pot-QTL2 and pot-QTL3. Field-QTL1 and field-QTL2 were

located on R10 and R03, respectively, and accounted for 30.7% and 27.6% of the bolting variation, respectively (Fig.2 and Table 2). BrFLC1 and BrFLC5 colocalized with field-QTL1 and field-QTL2, respectively .

To examine the effects of the two genes, we classified the F 2

progeny on the basis of the marker genotype for each

Fig. 2.Simple sequence repeat (SSR)-based linkage map constructed using genotyping data of the 96 F 2 plants derived from a cross between the parental lines A9709 and PL6 that were cultivated in a greenhouse in 2009. Linkage groups (LGs) are numbered according to the consensus LGs (R01 to R10) of the Multinational Brassica Genome Project (https://www.sodocs.net/doc/2410983350.html,/index.php), based on the common SSR markers. Marker names are indicated to the right of each LG. Markers with asterisks were used for the analysis of quantitative trait loci (QTLs) in the field experi-ment in 2009. Black and white boxes to the right of the LGs indicate QTLs detected in the greenhouse and field in 2009, respectively. The flowering-related genes, which are present in BAC sequences together with the mapped SSR markers, are shown in brackets.

QTLs for bolting time in Chinese cabbage 155

gene and correlated the stem length with the 9 different genotypes (Table 3). The F 2 progeny with the BrFLC1 and BrFLC5 alleles derived from the PL6 parent showed sig-nificant delays in stem elongation, except in the 09-fieldB experiment (Table 3). In the 09-fieldB experiment, BrFLC1appeared to predominantly determine late bolting whereas BrFLC5 had no effect. Because BrFLC1 and BrFLC5played important roles in determining the timing of bolting in the field experiments, we next investigated the effects of other genetic markers, namely BrFTa and KBrH068G07R,which affected the timing of bolting in the pot experiment (Fig.2). Although we failed to detect any QTLs for these

two markers in the field experiments, we observed a signifi-cant selection effect (Table 3). For example, in three of the four field experiments (09-fieldB, 10-fieldA and 10-fieldB),the F 2 progeny with the BrFTa and KBrH068G07R alleles from PL6 showed significantly shorter stem than did the F 2progeny with the BrFTa and KBrH068G07R alleles from A9709 (Table 3).

Expression analysis of BrFTa and BrFLC2

Several studies have shown that the QTL that controls flowering time is located near BrFLC2 in B. rapa (Li et al.2009, Lou et al. 2007, Osborn et al. 1997). Similarly,

Table 2.Results of QTL analysis for the late bolting of PL6Table 3.Relationship between two marker genotypes and the stem length (mean ±SD) in F 2 progeny

QTL linkage group Marker

candidate gene 09-pot experiment 09-fieldA experiment LOD Additive effect a Variance explained (%)

LOD

Additive effect b

Variance explained (%)

pot-QTL1R07KBrB092C03(BrFTa)BrFTa 6.09?1.725.3n.d.pot-QTL2R07KBrH068G07R n.d. 5.26?1.722.3n.d.pot-QTL3R02KBrH072H08F

n.d. 4.54

?1.6

19.6

n.d.

field-QTL1R10KBrH080A08(BrFLC1)BrFLC1n.d.7.64 3.830.7field-QTL2

R03

BrFLC5

BrFLC5

n.d.

5.98

7.7

27.6n.d., not-detected.

a Additive effect of A9709 allele in days to bolting.

b Additive effect A9709 allele in stem length.

