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Transcriptome sequencing and de novo analysis of a cytoplasmic male sterile line and its near-isogenic restorer line in chili pepper (Capsicum annuum L.).

Liu C, Ma N, Wang PY, Fu N, Shen HL - PLoS ONE (2013)

Bottom Line: Among these unigenes, 27,191 were identified as putative homologs of annotated sequences in the public protein databases, 4,326 and 7,061 unigenes were found to be highly abundant in lines 121A and 121C, respectively.Many of the differentially expressed unigenes represent a set of potential candidate genes associated with the formation or abortion of pollen.The results shed the lights on the occurrence and recovery of the disturbances in nuclear-mitochondrial interaction and provide clues for further investigations.

View Article: PubMed Central - PubMed

Affiliation: China Agricultural University, Beijing, China.

ABSTRACT

Background: The use of cytoplasmic male sterility (CMS) in F1 hybrid seed production of chili pepper is increasingly popular. However, the molecular mechanisms of cytoplasmic male sterility and fertility restoration remain poorly understood due to limited transcriptomic and genomic data. Therefore, we analyzed the difference between a CMS line 121A and its near-isogenic restorer line 121C in transcriptome level using next generation sequencing technology (NGS), aiming to find out critical genes and pathways associated with the male sterility.

Results: We generated approximately 53 million sequencing reads and assembled de novo, yielding 85,144 high quality unigenes with an average length of 643 bp. Among these unigenes, 27,191 were identified as putative homologs of annotated sequences in the public protein databases, 4,326 and 7,061 unigenes were found to be highly abundant in lines 121A and 121C, respectively. Many of the differentially expressed unigenes represent a set of potential candidate genes associated with the formation or abortion of pollen.

Conclusions: Our study profiled anther transcriptomes of a chili pepper CMS line and its restorer line. The results shed the lights on the occurrence and recovery of the disturbances in nuclear-mitochondrial interaction and provide clues for further investigations.

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Related in: MedlinePlus

Relative expressions of the twelve selected known function unigenes detected by real-time RT-PCR.The transcript levels were normalized with actin gene, and the level of each unigene in 121A was set at 1.0. Error bars represent the SE for three independent experiments. The unigene expression in Illumina experiment and the descriptions are listed in Table 2. The primers used for each gene are listed in Table S4.
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pone-0065209-g006: Relative expressions of the twelve selected known function unigenes detected by real-time RT-PCR.The transcript levels were normalized with actin gene, and the level of each unigene in 121A was set at 1.0. Error bars represent the SE for three independent experiments. The unigene expression in Illumina experiment and the descriptions are listed in Table 2. The primers used for each gene are listed in Table S4.

Mentions: To evaluate the validity of Illumina analysis and to further assess the patterns of differential gene expression, several unigenes from our sequencing results were selected and detected by RT-PCR and qRT-PCR (Table 2) with unigene-specific primers (Table S4). For real-time RT-PCR (Figure 6), this study selected unigenes with known function like ATP binding protein, pentatricopeptide (PPR) repeat-containing protein, MADS-box transcription factor and the others. In contrast to the Illumina data, the highest up-regulation of comp66553_c0_seq1 was observed with almost 263-fold in 121C, while the transcript abundance of comp62432_c0_seq1 was induced by approximately 188-fold, lower than that of comp66553_c0_seq1. Most selected unigenes (e.g. comp215802_c0_seq1, comp198237_c0_seq1, comp54012_c0_seq1, comp56601_c0_seq1, comp71609_c0_seq1, comp66553_c0_seq1, comp62048_c0_seq1 and comp62432_c0_seq1) showed lower changes when compared with Illumina sequencing. Furthermore, 8 “non-BLASTable” unigenes were selected randomly for relative RT-PCR (Figure 7). The log2ratios for comp54630_c1_seq1 and comp60513_c2_seq1 were −2.6 and −7.9, respectively. In contrast to the Illumina data, expression difference of comp54630_c1_seq1 between the two materials was more obvious than that of comp60513_c2_seq1 when using relative RT-PCR. However, the trend of higher abundance in the restorer line was consistent between sequencing and relative RT-PCR. Taken together, the expression patterns of these genes in 121A and 121C are consistent with the Illumina data. RT-PCR results basically confirmed the reliability of our transcriptome analysis. However, the two techniques essentially use different algorithms, which may explain the above-mentioned some inconsistent results [49]–[51].


