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Article

Allelic Variation of Puroindolines Genes in Iranian Common Wheat Landraces

by
Ana B. Huertas-García
1,
Carlos Guzmán
1,*,
Facundo Tabbita
1,2 and
Juan B. Alvarez
1
1
Departamento de Genética, Escuela Técnica Superior de Ingeniería Agronómica y de Montes, Edificio Gregor Mendel, Campus de Rabanales, Universidad de Córdoba, CeiA3, ES-14071 Córdoba, Spain
2
Instituto Nacional de Tecnología Agropecuaria (INTA), Instituto de Recursos Biológicos, Las Cabañas y los Reseros s/n, Buenos Aires, Hurlingham 1686, Argentina
*
Author to whom correspondence should be addressed.
Agriculture 2022, 12(8), 1196; https://doi.org/10.3390/agriculture12081196
Submission received: 11 July 2022 / Revised: 7 August 2022 / Accepted: 8 August 2022 / Published: 10 August 2022
(This article belongs to the Section Genotype Evaluation and Breeding)

Abstract

:
Wheat is one of the most widely grown crops in the world. One of the traits that defines wheat quality is grain hardness, which is determined by puroindolines (PINA and PINB) proteins encoded with Pina-D1 and Pinb-D1 genes. In this study, the diversity of Pina-D1 and Pinb-D1 was evaluated in a collection of 271 Iranian common wheat (Triticum aestivum L. ssp. aestivum) landraces, whose kernels had previously been classified as hard or semi-hard based on PSI analysis. Three alleles previously described as associated with hard grain were detected in the collection: Pinb-D1b in 11 accessions, Pinb-d1ab in 175 accessions, and Pinb-d1p in 80 accessions. In addition, a novel allele tentatively named Pinb-d1ak was detected in Pinb-D1 and was characterized by a change at position 140 of the deduced protein (cysteine/tyrosine). On average, the accessions with this allele showed a lower PSI value than the accessions with other Pin allele. This means that this novel allele may be associated with harder grains than other Pin alleles and could be used by breeding programs targeting different grain hardness levels. This study highlights the importance of conserving and characterizing wheat genetic resources that could be used as sources of genetic variability in breeding programs.

1. Introduction

Wheat (Triticum sp.) is one of the three main crops grown around the world, with a field area of 219 million hectares that represents a quarter of total cereal production, with 760 million tons produced in 2020 [1].
In wheat, grain quality is one important factor because it determines the market value and its subsequent use. Wheat quality is determined by grain composition and the characteristics derived from it: hardness, gluten strength, and starch. Grain hardness is the most important single trait determining end-use quality [2], as it is related to the amount of damaged starch produced during the milling process. Since hard grain produces more damaged starch than soft grain, hard wheat is used for bread-making, which requires high water absorption for the correct development of fermented dough, while soft wheat is preferred for making cookies and pastries, which requires less hydrophilic flour, so that more water is available for the sugar to form a syrup, resulting in greater cookie spread [3].
In common wheat (Triticum aestivum L. ssp. aestivum), grain hardness is genetically controlled by two genes (Pina-D1 and Pinb-D1) that codify for puroindolines (PINA and PINB, respectively). Puroindolines are small (~13 kDa), cysteine-rich, lipid-binding proteins consisting of 148 amino acids [4]. These proteins exhibit a tryptophan-rich domain, which consists of five tryptophan residues in PINA and three residues in PINB [2], and both PINA and PINB contain a backbone of 10 cysteine residues [5]. The Pin-D1 genes are located at the hardness locus (Ha) in the distal end of the 5DS chromosome [6]. Both genes have a 447 bp coding sequence [7], and these sequences are 70.2% similar to each other [8]. The Ha locus includes also the Gsp-1 gene (495 bp) that codes for the grain softness protein-1 [9].
The presence of Pina-D1a and Pinb-D1a alleles in common wheat is associated with a soft texture, while nucleotide changes in coding regions affecting the original protein or deletions of entire Pin-D1 genes (null alleles) are correlated with a hard texture [5]. Since allopolyploidization resulting in durum wheat (T. turgidum ssp. durum (Desf.) Husn) caused the loss of the puroindolines genes in the A and B genomes, durum wheat lacks puroindolines genes, and its grains are very hard [5]. The Pin-D1 genes’ polymorphisms led to differences in the degree of hardness and in processing and end-use quality traits [10]. Several studies were conducted comparing the effects of the Pina-D1b and Pinb-D1b alleles, which are the two most common Pin-D1 alleles with hard endosperm. Genotypes with Pina-D1b/Pinb-D1a showed harder kernels than those with Pina-D1a/Pinb-D1b [11]. In addition to this, the Pinb-D1b allele was associated with higher milling yield, higher dough extensibility, and better baking quality (higher loaf volume and better crumb) than the Pina-D1b allele [12]. These studies clearly illustrate the importance of the Pin-D1 genes not only for hardness but also for processing and end-use quality.
Wheat landraces possess variability in different genes that are not present in modern cultivars [13]. For wheat quality improvement, this variability could be useful in generating materials with novel properties. In fact, several puroindoline alleles have been found in different collections of genetic resources worldwide. For example, the allele Pinb-D1d was first detected when researchers were analyzing Pin-D1 genes of cultivars from northern Europe [14], and later the same allele was found in cultivars with Spanish origins [15]. Chinese landraces have been showed to carry alleles very rare in other germplasm pools, such as Pina-D1r, Pina-D1s, and Pinb-D1p [16] or Pinb-D1l [17]. Similarly, cultivars with origins in India were found to be good sources for the variability of the Pin-D1 genes by Kumar et al. [18], who identified five novel alleles in materials from that country.
Iran is one of the main habitats of wheat ancestors and is therefore a reservoir of new alleles [19]. In the Seeds of Discovery project at CIMMYT (International Maize and Wheat Improvement Center, Mexico), 30,000 wheat accessions (including a collection of 6800 Iranian wheat landrace accessions [19]) were characterized for quality traits [20], including grain hardness, using the PSI method [21].
The objective of our study was to assess the diversity for the Pina-D1 and Pinb-D1 genes in a collection of 271 common wheat landraces from Iran, in order to detect new alleles of the puroindoline genes associated with different grain hardnesses.