Marker genotype*09-fieldA 09-fieldB 10-fieldA 10-fieldB KBrH080A08 (BrFLC1)BrFLC5No. of plants stem length (cm)No. of plants stem length (cm)No. of plants stem length (cm)No. of plants stem length (cm)AA AA 521.2±7.8a 211.6±5.3ab 711.3±1.7a 411.3±1.3a AA AB 918.1±1.8ab 1115.9±5.8a 219.5±2.7a 109.3±1.8a AA BB 411.9±3.0bcd 1011.4±2.6ab 67.8±0.8ab 67.2±2.3abc AB AA 1114.8±3.8bc 1413.3±4.5ab 89.8±4.7a 58.2±2.4ab AB AB 2112.0±3.5cd 2511.1±3.9b 27 6.7±1.7b 247.4±1.8bc AB BB 229.0±2.4d 1111.5±3.6ab 16 5.9±1.5b 9 5.9±1.6bc BB AA 414.0±4.2bcd 39.0±1.6ab 4 5.5±1.3b 57.2±2.4abc BB AB 169.6±2.7d 149.0±4.0b 13 5.9±1.0b 15 6.0±1.9bc BB

BB

48.0±1.8d 69.3±3.6b 7 5.1±0.7b 3 4.3±1.2c KBrB092C03

(BrFTa )KBrH068G07R

No. of plants stem length (cm)No. of plants stem length (cm)No. of plants stem length (cm)No. of plants stem length (cm)AA AA 1013.8±6.8ab 716.3±2.6a 311.7±8.1a 129.3±1.8a AA AB 1113.0±6.8ab 1212.6±5.1ab 167.9±2.5ab 97.0±2.3ab AA BB 39.0±1.7ab 510.5±6.3ab 57.2±1.8ab 27.0±1.4ab AB AA 1113.4±3.8ab 1113.8±5.3ab 157.9±2.7ab 38.7±2.1ab AB AB 2713.8±4.5a 2911.1±4.0ab 317.4±2.8ab 218.1±1.9ab AB BB 810.9±4.2ab 1810.5±3.1ab 8 6.6±2.4ab 10 5.9±1.8b BB AA 411.6±3.4ab 212.0±0.9ab 3 6.7±1.2ab 37.3±3.1ab BB AB 1011.4±2.8ab 710.2±3.9ab 177.7±2.3ab 8 6.3±1.5ab BB

BB

107.8±1.6b 47.0±1.3b 8 5.8±1.7b 13 5.8±2.6b A9709 (AA)333.7±4.83Flowered

348.5±21.5237.0PL6 (BB)

3

5.7±1.7

–

3

4.5±0.9

3

4.7±0.6

* AA, genotype of A9709 (early-bolting); BB, genotype of PL6 (late-bolting).

Values followed by the same letter are not significantly different at the 5% level, as determined by Tukey-Kramer HSD test.

Kakizaki, Kato, Fukino, Ishida, Hatakeyama and Matsumoto

156Okazaki et al. (2007) reported that BoFLC2 plays a central role in the repression of flowering in B. oleracea (Okazaki et al. 2007). Wang et al. (2009) reported that BnC6FTa , the orthologous gene of BrFTa , was associated with the major QTL for flowering time in B. napus . Furthermore, our QTL analysis showed that BrFTa was located near QTL for bolt-ing time in B. rapa (Fig.2). Next, we quantified the expres-sion of BrFTa and BrFLC2 in the pot experiment at 7-day intervals for 49days by real-time RT-PCR. The expression of BrFTa increased gradually in A9709, whereas its expres-sion was stable in PL6 (Fig.3A). To investigate the effect of BrFLC2 on bolting after vernalization under artificial condi-tions, we monitored the expression of BrFLC2 during ver-nalization (Fig.3B). However, we were unable to detect any

significant differences in BrFLC2 expression between PL6and A9709 under these conditions. In addition to BrFLC2expression analysis, no peaks of LOD scores were observed in nearby regions of BrFLC2 in the QTL analysis. These results suggest that late bolting in PL6 under conditions of continuous exposure to low temperatures is primarily deter-mined by the repression of BrFTa during vernalization and is independent of BrFLC2.

