Identification and molecular characterization of mutant line deficiency in three waxy proteins of common wheat (Triticum aestivum L.)

Starch is the main component of wheat (Triticum aestivum L.) grain and a key factor in determining wheat processing quality. The Wx gene is the gene responsible for amylose synthesis. An ethyl methanesulfonate (EMS) mutagenized population was generated using common wheat cv. Gao 8901, a popular and high-quality cultivar in China. A waxy mutant (Wx-null) was isolated by screening M3 seeds with KI-I2 staining of endosperm starch. No obvious waxy proteins in Wx-null line were detected using Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). DNA sequencing revealed three SNPs and a 3-bp InDel in the first exon, and a 16-bp InDel at the junction region of the first Wx-A1 intron from the Wx-null line. Six SNPs were identified in Wx-B1 gene of Wx-null line compared to the wild-type Gao 8901, including four missense mutations. One nonsense mutation was found at position 857 in the fourth exon, which resulted in a premature stop codon. Expression levels of Wx genes were dramatically reduced in the Wx-null line. There were no detectable differences in granule size and morphology between Wx-null and wild-type, but the Wx-null line contained more B-type starch granules. The amylose content of the Wx-null line (0.22%) was remarkably lower compared to the wild-type Gao 8901 (24.71%). Total starch is also lower in the Wx-null line. The Wx-null line may provide a potential waxy material with high agronomic performance in wheat breeding programs.

www.nature.com/scientificreports/ rheological characteristics of starch gels 12 . As a result, changes in amylose or amylopectin content have profound impacts on end-use quality of wheat 13 . Compared with wild-type wheat starch, waxy wheat starch generally has higher peak viscosity and swelling volume, which can improve the quality of noodles including Japanese udon, Chinese-style ra-men and yellow alkaline noodles [14][15][16] . Waxy starch can also influence dough mixing characteristics, bread-making quality and bread shelf-life 17 .
The development and selection of waxy wheat are of importance in wheat breeding programs, but natural waxy wheat does not occur in nature. Identification of waxy protein-deficient lines play an important role in developing waxy wheat. Firstly, two cultivars with lower amylose content, Kanto l07 (K107) and K79, were found among Japanese wheat cultivars and shown to be Wx-A1/Wx-B1 double null lines 7,18 . Later a worldwide sample screening of 1960 wheat cultivars was performed, and one Chinese wheat (BaiHuo) was identified as null at the Wx-D1 locus 5 . By crossing K107 and BaiHuo, Nakamura 8 succeeded in producing the world's first completely waxy hexaploid wheat that lacked all isoforms of GBSS I. Since then, searches for novel Wx alleles in common wheat continued, with the goal of finding genetic variability to enable breeders to develop wheat lines with diverse starch properties and end-use quality characteristics. Ninteen null alleles were found in Wx-A1, 17 alleles in Wx-B1, and 7 alleles in Wx-D1, respectively 10,[19][20][21][22][23][24][25][26][27][28][29] . However, no cultivars lacking both Wx-A1 and Wx-D1 proteins, both Wx-B1 and Wx-D1 proteins, or all three Wx proteins had been detected 5,30 . Mutagenesis is a popular and effective way to create genetic variations in crops. Many Wx gene mutants have been isolated using EMS-induced assays 29,31-34 . Slade and colleagues 35 first time produced a wx-null genotype using a TILL-ING approach in bread wheat. Later Botticella 36 reported another TILLING wx-null genotype. In the present study, by screening about 9000 seeds from a mutant population of bread wheat cultivar Gao 8901 treated with EMS solution we were lucky to obtain a triple mutant of Wx gene. The Wx null line provides breeders valuable germplasms suitable for the production of bread and high-quality salted noodles. It may also be used to provide one or more waxy null alleles with high agronomic performance in wheat breeding programs.

Results
Identification of the waxy protein null mutant line. Seeds of common wheat cv. Gao 8901 were treated with a 1.0% EMS solution, with a survival rate of M1 plants of 50-60%. M1 plants were self-fertilized to produce the M2 generation. Half-seed staining using iodine potassium iodine solution (0.1% I 2 /1% KI) was employed to screen for waxy protein mutants. About 9000 seeds from the M3 generation were screened. One seed showed brown-red color stained with I 2 -KI solution while others were dark blue ( Supplementary Fig. S1). We hypothesized that this seed was a waxy protein-deficient mutant and named it Wx-null.