2. Materials and Methods

2.1. Plant Materials

In this study, 271 Iranian common wheat landraces, which were provided by the CIMMYT’s germplasm bank and known from a previous study to have hard and semi-hard grains, were used [20]. The hardness data of all of them were determined by near infrared spectroscopy (NIRS, Antaris II FT-Analyzer, Thermo Scientific, Waltham, MA, USA), calibrated on the basis of AACC methods [21] (Supplementary Table S1).

2.2. Genomic DNA Extraction

Genomic DNA was extracted from the young leaves of approximately two-week-old seedlings grown using the modified CTAB method described by Stacey and Isaac [22], and the samples were diluted to a final concentration of 20 ng/mL.

2.3. Amplification and Digestion of Pina-D1 and Pinb-D1 Genes

The Pina-D1 gene was amplified with the primers designed by Lillemo et al. [23] (LIL1-Fw: 5′-CATCTATTCATCTCCACCTGC-3′ and LIL1-Rv: 5′-GTGACAGTTTATTAGCTAGTC-3′) and with the primers designed by Massa et al. [24] (MAS-Fw: 5′-GGTGTGGCCTCATCTCATCT-3′ and MAS-Rv: 5′-AAATGGAAGCTACATCACCAGT-3′). For the amplification of the Pinb-D1 gene, we used the primers designed by Lillemo et al. [23] (LIL2-Fw: 5′-GAGCCTCAACCCATCTATTCATC-3′ and LIL2-Rv: 5′-CAAGGGTGATTTTATTCATAG-3′).
Each 15 mL reaction included 40 ng of DNA, 1.5 mM MgCl2, 0.3 mM of each primer, 0.2 mM dNTPs, 3 μL of 5× PCR buffer, and 0.75 U of GoTaq® G2 Flexi DNA polymerase (Promega). The PCR conditions included an initial denaturation for 3 min at 94 °C followed by 35 subsequent cycles: 45 s at 94 °C, 1 min 30 s at 60 °C, and then 45 s at 72 °C for the primers LIL1 and LIL2; and 45 s at 94 °C, 30 s at 64 °C, and then 45 s at 72 °C for the primer MAS. After the 35 cycles, all reactions included a final extension of 5 min at 72 °C. The amplification products were fractionated on vertical PAGE gels in a discontinuous Tris-HCl buffer system (pH 6.8/8.8) with 8% (w/v, C: 1.28%) polyacrylamide concentration.
To detect some mutations already described in Pina-D1 and Pinb-D1 genes, the amplicons of these genes were digested with endonucleases or restriction enzymes. The Pina-D1 amplicons were digested with MspI (Biolabs) to detect the Pina-D1m allele [25] and with DdeI (Biolabs) to see if there were differences in the pattern when digesting the different accessions. The Pinb-D1 amplicons were digested with BsrBI (Biolabs) to detect the Pinb-D1b allele [26], with PvuII (Biolabs) to detect the Pinb-D1c allele [14], with BstXI (Biolabs) to detect the Pinb-D1e allele [26], with ApaI (Biolabs) to detect the Pinb-d1ab allele [26], and with PflMI (Biolabs) to detect the Pinb-D1p allele [27]. Digests were performed in a total volume of 10 μL, with 1 μL of buffer (1×), 0.25 U of enzyme, and 5 μL of PCR product for 2 h at 37 °C. The products resulting from this digestion were analyzed using electrophoresis on 10% polyacrylamide concentration gels (w/v; C: 3%).