Genomic structure and expression of BrFLC genes

To elucidate the genome structure of BrFLC1 and BrFLC5, we determined the nucleotide sequences from the 5′-UTR to exon 7 of BrFLC1 and from exon 4 to exon 7 of BrFLC5, respectively (Fig.4A). A sequence comparison of the BrFLC1 alleles from A9709 and PL6 identified a single nucleotide polymorphism in exon 2. In BrFLC5, a 17-bp de-letion was identified in intron 6 of the A9709 allele. To in-vestigate the effect of these nucleotide polymorphisms on BrFLC1 expression, we performed RT-PCR for 35 cycles with primers in the 5′-UTR and exon 7. Surprisingly, a radi-cal reduction in the BrFLC1 transcript level was observed in A9709, and an alternatively spliced transcript was identified (Fig.4B). According to the existing literature, alternative splice variants generated by a G-to-A polymorphism at the 5′ splice site in intron 6 of BrFLC1 are associated with a flowering phenotype (Yuan et al. 2009). However, although one SNP site was detected at exon 2, no differences were detected in intron 6 between A9709 and PL6 according to genomic sequencing. Next, we cloned and sequenced this alternative transcript derived from A9709. In the longer tran-script, intron 3, consisting of 77 nucleotides, was retained in the mature mRNA. This resulted in a frame-shift mutation and introduced a stop codon, giving rise to a truncated protein of 137 amino acids (Fig.4C). To investigate the response of 4 BrFLC paralogs under cold conditions, we performed RT-PCR analysis using specific primer pairs that amplify exon 4 to exon 7 of each BrFLC gene (Fig.4D).During the cold treatment period, the expression of the four BrFLC genes was decreased. However, the expression levels of BrFLC1 and BrFLC5 were higher in PL6 than in A9709during the cold treatment, and the expression was slightly detected even after seven weeks of cold treatment. These re-sults suggest that the late bolting of PL6 was controlled by BrFLC1 and BrFLC5.

Discussion

The timing of bolting and flowering is regulated by endoge-nous factors and environmental stimuli. Many genes con-trolling this pathway have been isolated and characterized in Arabidopsis (Crevillen and Dean 2010, Dennis and Peacock 2007). To obtain information regarding the marker-assisted selection of Brassica crops, we mapped vernalization genes and flowering-related genes on a genetic map constructed using an F 2 population derived from two parental lines that

show clearly different bolting times. Our data suggest that

Fig. 3.Expression analysis of BrFTa and BrFLC2 in response to ver-nalization. Two parental lines (A9709; closed circles and PL6; open circles) of 15-day-old plants were vernalized at 5°C for up to 49days under a 12-h light cycle. The BrFTa (A) and BrFLC2 (B) transcripts were examined in young leaves at the end of the light period. Tran-script levels were analyzed by real-time RT-PCR and normalized to the levels of BrACTIN (Li et al. 2009). Error bars represent standard deviations of the mean.

QTLs for bolting time in Chinese cabbage157

known genes involved in the vernalization pathway in Arabidopsis can be mapped at QTL positions. Unexpectedly, we were unable to detect any common QTLs for different environmental conditions, i.e., greenhouse and open field. Alternative transcripts of BrFLC1

In this study, we showed that BrFLC1 was colocalized with the field-QTL1, and its allele from PL6 delayed bolting. Although we identified a nucleotide substitution at 2675bp in exon 2 of BrFLC1 (causing a nonsynonymous change (Q69R) in the PL6 allele), this did not directly explain the difference in bolting times between PL6 and A9709. Inter-estingly, we detected an alternative BrFLC1 transcript in A9709 (Fig.4B). This alternative transcript retains intron 3 and gives rise to a truncated protein (Fig.4C). Yuan et al. (2009) reported that a substitution at the 5′ splice site in in-tron 6 of BrFLC1 causes alternative splicing and affects flowering time. Alternative splicing can result in the genera-tion of protein with new functions through exon sliding (Tarrio et al. 2008). However, it often creates transcripts with premature stop codons, which are generally degraded by nonsense-mediated decay (Barbazuk et al. 2008). Al-though it is unclear whether splicing is influenced by SNPs in exon 2 of BrFLC1, the possibility of an SNP in the exon causing the alternative splicing has been suggested in wheat (Sun et al. 2010).

