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Rapid alkalinization factor (RALF) genes are related to genic male sterility in Chinese cabbage (Brassica rapa L. ssp. pekinensis)
Scientia Horticulturae 225 (2017) 480–489
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Research Paper
Rapid alkalinization factor (RALF) genes are related to genic male sterility in Chinese cabbage (Brassica rapa L. ssp. pekinensis)
MARK
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Fengyan Shi1, Xue Zhou1, Zhiyong Liu, Hui Feng
Department of Horticulture, Shenyang Agricultural University, Shenyang 110866, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Brassica rapa L. ssp. pekinensis RALF gene family Genic male sterile RNA-Seq qRT-PCR
Male sterility is commonly and widely used for hybrid seed production in Brassica rapa L. ssp. pekinensis (B. rapa), and some genes related to anther and pollen development may play significant roles in pollen fertility. Hence, in this study, we performed whole-genome scanning and identified 38 genes from the RALF gene family in B. rapa, which were grouped into four clades according to the phylogenetic tree. Thirty-six of the sequences contained only exons; the other two contained both exonic and intronic sequences. Conserved motifs of the 38 RALF genes were identified. The Ka/Ks ratios of duplicated RALF gene pairs were less than 1, indicating that they had gone through purifying selection. Moreover, a total of 14 RALF-like differentially expressed genes (DEGs) were identified by RNA-Seq between male fertile and male sterile buds, and all were highly or specifically expressed in male fertile buds of the AB line ‘AB01’. A qRT-PCR analysis confirmed that the expression patterns of the 14 RALF DEGs in the AB line ‘AB01’ were similar to those in the other three genic male sterility (GMS) AB lines ‘AB02’, ‘AB03’, and ‘AB04’, indicating that the RALF genes may be related to pollen development in B. rapa. Meanwhile, the results of qRT-PCR validated the RNA-Seq transcriptome as accurate and reliable. This study will provide valuable information for exploring the molecular mechanism underlying sterility in GMS AB lines of B. rapa.
1. Introduction Chinese cabbage (Brassica rapa L. ssp. pekinensis) is a typical crosspollinated crop species that shows obvious heterosis. In China, this crop is grown widely on a significant area of farmland. In attempts to improve the quality and yield of this crop, male sterile lines have been found to be an effective means of hybrid seed production (Bino, 1985). Genic male sterility (GMS) is a type of male sterility in Chinese cabbage, and it is genetically stable (Van der meer, 1987). In previous studies, a stable GMS male sterile line was obtained by crossing male sterile plants of the AB line to a temporary maintainer line (Feng et al., 1995, 1996), and a model of multiple allele inherited male sterility was put forward on the basis of this work. In this model, male sterility is regulated by a single locus that has three alleles: Ms, the male sterile gene; ms, the fertile gene; and, Msf, a fertility restoring gene. The dominance-recessive relationship of the three genes is Msf > Ms > ms. Based on this model, new male sterile lines and temporary maintainer lines have been derived from B. rapa AB lines, and several of them have been used in breeding programs (Feng et al., 2007; ; Wang et al., 2005).
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Corresponding author. E-mail address: [email protected] (H. Feng). Fengyan Shi and Xue Zhou contributed equally to this work and share first authorship.
http://dx.doi.org/10.1016/j.scienta.2017.07.041 Received 19 June 2017; Received in revised form 20 July 2017; Accepted 25 July 2017 0304-4238/ © 2017 Elsevier B.V. All rights reserved.
Polypeptide hormone genes play an important role in plant developmental and defense processes (Albert, 2013; Huffaker et al., 2013; Matsubayashi, 2014; Grienenberger and Fletcher, 2015). One such gene is rapid alkalinization factor (RALF), which is a type of small polypeptide hormone gene that was first discovered in tobacco by Pearce et al. (2001). Expression of RALF in a plant tissue culture can lead to the alkalinization of the medium, and plantlet root growth and development can be inhibited by RALF expression (Pearce et al., 2001). RALF plays a very important role in the growth of the radicle and in regulating the development of floral organs (Olsen et al., 2002; Haruta et al., 2003). A study of RALF genes in sugarcane (SacRALF) suggested that they play a vital role in plant development, especially in regulating tissue expansion (Mingossi et al., 2010). RALF also affects fruit formation (Germain et al., 2005). Cao and Shi (2001) showed that the RALF gene family expanded during evolution. RALF genes are widely distributed in many plant species, with variations among species in the number of genes: 39, 18, 43, and 34 RALF genes have been identified in Arabidopsis, soybean, rice, and maize, respectively (Sharma et al., 2016). Investigation of pollen development is essential to elucidate the
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Fig 1. Phenotypic and Morphological characteristics of GMS AB lines. a, Phenotypic characterization of fertile and sterile flower buds in ‘AB01’. b–e, Morphological characteristics of plants and a comparison of male sterile and male fertile flowers of ‘AB01’, ‘AB02’, ‘AB03’, and ‘AB04’. F: male fertile flower; S: male sterile flower.
GMS ‘AB01’ line of B. rapa and identified 14 RALF DEGs. qRT-PCR was employed to analyze the expression of RALF DEGs in four different genic male sterility AB lines of B. rapa. We believe that this study enhances our understanding of the characteristics of the RALF gene family and its role in male sterility in B. rapa.
genetic mechanism of male sterility. Many studies have shown that some RALF genes are specifically expressed in pollen such as RALFLIKE10 in Arabidopsis (Becker et al., 2003). The interaction of FER, a receptor protein, with RALF1 can result in pollen tube rupture and cause sperm cell release during fertilization (Wolf and Höfte, 2014). Two RALF genes (RALF14 and RALF18) are specifically expressed in synergid cells, which are important for pollen development (Wuest et al., 2010). A synthetic analysis of eight plant species showed that RALF peptides have important functions during plant development; for instance, SIPRALF, a pollen-specific gene that can inhibit the elongation of the pollen tube, and ScRALF1, 2, 3, 4, and 5, which have effects on sexual reproduction and development in Solanum chacoense (Murphy and De Smet, 2014). Currently, the relationship between differentially expressed RALF genes and male sterility has not been reported for GMS AB lines of B. rapa. RNA-Seq is an important tool for the exploration of complex transcriptome data (Mortazavi et al., 2008; Nagalakshmi et al., 2008) as it can detect a large number of DEGs with high fold-changes (Zhao et al., 2014). Recently, RNA-Seq has been applied to some horticultural crops, including Cucumis sativus (Zhang et al., 2014), Brassica napus (Yan et al., 2013; An et al., 2014), and B. rapa (Tong et al., 2013). Moreover, transcriptome analyses have provided valuable information on signal transduction in fertile and sterile pollen development in olive (Iaria et al., 2016), Arabidopsis (Pina et al., 2005; Wang et al., 2008; Boavida et al., 2011), and soybean (Li et al., 2015). For a better understanding of the roles the RALF gene family play in B. rapa, we performed a genome-wide analysis to examine gene structures, phylogeny, chromosomal distribution, conserved motifs, and selection patterns. Meanwhile, we used the Illumina HiSeq 2000 platform to sequence RNAs of the male fertile and male sterile buds from the
2. Materials and methods 2.1. Plant materials We obtained three GMS AB lines (Brassica rapa ssp. parachinensis, ‘AB02’; Brassica rapa ssp. chinensis, ‘AB03’; and Brassica rapa var. purpuraria, ‘AB04’,) by backcrossing with a B. rapa L. ssp. pekinensis line (‘AB01’). All GMS AB lines included sterile (MsMs) and fertile (MsfMs) plants segregating at a ratio of 1:1. The lines were planted at the experimental base of Shenyang Agricultural University. On March 10, 2016, seeds were sown on culture dishes with two layers of filter paper and kept at 4 °C for 20 days. Subsequently, seedlings were planted in pots and grown in a greenhouse. When flowers opened, the plants were identified as either male sterile or male fertile. Firstly, we removed the open flowers and then mixed buds from the four kinds of male sterile and male fertile plants were sampled. Some of the buds from ‘AB01’ were used for RNA-Seq, and each treatment had three biological replicates. Other buds from ‘AB01’, ‘AB02’, ‘AB03’, and ‘AB04’ were selected for qRT-PCR. All of the buds were stored at −80 °C until used for RNA extraction. 2.2. Phylogenetic and sequence analysis of RALF genes in B. rapa The B. rapa RALF gene family was identified by homology analysis 481
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Table 1 Properties of the B. rapa RALF genes and proteins. Gene ID
Chr. no.