2.4. Cloning of Pinb-D1 Genes

The Pinb-D1 amplicons were then purified with SureClean Plus (Bioline Reagents, London, UK) and cloned into the vector pSpark TA Done (Canvax, Cordoba, Spain). The nucleotide sequences were obtained from three positive clones. These sequences were analyzed and compared with the common wheat sequences available in the NCBI database for Pinb-D1a: DQ363913 using the Geneious Pro software version 2020.2.4 (Biomatters Ltd., Auckland, New Zealand). To evaluate the impact of the allelic variations in the PINB protein’s function, the potential effects of amino acid changes were analyzed with PROVEAN (Protein Variation Effect Analyzer) [28]. In PROVEAN (http://provean.jcvi.org, accesed on 1 June 2022), variants with values below −2.5 are predicted as deleterious, while values above −2.5 are considered neutral [28]. Finally, the novel sequence found in the current study is available from the Genbank database (NCBI ID: ON723901).

3. Results

3.1. Variation in Grain Hardness

A large set of Iranian common wheat landraces were evaluated previously for grain hardness using the PSI test [20]. For the current study, a subset of those accessions was selected based on their PSI values. The selected accessions showed an average PSI value of 47.14 ± 2.74% and values ranging between 40 and 50%. These values correspond to those of hard and semi-hard grains: 221 accessions showed a semi-hard grain texture ranging between 45 and 50%, while 50 accessions were classified as semi-hard grain texture since they ranged between 45 and 40% (Figure 1). According to the literature [5], these accessions should carry mutations in either Pina-D1 or Pinb-D1 that lead to their non-soft grain texture.

3.2. PCR Analysis of the Pina-D1 and Pinb-D1 Genes

The Pina-D1 and Pinb-D1 genes were amplified to detect possible differences in their size, together with the presence/absence of amplicons. For the Pina-D1 gene using primer LIL1, all of the accessions produced a PCR product of 524 bp in size (Figure 2A). When the primer MAS was used, the results for Pina-D1 amplification presented two amplicons (Figure 2B), one of them with 516 bp (Pina-D1) and the other with 564 bp that corresponded to the grain softness protein (Gsp-1 gene). All accessions showed the expected amplification product of 597 bp for the Pinb-D1 gene (Figure 2C). Therefore, no apparent polymorphism was detected in any of the accessions, and the amplicons seemed similar to the ones of cv. Chinese Spring (Pina-D1a, Pinb-D1a).

