Identi fi cation of Alleles of Puroindoline Genes and Their Effect on Wheat ( Triticum aestivum L . ) Grain Texture

Wheat seeds contain the group of proteins called puroindolines (Pin). They belong to the broad superfamily of plant proteins consisting of a number of other cereal proteins. They are characterised by the presence of short sequences rich in the amino acid tryptophan (4). Grain hardness is primarily controlled by the complex hardness (Ha) locus, which consists of three closely-linked genes Gsp-1, Pina and Pinb (5). Wheat includes two types of protein: puroindoline a (Pina) and puroindoline b (Pinb). The milling of wheat is strongly infl uenced by grain hardness due to the presence or absence of the polypeptides Pina and Pinb (6). The key role of the Pina and Pinb genes is to determine the structure of the proteins in wheat grain and also possible antimicrobial eff ects (7).


Introduction
Common wheat (Triticum aestivum L.) is one of the most important food crops in the world.Grain texture is a major characteristic and a determinant of end product quality, especially important in baking and noodle making (1,2).Flour from hard wheat is best for making bread, while fl our from soft wheat is mainly used for making cakes, pastries and biscuits (3).
Wheat seeds contain the group of proteins called puroindolines (Pin).They belong to the broad superfamily of plant proteins consisting of a number of other cereal proteins.They are characterised by the presence of short sequences rich in the amino acid tryptophan (4).Grain hardness is primarily controlled by the complex hardness (Ha) locus, which consists of three closely-linked genes Gsp-1, Pina and Pinb (5).Wheat includes two types of protein: puroindoline a (Pina) and puroindoline b (Pinb).The milling of wheat is strongly infl uenced by grain hardness due to the presence or absence of the polypeptides Pina and Pinb (6).The key role of the Pina and Pinb genes is to determine the structure of the proteins in wheat grain and also possible antimicrobial eff ects (7).
The endosperm texture can be vitreous (steely, fl inty, glassy or corneous) or mealy (starchy or chalky Wheat hardness is aff ected especially by genetic factors (9).Starch granules of diff erent sizes are coated by a protein matrix created predominantly by gluten proteins.Diff erences in wheat hardness are due to the adhesion of storage proteins to starch granules (10).Cultivars with soft er endosperm texture have bigger starch granules and harder wheat has smaller starch granules.Smaller granules have a larger surface available for non-covalent bonds with endosperm proteins and can be packed more eff ectively, which ensures harder endosperm.
The main aim of this work is the identifi cation of markers for puroindoline genes (Pina and Pinb) in wheat using polymerase chain reaction and determination of endosperm texture of wheat.

Materials and Methods
Common wheat (Triticum aestivum L.) genotypes with unusual grain and endosperm colour (Table 1) from the 2014 harvest of the Agricultural Research Institute Kroměříž, Ltd., Kroměříž, Czech Republic, were investigated in the present study.
Genomic DNA was isolated from young leaf plant tissues using a DNeasy Plant Mini Kit (Qiagen, Hilden, Germany).For the identifi cation of puroindoline genes, primers for Pina and Pinb (3) were used.The PCR analysis for determination of puroindoline genes was performed as follows: initial denaturation for 5 min at 94 °C, then 35 cycles for 30 s at 95 °C, 30 s at 60 °C, 90 s at 72 °C and a final extension step of 10 min at 72 °C.The total volume in one reaction was 25 μL.The visualisation of PCR products was carried out in 1.5 % agarose gel.
A light transfl ectance meter (LTm; Brewing Research International (BRi), Nutfi eld, UK) was used for the evaluation of vitreousness and mealiness of caryopses (11).This non-destructive method is based on the quantitative measurement of laser beam propagation through a barley or wheat caryopsis.Ninety-seven caryopses of each genotype were used for one experiment.Mealy caryopses are not transparent to light and vitreous caryopses allow The hardness of wheat was determined at the University of Veterinary and Pharmaceutical Sciences in Brno, Czech Republic.The texture analysis was performed with TA.XTplus texture analyser (Stable Micro Systems, Godalming, Surrey, UK).The samples were examined using Exponent v. 5.0 soft ware (Stable Micro Systems).A three--inch compression plate was installed in the 25-kg load cell of the analyser.A 5-kg weight was used to calibrate the 25-kg load cell prior to analysis and the sett ing was adjusted at a pretest, test and postt est speed of 1 mm/s.All samples were compressed once to 60 % of their original height using an upper fracture wedge piston.The obtained texture profi les were used to measure the instrumental hardness.The maximum positive force is the force required to penetrate the sample to the specifi ed distance.The higher this value, the harder the sample.The maximum hardness force was measured during the fi rst compression cycle.