Gene start
Gene end
A. thaliana
Annotation
Strand direction
Length (aa)
CDS
MW (kDa)
pI
Bra031425 Bra027081 Bra005473 Bra032888 Bra010886 Bra030081 Bra014983 Bra028344 Bra015112 Bra012130 Bra037127 Bra032754 Bra033111 Bra012764 Bra005549 Bra021816 Bra022922 Bra039434 Bra040217 Bra001149 Bra033357 Bra030513 Bra022168 Bra001636 Bra014003 Bra036617 Bra036802 Bra039403 Bra038962 Bra023451 Bra008511 Bra025405 Bra033112 Bra001920 Bra012569 Bra040399 Bra040400 Bra035990
A01 A09 A05 A09 A08 A07 A07 A01 A07 A07 A09 A04 A02 A03 A05 A04 A03 A05 Scaffold000191 A03 A10 A08 A05 A03 A08 A09 A09 Scaffold000164 A08 A02 A02 A06 A02 A03 A03 Scaffold000203 Scaffold000203 Scaffold000111
17422490 8740248 5692557 23272745 16178742 8941729 6806949 18229367 5599353 12090454 4575174 5229364 22734918 22395966 6158137 14769687 7792849 24294040 158302 15009497 2733550 20738311 20071938 17635934 4701009 2547750 27227124 301082 8668705 2396208 15086828 20713697 22735747 19368078 23518885 32104 35236 328659
17422717 8740487 5692889 23273080 16179077 8943101 6807302 18229822 5599577 12090843 4575557 5229723 22735265 22396301 6158367 14769854 7793076 24294406 158661 15009853 2733918 20738676 20072321 17636335 4701341 2548100 27227522 301333 8669052 2396423 15087136 20714044 22735980 19368272 23519097 32355 35496 328979
AT2G22055 AT1G61566 AT2G33775 AT1G28270 AT1G28270 AT1G28270 AT3G23805 AT3G23805 AT3G25170 AT5G67070 AT5G67070 AT4G14010 AT3G29780 AT4G15800 AT2G32890 AT2G32890 AT2G32890 AT3G05490 AT3G05490 AT3G05490 AT1G02900 AT1G02900 AT3G16570 AT3G16570 AT4G13950 AT3G05490 AT5G67070 AT2G19020 AT4G14020 AT1G61566 AT2G33130 AT3G29780 AT3G29780 AT4G14010 AT3G25170 AT4G11510 AT4G11510 AT2G33130
RALFL15; RALF-like 15 RALFL9; RALF-like 9 RALFL19; RALF-like1 9 RALFL4; RALF-like 4 RALFL4; RALF-like 4 RALFL4; RALF-like 4 RALFL24; RALF-like24 RALFL24; RALF-like24 RALFL26; RALF-like26 RALFL34; RALF-like34 RALFL34; RALF-like34 RALFL32; RALF-like32 RALFL27; RALF-like27 RALFL33; RALF-like33 RALFL17; RALF-like17 RALFL17; RALF-like17 RALFL17; RALF-like17 RALFL22; RALF-like22 RALFL22; RALF-like22 RALFL22; RALF-like22 RALFL1; RALF-like1 RALFL1; RALF-like1 RALFL23; RALF-like23 RALFL23; RALF-like23 RALFL31; RALF-like31 RALFL22; RALF-like22 RALFL34; RALF-like34 RALFL10; RALF-like10 RALF family protein RALFL9; RALF-like9 RALFL18; RALF-like18 RALFL27; RALF-like27 RALFL27; RALF-like27 RALFL32; RALF-like32 RALFL26; RALF-like26 RALFL28; RALF-like28 RALFL28; RALF-like28 RALFL18; RALF-like18
+ + − − − − − − − − − + + + + − + − − + + + − + + + − + − + + − + + + + + +
75 79 110 111 111 146 116 150 74 129 127 119 115 111 76 55 75 96 119 118 122 121 127 133 109 116 132 83 115 71 102 115 77 64 70 83 86 106
228 240 333 336 336 169 354 456 225 390 384 360 348 336 231 168 228 70 360 357 369 366 384 402 333 351 399 399 348 216 309 348 234 195 213 252 261 321
8.38 8.76 12.44 12.73 12.7 16.92 13.15 16.71 7.97 14.37 14.45 13.48 12.38 12.3 8.35 6.01 8.41 10.09 13.04 12.91 13.26 13.17 13.88 14.56 12.46 12.72 15.03 9 13.33 7.89 11.64 12.21 8.4 7.47 7.81 8.64 9.17 12
8.98 6.91 9.97 10.3 10.2 10.12 8.92 9.18 8.86 7.95 7.95 8.12 9.68 8.69 4.15 4.69 4.1 4.17 8.59 8.81 8.12 7.75 8.83 8.81 5.7 9.28 6.76 8.78 6.68 8.66 7.85 9.36 4.46 10.46 10.34 9.11 9.74 9.32
was calculated as T = Ks/2R, where T indicates divergence time, R is the rate of divergence of nuclear genes (the value of R is generally considered to be 1.5 × 10−8 per site per year in dicotyledonous plants), and Ks is the number of synonymous substitutions per site (Koch et al., 2000).
and a blast search from the Brassica database (http://brassicadb.org/ brad/;V1.5) and the PFAM protein family database (Finn et al., 2014). Gene and protein sequences of the RALF gene family were aligned using Clustal X in both B. rapa and Arabidopsis (http://www.arabidopsis.org/) (Thompson et al., 1997), and MEGA 6.0 was used to analyze the phylogenetic and biological evolutionary relationships of B. rapa and Arabidopsis (Tamura et al., 2013). RALF protein sequences were used to construct a phylogenetic tree by the neighbor-joining (NJ) method (Saitou and Nei, 1987) with 1000 bootstrap replications (Felsenstein, 1985). To investigate the major structures of B. rapa RALF proteins, molecular weights and isoelectric points were determined using the ProtParam tool (http://web.expasy.org/protparam/). The full RALF protein sequences were used to identify conserved motifs using the MEME program (Bailey et al., 2006). The parameter settings for the MEME analysis were as follows: width of optimum motif ≥ 6 and ≤ 50 and the maximum number of motifs for confirmation = 12. The numbers and positions of intron and exon sequences in B. rapa RALF genes (BrRALFs) were determined by the Gene Structure Display Server program (Guo et al., 2007).
2.4. RNA preparation The TRIzol reagent was used to extract the total RNA from male sterile and male fertile buds of four GMS AB lines (‘AB01’, ‘AB02’, ‘AB03’, and ‘AB04’) in B. rapa according to the manufacturer’s instructions. The quality and purity of total RNA were confirmed using an Agilent 2100 Bioanalyzer and 0.8% agarose gel electrophoresis. RNA concentration was determined by a Qubit RNA Assay Kit and a Qubit 2.0 Fluorometer. The integrity of the RNA was tested using an RNA Nano 6000 Assay Kit with a Bioanalyzer 2100. Total RNA of male sterile and male fertile buds from ‘AB01’ was used for RNA-Seq. 2.5. Construction of cDNA library and RNA-Seq Sequencing libraries were constructed using an Illumina TruSeq RNA Sample Preparation Kit. mRNA from total RNA was purified using oligo-dT magnetic beads. First-strand cDNA was synthesized using RNase H− and random oligonucleotides. Second-strand cDNA was synthesized using RNase H and DNA polymerase I. To obtain 150–200 bp cDNA fragments, an AMPure XP system was used to purify the library fragments. An Illumina PCR Primer Cocktail kit was used to enrich the DNA fragments. Finally, the purity and quality of PCR
2.3. Chromosomal localizations and selection patterns analysis of BrRALFs The positions of the BrRALFs on B. rapa chromosomes and subgenomic information were acquired from the Brassica database (Cheng et al., 2011). We calculated the rates of non-synonymous (Ka) and synonymous (Ks) substitutions and the Ka/Ks ratios of duplicated RALF gene pairs using PAML (Goldman and Yang, 1994). Divergence time 482
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Fig. 2. Phylogenetic relationships of Brassica rapa and Arabidopsis RALF gene families. The phylogenetic tree was constructed using the NJ method with 1000 bootstrap replicates in MEGA 6.0. The RALF proteins fell into four clades and ten subclades.
A TruSeq PE Cluster Kit v3-cBOT-HS (Illumina) was used to perform clustering of the index-coded samples on a cBot Cluster Generation System according to the manufacturer’s instructions. The prepared library samples were sequenced on an Illumina HiSeq 2000 platform and 100-bp paired-end reads were generated.
synthesis system according to the manufacturer’s protocol. Diluted cDNA (1:10) was used for the qRT-PCR reaction with gene-specific primers designed using Primer 5.0. The gene BrActin was used as an internal control. qRT-PCR was carried using a Bio-Rad IQ5 system with the following program settings: 95 °C for 5 min; 35 cycles of 95 °C for 30 s, 60 °C for 30 s, 72 °C for 1 min; and a final extension at 72 °C for 7 min. Relative gene expression levels were quantified using the formula 2−ΔΔCt (Livak and Schmittgen, 2001). Each treatment was repeated three times, and the data were analyzed using Bio-Rad IQ5 software.