As the chance of acquiring a triple mutant is really low, it was important to make sure it was not a contaminating seed. The wild-type (Gao 8901) has obvious pubescence on its glume, which can easily distinguish it from other cultivars. Morphologically, the Wx-null line has the same characteristics as the wild type, such as plant height, plant shape, leaf shape, leaf color, spike shape, etc. Seven pairs of SSR primers were used to detect the mutant line and wild-type; they were Xbarc80 (1BL), Xgwm294 (2AL), Xgdm72 (3DL), Xbarc91 (4DL), Xgwm67 (5BS), Xgwm334 (6AS) and Xgwm333 (7BL). The mutant line displayed the same band patterns as the wild type ( Supplementary Fig. S2). So, it was determined that the mutant line was from the wild-type Gao 8901.
Waxy protein pattern analysis by SDS-PAGE electrophoresis. The waxy protein pattern of the Wxnull line (M5 generation) was analyzed by SDS-PAGE electrophoresis. The wild-type Gao 8901 and two Wx-A1 null lines were used as controls. The molecular weight of three GBSS I isotypes (Wx-A1, Wx-B1, and Wx-D1 proteins) are about 62.8, 58.7 and 56.7 kD, respectively. Because the molecular weight difference between Wx-B1 and Wx-D1 proteins is small, the two bands are very close. The wild-type Gao 8901 showed three waxy protein bands, otherwise, no obvious bands were seen in the Wx-null mutant line ( Supplementary Fig. S3).
Amylose content and GBSS I activity decreased significantly in Wx-null line. Total starch, amylose content and GBSS I activity were determined for wild-type Gao 8901 and mutant line Wx-null (Fig. 1). Total starch content in both wild-type and the Wx-null line increased continuously from 7 to 35 DAF, but the final starch content in Wx-null (61.4%) was lower than in the wild-type (64.3%) (P < 0.05). The amylose content in the Wx-null line (0.22%) was also significantly lower than in the wild-type (24.7%) (P < 0.01). The GBSS I activity reached the maximum value (4.54 ± 0.05) at 28 DAF, then decreased lightly. Weak GBSS I activity can be detected in the Wx-null line, showing a similar trend as wild-type; However, the activity at 28 DAF was only 0.41 ± 0.02.
Starch granule characteristics of the Wx-null line. Starch granule staining and scanning electron microscopy were employed to assess the effect of waxy protein deficiency on Wx-null line starch granule characteristics. When stained with Lugol, starch granules of wild-type Gao 8901 were dyed blue ( Fig. 2A), while Wxnull line granules were stained red-brown (Fig. 2B). SEM observation of wild-type and Wx-null starch shapes revealed similar granule morphological features and size. A-type starch granules were over 10 μm in diameter and lenticular in shape, and B-type starch granules were less than 10 μm in diameter and roughly spherical in both the wild-type and the Wx-null line (Fig. 2C,D). The numbers of A-type and B-type starch granules in 5 fields of microscope view were counted, showing that the ratio of B-type/A-type of Wx-null (12.41 ± 3.56) was much higher than wild-type (5.19 ± 2.42).  www.nature.com/scientificreports/ introns. The gDNA sequences of Wx genes of the Wx-null line were also analyzed to detect mutant sites. DNA sequencing of the Wx-A1 gene from the Wx-null line revealed three SNPs and a 3-bp insertions and deletions (InDel) in the first exon. The G/C mutant at position 126 and A/T mutant at position 220 resulted in amino acid changes from methionine to isoleucine and serine to cystine, respectively. The 3-bp InDel (CAT) and the third SNP occurred at positions 315-317 and 320, respectively. Furthermore, a 16-bp InDel (GGC CGT AAG CTT GCG CCA C) was found at the junction region in the first intron (Fig. 3). The Wx-null Wx-A1 gene was named Wx-A1-null (GenBank accession EU719609). There was a total of six SNPs in the Wx-B1 gene from the Wx-null line compared to wild-type Gao 8901, three of which were present in the first exon, one in the second, one in the third, and the last in the third intron (Fig. 4).    Fig. S4).

