ГЕНЕТИКА, 2015, том 51, № 8, с. 905-914
ANALYSIS OF GENOMIC DNA METHYLATION AND GENE EXPRESSION IN CHINESE CABBAGE (Brassica rapa L. ssp. pekinensis) AFTER CONTINUOUS SEEDLING BREEDING
© 2015 L. Taoa, X. L. Wrngc, M. H. Guoa, and Y. W. Zhanga b
aCollege of Horticulture, Northeast Agricultural University, Harbin, 150030 China e-mail: firstname.lastname@example.org bKey Laboratory of Biology and Genetic Improvement of Horticultural Crops (Northeast Region), Ministry of Agriculture,
Harbin, 150030 China cChengguan Middle School, Linqu County, 262600 China Received September 05, 2014
Vernalization plays a key role in the bolting and flowering of Chinese cabbage (Brassica rapa L. ssp. pekinensis). Plants can switch from vegetative to reproductive growth and then bolt and flower under low temperature induction. The economic benefits of Chinese cabbage will decline significantly when the bolting happens before the vegetative body fully grows due to a lack of the edible value. It was found that continuous seedling breeding reduced the heading of Chinese cabbage and led to bolt and flower more easily. In the present study, two inbred lines, termed A161 and A105, were used as experiment materials. These two lines were subjected to vernalization and formed four types: seeds-seedling breeding once, seedling breeding twice, seedling breeding thrice and normal type. Differences in plant phenotype were compared. DNA methylation analysis was performed based on MSAP method. The differential fragments were cloned and analyzed by qPCR. Results showed that plants after seedling breeding thrice had a loosen heading leaves, elongated center axis and were easier to bolt and flower. It is suggested that continuous seedling breeding had a weaker winterness. It was observed that genome methylation level decreased with increasing generation. Four differential genes were identified, short for BraAPCl, BraEMP3, BraUBC26, and BraAL5. Fluorescent qPCR analysis showed that expression of four genes varied at different reproduction modes and different vernalization time. It is indicated that these genes might be involve in the development and regulation of bolting and flowering of plants. Herein, the molecular mechanism that continuous seedling breeding caused weaker winterness was analyzed preliminarily. It plays an important guiding significance for Chinese cabbage breeding.
Chinese cabbage (Brassica rapa L. ssp. pekinensis) belongs to cruciferous biennial plant whose bolting and flowering are mainly influenced by low temperature, also known as vernalization. Chinese cabbage is a seed-vernalization-type plant which can feel low temperature at any period of seed germination and growth. Plants can transmit vernalization signal by cell division and then bolt and flower under certain lighting conditions. The cabbage was usually divided into four groups: springness, weak-winterness, middle-winterness, and strong-winterness based on the need of low temperature accumulation value.
With the development of molecular biology, people pay more attention to the mechanism of bolting and flowering in Arabidopsis and Chinese cabbage [1—5]. The emergence of epigenetics broadened research direction of flowering regulation. It was found that flowering time was much earlier when Arabidopsis was treated with 5-aza-CdR . Methylation level was significantly lower than that in control group when the plants got vernalized or been treated with 5-aza-CdR,
and both methods could promote plant flowering. It was believed that demethylation was the molecular basis of vernalization. The lower methylation level made it easier to flower. Li et al. found that DNA methylation level decreased after the vernalization . It was suggested that vernalization led to the decrease in plant DNA methylation level. Finnegan et al. transferred anti-methyl transferase gene into Arabidopsis, found that methylation level was significantly lower than that in control and had an early flowering time, indicating that the decrease of the methylation level could promote the flowering .
It has been a hot topic about DNA methylation with the increase of people's attention and many methods were developed to detect DNA methylation . Methylation sensitive amplification polymorphism (MSAP) is a molecular marker based on AFLP which is used to analyze genome DNA methylation level. MSAP has many advantages, such as less dosage of DNA, low cost and simple operation. It can detect cytosine methylation changes at all CCGG site across
Table 1. List of adapters and primers used for MSAP analysis
Primer and adapter
5'-CTCGTAGACTGCGTACC-3' 5'-AATTGGTACGCAGTCTAC-3' 5'-CGAGCAGGACTCATGA-3' 5'-GATCATGAGTCCTGCT-3'
genome. MSAP technology does not require the sequence information of the tested DNA and is universal among different species. At present, the MSAP technology has been widely used in potato , Oryza sativa [11, 12], Arabidopsis , Rape [13, 14], Zea mays  for the study on the pattern and level of DNA me-thylation across the genome.
