Molecular Genetic Analysis of Cereal β-Amylase Genes Using Exon-Primed Intron-Crossing ( EPIC ) PCR

The proteins encoded by cereal β-amylase genes Bamy1 and Bamy2 genes play an important role in seedling germination and in the brewing process. Here, we use exon-primed intron-crossing (EPIC) to analyse Bamy1 and Bamy2 genetic diversity among 38 accessions belonging to six Poaceae tribes. DNA sequence alignment of multiple Poaceae species β-amylase sequences allowed design of EPIC primers that simultaneously amplify Bamy1 and Bamy2 in all the cereal species investigated. The genetic variation observed in the samples investigated is analysed and discussed, and illustrates the effectiveness of this approach for intraand interspecific analysis in plant species.


Introduction
Due to the growing volume of data provided by genome sequencing projects, and the presence of regulatory regions within introns, the study of intronic polymorphism is emerging as an important aspect of genetic research in agricultural crops (Braglia et al. 2010).Compared to exonic regions, introns are more variable due to reduced selective pressure, i.e. the rate of mutation accumulation in these regions is comparatively high (Ludwig 2002, Morello & Breviario 2008, Yang et al. 2007).Introns often contain a variety of functional elements, including enhancers, silencers, regulators of alternative splicing, trans-splicing elements and other regulatory elements (some of which can be found in the expanded Ratar. Povrt. 51:3 (2014) 175-189 introns).Furthermore, intron characteristics such as length variability, position within the gene, dependency on the length of the exons, can also modulate gene properties such as expression, transcription, splicing and microRNA lifetime (Braglia et al. 2010, Ludwig 2002, Rose & Beliakoff 2000).In plants, investigation into intron function has highlighted their role in gene expression, and specifically in tissue-or stagespecific expression (Morello & Breviario 2008).Analysis of plant intron sequences has been applied in numerous studies (Chetelat et al. 1995, Fridman et al. 2000, Holland et al. 2001, Hongtrakul V 1998), with a well-studied example in cereal species being the role of putative ciselements in intron 1 of vernalization response (Vrn) genes.Deletions spanning this region are thought to result in vernalization nonresponsiveness (Cockram et al. 2007), and underlie the creation of globally important springsown cereal cultivars.
The polymerase chain reaction (PCR) based technique, 'exon-primed intron-crossing' (EPIC-PCR) (Palumbi & Baker 1994) has gained favour in plant and animal studies, and relies on design of primers selected to anneal to highly-conserved regions of the exons.For example, it has been used to study conserved regions within eukaryotic 18S and 28S ribosomal genes and prokaryotic 16S and 23S ribosomal genes, for amplification of variable intergenic regions known as internal transcribed spacers (ITS), containing 5.8S ribosomal gene (Gardes 1993).
EPIC has a number of experimental advantages, including, (i) primers complementary to conserved regions within exons can be used across a wide taxonomic range, (ii) homologous amplified sequences can be easily defined by comparing their exonic and intronic regions depending on genetic distance between the taxons, and (iii) exonic and intronic fragments can help in the simultaneous studies of genetic variety at intraspecific and interspecific levels (Bierne et al. 2000, Ishikawa et al. 2007, Lessa 1992, Li et al. 2010).A further advantage of EPIC is that if avoids the need to develop DNA markers on a species -by-species basis, which can be costly and time consuming.
In cereal species (tribe: Triticeae), β-amylase hydrolyses starch by splitting α-1,4-glycosidic bonds, resulting in the formation of the high molecular weight dextrin and maltose -a disaccharide, that easily diffuses and can be used by the growing embryo (James et al. 2003).It is also an important component of the mash, a process used in brewing by which grain and water are combined and heated allowing enzymatic degradation of starch into sugars for fermentation (Priest 1986).Numerous species within the Triticeae can be used for brewing, including barley, rye, triticale, wheat, oat, and millet (Briggs 1998).Only members of the Triticeae are reported to have two different forms of β-amylase (Mason-Gamer 2005), differing significantly in their patterns of gene expression: Bamy1 is specific to the endosperm (endospermal β-amylase), while Bamy2 is expressed in all tissues (ubiquitous β-amylase).These genes are paralogs, and both consist of 7 exons and 6 introns.
In this study, EPIC-PCR was used to investigate Bamy1 and Bamy2 intronic genetic variation within representative species belonging to the Poaceae.Due to their importance within cereal species, molecular genetic characterisation of cereal Bamy1 and Bamy2 will provide new information on the range of inter-and intra-specific genetic variation present.This data will help inform how Triticeae germplasm resources may be used as important sources of qualitative character enhancement, allowing enhancement of β-amylase activity in cereals.