Differences in bolting between pot and field experiments We identified three QTLs in pot experiments (pot-QTL1, pot-QTL2 and pot-QTL3) and two in field experiments (field-QTL1 and field-QTL2). Three of the five QTLs colo-calized with homologs of well-known flowering-related genes from Arabidopsis, such as FT and FLC (Fig.2). Un-expectedly, no QTLs involved in bolting variation were de-tected in both the pot and field experiments. Several studies involving QTL analyses have suggested that Brassica possesses four FLC orthologs that control the vernalization response (Li et al. 2009, Osborn et al. 1997, Schranz et al. 2002, Yuan et al. 2009). However, in the pot experiment, no major QTL was detected in any of the four FLC orthologs. Real-time RT-PCR results indicated that one of the FLC genes in Brassica, BrFLC2, showed similar expression pat-terns in A9709 and PL6. In contrast, BrFTa was dramatical-ly induced after 35-day cold treatment in only A9709 (Fig.3A).

In the pot experiment, the temperature was maintained at Fig.4.Nucleotide polymorphisms and gene expression analysis of BrFLC genes. (A) DNA sequences of BrFLC1 and BrFLC5 from exon 1 to exon 7 and from exon 4 to exon 7, respectively, were determined. Exons, introns, and the 5′-UTR are shown as open boxes, black bars, and a dotted line, respectively. The position of the translational start site is indicated as +1. Polymorphisms between A9709 and PL6 are in-dicated with asterisks. (B) RT-PCR amplification of BrFLC1. Leaf samples were collected from 15-day-old plants. Primer annealing sites are indicated in (A) by black triangles located at the 5′-UTR and exon 7 of BrFLC1 (Yuan et al. 2009). The alternative and normal transcripts in A9709 are indicated by an asterisk and a white triangle, respective-ly. BrACTIN was amplified as an internal control. A: A9709; P: PL6.

(C) Nucleotide and deduced amino acid sequences of the region giving rise to alternative splicing (exon 3 to exon 4) of BrFLC1 in A9709. The normal transcript (PL6) is shown in the upper lane, and the alter-native transcript (A9709) is shown in the lower lane. The nucleotide sequence of intron 3 is shown in lower case. The position of the trans-lational start site in the BrFLC1 cDNA is indicated as +1. The pre-mature stop codon (TAA) is indicated with an asterisk. (D) Expression of BrFLC genes during cold treatment, harvested after 0, 1, 3, or 7weeks of growth at 5°C, as indicated in above the figure. BrACTIN was amplified as an internal control.

Kakizaki, Kato, Fukino, Ishida, Hatakeyama and Matsumoto 158

5°C for 49days during the cold treatment. In the field exper-iment, the temperature showed daily fluctuations during the winter. Although the expression of VIN3, which is the activator of FLC repression, is upregulated by cold treatment for 20days, its expression is repressed once the plant is again exposed to normal temperatures (Sung and Amasino 2004). To accomplish robust repression of FLC, Arabidopsis requires continuous cold treatment for at least 3weeks (Gendall et al. 2001). Probably because a temperature suffi-ciently low for FLC repression was achieved in the pot ex-periment, QTLs were not detected near FLC but were detect-ed near flowering-promoting genes such as FT (Fig.2 and Table2). In the field experiments, which involved tempera-ture oscillations and a gradual temperature decrease, we de-tected QTLs for genes involved in the vernalization pathway (Fig.2 and Table2).

Although the QTLs at BrFTa and KBrH068G07R were detected only in the pot experiment, their effects on the se-lection process were also observed in the field experiment (Table3). This suggests the existence of minor QTLs with a slight but distinctive effect on the differences in bolting be-tween PL6 and A9709. In fact, we did not isolate any F2 progeny that bolted as early as A9709 in any of the experi-ments. This indicates that many loci are involved in bolting in B. rapa, some of which remained undetected in this study.