Transcriptome sequencing and de novo analysis of a cytoplasmic male sterile line and its near-isogenic restorer line in chili pepper (Capsicum annuum L.).

Liu C, Ma N, Wang PY, Fu N, Shen HL - PLoS ONE (2013)

Relative expressions of the twelve selected known function unigenes detected by real-time RT-PCR.The transcript levels were normalized with actin gene, and the level of each unigene in 121A was set at 1.0. Error bars represent the SE for three independent experiments. The unigene expression in Illumina experiment and the descriptions are listed in Table 2. The primers used for each gene are listed in Table S4.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3672106&req=5

pone-0065209-g006: Relative expressions of the twelve selected known function unigenes detected by real-time RT-PCR.The transcript levels were normalized with actin gene, and the level of each unigene in 121A was set at 1.0. Error bars represent the SE for three independent experiments. The unigene expression in Illumina experiment and the descriptions are listed in Table 2. The primers used for each gene are listed in Table S4.
Mentions: To evaluate the validity of Illumina analysis and to further assess the patterns of differential gene expression, several unigenes from our sequencing results were selected and detected by RT-PCR and qRT-PCR (Table 2) with unigene-specific primers (Table S4). For real-time RT-PCR (Figure 6), this study selected unigenes with known function like ATP binding protein, pentatricopeptide (PPR) repeat-containing protein, MADS-box transcription factor and the others. In contrast to the Illumina data, the highest up-regulation of comp66553_c0_seq1 was observed with almost 263-fold in 121C, while the transcript abundance of comp62432_c0_seq1 was induced by approximately 188-fold, lower than that of comp66553_c0_seq1. Most selected unigenes (e.g. comp215802_c0_seq1, comp198237_c0_seq1, comp54012_c0_seq1, comp56601_c0_seq1, comp71609_c0_seq1, comp66553_c0_seq1, comp62048_c0_seq1 and comp62432_c0_seq1) showed lower changes when compared with Illumina sequencing. Furthermore, 8 “non-BLASTable” unigenes were selected randomly for relative RT-PCR (Figure 7). The log2ratios for comp54630_c1_seq1 and comp60513_c2_seq1 were −2.6 and −7.9, respectively. In contrast to the Illumina data, expression difference of comp54630_c1_seq1 between the two materials was more obvious than that of comp60513_c2_seq1 when using relative RT-PCR. However, the trend of higher abundance in the restorer line was consistent between sequencing and relative RT-PCR. Taken together, the expression patterns of these genes in 121A and 121C are consistent with the Illumina data. RT-PCR results basically confirmed the reliability of our transcriptome analysis. However, the two techniques essentially use different algorithms, which may explain the above-mentioned some inconsistent results [49]–[51].

Bottom Line: Among these unigenes, 27,191 were identified as putative homologs of annotated sequences in the public protein databases, 4,326 and 7,061 unigenes were found to be highly abundant in lines 121A and 121C, respectively.Many of the differentially expressed unigenes represent a set of potential candidate genes associated with the formation or abortion of pollen.The results shed the lights on the occurrence and recovery of the disturbances in nuclear-mitochondrial interaction and provide clues for further investigations.

View Article: PubMed Central - PubMed

Affiliation: China Agricultural University, Beijing, China.

ABSTRACT

Background: The use of cytoplasmic male sterility (CMS) in F1 hybrid seed production of chili pepper is increasingly popular. However, the molecular mechanisms of cytoplasmic male sterility and fertility restoration remain poorly understood due to limited transcriptomic and genomic data. Therefore, we analyzed the difference between a CMS line 121A and its near-isogenic restorer line 121C in transcriptome level using next generation sequencing technology (NGS), aiming to find out critical genes and pathways associated with the male sterility.

Results: We generated approximately 53 million sequencing reads and assembled de novo, yielding 85,144 high quality unigenes with an average length of 643 bp. Among these unigenes, 27,191 were identified as putative homologs of annotated sequences in the public protein databases, 4,326 and 7,061 unigenes were found to be highly abundant in lines 121A and 121C, respectively. Many of the differentially expressed unigenes represent a set of potential candidate genes associated with the formation or abortion of pollen.

Conclusions: Our study profiled anther transcriptomes of a chili pepper CMS line and its restorer line. The results shed the lights on the occurrence and recovery of the disturbances in nuclear-mitochondrial interaction and provide clues for further investigations.

Show MeSH
Related in: MedlinePlus