3.3. Enzymatic Digestion and Sequencing of Pinb-D1 Alleles

Several specific restriction enzymes were used to digest the amplicons of Pina-D1 and Pinb-D1 in order to screen the Iranian landraces that were tested for the most common mutations for Pina-D1 and Pinb-D1. The Pina-D1 amplicons were digested with MspI (used to detect the Pina-D1m allele), but no accession provided a positive result for this allele (Figure 3). Additionally, the Pina-D1 amplicons were also digested with DdeI (used by Guzmán et al. [29] to detect polymorphism in Pina-D1), but again no polymorphism was detected (data not shown).
However, several Pinb-D1 alleles were found in our accessions using specific restriction enzymes (Figure 4). We found 11 accessions with the Pinb-D1b allele (positive for BsrBI digestion), 80 accessions with Pinb-d1p (negative for PflMI digestion), and 175 accessions with the Pinb-D1ab allele (negative for ApaI digestion) (Table 1). The alleles Pinb-D1c and Pinb-D1e (digestions with PvuII and BstXI, respectively) were not found in any of our accessions (data not shown).
To detect more variability in the Pinb-D1 gene, the remaining five accessions that did not show any differences when compared with those of cv. Chinese Spring in the above-mentioned analysis were selected for the sequencing of their Pinb-D1 gene. The sequences obtained were compared with the sequence of the Pinb-D1a allele of cv. Chinese Spring (NCBI ID: DQ363913). All these accessions showed a G/A transition, resulting in a codon change at position 419 (TGC → TAG). According to a BLAST analysis performed and the literature reviewed, this mutation was not reported previously, and therefore we consider this allele to be novel. Following the order of the Wheat Gene Catalogue, we tentatively proposed to name this allele Pinb-D1ak. In addition, the results obtained with the restriction enzymes were corroborated by sequencing the Pinb-D1 gene in few of the accessions that resulted in a positive for other alleles (Pinb-D1p and Pinb-D1ab). As all the accessions used in the study showed a mutation in Pinb-D1 gene that explained their hard grain texture, it was assumed that they carried the Pina-D1a allele for Pina-D1 gene.

3.4. Amino Acid Sequence Analysis and Relationship with Grain Hardness

Both PIN proteins are synthesized as precursors or preproteins and contain four domains: a signal-peptide, N-terminal domain, mature protein, and C-terminal domain [7]. Another distinctive feature of these proteins is the presence of a highly conservative cysteine backbone made up of ten cysteines and a tryptophan-rich domain. In the novel allele identified (Pinb-D1ak), which causes an amino acid change from cysteine to tyrosine at position 140 of the amino acid sequence, the cysteine change coincided with the tenth cysteine of the cysteine backbone of the PINB protein (Figure 5). In addition to this, the PROVEAN score obtained for the Pinb-D1ak allele (−4.2) indicated that this mutation likely has a strong effect on the protein function.
The relationship between the grain hardness of the accessions with the new Pinb-D1ak allele and the rest of the accessions identified with the Pinb-D1b, Pinb-D1ab, and Pinb-D1p alleles was studied by analyzing the mean PSI of the group of accessions that had each allele. It was observed that the accessions with the Pinb-d1ak allele had a mean PSI value of 44.2 ± 2.93, slightly lower than that of the rest of the accessions groups with the Pinb-D1b, Pinb-D1ab, and Pinb-D1p alleles with mean PSI values of 45.91 ± 3.58, 46.91 ± 2.77, and 47.11 ± 2.64, respectively (Table 2). Therefore, it was observed that the accessions with the Pinb-d1ak allele had harder grains than those of the accessions with other mutations, as lower values of the PSI (%) correspond to harder grains [7].