In summary, we identified three QTLs at the periphery of orthologous genes of well-known flowering genes from Arabidopsis, including FLC and FT. We expect the BrFLC genes to be mainly involved in the low-temperature require-ment, whereas BrFTa and KBrH068G07R affect elongation of the stem after exposure to low temperature. We used two different environments to identify four genetic markers that almost completely explain the late bolting feature of PL6. Marker-assisted selection by combining these four markers will enable the selection of a late-bolting Chinese cabbage. Acknowledgments

We are grateful to Ms. Satomi Negoro and Shuko Toyoda for their technical assistance. This work was supported by NARO Research Project No.211 “Establishment of Integrat-ed Basis for Development and Application of Advanced Tools for DNA Marker-Assisted Selection in Horticultural Crops.”

Literature Cited

Abe,M., Y.Kobayashi, S.Yamamoto, Y.Daimon, A.Yamaguchi, Y.I keda, H.I chinoki, M.N otaguchi, K.G oto and T.A raki (2005) FD, a bZI P protein mediating signals from the floral pathway integrator FT at the shoot apex. Science 309: 1052–1056. Barbazuk,W.B., Y.F u and K.M.M cGinnis (2008) Genome-wide anal-yses of alternative splicing in plants: Opportunities and challenges.

Genome Res. 18: 1381–1392.

Bastow,R., J.S.Mylne, C.Lister, Z.Lippman, R.A.Martienssen and

C.Dean (2004) Vernalization requires epigenetic silencing of FLC

by histone methylation. Nature 427: 164–167.Choi,S.R., G.R.T eakle, P.P laha, J.H.K im, C.J.A llender, E.B eynon, Z.Y.P iao, P.S oengas, T.H.H an, G.J.K ing et al. (2007) The refer-ence genetic linkage map for the multinational Brassica rapa ge-nome sequencing project. Theor. Appl. Genet. 115: 777–792. Crevillén,P. and C.D ean (2011) Regulation of the floral repressor gene FLC: the complexity of transcription in a chromatin context. Curr.

Opin. Plant Biol. 14: 38–44.

Dennis,E.S. and W.J.P eacock (2007) Epigenetic regulation of flower-ing. Curr. Opin. Plant Biol. 10: 520–527.

Fukuoka,H., T.Nunome, Y.Minamiyama, .Kono, N.Namiki and

A.Kojima (2005) Read2Marker: a data processing tool for micro-

satellite marker development from a large data set. Biotechniques 39: 472: 474–476.

Gendall,A.R., Y.Y.L evy, A.W ilson and C.D ean (2001) The VERNAL-IZATION 2 gene mediates the epigenetic regulation of vernaliza-tion in Arabidopsis. Cell 107: 525–535.

Hatakeyama,K., A.Horisaki, S.Niikura, Y.Narusaka, H.Abe, H.

Yoshiaki, M.I shida, H.F ukuoka and S.M atsumoto (2010) Mapping of quantitative trait loci for high level of self-incompatibility in Brassica rapa L. Genome 53: 257–265.

Helliwell,C.A., C.C.Wood, M.Robertson, W.J.Peacock and E.S.

Dennis (2006) The Arabidopsis FLC protein interacts directly in vivo with SOC1 and FT chromatin and is part of a high-molecular-weight protein complex. Plant J. 46: 183–192.

Johnston,J.S., A.E.P epper, A.E.H all, Z.J.C hen, G.H odnett, J.D rabek, R.Lopez and H.J.Price (2005) Evolution of genome size in Brassicaceae. Annal. Bot. 95: 229–235.

Kardailsky,I., V.K.S hukla, J.H.A hn, N.D agenais, S.K.Christensen, J.T.N guyen, J.C hory, M.J.H arrison and D.W eigel (1999) Activa-tion tagging of the floral inducer FT. Science 286: 1962–1965. Kobayashi,Y., H.K aya, K.G oto, M.I wabuchi and T.A raki (1999) A pair of related genes with antagonistic roles in mediating flowering signals. Science 286: 1960–1962.

