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A high-throughput pipeline for detecting locus-specific polymorphism in hexaploid wheat (Triticum aestivum L.).

Ma J, Stiller J, Zheng Z, Liu YX, Wei Y, Zheng YL, Liu C - Plant Methods (2015)

Bottom Line: This pipeline was successfully employed in retrieving and aligning homoeologous sequences and 83% of the primers designed based on the pipeline successfully amplified fragments from the targeted subgenomes.In addition to generating locus-specific markers, the pipeline was also used in our laboratory to identify differentially expressed genes among the three subgenomes of bread wheat.Importantly, the pipeline is not only valuable for research in wheat but should also be applicable to other allopolyploid species.

View Article: PubMed Central - PubMed

Affiliation: Triticeae Research Institute, Sichuan Agricultural University, Wenjiang, Chengdu, 611130 China ; CSIRO Agriculture Flagship, 306 Carmody Road, St Lucia, QLD 4067 Australia.

ABSTRACT

Background: Bread wheat (Triticum aestivum L., 2n = 6x = 42) is an allohexaploid with a huge genome. Due to the presence of extensive homoeologs and paralogs, generating locus-specific sequences can be challenging, especially when a large number of sequences are required. Traditional methods of generating locus-specific sequences are rather strenuous and time-consuming if large numbers of sequences are to be handled.

Results: To improve the efficiency of isolating sequences for targeted loci, a time-saving and high-throughput pipeline integrating orthologous sequence alignment, genomic sequence retrieving, and multiple sequence alignment was developed. This pipeline was successfully employed in retrieving and aligning homoeologous sequences and 83% of the primers designed based on the pipeline successfully amplified fragments from the targeted subgenomes.

Conclusions: The high-throughput pipeline developed in this study makes it feasible to efficiently identify locus-specific sequences for large numbers of sequences. It could find applications in all research projects where locus-specific sequences are required. In addition to generating locus-specific markers, the pipeline was also used in our laboratory to identify differentially expressed genes among the three subgenomes of bread wheat. Importantly, the pipeline is not only valuable for research in wheat but should also be applicable to other allopolyploid species.

No MeSH data available.


Examples of multiple alignments for two (a), three (b), and five (c) homoeologous sequences for identifying allele-specific sequences for marker development.
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Fig1: Examples of multiple alignments for two (a), three (b), and five (c) homoeologous sequences for identifying allele-specific sequences for marker development.

Mentions: The percentages of query gene sequences which detected two or more orthologous sequences from chromosome shotgun sequences (CSSs) were about 90% from Brachypodium, 93% from Ae. tauschii and 95% from T. urartu (Table 1). Examples of these stringent alignments containing two orthologous sequences (e.g. Bradi2g16370.1), three homoelogous sequences (e.g. Bradi2g33190.1), and more than four homoeologous sequences (e.g. Bradi2g14840.1) are shown in Fig. 1. Each of the alignments with the suffix ‘.htm’ is easily readable by any web browser. The alignments generated and described in ‘Methods’ can be directly used to check possible allele-specific loci for isolating genes in hexaploid wheat.Table 1


A high-throughput pipeline for detecting locus-specific polymorphism in hexaploid wheat (Triticum aestivum L.).

Ma J, Stiller J, Zheng Z, Liu YX, Wei Y, Zheng YL, Liu C - Plant Methods (2015)

Examples of multiple alignments for two (a), three (b), and five (c) homoeologous sequences for identifying allele-specific sequences for marker development.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4524443&req=5

Fig1: Examples of multiple alignments for two (a), three (b), and five (c) homoeologous sequences for identifying allele-specific sequences for marker development.
Mentions: The percentages of query gene sequences which detected two or more orthologous sequences from chromosome shotgun sequences (CSSs) were about 90% from Brachypodium, 93% from Ae. tauschii and 95% from T. urartu (Table 1). Examples of these stringent alignments containing two orthologous sequences (e.g. Bradi2g16370.1), three homoelogous sequences (e.g. Bradi2g33190.1), and more than four homoeologous sequences (e.g. Bradi2g14840.1) are shown in Fig. 1. Each of the alignments with the suffix ‘.htm’ is easily readable by any web browser. The alignments generated and described in ‘Methods’ can be directly used to check possible allele-specific loci for isolating genes in hexaploid wheat.Table 1

Bottom Line: This pipeline was successfully employed in retrieving and aligning homoeologous sequences and 83% of the primers designed based on the pipeline successfully amplified fragments from the targeted subgenomes.In addition to generating locus-specific markers, the pipeline was also used in our laboratory to identify differentially expressed genes among the three subgenomes of bread wheat.Importantly, the pipeline is not only valuable for research in wheat but should also be applicable to other allopolyploid species.

View Article: PubMed Central - PubMed

Affiliation: Triticeae Research Institute, Sichuan Agricultural University, Wenjiang, Chengdu, 611130 China ; CSIRO Agriculture Flagship, 306 Carmody Road, St Lucia, QLD 4067 Australia.

ABSTRACT

Background: Bread wheat (Triticum aestivum L., 2n = 6x = 42) is an allohexaploid with a huge genome. Due to the presence of extensive homoeologs and paralogs, generating locus-specific sequences can be challenging, especially when a large number of sequences are required. Traditional methods of generating locus-specific sequences are rather strenuous and time-consuming if large numbers of sequences are to be handled.

Results: To improve the efficiency of isolating sequences for targeted loci, a time-saving and high-throughput pipeline integrating orthologous sequence alignment, genomic sequence retrieving, and multiple sequence alignment was developed. This pipeline was successfully employed in retrieving and aligning homoeologous sequences and 83% of the primers designed based on the pipeline successfully amplified fragments from the targeted subgenomes.

Conclusions: The high-throughput pipeline developed in this study makes it feasible to efficiently identify locus-specific sequences for large numbers of sequences. It could find applications in all research projects where locus-specific sequences are required. In addition to generating locus-specific markers, the pipeline was also used in our laboratory to identify differentially expressed genes among the three subgenomes of bread wheat. Importantly, the pipeline is not only valuable for research in wheat but should also be applicable to other allopolyploid species.

No MeSH data available.