Results and Discussion
The genes for Pina-D1 and Pinb-D1 are located on chromosome 5D and are the main determinants of grain texture in hexaploid wheat.All wheat with hard endosperm is characterised by a sequence mutation in either Pina or Pinb.The result is a change in kernel hardness from soft to hard (6,12,13).
We analysed nine markers for Pina and four combinations of markers for Pinb with diff erent product sizes (Table 2).Two alleles of Pina for each genotype were analysed.Only one allele, Pina-D1a, was detected (Table 1).Three alleles of Pinb were found: Pinb-D1a, Pinb-D1b and Pinb-D1d (Table 1), while allele Pinb-D1c was not detected.In genotypes Novosibirskaya 67, ANK-28A and ANK--28B unknown PCR products were observed (Figs. 1 and  2).A mutation in the locus for annealing primer temperature for Pinb-D1, which resulted in hard texture (2), was detected.Mutations in the Pina-D1 and Pinb-D1 genes have individually been associated with grain hardness,  but it is not known if mutations at both loci may further increase hardness or if additional copies may reduce it (14).
In Novosibirskaya 67 genotype and its isogenic ANK lines with purple caryopses, alleles were not identifi ed even when testing the primer combinations described by Gautier et al. (15).It is necessary to design primers for a sequence analysis of these genotypes, which would allow the identifi cation of relevant alleles for Pina and Pinb loci.
The development of mealiness appears to depend on maturation.Immature grains of all wheat types are mealy.Vitreous grains are found in plants that grow and ripen quickly and mealy grains are characteristic of varieties that grow slowly and have a long maturation period.This means that vitreousness is characteristic of a short vegetation period.The mealy or vitreous character is hereditary but it is also aff ected by environment (16).The mealiness of caryopses ranged from 0 to 100 %.Eight genotypes had less than 50 % mealy caryopses.The lowest value was in Citrus genotype (0 % mealy endosperm).Mealy endosperm was observed in 17 genotypes, eight of which had 90 % or more mealy caryopses.
Mealiness is closely related to hardness.The hardness of grains is used for the evaluation of breeding material, especially for wheat and barley.Values of wheat hardness varied from 15.10 to 26.87 N. The highest and lowest values were observed in Heroldo genotype with white pericarp and Purple genotype with purple pericarp, respectively.The results for mealiness and hardness are shown in Table 1.
Nonsignifi cant diff erences were found for grain hardness.Starch grains in the endosperm of the grain are bound by diff erent molecules of puroindoline that exhibit diff erent grain hardness.High values are characteristic of vitreous endosperm and vice versa (7).Gaines et al. (17) found that soft wheat had a higher content of amylose bound to lipids and a lower total starch content.
The nonstandard colours of wheat caryopsis are caused by the presence of anthocyanins, and the interest in such wheat is mainly because of its positive eff ects on the health of consumers.The hardness of wheat endosperm is critical in determining the suitability of wheat for various end products and infl uences the processing and milling of wheat.The results indicate that there is no general relationship between the colour of wheat endosperm and its hardness.

Conclusion
We identifi ed puroindoline genes in wheat and their eff ect on mealiness and hardness.Only Pinb-D1c allele was not found.Three genotypes did not have amplifi ed PCR products, which is caused by mutation.Therefore, further studies are required for the identifi cation of alleles (preferably DNA sequencing).The knowledge of the genetic determination of Pina and Pinb loci in wheat with nonstandard coloured caryopses can be used in breeding for marker-assisted selection of bread-making quality wheat genotypes.The grain hardness ranged from 15.10 to 26.87 N per sample.Hard wheat is considered of higher quality and suitable for bread making, while most cakes are made from soft wheat fl our.Bread-making quality is essential because it determines other physical characteristics such as the volume of dough and sensory att ributes.The endosperm texture of wheat showed diff erences among the studied genotypes in the mealiness and hardness of grains.The colour does not have any effect on grain hardness.

Table 1 .
Investigated genotypes, grain colour and texture, and puroindoline alleles Table1shows the percentage of mealy caryopses.Vitreous endosperm has lower percentage of mealiness than mealy endosperm.
Hardness is expressed as mean value±standard deviation, n.d.=not detected more light to transmit.

Table 2 .
Markers for identifi cation of puroindoline Pina and Pinb genes and their product size