Lee,H., S.S.Suh, E.Park, E.Cho, J.H.Ahn, S.G.Kim, J.S.Lee, Y.M.Kwon and I.L ee (2000) The AGAMOUS-LI KE 20 MADS domain protein integrates floral inductive pathways in Arabidopsis.

Genes Dev. 14: 2366–2376.

Levy,Y.Y., S.M esnage, J.S.M ylne, A.R.G endall and C.D ean (2002) Multiple roles of Arabidopsis VRN1 in vernalization and flowering time control. Science 297: 243–246.

Li,F., H.K itashiba, K.I naba and T.N ishio (2009) A Brassica rapa linkage map of EST-based SNP markers for identification of candi-date genes controlling flowering time and leaf morphological traits.

DNA Res. 16: 311–323.

Lou,P., J.J.Zhao, J.S.Kim, S.X.Shen, D.P.Del Carpio, X.F.Song, M.N.Jin, D.V reugdenhil, X.W.W ang, M.K oornneef et al. (2007) Quantitative trait loci for flowering time and morphological traits in multiple populations of Brassica rapa. J. Exp. Bot. 58: 4005–4016.

Lowe,A.J., C.Moule, M.Trick and K.J.Edwards (2004) Efficient large-scale development of microsatellites for marker and mapping applications in Brassica crop species. Theor. Appl. Genet. 108: 1103–1112.

Michaels,S.D. and R.M.Amasino (1999) FLOWERING LOCUS C encodes a novel MADS domain protein that acts as a repressor of flowering. Plant Cell 11: 949–956.

Nishio,T., M.K usaba, M.W atanabe and K.H inata (1996) Registration of S alleles in Brassica campestris L by the restriction fragment sizes of SLGs. Theor. Appl. Genet. 92: 388–394.

Okazaki,K., K.Sakamoto, R.Kikuchi, A.Saito, E.Togashi, Y.

Kuginuki, S.Matsumoto and M.Hirai (2007) Mapping and

QTLs for bolting time in Chinese cabbage159

characterization of FLC homologs and QTL analysis of flowering

time in Brassica oleracea. Theor. Appl. Genet. 114: 595–608.

Osborn,T.C., C.Kole, .A.P.Parkin, A.G.Sharpe, M.Kuiper, D.J.

Lydiate and M.T rick (1997) Comparison of flowering time genes

in Brassica rapa, B. napus and Arabidopsis th aliana. Genetics

146: 1123–1129.

Samach,A., H.Onouchi, S.E.Gold, G.S.Ditta, Z.Schwarz-Sommer,

M.F.Y anofsky and G.C oupland (2000) Distinct roles of CONSTANS

target genes in reproductive development of Arabidopsis. Science

288: 1613–1616.

Schranz,M.E., P.Quijada, S.B.Sung, L.Lukens, R.Amasino and

T.C.Osborn (2002) Characterization and effects of the replicated

flowering time gene FLC in Brassica rapa. Genetics 162: 1457–

1468.

Sheldon,C.C., J.E.Burn, P.P.Perez, J.Metzger, J.A.Edwards, W.J.

Peacock and E.S.D ennis (1999) The FLF MADS box gene: A

repressor of flowering in Arabidopsis regulated by vernalization

and methylation. Plant Cell 11: 445–458.

Sheldon,C.C., D.T.R ouse, E.J.F innegan, W.J.P eacock and E.S.D ennis

(2000) The molecular basis of vernalization: The central role of

FLOWERING LOCUS C(FLC). Proc. Natl. Acad. Sci. USA 97:

3753–3758.

Sun,Y., Z.H e, W.M a and X.X ia (2011) Alternative splicing in the cod-

ing region of Ppo-A1 directly influences the polyphenol oxidase

activity in common wheat (Triticum aestivum L.). Funct. I ntegr.

Genomics 11: 85–93.

Sung,S.B. and R.M.Amasino (2004) Vernalization in Arabidopsis

thaliana is mediated by the PHD finger protein VIN3. Nature 427:

159–164.