2.7. Quantification of gene expression levels and analysis of DEGs
3. Results
For the RNA-Seq analysis, RPKMs (reads per kilobase per million reads) were used to estimate gene expression levels based on the different gene lengths and read number (Mortazavi et al., 2008). In addition, differential gene expression between the two samples was identified using the DESeq R package (1.10.1). The P-values were adjusted to control the FDR (false discovery rate) and a P-value < 0.05 was used as the threshold for differential expression (Anders and Huber, 2012).
3.1. Morphological features of male fertile and male sterile plants in B. rapa
products and library quality were tested by an AMPure XP system and an Agilent Bioanalyzer 2100, respectively. 2.6. Clustering and resequencing
As for male sterile flowers from GMS AB lines in B. rapa, the filaments were short, and the anthers were small, dry, and without pollen, but the pistils were normal. However, for male fertile flowers, the filaments were of normal size, and the anthers were full, yellow, and had an abundance of pollen (Fig. 1a). In this study, we used four different GMS B. rapa AB lines as our test materials, including ‘AB01’, ‘AB02’, ‘AB03’, and ‘AB04’. Morphological features of these plants and their flowers are shown in Fig. 1b–e. The GMS AB lines generated a 1:1 proportion of sterile and fertile plant progeny, which were ideal materials for research on the molecular mechanism of sterility in B. rapa.
2.8. qRT-PCR analysis Based on analysis of DEGs in the transcriptome data, a qRT-PCR analysis was performed on 14 RALF DEGs from the four GMS AB B. rapa lines to determine relative expression levels and to verify the RNA-Seq data. After isolating RNA from buds of male fertile and male sterile plants, cDNA was synthesized using the SuperScript®III First strand
3.2. Sequence analysis of the RALF gene family in B. rapa In total, 38 BrRALFs were identified and analyzed. Coding DNA 483
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Fig. 3. The exon-intron structure distribution and positions in B. rapa RALF genes.
sequences (CDS) of BrRALFs ranged in size from 70 to 456 bp. Most RALF proteins (37/38) contained 64–150 amino acids, although Bra021816 had 55 amino acids. The molecular weight of the RALF proteins varied from 6.01 to 16.92 KDa and the theoretical isoelectric point (PI) ranged from 4.1 to 10.46 (Table 1).
proteins, respectively; motifs 2 and 3 were present in 19 proteins; and, motifs 5 and 8 were present in 8 proteins. Some motifs only appeared in specific RALF proteins. For example, motifs 6, 7, 9, 10, 11, and 12 occurred three times in the 38 RALF proteins; Bra021816 only contained motif 6 (Fig. 4).
3.3. Evolution and gene structure analysis of the B. rapa RALF gene family
3.4. Chromosomal mapping, synteny, and selection patterns of BrRALFs
We investigated the evolutionary relationships of the BrRALFs in a comparison with the Arabidopsis RALF genes (AtRALFs). A phylogenetic tree was constructed using the protein sequences of 38 BrRALFs and 39 AtRALFs, which had four clades (I, II, III, and IV) and ten subclades. The low bootstrap values in the phylogenetic tree were a result of the low rate of divergence in the RALF sequences between the two species. The phylogenetic tree showed substantial differences among the subclades, indicating that the RALF gene family has changed markedly during the evolution of B. rapa and Arabidopsis (Fig. 2). Analysis of the intron and exon structures in the BrRALFs using the Gene Structure Display Server program showed that 36 BrRALFs contained only exonic sequences and that Bra030081 and Bra039434 contained both exonic and intronic sequences (Fig. 3). Conserved motifs were identified using the MEME program in the 38 BrRALF proteins. Twelve conserved motifs were identified and named motifs 1–12. Motifs 1 and 4 were present in 33 and 34 BrRALF
Considering that gene duplication plays an important role in the evolution of gene families (De Bodt et al., 2005), we performed a synteny analysis of the BrRALFs and compared their distribution in the subgenomes (LF, MF1, and MF2) of B. rapa. Eleven syntenic genes were found in the LF subgenome, 16 in the MF1 subgenome, and 11 in the MF2 subgenome. However, 12 BrRALFs (Bra014003, Bra036617, Bra036802, Bra039403, Bra023451, Bra008511, Bra033112, Bra001920, Bra012569, Bra040401, Bra035990, and Bra040399) had no syntenic relationships with Arabidopsis RALF genes (Table 2). A search of the Brassica database showed that 33 of the BrRALFs were located on the 10 chromosomes in B. rapa; however, Bra040217, Bra039403, Bra040399, Bra040400, and Bra035990 mapped to Scaffold000191, Scaffold000164, Scaffold000203, Scaffold000203, and Scaffold000111, respectively. Chromosome A03 had 6 RALF genes; chromosome A09 had 5 genes; chromosomes A02, A05, A07, and A08 each had 4 genes; chromosomes A01 and A04 each had 2 RALF genes; 484
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Fig. 4. Conserved motifs in B. rapa RALF proteins identified using the MEME program. a, Amino acid sequences of the conserved residues. b, The position of the various motifs in the proteins. Different colors represent different motifs.
chromosomes A06 and A10 each had one gene (Fig. 5). The Ka/Ks ratios of duplicated RALF gene pairs were less than 1, indicating that they had gone through purifying selection. The estimated divergence time of the duplicated RALF gene pairs ranged from 6.05 to 22.91 MYA and averaged 12.28 MYA (Table 3). 3.5. Analysis of DEGs in male fertile and male sterile buds Differentially expressed genes between male fertile and male sterile buds of the AB line ‘AB01’ cultivar from B. rapa were analyzed using the Illumina HiSeq 2000 system. We obtained a total of 37,782,951 and 40,785,524 raw reads on average from the male fertile and male sterile buds, respectively. Based on strict quality assessment, 36,860,509 and 39,528,812 clean reads were obtained, respectively, and more than 80% unique clean reads were mapped to the B. rapa genome; very few of the reads were mapped to multiple locations (Supplementary Table S1). We conducted hierarchical clustering of the DEGs using the log10 RPKM values between the two samples to observe the overall pattern of gene expression (Fig. 6; high gene expression levels in red and low levels of expression in blue). The DEG expression profiles were investigated by a cluster analysis using the k-means method (Hartigan and Wong, 1979). In this study, we identified similar and different patterns of expression among DEGs. Six major expression clusters of DEGs were identified; two of these showed up-regulation, and the other four showed down-regulation (Supplementary Fig. S1). Subclusters 1 and 3 consisted of 146 and 50 genes, respectively, with expression exhibiting a continuous positive slope in the male sterile buds; by contrast, the DEGs in subclusters 2, 4, 5, and 6—with 399, 262, 366, and 222 genes, respectively—consistently exhibited a negative slope. To study the molecular mechanisms of male sterility in B. rapa, we used DESeq software to identify differentially expressed tags. Under the restriction of Padj < 0.05 and log2 |(fold-change)| ≥ 2, a total of 4795 genes were identified as differentially expressed: 4096 were downregulated and 699 were up-regulated in male sterile buds. Based on their annotations, fourteen BrRALFs were identified, and all were highly or specifically expressed in male fertile buds. Moreover, these BrRALFs may be involved in anther and pollen development in B. rapa
Fig. 4. (continued)
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Bra021816, Bra023451, Bra030081, Bra010886, Bra012569, Bra015112) was found in male fertile buds from ‘AB01’, four (Bra023451, Bra010886, Bra031425, Bra005473) in ‘AB02’, nine (Bra027081, Bra032888, Bra021816, Bra023451, Bra030081, Bra010886, Bra012569, Bra015112, Bra031425) in ‘AB03’, and five (Bra032888, Bra021816, Bra010886, Bra012569, Bra031425) in ‘AB04’, and the remaining genes were highly expressed in fertile buds (Fig. 7). Meanwhile, the results verified the accuracy of the RNA-Seq data. These results indicated that the BrRALFs detected in this study were probably associated with pollen development, which may provide the basis for the study of the molecular mechanism underlying sterility in B. rapa.
