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Molecular characterization and evolutionary origins of farinin genes in Brachypodium distachyon L.

  • Plant Genetics • Original Paper
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Abstract

Farinins are one of the oldest members of the gluten family in wheat and Aegilops species, and they influence dough properties. Here, we performed the first detailed molecular genetic study on farinin genes in Brachypodium distachyon L., the model species for Triticum aestivum. A total of 51 b-type farinin genes were cloned and characterized, including 27 functional and 24 non-functional pseudogenes from 14 different B. distachyon accessions. All genes were highly similar to those previously reported from wheat and Aegilops species. The identification of deduced amino acid sequences showed that b-type farinins across Triticeae genomes could be classified as b1-, b2-, b3-, and b4-type farinins; however, B. distachyon had only b3- and b4-type farinins. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) revealed that farinin genes are transcribed into mRNA in B. distachyon at much lower levels than in Triticeae, despite the presence of cis-acting elements in promoter regions. Phylogenetic analysis suggested that Brachypodium farinins may have closer relationships with common wheat and further confirmed four different types of b-type farinins in Triticeae and Brachypodium genomes, corresponding to b1, b2, b3 (group 1), and b4 (group 2). A putative evolutionary origin model of farinin genes in Brachypodium, Triticum, and the related species suggests that all b-type farinins diverged from their common ancestor ~3.2 million years ago (MYA). The b3 and b4 types could be considered older in the farinin family. The results explain the loss of b1- and b2-type farinin alleles in Brachypodium.

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Acknowledgments

The authors are grateful to Ms. Sarah Hancock for the language editing from the Department of Communications and Agricultural Education, Kansas State University Research and Extension. This research was financially supported by grants from the National Natural Science Foundation of China (31471485), Natural Science Foundation of Beijing City and the Key Developmental Project of Science and Technology, Beijing Municipal Commission of Education (KZ201410028031), International Science and Technology Cooperation Program of China (2013DFG30530), and the National Key Project for Transgenic Crops in China (2014ZX08009-003). This is contribution number 14-301-J from the Kansas Agricultural Experiment Station.

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This manuscript has no financial or non-financial competing interests.

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Authors and Affiliations

  1. College of Life Science, Capital Normal University, Beijing, 100048, China

    Saminathan Subburaj, Nana Luo, Xiaobing Lu, Xiaohui Li, Hui Cao, Yingkao Hu & Yueming Yan

  2. Hubei Collaborative Innovation Center for Grain Industry (HCICGI), 434025, Jingzhou, China

    Yueming Yan

  3. Department of Plant Pathology, Kansas State University, Manhattan, KS, 66506, USA

    Jiarui Li

Authors
  1. Saminathan Subburaj
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  2. Nana Luo
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  3. Xiaobing Lu
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  4. Xiaohui Li
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  5. Hui Cao
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  6. Yingkao Hu
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  7. Jiarui Li
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  8. Yueming Yan
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Corresponding authors

Correspondence to Yingkao Hu or Yueming Yan.

Additional information

Communicated by: Andrzej Górny

Saminathan Subburaj and Nana Luo contributed equally to this work.

Electronic supplementary material

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Supplementary Figure 1

Chromosomal location of farinin-like genes from blast results in the Brachypodium genome (diploid line Bd21) from websites http://www.phytozome.net and http://www.brachypodium.org/. The black lines show the location of farinin-like genes in chromosomes with their corresponding megabase (Mb) position. The blue and orange triangles indicate the upward and downward directions of transcription of corresponding locus genes, respectively. (GIF 41 kb)

High-resolution image (TIFF 581 kb)

Supplementary Figure 2

Characterization of b-type farinin genes isolated from Brachypodium. a Polymerase chain reaction (PCR) amplification of b3-type farinins. b PCR amplification of b4-type farinin genes. c 5′ promoter regions from b-type farinin genes. Lane 1, Chinese Spring; lane 2, Bd21; lane 3, Bd21-3; lane 4, Bd2; lane 5, Bd3; lane 6, Bd4; lane 7, Bd10; lane 8, Bd347; lane 9 Bd14; M is the 1-Kb DNA ladder. Target bands are indicated by arrows. (GIF 53 kb)

High-resolution image (TIFF 7927 kb)