Sung,S.B., R.J.S chmitz and R.M.A masino (2006) A PHD finger pro-

tein involved in both the vernalization and photoperiod pathways

in Arabidopsis. Genes Dev. 20: 3244–3248.

Suwabe,K., H.I ketani, T.N unome, T.K age and M.H irai (2002) Isola-

tion and characterization of microsatellites in Brassica rapa L.

Theor. Appl. Genet. 104: 1092–1098.

Suwabe,K., H.

I ketani, T.Nunome, A.Ohyama, M.Hirai and H.

Fukuoka (2004) Characteristics of Microsatellites in Brassica rapa genome and their potential utilization for comparative genomics in Cruciferae. Breed. Sci. 54: 85–90.

Suwabe,K., H.T sukazaki, H.I ketani, K.H atakeyama, M.K ondo, M.

Fujimura, T.Nunome, H.Fukuoka, M.Hirai and S.Matsumoto (2006) Simple sequence repeat-based comparative genomics be-tween Brassica rapa and Arabidopsis thaliana: the genetic origin of clubroot resistance. Genetics 173: 309–319.

Tarrio,R., F.J.A yala and F.R odriguez-Trelles (2008) Alternative splic-ing: a missing piece in the puzzle of intron gain. Proc. Natl. Acad.

Sci. USA 105: 7223–7228.

van Ooijen,J.W. (2004) MapQTL version 5.0: software for the map-ping of quantitative trait loci in experiment population. Plant Research International, Wageningen, Netherlands.

van Ooijen,J.W. (2006) JoinMap? 4.0: Software for the calculation of genetic linkage maps in experimental populations. Kyazma BV, Wageningen, Netherlands.

Wang,J., Y.L ong, B.W u, J.L iu, C.J iang, L.S hi, J.Z hao, G.J.K ing and J.M eng (2009) The evolution of Brassica napus FLOWERING LOCUS T paralogues in the context of inverted chromosomal du-plication blocks. BMC Evol. Biol. 9: 271.

Yang,T.J., J.S.K im, S.J.K won, K.B.L im, B.S.C hoi, J.A.K im, M.J in, J.Y.P ark, M.H.L im, H.I.K im et al. (2006) Sequence-level analysis of the diploidization process in the triplicated FLOWERING LOCUS C region of Brassica rapa. Plant Cell 18: 1339–1347. Yang,Y.W., K.N.L ai, P.Y.T ai and W.H.L i (1999) Rates of nucleotide substitution in angiosperm mitochondrial DNA sequences and dates of divergence between Brassica and other angiosperm lin-eages. J. Mol. Evol. 48: 597–604.

Yuan,Y.X., J.W u, R.F.S un, X.W.Z hang, D.H.X u, G.B onnema and X.W.W ang (2009) A naturally occurring splicing site mutation in the Brassica rapa FLC1 gene is associated with variation in flow-ering time. J. Exp. Bot. 60: 1299–1308.

关于时间管理的英语作文 manage time

How to manage time Time treats everyone fairly that we all have 24 hours per day. Some of us are capable to make good use of time while some find it hard to do so. Knowing how to manage them is essential in our life. Take myself as an example. When I was still a senior high student, I was fully occupied with my studies. Therefore, I hardly had spare time to have fun or develop my hobbies. But things were changed after I entered university. I got more free time than ever before. But ironically, I found it difficult to adjust this kind of brand-new school life and there was no such thing called time management on my mind. It was not until the second year that I realized I had wasted my whole year doing nothing. I could have taken up a Spanish course. I could have read ten books about the stories of successful people. I could have applied for a part-time job to earn some working experiences. B ut I didn’t spend my time on any of them. I felt guilty whenever I looked back to the moments that I just sat around doing nothing. It’s said that better late than never. At least I had the consciousness that I should stop wasting my time. Making up my mind is the first step for me to learn to manage my time. Next, I wrote a timetable, setting some targets that I had to finish each day. For instance, on Monday, I must read two pieces of news and review all the lessons that I have learnt on that day. By the way, the daily plan that I made was flexible. If there’s something unexpected that I had to finish first, I would reduce the time for resting or delay my target to the next day. Also, I would try to achieve those targets ahead of time that I planed so that I could reserve some more time to relax or do something out of my plan. At the beginning, it’s kind of difficult to s tick to the plan. But as time went by, having a plan for time in advance became a part of my life. At the same time, I gradually became a well-organized person. Now I’ve grasped the time management skill and I’m able to use my time efficiently.