Table 2 Syntenic RALF genes in Brassica rapa and Arabidopsis. Brassica rapa Gene tPCK Chr
Block
Arabidopsis Gene
LF
MF1
MF2
tPCK7 tPCK3 tPCK1 tPCK2 tPCK2 tPCK7 tPCK4 tPCK7 tPCK4 tPCK3 tPCK2 tPCK1 tPCK2 tPCK4 – – – – – – – – – – – –
D J B F F X T L T J F A F T – – – – – – – – – – – –
AT1G61563 AT2G33775 AT1G28270 AT3G23805 AT3G25170 AT5G67070 AT4G14010 AT3G29780 AT4G15800 AT2G32890 AT3G05490 AT1G02900 AT3G16570 AT4G14020 – – – – – – – – – – – –
– Bra005473 Bra032888 Bra014983 Bra015112 Bra012130 – Bra025405 – Bra005549 Bra039434 Bra033357 Bra022168 – – – Bra036802 – – – – – – – – –
Bra031425 – Bra010886 Bra028344 – – Bra032754 Bra033111 Bra012764 Bra021816 Bra040217 Bra030513 – – Bra014003 – – Bra039403 – – Bra033112 – Bra012569 Bra040400 Bra035990 Bra040399
Bra027081 – Bra030081 – – Bra037127 – – – Bra022922 Bra001149 – Bra001636 Bra038962 – Bra036617 – – Bra023451 Bra008511 – Bra001920 – – – –
4. Discussion This study was initiated to analyze the characteristics of the RALF gene family in B. rapa and to identify RALF DEGs in male fertile and male sterile buds to elucidate their roles in pollen development. The RALF gene family is a type of polypeptide growth regulator and has a significant role in plant growth and development (Pearce et al., 2001; Germain et al., 2005). During evolution, B. rapa experienced whole genome triplication (WGT) and diverged from Arabidopsis (Saha et al., 2015). Considering that Arabidopsis has 39 RALF genes (Sharma et al., 2016), then B. rapa might be expected have three times as many. Here, however, we only found 38 BrRALFs, suggesting that there may have been extensive gene loss during genome evolution and duplication (Cheng et al., 2013). There are similar cases of this occurring in other gene families of B. rapa, such as the MAPK gene family (Lu et al., 2015) and the WRKY gene family (Tang et al., 2014). Most RALF proteins (37/ 38) contained 64–150 amino acids, which is similar to that of a previous study of Arabidopsis and rice (Sharma et al., 2016). According to the phylogenetic tree, the BrRALFs were divided into four clades and ten subclades, indicating that the RALF gene family has changed greatly during evolution as in Arabidopsis and other species (Campbell and Turner, 2017). Genome duplication plays an important role in producing diverse gene functions and expanding the genome, and a duplication event may confer new functions to genes (Lu et al., 2015). Massive genome-scale events may cause segmental duplication and lead to numerous homologs on different chromosomes (Cannon et al., 2004; Freeling, 2009). The RALF gene duplication probably occurred due to whole genome duplication or segmental duplication. The Ka/Ks ratios
(Supplementary Table S2). 3.6. Comparison of RALF gene expression in male fertile and male sterile buds The expression patterns of RALF genes were compared between male fertile and male sterile buds by qRT-PCR in four GMS AB lines (‘AB01’, ‘AB02’, ‘AB03’, and ‘AB04’). The sequences of the gene-specific primers used for these analyses are shown in Supplementary Table S3. The results showed that the expression levels of the 14 RALF DEGs were much higher in male fertile buds than in male sterile buds. Specific expression of nine genes (Bra027081, Bra032888, Bra039403,
Fig. 5. Distribution of RALF genes on the 10 chromosomes of the B. rapa genome. The name of each chromosome is given at the top. Duplicated segments of RALF genes are joined by red lines and BrRALFs are shown on the right of each chromosome. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Table 3 Ka/Ks ratios and the estimated divergence times of duplicated RALF gene pairs in B. rapa. Duplicated gene pairs
Ka
Ks
Ka/Ks
Purifying selection
Time (MYA)
Bra031425-Bra027081 Bra032888-Bra010886 Bra010886-Bra030081 Bra032888-Bra030081 Bra014983-Bra028344 Bra012130-Bra037127 Bra025405-Bra033111 Bra005549-Bra021816 Bra021816-Bra022922 Bra005549-Bra022922 Bra039434-Bra040217 Bra039434-Bra001149 Bra040217-Bra001149 Bra033357-Bra030513 Bra022168-Bra001636
0.2119 0.0079 0.0235 0.0283 0.0668 0.0667 0.1998 0.1578 0.1364 0.0826 0.1925 0.1819 0.0285 0.0603 0.0476
0.3864 0.2326 0.5235 0.4674 0.2326 0.2457 0.3206 0.5025 0.3782 0.3245 0.6872 0.5120 0.2905 0.2421 0.1815
0.5484 0.0340 0.0449 0.0605 0.2872 0.2715 0.6232 0.3140 0.3606 0.2545 0.2801 0.3553 0.0981 0.2490 0.2623
Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes
12.88 7.75 17.45 15.58 7.75 8.19 10.69 16.75 12.61 10.82 22.91 17.07 9.68 8.07 6.05
Ka, non-synonymous substitution rate; Ks, synonymous substitution rate; MYA, million years ago.
and Cao, 2008). ScRALF3 is a vital peptide regulator of the cell–cell communication between the sporophyte and the female gametophyte, and it plays a vital role in pollen development (Germain et al., 2005; Chevalier et al., 2013). To determine which of the BrRALF genes were related to pollen development in GMS AB lines of B. rapa, fourteen RALF genes were highly and specifically expressed in male fertile buds according to the RNA-Seq results. In previous studies, these RALF-LIKE genes were reported in different plants. Covey et al. (2010) reported that a pollenspecific tomato RALF (SlPRALF) inhibited pollen tube germination; AtRALF4 is the Arabidopsis homolog of SlPRALF, and it has a high level of expression in inflorescences but inhibits pollen germination (Morato et al., 2013). Additionally, several RALF genes (At4g14020, RALFL4, RALFL9, RALFL18, RALFL26) have been enriched in female gametic cells and show high levels of expression during the later stages of pollen tube growth and the pollen-pistil interactions in the ovary (JonesRhoades et al., 2007; Wuest et al., 2010; Boavida et al., 2011). BoRALF1 of Brassica oleracea is a homolog of RALF-LIKE19 and gene expression analysis has shown that BoRALF1 is specifically expressed in mature pollen grains and fertile flowers, and inhibiting the expression of this gene causes development of abnormal pollen grains (Zhang et al., 2010). In soybean, RALF-LIKE11 and RALF-LIKE19 are selectively expressed in pollen indicating the essential role of RALF-LIKE signaling peptides in pollen growth (Haerizadeh et al., 2009). To summarize, the 14 RALF DEGs that we obtained here were likely related to pollen development in GMS AB lines in B. rapa. Finally, we analyzed expression patterns of the 14 RALF DEGs in four different kinds of GMS AB lines by qRT-PCR and found that their expression levels in male fertile buds were higher than they were in male sterile buds. Analyses of these expression patterns in male fertile and sterile buds lay the foundation for further study of the molecular mechanism underlying sterility in B. rapa. Cloning of the 14 RALF DEGs in other crops and validation of transgenic functions will provide further insights into their roles in pollen fertility.
5. Conclusion
Fig. 6. Cluster analysis of DEGs using the RNA-Seq data derived from two samples (male fertile and male sterile buds) based on log10 RPKM values. Each row represents an individual gene.
We identified 38 BrRALFs by whole-genome scanning, and a phylogenetic analysis identified four clades. Thirty-six of the RALF genes only contained exonic sequences, while the other two contained both exonic and intronic sequences. The chromosomal locations of 33 RALF genes were identified, and the other five mapped to scaffolds. The Ka/ Ks ratios of duplicated RALF gene pairs were less than 1 indicating that occurrence of purifying selection. A total of 14 RALF DEGs were highly or specifically expressed in male fertile buds by RNA-Seq, and the expression levels of these DEGs from the GMS AB line ‘AB01’ were higher
of all RALF gene pairs were less than 1, which is similar to the selection patterns that has been identified in Arabidopsis and rice RALF gene families (Cao and Shi, 2012). Previous study has shown that RALF genes are linked to pollen development (Nie et al., 2015). BcMF14 (BcRALF1) is active during the late flowering stage in male fertile Brassica campestris var. purpurea (Li 487
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Fig. 7. Comparison of BrRALF expression patterns in male sterile and male fertile buds of ‘AB01’, ‘AB02’, ‘AB03’, and ‘AB04’. BrActin was used as the internal control. Relative expression was calculated using the formula 2−ΔΔCt. a, The relative expression level of 14 RALF DEGs in ‘AB01’. b, The relative expression level of 14 RALF DEGs in ‘AB02’. c, The relative expression level of 14 RALF DEGs in ‘AB03’. d, The relative expression level of 14 RALF DEGs in ‘AB04’.
from the National Natural Science Foundation of China (31672144).