It was found that continuous seedling breeding caused Chinese cabbage a weaker winterness in practice. We deduced that continuous vernalization might changed the expression of some genes related to flowering. In this report, we detected the genomic DNA methylation levels in four types of cabbage mentioned above by MSAP. The differential fragments were cloned and analyzed by BLAST on the Brassica database (http://brassicadb.org/brad/) to find the target genes. Then, gene expression of different reproductive modes was detected by qPCR method. By analyzing the relationship between continuous vernalization and winterness changes, the results can provide more theoretical basis for molecular mechanism of bolting and
flowering in Chinese cabbage as well as for Chinese cabbage breeding.
MATERIALS AND METHODS
The seeds of two Chinese cabbage inbred lines (A161 and A105) which were bred at Northeast Agricultural University were harvested in March, 2011, and then seeded in 8 x 8 cm pots with nutrient-rich soil on March 25, 2011. The seedlings with unfold cotyledon were treated by means of vernalization in an illuminated incubator under control conditions (8/3°C (day/night), 16/8 h (light/dark) photoperiod and 10000 lux light intensity) for 25 d. The well-developed plants were transplanted in 25 x 25 cm pots to obtain the vernalization-once seeds after mixed pollination. The continuous vernalization-twice and continuous vernalization-thrice seeds were got with the same method. These four types of seeds obtained from inbred lines (A161 and A105) were seeded on July 15, 2011 in 8 x 8 cm pots with nutrient-rich soil in an illuminated incubator. The young leaves whose lengths were less than 1.5 cm were harvested, marked as C0, C1, C2, and C3, and then frozen in liquid nitrogen for the isolation of DNA and RNA. The cabbage seedlings in the five-leaves period were transplanted to the field with 65 cm row spacing and 35 cm plant spacing for the analysis of winterness. The normal type seeds of inbred line A105 were seeded in 8 x 8 cm pots with nutrient-rich soil. The seedlings in two leaves period were subjected to vernalization in an illuminated incubator at 8/3°C (day/night) with 16/8 h (light/dark) photo-period and 10000 lux light intensity. Sampling was conducted after 0, 7, 14, 21, 28 d, marked as B0, B1, B2, B3, and B4, respectively and then stored at —80°C for the extraction of RNA. All treatments were repeated three times.
To measure the heading percentage and the length of the center axis, four types of the seedlings were grown under natural conditions for 60 d and field survey was carried out on October 12, 2013.
Genomic methylation analysis by MSAP method
DNA was extracted by CTAB method from the new leaves. MSAP analysis was performed as previously described . All DNA samples were digested with EcoRI/Mspl and EcoRI/Hpall and then connected with the adapter, respectively. The product was subjected to pre-amplification and selective amplification after restriction digestion and connection. The primers and adapters were shown in Table 1. Selective amplification was performed as follows: initially denatured at 94°C for 5 min followed by 12 cycles of dena-turation at 94°C for 30 s, annealing at 65°C for 30 s,
and extension at 72°C for 1 min, 23 cycles of denatur-ation at 94°C for 30 s, annealing at 56°C for 30 s, and extension at 72°C for 1 min; followed by a final extension at 72°C for 10 min. PCR products were analyzed by electrophoresis on a 6% polyacrylamide gel. According to the results of restriction digestion, the genomic DNA methylation level was analyzed and the ratio of hyper-methylation, full methylation and semi-methylation were calculated.
Cloning and sequence analysis of specific methylation fragments
The fragments which were same in the C0, C1, C2 group, but different in the C3 were obtained according to the results of 6% denaturing polyacrylamide gel electrophoresis and added into 100 |L ddH2O followed by 95°C bath for 10 min. The mixture was cen-trifuged at 12,000 rpm for 5 min. The supernatant was used as PCR template. PCR product was purified with agarose gel electrophoresis and sequenced using pEASY-T3 Cloning kit (TransGen Bio
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