4. Discussion

Landraces and old cultivars possess variability for different genes that may not be present in modern cultivars, and that knowledge could be useful for modern breeding programs to develop materials with novel differential properties. Because of this characteristic, efforts are being made to identify useful novel variations among the vast wheat genetic resources stored in germplasm banks. In this regard, the CIMMYT’s wheat germplasm bank holds a huge collection (more than 100,000 accessions) that is being characterized by different means [14,32] in order to possibly use its unexploited variation for the genetic improvement of the breeding program. Within the CIMMYT’s collection, there is a large set (6800 accessions) of landraces with Iranian origin since Iran is one of the centers of diversity for the common wheat. This set of materials has shown great genetic diversity [20], including interesting variations for grain quality traits related to both end-use and nutritional properties [33].
In this study, the grain texture of a set of Iranian bread wheat landraces was analyzed and characterized at the molecular level for puroindoline composition. Since grain hardness is one of the most important wheat quality traits, its genetic control has been extensively studied, and a lot of alleles of the two puroindoline genes involved (Pina-D1 and Pinb-D1) have been identified. The Pina-D1a and Pinb-D1a alleles are associated with a soft grain texture [6], while most of the other described alleles of Pina-D1 and Pinb-D1 are associated with a hard grain texture. The variability of these genes was studied before in germplasm collections of diverse origins. For example, Cane et al. [34] examined the polymorphism of these genes in Australian wheat cultivars; Ayala et al. [15] screened Mexican landraces, finding three different Pin-D1 alleles leading to hard grain; Ma et al. [16] characterized a large collection, mainly from China but with some cultivars from other countries (USA, Europe, Japan, etc.), to find six and nine different Pina-D1 and Pinb-D1 alleles, respectively. However, Iranian materials have been not studied in detail for the variability of these important genes. In our study, all the accessions were previously evaluated for grain hardness and had either hard or semi-hard grains. This indicated that the Iranian landraces selected for the current study likely carry mutations in the puroindoline genes that lead to a loss of function in their respective puroindoline proteins. Therefore, the probability of detecting mutations for the Pina-D1 or Pinb-D1 genes was rather high.
First, using specific restriction enzymes, we searched different alleles that were previously described to cause changes in the PINA or PINB protein sequence or their absence and lead to hard endosperm. The Pinb-D1b allele was found, which consists of a single nucleotide change at position 46 in the Pinb-D1 sequence that confers a glycine to serine change in the expressed protein. This change in the sequence resides near the tryptophan-rich domain and probably changes the secondary or tertiary structure of the protein, leading to hard endosperm [6]. The Pinb-D1b allele was detected in 11 accessions, which corresponded to a frequency of 4% of the total of our samples. This allele was found in different studies in landraces from different countries such as Spain [35], Mexico [15], and China [10,27] with a frequency similar to that of our study. However, in other collections such as in a study of landraces from Poland [36], Pinb-D1b had a frequency of 21% of the total, which was much higher than the frequency of our study. It was also very frequent in cultivars from Norway, Sweden, and Finland [14]. Therefore, we deduced that Pinb-d1b is widely distributed in America, Asia, and Europe, although in Europe the frequencies of Pinb-D1b in landraces are higher than in other regions.
Another allele found in our materials was Pinb-d1ab, which has a transition (C/T) at the 382 position, leading to the appearance of an early stop codon in the deduced mature protein (Gln → Stop) [17,21]. The presence of this premature stop codon causes the synthesis of a non-functional PINB protein, which explains the hard grain in genotypes with this mutation. This mutation is very similar to the ones present in Pinb-D1f, Pinb-D1e, and Pinb-D1g alleles [31,36]. These mutations lead to an increase in grain hardness by overriding the correct expression of the PINB protein through the appearance of premature stop codons in other regions of the gene. The Pinb-d1ab allele was detected in 175 accessions, equivalent to a frequency of 64.6% of our total collection. The Pinb-d1ab allele was first discovered in two accessions from Afghanistan [17]. Later, it was detected in landraces from China (in 19.1% of the landraces studied) [26] and from Pakistan (in 1% of the genotypes) [37]. Therefore, it seems that both the origin and distribution of the Pinb-d1ab allele is exclusive to the Asian continent.
The Pinb-D1p allele was also found in our collection; this allele consists of an adenine deletion at the 210 position, resulting in a lysine to asparagine change and then a premature stop codon in the mature protein [30]. The Pinb-D1u [10] and Pina-D1l [38] alleles have similar mutations, characterized by the deletion of a nucleotide in their sequence. The Pinb-D1p allele was found in 80 accessions, resulting in a frequency of 29.5% of the total of our collection. This frequency is lower than in the study of wheat landraces from Shandong (China), in which the frequency of the Pinb-D1p allele was 59.6% [27]. However, in other screenings with accessions from Pakistan and Afghanistan, this allele showed low frequency [17]. These studies indicate that this allele may have spread along the “Silk Road”. Therefore, it appears that its distribution is only across the Asian continent, as in the case of Pinb-d1ab.
Finally, the sequencing of the Pinb-D1 gene in the remaining accessions that did not show any of the mutations described above allowed for the detection of a novel allele (Pinb-D1ak) that had not been previously described. This allele caused a nucleotide change that generated a change from cysteine to tyrosine at the 140 position of the amino acid sequence. Several studies have shown that the conservation of domains in the PINA and PINB proteins is very important for the conservation of the soft texture of the grain [39,40], for which the backbone of cysteines (whose function is the stabilization of the structure of the puroindolines) is essential [41]. In this sense, it is understood that the Pinb-D1ak allele has a large effect on protein functionality according to the PROVEAN score obtained, because as previously mentioned, this allele causes a change in the tenth cysteine of the cysteine backbone. There are other alleles described with similar characteristics as they have changes affecting the cysteine backbone, such as Pinb-D1x, which also has an amino acid change (Cys57 → Tyr) [42]; in general, amino acid changes affecting the cysteine backbone are not very frequent. Other alleles in which a single amino acid change in the protein leads to an increase in grain hardness are Pinb-D1d [14] and Pinb-D1q [43]. In these cases, both alleles have an amino acid change within the tryptophan-rich domain: in Pinb-D1d, there is a change from tryptophan-44 to arginine, and in Pinb-D1q, there is a change from tryptophan-44 to leucine. All these alleles are particularly interesting since they encode a protein that could have a different functionality, which may lead to intermediate grain hardness levels between the hard endosperm caused by the Pin-D1 null alleles and the soft endosperm associated with the Pin-D1a alleles. In fact, several studies showed that genotypes with the Pina-D1b allele (null) are harder than genotypes with the Pinb-D1b allele (Gly46 → Ser) [11,12]. In the case of the novel allele Pinb-D1ak found in this study, it was preliminary associated with a slightly harder grain texture than that of the other alleles reported; further studies with more balanced populations in terms of the frequency of each Pin-D1 allele or with near isogenic lines are necessary to assess the level of grain hardness associated with this novel allele.