英语演讲稿：未来的工作

英语演讲稿：未来的工作 这篇《英语演讲稿范文：未来的工作》，是特地，希望对大家有所帮助！ 热门演讲推荐：竞聘演讲稿 | 国旗下演讲稿 | 英语演讲稿 | 师德师风演讲稿 | 年会主持词 | 领导致辞 everybody good afternoon:. first of all thank the teacher gave me a story in my own future ideal job. everyone has a dream job. my dream is to bee a boss, own a pany. in order to achieve my dreams, i need to find a good job, to accumulate some experience and wealth, it is the necessary things of course, in the school good achievement and rich knowledge is also very important. good achievement and rich experience can let me work to make the right choice, have more opportunities and achievements. at the same time, munication is very important, because it determines whether my pany has a good future development. so i need to exercise their municative ability. i need to use all of the free time to learn

英语48个音标中文谐音读法大全

英语国际音标共48个音素，其中元音音素20个，辅音音素28个。48个国际音标表 [ ] [] [ [i[ [u [ [] [ 元音音素： /i:/ 【衣发长点】 /I/ 【衣急促地发声】 /e/ 【哎发这个音的时候不要把“哎”的ī给读出来，嘴唇放松自然地读】

/?/【哎发这个音的时候不要把“哎”的ī给读出来，嘴唇扁平地发】 /?:/ 【额发长音】 /?/ 【额发短音】 /∧/ 【阿嘴巴微微地张开发出这个音就可以了】/a:/ 【啊嘴巴长到最大】 /?/ 【哦急促】 /?:/ 【哦声音拖长】 /u/ 【乌急促】 /u:/ 【乌声音拖长】 /eI/ 【有点像答应人的“诶”的声音】 /aI/ 【唉】 /?I/ 【哦－喂连着读】 /?u/ 【呕】 /au/ 【傲】 /I?/ 【衣－饿连着读】 /ε?/ 【哎－饿连着读】 /u?/ 【乌－饿连着读】 辅音音素： /p/ 【普不要把ǔ给发出来，轻音】 /b/ 【不不要把ù给发出来，浊音】

/t/ 【特不要把è给发出来，轻音】 /d/ 【得不要把é给发出来，浊音】 /k/ 【克不要把è给发出来，轻音】 /g/ 【各不要把è给发出来，浊音】 /f/ 【福不要把ú给发出来，轻音】 /v/ 【有点像摩托车启动的声音，“呜呜呜”地，但是是像发“vúvúvú”一样，不要把ú给发出来，浊音】 /s/ 【丝像蛇吐芯子发出的那种声音，不要把ī给读出来，轻音】 /z/ 【就是/s/的浊音】 /θ/ 【牙齿咬住舌头的轻音】 /δ/ 【牙齿咬住舌头的浊音】 /∫/ 【西不要把ī给发出来，轻音】 /з/ 【衣不要把ī给发出来，浊音】 /h/ 【喝不要把ē给发出来，轻音】 /r/ 【若不要把uò给发出来，浊音】 /t∫/ 【七不要把ī给发出来，轻音】 /dз/ 【姬不要把ī给发出来，浊音】 /tr/ 【缺不要把uē给发出来，轻音】 /dr/ 【撅不要把uē给发出来，浊音】 /ts/ 【次不要把ì给发出来，轻音】 /dz/ 【自不要把ì给发出来，浊音】

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关于工作的优秀英语演讲稿

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