Germplasm and DNA extraction
The 38 accessions investigated, belonging to six Poaceae tribes, are listed in Table 1.Ratar. Povrt. 51:3 (2014)  Genomic DNA was extracted from 5-day-old etiolated seedling or green leaves using CTAB buffer following established protocols (http://primerdigital.com/dna.html).DNA concentration was estimated by spectrophotometric analysis; DNA quality was determined by agarose gel electrophoresis.
DNA multiple sequence alignments of the βamylase genes identified regions of DNA conservation within exons, as well as variability within intronic regions (Supplemental File).The sequence alignment was used in conjunction with the program FastPCR (Kalendar et al. 2011) to design EPIC-primers which target the most conserved protein-coding regions of Bamy1 and Bamy2.It should be noted that the exonic regions chosen for primer annealing show high levels of conservation not only for cereals, but also for more distant plant species, thus demonstrating the flexibility of EPIC for plant molecular studies.

Interspecific Poaceae Bamy1 and Bamy2 genetic variation
To study interspecific genetic variability in Bamy1 and Bamy2, 38 Poaceae species were analysed (Tab.2).Detection of β-amylase genetic polymorphism was performed using EPIC primers 3162 and 3816 (complementary to exons 1 and 4 in barley, respectively), allowing simultaneous study of Bamy1 and Bamy2 amplicons from multiple species.Analysis of EPIC PCR products amplified from all species found a wide range of genetic variation (Fig. 1).The expected amplicon size of barley PCR amplicons fragments from H. vulgare was 2295 or 2196 bp for Bamy1 (due to a 126 bp MITE insertion in intron 3 (Erkkilä 1999), and 1365 bp for Bamy2.Amplification products of these sizes were identified in Hordeum vulgare (Fig. 1, samples 1-4), as well as for H. spontaneum, H. murinum and T. durum.Amplicons from the remaining species differed from these, ranging between 1700 and 2500 bp for Bamy1, and between 1300 and 1500 bp for Bamy2 (Fig. 1).Species belonging to the tribes Aveneae, Poeae and Brachypodieae (Phleum pratense, Zingeria biebersteiniana, Colpodium versicolor, Spartina alterniflora, Avena sativa, Brachypodium distachyon; samples 31-34, 36-37) contain just one βamylase gene, Bamy2.Similarly, for Bromus sterilis (which belongs to the Bromeae tribe), searches of public DNA databases found sequences for Bamy1 alone (HE565904, HE565905).However, the EPIC analysis undertaken here found B. sterilis to possess both Bamy1 and Bamy2 (Fig 1,sample 35), indicating that the presence of endospermal β-amylase genes is not exclusive to the Triticeae tribe.In total, ten different Bamy1 alleles (from one to three amplicons per sample) and four Bamy2 alleles (one amplicon per sample) were identified.EPIC-PCR analyses revealed both inter-and intraspecific variation in the samples analysed.Accordingly, the EPIC approach is appropriate for rapid analysis of genetic variety within the Poаceae family.