5. Conclusions

Grain hardness is of utmost importance for wheat processors, end-users, and those involved in wheat breeding and improvement. The materials used in this study showed remarkable variability for the puroindoline genes. One of the four mutations found in Pinb-D1 had not been previously described, was considered novel, and was named Pinb-D1ak. This mutation is in the tenth cysteine of the protein backbone, which is a highly conserved region. On average, accessions with the Pinb-D1ak allele showed harder grains than accessions with other Pinb-D1 alleles did. Thus, this new allele may be associated with harder kernels than other Pin alleles and could be used by breeding programs targeting different levels of kernel hardness. This study highlights the importance of conserving and characterizing wheat genetic resources that could be used as sources of genetic variability in breeding programs

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agriculture12081196/s1, Table S1: Samples used in this study.

Author Contributions

J.B.A. and C.G. conceived and designed the study. A.B.H.-G. performed the experiments. All authors analyzed the data and wrote the paper. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by grant RTI2018-093367-B-I00 from the State Research Agency (Ministry of Science and Innovation), co-funded by the European Regional Development Fund (FEDER) of the European Union. C.G. gratefully acknowledges the European Social Fund and the Spanish State Research Agency (Ministry of Science and Innovation) for financial funding through the Ramon y Cajal Program (RYC-2017-21891). In addition, A.B.H.-G. would like to thank the University of Cordoba for the economic funding to carry out the doctoral thesis through the reinforcement fund for participants of the Ramon y Cajal Program (RYC-2017-21891). F.T. would like to acknowledge the economic funding from the “María Zambrano Grants” financed by the European Union’s NextGenerationEU.

