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Transcriptome sequencing and analysis of major genes involved in calcium signaling pathways in pear plants (Pyrus calleryana Decne.).

Xu Y, Li X, Lin J, Wang Z, Yang Q, Chang Y - BMC Genomics (2015)

Bottom Line: This provided an ample foundation for characterizing the transcriptome of pear under salt stress.These results facilitate a better understanding of the molecular genetics and functional genomic mechanisms of salt stress in pear plants.Furthermore, they provide a valuable foundation for additional research on the molecular biology and functional genomics of pear and related species.

View Article: PubMed Central - PubMed

Affiliation: Jiangsu Academy of Agricultural Sciences; Jiangsu Key Laboratory for Horticultural Crop Genetic Improvement, Institute of Horticulture, Nanjing, 210014, People's Republic of China.

ABSTRACT

Background: Pears (Pyrus spp. L.) are an important genus of trees that produce one of the world's oldest fruit crops. Salinity stress is a common limiting factor for plant productivity that significantly affects the flavor and nutritional quality of pear fruits. Much research has shown that calcium signaling pathways, mediated by Calcineurin B-like proteins (CBLs) and their interacting kinases (CIPKs), are closely associated with responses to stresses, including salt. However, little is known about the molecular mechanisms that govern the relationship between salt stress and calcium signaling pathways in pear plants. The available genomic information for pears has promoted much functional genomic analysis and molecular breeding of the genus. This provided an ample foundation for characterizing the transcriptome of pear under salt stress.

Results: A high-throughput Illumina RNA-seq technology was used to identify a total of 78,695 unigenes that were successfully annotated by BLASTX analysis, using the publicly available protein database. Additionally, 2,855 novel transcripts, 218,167 SNPs, 23,248 indels and 18,322 alternative splicing events occurred. Assembled unique sequences were annotated and classified with Gene Ontology (GO), Clusters of Orthologous Group (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, which revealed that the main activated genes in pear are predominately involved in functions such as basic physiological processes, metabolic pathways, operation of cellular components, signal transduction mechanisms, and other molecular activities. Through targeted searches of the annotations, the majority of the genes involved in calcium signaling pathways were identified, among which, four genes were validated by molecular cloning, while 11 were validated by RT-qPCR expression profiles under salt stress treatment.

Conclusions: These results facilitate a better understanding of the molecular genetics and functional genomic mechanisms of salt stress in pear plants. Furthermore, they provide a valuable foundation for additional research on the molecular biology and functional genomics of pear and related species.

No MeSH data available.


Discovery of novel transcripts (a), SNP types (b), indels (c) and alternative splicing (d) in the unique sequence assembled and aligned with the pear genome.
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Fig6: Discovery of novel transcripts (a), SNP types (b), indels (c) and alternative splicing (d) in the unique sequence assembled and aligned with the pear genome.

Mentions: During the RNA-Seq experiment, mRNA-enriched RNAs were firstly broken into short segments by chemical methods and then sequenced. Read alignment to the reference genome was performed using the TopHat/Cufflinks/Bowtie/SOAP and Coding Potential Calculator [31]. Gene coverage was calculated as the percentage of a gene covered by reads. This value is equal to the ratio of the base number in a gene covered by unique mapping reads to the total number of bases in the coding region of that gene [32, 33]. A total of 78,023 (82 %) unique sequences with coverage between 90 %–100 % became the largest group, whereas no unique sequences were identified with coverage between 0 %–20 % (Fig. 5). After demultiplexing, merging and filtering of reads, about 26.8 million clean reads were obtained in the PDX sample of our experiment; 64.58 % of the unique transcripts were sequenced and successfully mapped to the pear genome, and 45.39 % were mapped to the de novo assembled results of pear (Table 2). As previously reported, an unprecedentedly high number of novel transcripts have been discovered in the genomes of humans, mice, and many plant species [31, 34]. In our research, a total of 2,855 transcripts were presumably displaying potentially novel isoforms for all the transcripts (Additional file 3). Additionally, results from Coding Potential Calculator analysis revealed that 1,720 potentially novel transcripts had the ability to code proteins, but the specific functions of these proteins remain to be determined (Fig. 6a). In order to provide the best genome coverage for the analysis of performance and production traits, numerous SNPs are needed [35, 36]. SNPs include transition and transversion of the nucleotide; in this study, among the 218,167 SNPs found, 60.5 % (132,070) were transitions between ‘A/G’ and ‘C/T’, whereas 39.5 % (86,097) were transversions among ‘A/C, A/T, C/G and G/T’ (Fig. 6b). The SNPs identified in this report provide a highly useful baseline for further genetic studies in pear, and will contribute to the development of a high density SNP array.Fig. 5


Transcriptome sequencing and analysis of major genes involved in calcium signaling pathways in pear plants (Pyrus calleryana Decne.).