than those in male sterile buds using qRT-PCR, which were similar to those in the ‘AB02’, ‘AB03’, and ‘AB04’ lines, indicating that the RALF genes may be related to pollen development in B. rapa. Meanwhile, the qRT-PCR results showed that our RNA-Seq data are reliable and accurate. This study will provide a basis for future research into the function of the RALF gene family regarding pollen fertility of GMS AB lines in B. rapa.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.scienta.2017.07.041. References
Conflicts of interest Albert, M., 2013. Peptides as triggers of plant defence. J. Exp. Bot. 64, 5269–5279. An, H., Yang, Z.H., Yi, B., Wen, J., Shen, J.X., Tu, J.X., Ma, C.Z., Fu, T.D., 2014. Comparative transcript profiling of the fertile and sterile flower buds of pol CMS in B. Napus. BMC Genom. 15, 258. Anders, S., Huber, W., 2012. Differential expression of RNA-Seq data at the gene level-the DESeq package. Embl. Bailey, T.L., Williams, N., Misleh, C., Li, W.W., 2006. MEME: discovering and analyzing DNA and protein sequence motifs. Nucleic Acids Res. 34, 369–373. Becker, J.D., Boavida, L.C., Carneiro, J., Haury, M., Feijó, J.A., 2003. Transcriptional
None. Acknowledgements We would like to thank Yinan Cui and Miwei Wang for their tremendous help with data analysis. This work was supported by grants 488
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Research Paper
Rapid alkalinization factor (RALF) genes are related to genic male sterility in Chinese cabbage (Brassica rapa L. ssp. pekinensis)
MARK
⁎
Fengyan Shi1, Xue Zhou1, Zhiyong Liu, Hui Feng
Department of Horticulture, Shenyang Agricultural University, Shenyang 110866, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Brassica rapa L. ssp. pekinensis RALF gene family Genic male sterile RNA-Seq qRT-PCR
Male sterility is commonly and widely used for hybrid seed production in Brassica rapa L. ssp. pekinensis (B. rapa), and some genes related to anther and pollen development may play significant roles in pollen fertility. Hence, in this study, we performed whole-genome scanning and identified 38 genes from the RALF gene family in B. rapa, which were grouped into four clades according to the phylogenetic tree. Thirty-six of the sequences contained only exons; the other two contained both exonic and intronic sequences. Conserved motifs of the 38 RALF genes were identified. The Ka/Ks ratios of duplicated RALF gene pairs were less than 1, indicating that they had gone through purifying selection. Moreover, a total of 14 RALF-like differentially expressed genes (DEGs) were identified by RNA-Seq between male fertile and male sterile buds, and all were highly or specifically expressed in male fertile buds of the AB line ‘AB01’. A qRT-PCR analysis confirmed that the expression patterns of the 14 RALF DEGs in the AB line ‘AB01’ were similar to those in the other three genic male sterility (GMS) AB lines ‘AB02’, ‘AB03’, and ‘AB04’, indicating that the RALF genes may be related to pollen development in B. rapa. Meanwhile, the results of qRT-PCR validated the RNA-Seq transcriptome as accurate and reliable. This study will provide valuable information for exploring the molecular mechanism underlying sterility in GMS AB lines of B. rapa.
1. Introduction Chinese cabbage (Brassica rapa L. ssp. pekinensis) is a typical crosspollinated crop species that shows obvious heterosis. In China, this crop is grown widely on a significant area of farmland. In attempts to improve the quality and yield of this crop, male sterile lines have been found to be an effective means of hybrid seed production (Bino, 1985). Genic male sterility (GMS) is a type of male sterility in Chinese cabbage, and it is genetically stable (Van der meer, 1987). In previous studies, a stable GMS male sterile line was obtained by crossing male sterile plants of the AB line to a temporary maintainer line (Feng et al., 1995, 1996), and a model of multiple allele inherited male sterility was put forward on the basis of this work. In this model, male sterility is regulated by a single locus that has three alleles: Ms, the male sterile gene; ms, the fertile gene; and, Msf, a fertility restoring gene. The dominance-recessive relationship of the three genes is Msf > Ms > ms. Based on this model, new male sterile lines and temporary maintainer lines have been derived from B. rapa AB lines, and several of them have been used in breeding programs (Feng et al., 2007; ; Wang et al., 2005).
⁎
1
Corresponding author. E-mail address: [email protected] (H. Feng). Fengyan Shi and Xue Zhou contributed equally to this work and share first authorship.
http://dx.doi.org/10.1016/j.scienta.2017.07.041 Received 19 June 2017; Received in revised form 20 July 2017; Accepted 25 July 2017 0304-4238/ © 2017 Elsevier B.V. All rights reserved.
Polypeptide hormone genes play an important role in plant developmental and defense processes (Albert, 2013; Huffaker et al., 2013; Matsubayashi, 2014; Grienenberger and Fletcher, 2015). One such gene is rapid alkalinization factor (RALF), which is a type of small polypeptide hormone gene that was first discovered in tobacco by Pearce et al. (2001). Expression of RALF in a plant tissue culture can lead to the alkalinization of the medium, and plantlet root growth and development can be inhibited by RALF expression (Pearce et al., 2001). RALF plays a very important role in the growth of the radicle and in regulating the development of floral organs (Olsen et al., 2002; Haruta et al., 2003). A study of RALF genes in sugarcane (SacRALF) suggested that they play a vital role in plant development, especially in regulating tissue expansion (Mingossi et al., 2010). RALF also affects fruit formation (Germain et al., 2005). Cao and Shi (2001) showed that the RALF gene family expanded during evolution. RALF genes are widely distributed in many plant species, with variations among species in the number of genes: 39, 18, 43, and 34 RALF genes have been identified in Arabidopsis, soybean, rice, and maize, respectively (Sharma et al., 2016). Investigation of pollen development is essential to elucidate the
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Fig 1. Phenotypic and Morphological characteristics of GMS AB lines. a, Phenotypic characterization of fertile and sterile flower buds in ‘AB01’. b–e, Morphological characteristics of plants and a comparison of male sterile and male fertile flowers of ‘AB01’, ‘AB02’, ‘AB03’, and ‘AB04’. F: male fertile flower; S: male sterile flower.
GMS ‘AB01’ line of B. rapa and identified 14 RALF DEGs. qRT-PCR was employed to analyze the expression of RALF DEGs in four different genic male sterility AB lines of B. rapa. We believe that this study enhances our understanding of the characteristics of the RALF gene family and its role in male sterility in B. rapa.
genetic mechanism of male sterility. Many studies have shown that some RALF genes are specifically expressed in pollen such as RALFLIKE10 in Arabidopsis (Becker et al., 2003). The interaction of FER, a receptor protein, with RALF1 can result in pollen tube rupture and cause sperm cell release during fertilization (Wolf and Höfte, 2014). Two RALF genes (RALF14 and RALF18) are specifically expressed in synergid cells, which are important for pollen development (Wuest et al., 2010). A synthetic analysis of eight plant species showed that RALF peptides have important functions during plant development; for instance, SIPRALF, a pollen-specific gene that can inhibit the elongation of the pollen tube, and ScRALF1, 2, 3, 4, and 5, which have effects on sexual reproduction and development in Solanum chacoense (Murphy and De Smet, 2014). Currently, the relationship between differentially expressed RALF genes and male sterility has not been reported for GMS AB lines of B. rapa. RNA-Seq is an important tool for the exploration of complex transcriptome data (Mortazavi et al., 2008; Nagalakshmi et al., 2008) as it can detect a large number of DEGs with high fold-changes (Zhao et al., 2014). Recently, RNA-Seq has been applied to some horticultural crops, including Cucumis sativus (Zhang et al., 2014), Brassica napus (Yan et al., 2013; An et al., 2014), and B. rapa (Tong et al., 2013). Moreover, transcriptome analyses have provided valuable information on signal transduction in fertile and sterile pollen development in olive (Iaria et al., 2016), Arabidopsis (Pina et al., 2005; Wang et al., 2008; Boavida et al., 2011), and soybean (Li et al., 2015). For a better understanding of the roles the RALF gene family play in B. rapa, we performed a genome-wide analysis to examine gene structures, phylogeny, chromosomal distribution, conserved motifs, and selection patterns. Meanwhile, we used the Illumina HiSeq 2000 platform to sequence RNAs of the male fertile and male sterile buds from the
2. Materials and methods 2.1. Plant materials We obtained three GMS AB lines (Brassica rapa ssp. parachinensis, ‘AB02’; Brassica rapa ssp. chinensis, ‘AB03’; and Brassica rapa var. purpuraria, ‘AB04’,) by backcrossing with a B. rapa L. ssp. pekinensis line (‘AB01’). All GMS AB lines included sterile (MsMs) and fertile (MsfMs) plants segregating at a ratio of 1:1. The lines were planted at the experimental base of Shenyang Agricultural University. On March 10, 2016, seeds were sown on culture dishes with two layers of filter paper and kept at 4 °C for 20 days. Subsequently, seedlings were planted in pots and grown in a greenhouse. When flowers opened, the plants were identified as either male sterile or male fertile. Firstly, we removed the open flowers and then mixed buds from the four kinds of male sterile and male fertile plants were sampled. Some of the buds from ‘AB01’ were used for RNA-Seq, and each treatment had three biological replicates. Other buds from ‘AB01’, ‘AB02’, ‘AB03’, and ‘AB04’ were selected for qRT-PCR. All of the buds were stored at −80 °C until used for RNA extraction. 2.2. Phylogenetic and sequence analysis of RALF genes in B. rapa The B. rapa RALF gene family was identified by homology analysis 481
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Table 1 Properties of the B. rapa RALF genes and proteins. Gene ID
Chr. no.
