Limits...
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.


Validation of marker locations using a DH (doubled haploid) population. Orthologous sequences of Bradi1g54730.1 and Bradi1g55847.1 were amplified from the two parents of the DH population, B (‘Batavia’) and E (‘Ernie’), and sequenced. The single nucleotide polymorphism (in red and green) and restriction enzyme sites (underlined) were identified between B and E for Bradi1g54730.1 with restriction enzyme AciI (a) and Bradi1g55847.1 with HaeIII (b). The amplified products of the two parents and 14 of the DH lines were digested and separated on agarose gels. Map positions of the two new markers on chromosome 2B (c) were calculated based on the linkage map published by Li et al. [21].
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Fig3: Validation of marker locations using a DH (doubled haploid) population. Orthologous sequences of Bradi1g54730.1 and Bradi1g55847.1 were amplified from the two parents of the DH population, B (‘Batavia’) and E (‘Ernie’), and sequenced. The single nucleotide polymorphism (in red and green) and restriction enzyme sites (underlined) were identified between B and E for Bradi1g54730.1 with restriction enzyme AciI (a) and Bradi1g55847.1 with HaeIII (b). The amplified products of the two parents and 14 of the DH lines were digested and separated on agarose gels. Map positions of the two new markers on chromosome 2B (c) were calculated based on the linkage map published by Li et al. [21].

Mentions: Given that primers designed based on a single nucleotide polymorphisms (SNP) did not always amplify a specific fragment in our previous studies, primers designed in this study were based on two or more SNPs or indels (Additional file 1: Table S1). Of the 36 primer pairs designed for selected loci, 30 (83%) amplified a product on the expected chromosomes, two failed to amplify any PCR products, and the other four generated locus-specific fragments (Fig. 2 and Additional file 1: Table S1). Eleven of the 30 pairs of primers were further assessed against other bread wheat genotypes (Additional file 1: Table S1). Sequence alignments indicated that, without exception, they all amplified sequences homologous with those from the expected chromosomes as shown in ‘Chinese Spring’ (‘CS’) (Additional file 1: Table S1). Four of these primer pairs generated polymorphic fragments between the parents of the mapping population used in this study. The polymorphic sequences were used to develop cleaved amplified polymorphic sequence (CAPS) markers. Each of the four CAPS markers was successfully mapped to the anticipated chromosome as originally detected using ‘CS’ aneuploids (Fig. 3, Additional file 2: Fig. S1 and Additional file 3: Fig. S2).Fig. 2


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)

Validation of marker locations using a DH (doubled haploid) population. Orthologous sequences of Bradi1g54730.1 and Bradi1g55847.1 were amplified from the two parents of the DH population, B (‘Batavia’) and E (‘Ernie’), and sequenced. The single nucleotide polymorphism (in red and green) and restriction enzyme sites (underlined) were identified between B and E for Bradi1g54730.1 with restriction enzyme AciI (a) and Bradi1g55847.1 with HaeIII (b). The amplified products of the two parents and 14 of the DH lines were digested and separated on agarose gels. Map positions of the two new markers on chromosome 2B (c) were calculated based on the linkage map published by Li et al. [21].
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig3: Validation of marker locations using a DH (doubled haploid) population. Orthologous sequences of Bradi1g54730.1 and Bradi1g55847.1 were amplified from the two parents of the DH population, B (‘Batavia’) and E (‘Ernie’), and sequenced. The single nucleotide polymorphism (in red and green) and restriction enzyme sites (underlined) were identified between B and E for Bradi1g54730.1 with restriction enzyme AciI (a) and Bradi1g55847.1 with HaeIII (b). The amplified products of the two parents and 14 of the DH lines were digested and separated on agarose gels. Map positions of the two new markers on chromosome 2B (c) were calculated based on the linkage map published by Li et al. [21].
Mentions: Given that primers designed based on a single nucleotide polymorphisms (SNP) did not always amplify a specific fragment in our previous studies, primers designed in this study were based on two or more SNPs or indels (Additional file 1: Table S1). Of the 36 primer pairs designed for selected loci, 30 (83%) amplified a product on the expected chromosomes, two failed to amplify any PCR products, and the other four generated locus-specific fragments (Fig. 2 and Additional file 1: Table S1). Eleven of the 30 pairs of primers were further assessed against other bread wheat genotypes (Additional file 1: Table S1). Sequence alignments indicated that, without exception, they all amplified sequences homologous with those from the expected chromosomes as shown in ‘Chinese Spring’ (‘CS’) (Additional file 1: Table S1). Four of these primer pairs generated polymorphic fragments between the parents of the mapping population used in this study. The polymorphic sequences were used to develop cleaved amplified polymorphic sequence (CAPS) markers. Each of the four CAPS markers was successfully mapped to the anticipated chromosome as originally detected using ‘CS’ aneuploids (Fig. 3, Additional file 2: Fig. S1 and Additional file 3: Fig. S2).Fig. 2

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.