Xu Y, Li X, Lin J, Wang Z, Yang Q, Chang Y - BMC Genomics (2015)

Discovery of novel transcripts (a), SNP types (b), indels (c) and alternative splicing (d) in the unique sequence assembled and aligned with the pear genome.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4590731&req=5

Fig6: Discovery of novel transcripts (a), SNP types (b), indels (c) and alternative splicing (d) in the unique sequence assembled and aligned with the pear genome.
Mentions: During the RNA-Seq experiment, mRNA-enriched RNAs were firstly broken into short segments by chemical methods and then sequenced. Read alignment to the reference genome was performed using the TopHat/Cufflinks/Bowtie/SOAP and Coding Potential Calculator [31]. Gene coverage was calculated as the percentage of a gene covered by reads. This value is equal to the ratio of the base number in a gene covered by unique mapping reads to the total number of bases in the coding region of that gene [32, 33]. A total of 78,023 (82 %) unique sequences with coverage between 90 %–100 % became the largest group, whereas no unique sequences were identified with coverage between 0 %–20 % (Fig. 5). After demultiplexing, merging and filtering of reads, about 26.8 million clean reads were obtained in the PDX sample of our experiment; 64.58 % of the unique transcripts were sequenced and successfully mapped to the pear genome, and 45.39 % were mapped to the de novo assembled results of pear (Table 2). As previously reported, an unprecedentedly high number of novel transcripts have been discovered in the genomes of humans, mice, and many plant species [31, 34]. In our research, a total of 2,855 transcripts were presumably displaying potentially novel isoforms for all the transcripts (Additional file 3). Additionally, results from Coding Potential Calculator analysis revealed that 1,720 potentially novel transcripts had the ability to code proteins, but the specific functions of these proteins remain to be determined (Fig. 6a). In order to provide the best genome coverage for the analysis of performance and production traits, numerous SNPs are needed [35, 36]. SNPs include transition and transversion of the nucleotide; in this study, among the 218,167 SNPs found, 60.5 % (132,070) were transitions between ‘A/G’ and ‘C/T’, whereas 39.5 % (86,097) were transversions among ‘A/C, A/T, C/G and G/T’ (Fig. 6b). The SNPs identified in this report provide a highly useful baseline for further genetic studies in pear, and will contribute to the development of a high density SNP array.Fig. 5

Bottom Line: This provided an ample foundation for characterizing the transcriptome of pear under salt stress.These results facilitate a better understanding of the molecular genetics and functional genomic mechanisms of salt stress in pear plants.Furthermore, they provide a valuable foundation for additional research on the molecular biology and functional genomics of pear and related species.

View Article: PubMed Central - PubMed

Affiliation: Jiangsu Academy of Agricultural Sciences; Jiangsu Key Laboratory for Horticultural Crop Genetic Improvement, Institute of Horticulture, Nanjing, 210014, People's Republic of China.

ABSTRACT

Background: Pears (Pyrus spp. L.) are an important genus of trees that produce one of the world's oldest fruit crops. Salinity stress is a common limiting factor for plant productivity that significantly affects the flavor and nutritional quality of pear fruits. Much research has shown that calcium signaling pathways, mediated by Calcineurin B-like proteins (CBLs) and their interacting kinases (CIPKs), are closely associated with responses to stresses, including salt. However, little is known about the molecular mechanisms that govern the relationship between salt stress and calcium signaling pathways in pear plants. The available genomic information for pears has promoted much functional genomic analysis and molecular breeding of the genus. This provided an ample foundation for characterizing the transcriptome of pear under salt stress.

Results: A high-throughput Illumina RNA-seq technology was used to identify a total of 78,695 unigenes that were successfully annotated by BLASTX analysis, using the publicly available protein database. Additionally, 2,855 novel transcripts, 218,167 SNPs, 23,248 indels and 18,322 alternative splicing events occurred. Assembled unique sequences were annotated and classified with Gene Ontology (GO), Clusters of Orthologous Group (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, which revealed that the main activated genes in pear are predominately involved in functions such as basic physiological processes, metabolic pathways, operation of cellular components, signal transduction mechanisms, and other molecular activities. Through targeted searches of the annotations, the majority of the genes involved in calcium signaling pathways were identified, among which, four genes were validated by molecular cloning, while 11 were validated by RT-qPCR expression profiles under salt stress treatment.

Conclusions: These results facilitate a better understanding of the molecular genetics and functional genomic mechanisms of salt stress in pear plants. Furthermore, they provide a valuable foundation for additional research on the molecular biology and functional genomics of pear and related species.

No MeSH data available.