Gene start
Gene end
A. thaliana
Annotation
Strand direction
Length (aa)
CDS
MW (kDa)
pI
Bra031425 Bra027081 Bra005473 Bra032888 Bra010886 Bra030081 Bra014983 Bra028344 Bra015112 Bra012130 Bra037127 Bra032754 Bra033111 Bra012764 Bra005549 Bra021816 Bra022922 Bra039434 Bra040217 Bra001149 Bra033357 Bra030513 Bra022168 Bra001636 Bra014003 Bra036617 Bra036802 Bra039403 Bra038962 Bra023451 Bra008511 Bra025405 Bra033112 Bra001920 Bra012569 Bra040399 Bra040400 Bra035990
A01 A09 A05 A09 A08 A07 A07 A01 A07 A07 A09 A04 A02 A03 A05 A04 A03 A05 Scaffold000191 A03 A10 A08 A05 A03 A08 A09 A09 Scaffold000164 A08 A02 A02 A06 A02 A03 A03 Scaffold000203 Scaffold000203 Scaffold000111
17422490 8740248 5692557 23272745 16178742 8941729 6806949 18229367 5599353 12090454 4575174 5229364 22734918 22395966 6158137 14769687 7792849 24294040 158302 15009497 2733550 20738311 20071938 17635934 4701009 2547750 27227124 301082 8668705 2396208 15086828 20713697 22735747 19368078 23518885 32104 35236 328659
17422717 8740487 5692889 23273080 16179077 8943101 6807302 18229822 5599577 12090843 4575557 5229723 22735265 22396301 6158367 14769854 7793076 24294406 158661 15009853 2733918 20738676 20072321 17636335 4701341 2548100 27227522 301333 8669052 2396423 15087136 20714044 22735980 19368272 23519097 32355 35496 328979
AT2G22055 AT1G61566 AT2G33775 AT1G28270 AT1G28270 AT1G28270 AT3G23805 AT3G23805 AT3G25170 AT5G67070 AT5G67070 AT4G14010 AT3G29780 AT4G15800 AT2G32890 AT2G32890 AT2G32890 AT3G05490 AT3G05490 AT3G05490 AT1G02900 AT1G02900 AT3G16570 AT3G16570 AT4G13950 AT3G05490 AT5G67070 AT2G19020 AT4G14020 AT1G61566 AT2G33130 AT3G29780 AT3G29780 AT4G14010 AT3G25170 AT4G11510 AT4G11510 AT2G33130
RALFL15; RALF-like 15 RALFL9; RALF-like 9 RALFL19; RALF-like1 9 RALFL4; RALF-like 4 RALFL4; RALF-like 4 RALFL4; RALF-like 4 RALFL24; RALF-like24 RALFL24; RALF-like24 RALFL26; RALF-like26 RALFL34; RALF-like34 RALFL34; RALF-like34 RALFL32; RALF-like32 RALFL27; RALF-like27 RALFL33; RALF-like33 RALFL17; RALF-like17 RALFL17; RALF-like17 RALFL17; RALF-like17 RALFL22; RALF-like22 RALFL22; RALF-like22 RALFL22; RALF-like22 RALFL1; RALF-like1 RALFL1; RALF-like1 RALFL23; RALF-like23 RALFL23; RALF-like23 RALFL31; RALF-like31 RALFL22; RALF-like22 RALFL34; RALF-like34 RALFL10; RALF-like10 RALF family protein RALFL9; RALF-like9 RALFL18; RALF-like18 RALFL27; RALF-like27 RALFL27; RALF-like27 RALFL32; RALF-like32 RALFL26; RALF-like26 RALFL28; RALF-like28 RALFL28; RALF-like28 RALFL18; RALF-like18
+ + − − − − − − − − − + + + + − + − − + + + − + + + − + − + + − + + + + + +
75 79 110 111 111 146 116 150 74 129 127 119 115 111 76 55 75 96 119 118 122 121 127 133 109 116 132 83 115 71 102 115 77 64 70 83 86 106
228 240 333 336 336 169 354 456 225 390 384 360 348 336 231 168 228 70 360 357 369 366 384 402 333 351 399 399 348 216 309 348 234 195 213 252 261 321
8.38 8.76 12.44 12.73 12.7 16.92 13.15 16.71 7.97 14.37 14.45 13.48 12.38 12.3 8.35 6.01 8.41 10.09 13.04 12.91 13.26 13.17 13.88 14.56 12.46 12.72 15.03 9 13.33 7.89 11.64 12.21 8.4 7.47 7.81 8.64 9.17 12
8.98 6.91 9.97 10.3 10.2 10.12 8.92 9.18 8.86 7.95 7.95 8.12 9.68 8.69 4.15 4.69 4.1 4.17 8.59 8.81 8.12 7.75 8.83 8.81 5.7 9.28 6.76 8.78 6.68 8.66 7.85 9.36 4.46 10.46 10.34 9.11 9.74 9.32
was calculated as T = Ks/2R, where T indicates divergence time, R is the rate of divergence of nuclear genes (the value of R is generally considered to be 1.5 × 10−8 per site per year in dicotyledonous plants), and Ks is the number of synonymous substitutions per site (Koch et al., 2000).
and a blast search from the Brassica database (http://brassicadb.org/ brad/;V1.5) and the PFAM protein family database (Finn et al., 2014). Gene and protein sequences of the RALF gene family were aligned using Clustal X in both B. rapa and Arabidopsis (http://www.arabidopsis.org/) (Thompson et al., 1997), and MEGA 6.0 was used to analyze the phylogenetic and biological evolutionary relationships of B. rapa and Arabidopsis (Tamura et al., 2013). RALF protein sequences were used to construct a phylogenetic tree by the neighbor-joining (NJ) method (Saitou and Nei, 1987) with 1000 bootstrap replications (Felsenstein, 1985). To investigate the major structures of B. rapa RALF proteins, molecular weights and isoelectric points were determined using the ProtParam tool (http://web.expasy.org/protparam/). The full RALF protein sequences were used to identify conserved motifs using the MEME program (Bailey et al., 2006). The parameter settings for the MEME analysis were as follows: width of optimum motif ≥ 6 and ≤ 50 and the maximum number of motifs for confirmation = 12. The numbers and positions of intron and exon sequences in B. rapa RALF genes (BrRALFs) were determined by the Gene Structure Display Server program (Guo et al., 2007).
2.4. RNA preparation The TRIzol reagent was used to extract the total RNA from male sterile and male fertile buds of four GMS AB lines (‘AB01’, ‘AB02’, ‘AB03’, and ‘AB04’) in B. rapa according to the manufacturer’s instructions. The quality and purity of total RNA were confirmed using an Agilent 2100 Bioanalyzer and 0.8% agarose gel electrophoresis. RNA concentration was determined by a Qubit RNA Assay Kit and a Qubit 2.0 Fluorometer. The integrity of the RNA was tested using an RNA Nano 6000 Assay Kit with a Bioanalyzer 2100. Total RNA of male sterile and male fertile buds from ‘AB01’ was used for RNA-Seq. 2.5. Construction of cDNA library and RNA-Seq Sequencing libraries were constructed using an Illumina TruSeq RNA Sample Preparation Kit. mRNA from total RNA was purified using oligo-dT magnetic beads. First-strand cDNA was synthesized using RNase H− and random oligonucleotides. Second-strand cDNA was synthesized using RNase H and DNA polymerase I. To obtain 150–200 bp cDNA fragments, an AMPure XP system was used to purify the library fragments. An Illumina PCR Primer Cocktail kit was used to enrich the DNA fragments. Finally, the purity and quality of PCR
2.3. Chromosomal localizations and selection patterns analysis of BrRALFs The positions of the BrRALFs on B. rapa chromosomes and subgenomic information were acquired from the Brassica database (Cheng et al., 2011). We calculated the rates of non-synonymous (Ka) and synonymous (Ks) substitutions and the Ka/Ks ratios of duplicated RALF gene pairs using PAML (Goldman and Yang, 1994). Divergence time 482
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Fig. 2. Phylogenetic relationships of Brassica rapa and Arabidopsis RALF gene families. The phylogenetic tree was constructed using the NJ method with 1000 bootstrap replicates in MEGA 6.0. The RALF proteins fell into four clades and ten subclades.
A TruSeq PE Cluster Kit v3-cBOT-HS (Illumina) was used to perform clustering of the index-coded samples on a cBot Cluster Generation System according to the manufacturer’s instructions. The prepared library samples were sequenced on an Illumina HiSeq 2000 platform and 100-bp paired-end reads were generated.
synthesis system according to the manufacturer’s protocol. Diluted cDNA (1:10) was used for the qRT-PCR reaction with gene-specific primers designed using Primer 5.0. The gene BrActin was used as an internal control. qRT-PCR was carried using a Bio-Rad IQ5 system with the following program settings: 95 °C for 5 min; 35 cycles of 95 °C for 30 s, 60 °C for 30 s, 72 °C for 1 min; and a final extension at 72 °C for 7 min. Relative gene expression levels were quantified using the formula 2−ΔΔCt (Livak and Schmittgen, 2001). Each treatment was repeated three times, and the data were analyzed using Bio-Rad IQ5 software.