Supplementary Figure 3

Multiple alignments of b-type farinins from Triticum and Aegilops, along with cloned Brachypodium b-type farinins. (a, b, c). Various protein domains (signal, N-terminal, repeat: R1, R2, and C-terminal) are indicated by black, blue, and orange arrows. The significant residue variations help distinguish two groups (1 and 2) and four b (b1, b2, b3 and b4) farinins; those from Triticum, Aegilops, and Brachypodium are highlighted by yellow-shaded boxes in the alignment. (PDF 4284 kb)

Supplementary Figure 4

Comparison of the 5′ flanking region from five different Bd accessions (KF933340, KF933342, KF933336, KF933345, and KF933346), including diploid, hexaploid, and tetraploid genotypes with the T. aestivum promoter (JN622144) region. The detected cis-regulatory elements such as CAAT-box, TATA-box, endosperm motif, GCN4-like motif (GLM), RY motif, and G-box are indicated by boxes. (PDF 754 kb)

Supplementary Figure 5

Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of b-type farinin genes. a–f Amplification efficiency and g–l dissociation melt curves for the amplicons of various reference genes (Ubiquitin, SAM-DC, and GAPDH), along with target genes (b3- and b4-type farinin) in CS (Chinese Spring) and Bd21 (B. distachyon) by qRT-PCR primers were tested. The non-template control (NTC) was confirmed by g–l gene-specific amplification and (M) single band amplification during qRT-PCR. (PDF 1185 kb)

Supplementary Figure 6

qRT-PCR analysis of b-type farinin genes. a–l Representation of Ct (threshold cycle) values for reference genes (Ubiquitin, SAM-DC, and GAPDH), along with target genes (b3- and b4-type farinins) during qRT-PCR analysis. (PDF 871 kb)

Supplementary Figure 7

Estimated divergence time between b-type farinin genes in Triticum family members based on aligned amino acid sequences using Bayesian Markov chain Monte Carlo (MCMC) analysis. A relaxed clock lognormal approach, Yule model of speciation process, and the WAG model of amino acid substitution were used. BEAST 2.1.3 was run for three independent times, each with 20,000,000 MCMC steps, and sampled once every 1000th generation. The molecular clock was calibrated using the divergence of B. distachyon–T. aestivum [35 ± 3 million years ago (MYA)] and A. speltoides–T. aestivum (0.87 ± 0.49 MYA). Estimated divergent time represented in MYA at each node. The green circle indicates the duplication event in the ancestors of both group 1 and group 2 farinins, and those within group 1 farinins are represented by a yellow circle. Farinin genes identified in this study are underlined. (PDF 93 kb)

Supplementary Table 1

Summary of blast results from the Phytozome and NCBI databases. The a- and b-type farinin genes cloned in this study were used to blast the Brachypodium genome database. Blast-annotated sequences are summarized here. (DOCX 16 kb)

Supplementary Table 2

List of primers used in this study to clone the farinin gene sequences and their expression under the heterologous Escherichia coli system. (DOCX 14 kb)

Supplementary Table 3

List of protein/peptide sequences identified from sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) bands of Fig. 4a (lanes 4 and 6) through LC-MS/MS analysis and searching the NCBInr database using MASCOT. (DOCX 14 kb)

Supplementary Table 4

Complete list of protein/peptide sequences identified from SDS-PAGE bands of Fig. 4a (lanes 4 and 6) through LC-MS/MS analysis and searching the NCBInr database using MASCOT. (XLS 51 kb)

Supplementary Table 5

List of primers designed and used for real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of mRNA expression studies of farinin genes. (DOCX 13 kb)

Supplementary Table 6

Absolute quantification of mRNA expression level of Ubiquitin, SAM-DC, and GAPDH genes in Brachypodium distachyon (Bd21) and Chinese Spring (CS) during the qRT-PCR analysis of target genes (b3- and b4-type farinins). Values are expressed as cDNA copies/μg of reverse transcribed total RNA. Data are shown as the mean ± standard deviation. (DOCX 17 kb)

Supplementary Table 7

The b-type farinin genes and its 5′ promoter from the current study and the previously reported HMW-GS, LMW-GS, and gliadins from Bd21 were used to blast against the unassembled Brachypodium genome sequences of Bd21-3, Bd3-1, Bd30-1, Bd1-1, BdKOZ, and BdTRC (ftp://brachypodium.org/brachypodium.org/NaturalVariation/). Blast-annotated sequences are summarized here. (XLSX 407 kb)

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Subburaj, S., Luo, N., Lu, X. et al. Molecular characterization and evolutionary origins of farinin genes in Brachypodium distachyon L.. J Appl Genetics 57, 287–303 (2016). https://doi.org/10.1007/s13353-015-0316-3

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