Triticeae β-amylase genetic variation
EPIC analyses of β-amylase intraspecific genetic variation for four members of the Triticеае tribe were undertaken: Triticum dicoccoides, Aegilops speltoides, rye and triticale.Ratar. Povrt. 51:3 (2014)   These species were selected for the following reasons: T. dicoccoides (emmer or spelt wheat) is the tetraploid parent of the cultivated hexaploid wheat T. aestivum (2n = 6x = 42, AABBDD), while A. speltoides (2n = 2x = 14, ВВ or SS) carries valuable agronomic attributes exploited within the artificial hybridization with bread wheat.As well as being a valuable crop in its own right, rye is used to form a hybrid with wheat to produce triticale, a crop which combines the valuable characteristics of these genera (Lukaszewski 2006, Yang et al. 2011).
Three EPIC amplicons were found to be common for all four species investigated: products of 2295 bp (as expected for Bamy1), ~1900 bp (included in the allelomorphic spectrum of Bamy1) and 1356 bp (Bamy2) (Fig. 2, 3, 4).It should be noted that EPIC amplicons of size 2295 and 1356 bp were also present in the allelomorphic spectrum of Hordeum (samples 1 -6) and T. durum (10) (Fig. 1).Analysis of Bamy1 polymorphism in triticale and rye showed that their level of common polymorphism is higher than those of the cultivars of barley (Fig. 1), in which only two Bamy1 alleles were found.Comparison of triticale and rye genotypes showed that the majority of the triticale allelomorphic spectrum was present in rye (including fragments of 2295 and 1356 bp).In addition, the allelomorphic spectrums of winter and spring triticale contained amplicons of ~2000 bp (Fig. 2 -samples no. 9, 17, 18, 22, 29 and 34).These were absent in rye, and as they were identified here in wheat, it is likely they originate from the wheat parent (Fig. 4 A).The 1850 bp amplicon found in the triticale samples was absent from rye and wheat, indicating this allele was not captured in the donor species samples screened in this study.Leontino;[37][38][39][40][41] Analysis of Cereal β-amylase Genes using EPIC PCR Ratar. Povrt. 51:3 (2014) [175][176][177][178][179][180][181][182][183][184][185][186][187][188][189] Up to eight Bamy1 polymorphic PCR products (maximum per accession = 4) and three Bamy2 variants (maximum per accession = 2) were detected in the samples of winter triticale analysed.For spring triticale, EPIC primers for Bamy1 and Bamy2 identified a total of six (maximum per accession = 3) and two amplicons (maximum per accession = 1), respectively.For triticale Bamy1, the frequency of the 2295 bp fragments varied with the seasonal growth habit (SGH) classification of the accessions studied: it is found in 86% of the winter cultivars, while it is present in just 48% of spring lines.The Bamy1 2000 bp fragment varied in frequency between SGH classes, being present in 52% of spring triticale, but found in just 13% of winter lines.Similarly, the 1900 bp Bamy1 amplicon is found in 56% of spring triticale cultivars, and in 27% of winter lines (Fig. 2 and  3).EPIC PCR products of ~1700 bp (found in cultivars Plains, Constant and Flavius) and ~1750 bp (cultivar Tatra) were found only in winter triticale.The most common Bamy2 variant identified in the triticale cultivars analysed was the 1356 bp fragment.For rye, six Bamy1 (from one to three amplicons per cultivar) and two Bamy2 (from one to two per cultivar) products were found.
EPIC analysis of β-amylase genes from Emmer wheat revealed that T. dicoccoides lines commonly possess two Bamy1 amplicons: the first (~ 1900 bp) was found to be present in all T. dicoccoides investigated, while amplicons of ~2000 bp and 2296 bp were observed in 79% and 16 % of samples, respectively (Fig. 4A).Among the 42 Aegilops speltoides accessions investigated, three and two amplicons were found for Bamy1 and Bamy2, respectively.In addition to amplicons of 2295 and 1900 bp (found also in T. turgidum, triticale, rye and barley), products of ~2600 bp (not found in any of the other species investigated) were also identified in A. speltoides, where it was found in 63% of accessions (Fig. 4В).Comparison of the molecular profiles of T. dicoccoides and Aegilops speltoides highlighted the presence of common Bamy1 amplicon sizes between these species, of 2295 and 1900 bp (Fig. 4B).The 1900 bp amplification fragment was found in 16% of A. speltoides accessions and in 100% of T. dicoccoides lines (Fig. 4А-B).Stratula O et al.

Figure S1
Figure S1Multiple alignments of β-amylase genes sequences.Exons are marked in red.

Table 1 .
Poaceae germplasm used in this study Stratula O et al.