2.7. Quantification of gene expression levels and analysis of DEGs
3. Results
For the RNA-Seq analysis, RPKMs (reads per kilobase per million reads) were used to estimate gene expression levels based on the different gene lengths and read number (Mortazavi et al., 2008). In addition, differential gene expression between the two samples was identified using the DESeq R package (1.10.1). The P-values were adjusted to control the FDR (false discovery rate) and a P-value < 0.05 was used as the threshold for differential expression (Anders and Huber, 2012).
3.1. Morphological features of male fertile and male sterile plants in B. rapa
products and library quality were tested by an AMPure XP system and an Agilent Bioanalyzer 2100, respectively. 2.6. Clustering and resequencing
As for male sterile flowers from GMS AB lines in B. rapa, the filaments were short, and the anthers were small, dry, and without pollen, but the pistils were normal. However, for male fertile flowers, the filaments were of normal size, and the anthers were full, yellow, and had an abundance of pollen (Fig. 1a). In this study, we used four different GMS B. rapa AB lines as our test materials, including ‘AB01’, ‘AB02’, ‘AB03’, and ‘AB04’. Morphological features of these plants and their flowers are shown in Fig. 1b–e. The GMS AB lines generated a 1:1 proportion of sterile and fertile plant progeny, which were ideal materials for research on the molecular mechanism of sterility in B. rapa.
2.8. qRT-PCR analysis Based on analysis of DEGs in the transcriptome data, a qRT-PCR analysis was performed on 14 RALF DEGs from the four GMS AB B. rapa lines to determine relative expression levels and to verify the RNA-Seq data. After isolating RNA from buds of male fertile and male sterile plants, cDNA was synthesized using the SuperScript®III First strand
3.2. Sequence analysis of the RALF gene family in B. rapa In total, 38 BrRALFs were identified and analyzed. Coding DNA 483
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Fig. 3. The exon-intron structure distribution and positions in B. rapa RALF genes.
sequences (CDS) of BrRALFs ranged in size from 70 to 456 bp. Most RALF proteins (37/38) contained 64–150 amino acids, although Bra021816 had 55 amino acids. The molecular weight of the RALF proteins varied from 6.01 to 16.92 KDa and the theoretical isoelectric point (PI) ranged from 4.1 to 10.46 (Table 1).
proteins, respectively; motifs 2 and 3 were present in 19 proteins; and, motifs 5 and 8 were present in 8 proteins. Some motifs only appeared in specific RALF proteins. For example, motifs 6, 7, 9, 10, 11, and 12 occurred three times in the 38 RALF proteins; Bra021816 only contained motif 6 (Fig. 4).
3.3. Evolution and gene structure analysis of the B. rapa RALF gene family
3.4. Chromosomal mapping, synteny, and selection patterns of BrRALFs
We investigated the evolutionary relationships of the BrRALFs in a comparison with the Arabidopsis RALF genes (AtRALFs). A phylogenetic tree was constructed using the protein sequences of 38 BrRALFs and 39 AtRALFs, which had four clades (I, II, III, and IV) and ten subclades. The low bootstrap values in the phylogenetic tree were a result of the low rate of divergence in the RALF sequences between the two species. The phylogenetic tree showed substantial differences among the subclades, indicating that the RALF gene family has changed markedly during the evolution of B. rapa and Arabidopsis (Fig. 2). Analysis of the intron and exon structures in the BrRALFs using the Gene Structure Display Server program showed that 36 BrRALFs contained only exonic sequences and that Bra030081 and Bra039434 contained both exonic and intronic sequences (Fig. 3). Conserved motifs were identified using the MEME program in the 38 BrRALF proteins. Twelve conserved motifs were identified and named motifs 1–12. Motifs 1 and 4 were present in 33 and 34 BrRALF
Considering that gene duplication plays an important role in the evolution of gene families (De Bodt et al., 2005), we performed a synteny analysis of the BrRALFs and compared their distribution in the subgenomes (LF, MF1, and MF2) of B. rapa. Eleven syntenic genes were found in the LF subgenome, 16 in the MF1 subgenome, and 11 in the MF2 subgenome. However, 12 BrRALFs (Bra014003, Bra036617, Bra036802, Bra039403, Bra023451, Bra008511, Bra033112, Bra001920, Bra012569, Bra040401, Bra035990, and Bra040399) had no syntenic relationships with Arabidopsis RALF genes (Table 2). A search of the Brassica database showed that 33 of the BrRALFs were located on the 10 chromosomes in B. rapa; however, Bra040217, Bra039403, Bra040399, Bra040400, and Bra035990 mapped to Scaffold000191, Scaffold000164, Scaffold000203, Scaffold000203, and Scaffold000111, respectively. Chromosome A03 had 6 RALF genes; chromosome A09 had 5 genes; chromosomes A02, A05, A07, and A08 each had 4 genes; chromosomes A01 and A04 each had 2 RALF genes; 484
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Fig. 4. Conserved motifs in B. rapa RALF proteins identified using the MEME program. a, Amino acid sequences of the conserved residues. b, The position of the various motifs in the proteins. Different colors represent different motifs.
chromosomes A06 and A10 each had one gene (Fig. 5). The Ka/Ks ratios of duplicated RALF gene pairs were less than 1, indicating that they had gone through purifying selection. The estimated divergence time of the duplicated RALF gene pairs ranged from 6.05 to 22.91 MYA and averaged 12.28 MYA (Table 3). 3.5. Analysis of DEGs in male fertile and male sterile buds Differentially expressed genes between male fertile and male sterile buds of the AB line ‘AB01’ cultivar from B. rapa were analyzed using the Illumina HiSeq 2000 system. We obtained a total of 37,782,951 and 40,785,524 raw reads on average from the male fertile and male sterile buds, respectively. Based on strict quality assessment, 36,860,509 and 39,528,812 clean reads were obtained, respectively, and more than 80% unique clean reads were mapped to the B. rapa genome; very few of the reads were mapped to multiple locations (Supplementary Table S1). We conducted hierarchical clustering of the DEGs using the log10 RPKM values between the two samples to observe the overall pattern of gene expression (Fig. 6; high gene expression levels in red and low levels of expression in blue). The DEG expression profiles were investigated by a cluster analysis using the k-means method (Hartigan and Wong, 1979). In this study, we identified similar and different patterns of expression among DEGs. Six major expression clusters of DEGs were identified; two of these showed up-regulation, and the other four showed down-regulation (Supplementary Fig. S1). Subclusters 1 and 3 consisted of 146 and 50 genes, respectively, with expression exhibiting a continuous positive slope in the male sterile buds; by contrast, the DEGs in subclusters 2, 4, 5, and 6—with 399, 262, 366, and 222 genes, respectively—consistently exhibited a negative slope. To study the molecular mechanisms of male sterility in B. rapa, we used DESeq software to identify differentially expressed tags. Under the restriction of Padj < 0.05 and log2 |(fold-change)| ≥ 2, a total of 4795 genes were identified as differentially expressed: 4096 were downregulated and 699 were up-regulated in male sterile buds. Based on their annotations, fourteen BrRALFs were identified, and all were highly or specifically expressed in male fertile buds. Moreover, these BrRALFs may be involved in anther and pollen development in B. rapa
Fig. 4. (continued)
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Bra021816, Bra023451, Bra030081, Bra010886, Bra012569, Bra015112) was found in male fertile buds from ‘AB01’, four (Bra023451, Bra010886, Bra031425, Bra005473) in ‘AB02’, nine (Bra027081, Bra032888, Bra021816, Bra023451, Bra030081, Bra010886, Bra012569, Bra015112, Bra031425) in ‘AB03’, and five (Bra032888, Bra021816, Bra010886, Bra012569, Bra031425) in ‘AB04’, and the remaining genes were highly expressed in fertile buds (Fig. 7). Meanwhile, the results verified the accuracy of the RNA-Seq data. These results indicated that the BrRALFs detected in this study were probably associated with pollen development, which may provide the basis for the study of the molecular mechanism underlying sterility in B. rapa.