Institutional Review Board Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

We thank the International Maize and Wheat Improvement Center (CIMMYT), Mexico, for supplying the analyzed material.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. PSI frequency distribution of the accessions evaluated. Blue bars indicate hard grain accessions; grey bars correspond to semi-hard grain accessions.
Figure 1. PSI frequency distribution of the accessions evaluated. Blue bars indicate hard grain accessions; grey bars correspond to semi-hard grain accessions.
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Figure 2. (A) Pina-D1 amplification with primer LIL1; (B) Pina-D1 amplification with primer MAS; (C) Pinb-D1 amplification with primer LIL2. Lanes represent as follows: (1) cv. Chinese Spring (Pina-D1a/Pinb-D1a); (2) CWI 67429; (3) CWI 57816; (4) CWI 67483; (5) CWI 73046; and (6) CWI 72689.
Figure 2. (A) Pina-D1 amplification with primer LIL1; (B) Pina-D1 amplification with primer MAS; (C) Pinb-D1 amplification with primer LIL2. Lanes represent as follows: (1) cv. Chinese Spring (Pina-D1a/Pinb-D1a); (2) CWI 67429; (3) CWI 57816; (4) CWI 67483; (5) CWI 73046; and (6) CWI 72689.
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Figure 3. Pina-D1 digestion with MspI endonuclease. Lanes represent as follows: (1) cv. Chinese Spring (Pina-D1a/Pinb-D1a); (2) CWI 67429; (3) CWI 57816; (4) CWI 67483; (5) CWI 73046; and (6) CWI 72689.
Figure 3. Pina-D1 digestion with MspI endonuclease. Lanes represent as follows: (1) cv. Chinese Spring (Pina-D1a/Pinb-D1a); (2) CWI 67429; (3) CWI 57816; (4) CWI 67483; (5) CWI 73046; and (6) CWI 72689.
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Figure 4. (A) Pinb-D1 digestion with BsrBI endonuclease; (B) Pinb-D1 digestion with PflMI endonuclease; (C) Pinb-D1 digestion with ApaI endonuclease. Lanes represent as follows: (1) cv. Chinese Spring (Pinb-D1a); (2) CWI 67429 (Pinb-D1ab); (3) CWI 57816 (Pinb-D1b); (4) CWI 67483 (Pinb-D1p); (5) CWI 73046 (Pinb-D1ab); (6) CWI 72689 (Pinb-D1p); (7) CWI 72663 (Pinb-D1a); (8) CWI 73075 (Pinb-D1ab); (9) CWI 66889 (Pinb-D1b); and (10) CWI 66921(Pinb-D1p).
Figure 4. (A) Pinb-D1 digestion with BsrBI endonuclease; (B) Pinb-D1 digestion with PflMI endonuclease; (C) Pinb-D1 digestion with ApaI endonuclease. Lanes represent as follows: (1) cv. Chinese Spring (Pinb-D1a); (2) CWI 67429 (Pinb-D1ab); (3) CWI 57816 (Pinb-D1b); (4) CWI 67483 (Pinb-D1p); (5) CWI 73046 (Pinb-D1ab); (6) CWI 72689 (Pinb-D1p); (7) CWI 72663 (Pinb-D1a); (8) CWI 73075 (Pinb-D1ab); (9) CWI 66889 (Pinb-D1b); and (10) CWI 66921(Pinb-D1p).
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Figure 5. PINB-D1 protein structure and Trp-domain. The ten cysteine residues are marked in grey. Amino acid change C140Y for Pinb-D1ak allele is indicated in black.
Figure 5. PINB-D1 protein structure and Trp-domain. The ten cysteine residues are marked in grey. Amino acid change C140Y for Pinb-D1ak allele is indicated in black.
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Table 1. List of Pinb-D1 alleles that were searched for specific restriction enzymes in our study.
Table 1. List of Pinb-D1 alleles that were searched for specific restriction enzymes in our study.
AlleleMutationNReference
Pinb-D1bGly/Ser at position 4611[6,26]
Pinb-D1abGln/Stop at position 382175[17,26]
Pinb-D1pLsy/Asn at position 21080[27,30]
Pinb-D1eTrp/Stop at position 390[26,31]
Pinb-D1cLeu/Pro at position 600[14]
Table 2. Allelic composition for puroindoline genes and the PSI values of the lines evaluated.
Table 2. Allelic composition for puroindoline genes and the PSI values of the lines evaluated.
Puroindoline CompositionNPSI (% ± s.d.)Range
Pina-D1a/Pinb-D1b1145.91 ± 3.58 AB*40–50
Pina-D1a/Pinb-D1ab17547.13 ± 2.70 A40–50
Pina-D1a/Pinb-D1p8047.48 ± 2.64 A41–50
Pina-D1a/Pinb-D1ak544.20 ± 2.93 B41–49
s.d.: Standard deviation; * letters indicate significantly (p < 0.05) different means groups.
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Huertas-García, A.B.; Guzmán, C.; Tabbita, F.; Alvarez, J.B. Allelic Variation of Puroindolines Genes in Iranian Common Wheat Landraces. Agriculture 2022, 12, 1196. https://doi.org/10.3390/agriculture12081196

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Huertas-García AB, Guzmán C, Tabbita F, Alvarez JB. Allelic Variation of Puroindolines Genes in Iranian Common Wheat Landraces. Agriculture. 2022; 12(8):1196. https://doi.org/10.3390/agriculture12081196

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Huertas-García, Ana B., Carlos Guzmán, Facundo Tabbita, and Juan B. Alvarez. 2022. "Allelic Variation of Puroindolines Genes in Iranian Common Wheat Landraces" Agriculture 12, no. 8: 1196. https://doi.org/10.3390/agriculture12081196

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