Table 2 Syntenic RALF genes in Brassica rapa and Arabidopsis. Brassica rapa Gene tPCK Chr
Block
Arabidopsis Gene
LF
MF1
MF2
tPCK7 tPCK3 tPCK1 tPCK2 tPCK2 tPCK7 tPCK4 tPCK7 tPCK4 tPCK3 tPCK2 tPCK1 tPCK2 tPCK4 – – – – – – – – – – – –
D J B F F X T L T J F A F T – – – – – – – – – – – –
AT1G61563 AT2G33775 AT1G28270 AT3G23805 AT3G25170 AT5G67070 AT4G14010 AT3G29780 AT4G15800 AT2G32890 AT3G05490 AT1G02900 AT3G16570 AT4G14020 – – – – – – – – – – – –
– Bra005473 Bra032888 Bra014983 Bra015112 Bra012130 – Bra025405 – Bra005549 Bra039434 Bra033357 Bra022168 – – – Bra036802 – – – – – – – – –
Bra031425 – Bra010886 Bra028344 – – Bra032754 Bra033111 Bra012764 Bra021816 Bra040217 Bra030513 – – Bra014003 – – Bra039403 – – Bra033112 – Bra012569 Bra040400 Bra035990 Bra040399
Bra027081 – Bra030081 – – Bra037127 – – – Bra022922 Bra001149 – Bra001636 Bra038962 – Bra036617 – – Bra023451 Bra008511 – Bra001920 – – – –
4. Discussion This study was initiated to analyze the characteristics of the RALF gene family in B. rapa and to identify RALF DEGs in male fertile and male sterile buds to elucidate their roles in pollen development. The RALF gene family is a type of polypeptide growth regulator and has a significant role in plant growth and development (Pearce et al., 2001; Germain et al., 2005). During evolution, B. rapa experienced whole genome triplication (WGT) and diverged from Arabidopsis (Saha et al., 2015). Considering that Arabidopsis has 39 RALF genes (Sharma et al., 2016), then B. rapa might be expected have three times as many. Here, however, we only found 38 BrRALFs, suggesting that there may have been extensive gene loss during genome evolution and duplication (Cheng et al., 2013). There are similar cases of this occurring in other gene families of B. rapa, such as the MAPK gene family (Lu et al., 2015) and the WRKY gene family (Tang et al., 2014). Most RALF proteins (37/ 38) contained 64–150 amino acids, which is similar to that of a previous study of Arabidopsis and rice (Sharma et al., 2016). According to the phylogenetic tree, the BrRALFs were divided into four clades and ten subclades, indicating that the RALF gene family has changed greatly during evolution as in Arabidopsis and other species (Campbell and Turner, 2017). Genome duplication plays an important role in producing diverse gene functions and expanding the genome, and a duplication event may confer new functions to genes (Lu et al., 2015). Massive genome-scale events may cause segmental duplication and lead to numerous homologs on different chromosomes (Cannon et al., 2004; Freeling, 2009). The RALF gene duplication probably occurred due to whole genome duplication or segmental duplication. The Ka/Ks ratios
(Supplementary Table S2). 3.6. Comparison of RALF gene expression in male fertile and male sterile buds The expression patterns of RALF genes were compared between male fertile and male sterile buds by qRT-PCR in four GMS AB lines (‘AB01’, ‘AB02’, ‘AB03’, and ‘AB04’). The sequences of the gene-specific primers used for these analyses are shown in Supplementary Table S3. The results showed that the expression levels of the 14 RALF DEGs were much higher in male fertile buds than in male sterile buds. Specific expression of nine genes (Bra027081, Bra032888, Bra039403,
Fig. 5. Distribution of RALF genes on the 10 chromosomes of the B. rapa genome. The name of each chromosome is given at the top. Duplicated segments of RALF genes are joined by red lines and BrRALFs are shown on the right of each chromosome. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Table 3 Ka/Ks ratios and the estimated divergence times of duplicated RALF gene pairs in B. rapa. Duplicated gene pairs
Ka
Ks
Ka/Ks
Purifying selection
Time (MYA)
Bra031425-Bra027081 Bra032888-Bra010886 Bra010886-Bra030081 Bra032888-Bra030081 Bra014983-Bra028344 Bra012130-Bra037127 Bra025405-Bra033111 Bra005549-Bra021816 Bra021816-Bra022922 Bra005549-Bra022922 Bra039434-Bra040217 Bra039434-Bra001149 Bra040217-Bra001149 Bra033357-Bra030513 Bra022168-Bra001636
0.2119 0.0079 0.0235 0.0283 0.0668 0.0667 0.1998 0.1578 0.1364 0.0826 0.1925 0.1819 0.0285 0.0603 0.0476
0.3864 0.2326 0.5235 0.4674 0.2326 0.2457 0.3206 0.5025 0.3782 0.3245 0.6872 0.5120 0.2905 0.2421 0.1815
0.5484 0.0340 0.0449 0.0605 0.2872 0.2715 0.6232 0.3140 0.3606 0.2545 0.2801 0.3553 0.0981 0.2490 0.2623
Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes
12.88 7.75 17.45 15.58 7.75 8.19 10.69 16.75 12.61 10.82 22.91 17.07 9.68 8.07 6.05
Ka, non-synonymous substitution rate; Ks, synonymous substitution rate; MYA, million years ago.
and Cao, 2008). ScRALF3 is a vital peptide regulator of the cell–cell communication between the sporophyte and the female gametophyte, and it plays a vital role in pollen development (Germain et al., 2005; Chevalier et al., 2013). To determine which of the BrRALF genes were related to pollen development in GMS AB lines of B. rapa, fourteen RALF genes were highly and specifically expressed in male fertile buds according to the RNA-Seq results. In previous studies, these RALF-LIKE genes were reported in different plants. Covey et al. (2010) reported that a pollenspecific tomato RALF (SlPRALF) inhibited pollen tube germination; AtRALF4 is the Arabidopsis homolog of SlPRALF, and it has a high level of expression in inflorescences but inhibits pollen germination (Morato et al., 2013). Additionally, several RALF genes (At4g14020, RALFL4, RALFL9, RALFL18, RALFL26) have been enriched in female gametic cells and show high levels of expression during the later stages of pollen tube growth and the pollen-pistil interactions in the ovary (JonesRhoades et al., 2007; Wuest et al., 2010; Boavida et al., 2011). BoRALF1 of Brassica oleracea is a homolog of RALF-LIKE19 and gene expression analysis has shown that BoRALF1 is specifically expressed in mature pollen grains and fertile flowers, and inhibiting the expression of this gene causes development of abnormal pollen grains (Zhang et al., 2010). In soybean, RALF-LIKE11 and RALF-LIKE19 are selectively expressed in pollen indicating the essential role of RALF-LIKE signaling peptides in pollen growth (Haerizadeh et al., 2009). To summarize, the 14 RALF DEGs that we obtained here were likely related to pollen development in GMS AB lines in B. rapa. Finally, we analyzed expression patterns of the 14 RALF DEGs in four different kinds of GMS AB lines by qRT-PCR and found that their expression levels in male fertile buds were higher than they were in male sterile buds. Analyses of these expression patterns in male fertile and sterile buds lay the foundation for further study of the molecular mechanism underlying sterility in B. rapa. Cloning of the 14 RALF DEGs in other crops and validation of transgenic functions will provide further insights into their roles in pollen fertility.
5. Conclusion
Fig. 6. Cluster analysis of DEGs using the RNA-Seq data derived from two samples (male fertile and male sterile buds) based on log10 RPKM values. Each row represents an individual gene.
We identified 38 BrRALFs by whole-genome scanning, and a phylogenetic analysis identified four clades. Thirty-six of the RALF genes only contained exonic sequences, while the other two contained both exonic and intronic sequences. The chromosomal locations of 33 RALF genes were identified, and the other five mapped to scaffolds. The Ka/ Ks ratios of duplicated RALF gene pairs were less than 1 indicating that occurrence of purifying selection. A total of 14 RALF DEGs were highly or specifically expressed in male fertile buds by RNA-Seq, and the expression levels of these DEGs from the GMS AB line ‘AB01’ were higher
of all RALF gene pairs were less than 1, which is similar to the selection patterns that has been identified in Arabidopsis and rice RALF gene families (Cao and Shi, 2012). Previous study has shown that RALF genes are linked to pollen development (Nie et al., 2015). BcMF14 (BcRALF1) is active during the late flowering stage in male fertile Brassica campestris var. purpurea (Li 487
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Fig. 7. Comparison of BrRALF expression patterns in male sterile and male fertile buds of ‘AB01’, ‘AB02’, ‘AB03’, and ‘AB04’. BrActin was used as the internal control. Relative expression was calculated using the formula 2−ΔΔCt. a, The relative expression level of 14 RALF DEGs in ‘AB01’. b, The relative expression level of 14 RALF DEGs in ‘AB02’. c, The relative expression level of 14 RALF DEGs in ‘AB03’. d, The relative expression level of 14 RALF DEGs in ‘AB04’.
from the National Natural Science Foundation of China (31672144).
than those in male sterile buds using qRT-PCR, which were similar to those in the ‘AB02’, ‘AB03’, and ‘AB04’ lines, indicating that the RALF genes may be related to pollen development in B. rapa. Meanwhile, the qRT-PCR results showed that our RNA-Seq data are reliable and accurate. This study will provide a basis for future research into the function of the RALF gene family regarding pollen fertility of GMS AB lines in B. rapa.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.scienta.2017.07.041. References
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None. Acknowledgements We would like to thank Yinan Cui and Miwei Wang for their tremendous help with data analysis. This work was supported by grants 488
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