Discussion
Pre-mRNA alternative splicing (AS) plays an important role in gene expression diversity in eukaryotes. It is also estimated that a significant fraction of genes (about 20%) undergo AS in plants 38,39 . InDel contributes to the generation of diversity in AS isoforms 40,41 , by affecting splicing efficiency 42 , the stability of the pre-mRNA structure, or the expression level of correctly spliced transcripts 43 . Through DNA sequencing, we identified three SNPs and a 3-bp InDel in the first exon, and a 16-bp InDel at the junction region in the first intron in the Wx-A1 gene of the Wx-null line (Fig. 3). The 16-bp InDel destroyed the GT-AT intron-exon boundary, which may have induced AS and loss of function of the Wx-A1 gene. The first reported null allele Wx-A1b (from K107) had a 23 bp deletion (in the second intron) also located at the exon-intron junction 44 . Additionally, Luo et al. 29 reported an SNP (G-A) at the splicing site within the eighth intron, which caused incorrect RNA splicing and gene inactivation, suggesting a similar molecular mechanism for the above null Wx-A1 alleles. Wx-D1 gene has the lowest variant frequency, and null Wx-D1 alleles are extremely rare in common wheat 45 . Previously, researchers described a 588-bp fragment deletion in Wx-D1b 44 , a 724-bp fragment deletion in Wx-D1h 46 and a nucleotide substitution in Wx-D1g 47 among others. In this study, molecular characterization of Wx-D1 gene of the Wx-null line showed one nonsense mutation (from CAG to TAG) in the fourth exon resulting in a premature stop and non-function waxy protein. Nonsense mutations on the allele Wx-D1 had also been reported in previous studies 35,48,49 .
The total starch content decreased in the Wx-null line (61.4%) compared to wild-type Gao 8901 (64.3%), probably because GBSS I enzyme deficiency causes a significant reduction in total starch biosynthesis though it does not affect amylopectin synthesis 50 . The amylose content was very low in the Wx-null line (0.22%), but it was not zero (Fig. 1). Furthermore, a faint band of Wx gene expression was observed in the Wx-null line in RT-PCR figure (Supplementary Fig. S4). Then, GBSS I activity analysis was performed to confirm the results. It can be seen that weak GBSS I activity can be detected in Wx-null line (Fig. 1). As the Wx-A1 and Wx-D1 were frameshift and nonsense mutations, respectively, the reason may be that the amino acid changes in Wx-B1 protein may dramatically reduce its activity, but can not cause complete loss of its function.  Approximately 5000 dry seeds of Gao 8901 were soaked in distilled water for 12 h before being treated with 1.0% EMS at room temperature (25-27 °C) for 6 h and then washed under running tap water for 4 h prior to seeding. In the subsequent growing season, treated seeds (M0) were individually sown and self-fertilized to produce the M1 generation. All the M1 plants were harvested and sown to produce the M2 generation. One spike per M2 plant was picked, mixed and threshed. The seeds (M3) were used for screening waxy protein mutants. Young leaves of Wx mutants were collected for DNA extraction and seeds were planted in a plant-to-row fashion to produce the M5 generation. The M5 seeds were used for RNA extraction, protein electrophoresis, amylose content determination, starch granule staining, and scanning electron microscopy analysis. All the field trials were conducted at the experimental station in Shijiazhuang, Hebei province.
Half-seed screening of Wx mutant lines. Waxy and non-waxy wheat seeds can be easily distinguished by staining. Starch from non-waxy wild-type containing amylose generates blue-black complexes with iodine, while starch from waxy protein mutants without amylose stains red-brown. M3 seeds were cut horizontally in two parts containing embryo and endosperm. The non-embryo half part of seed was dipped into an iodine potassium iodine solution (0.1% I 2 /1% KI) for 1 min. The seeds dyed red-brown were selected, and the embryo portion of the seed were used to grow to a plant.
Electrophoresis of waxy proteins. Waxy proteins were extracted following published methods 51 and separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in a discontinuous buffer system (pH: 6.8/7.8). The polyacrylamide concentrations of stacking gel and resolving gel were respectively 4.5% and 15%. Electrophoresis was performed at a constant voltage of 180 V for stacking gel and 220 V for resolving gel. Run time was approximately 4 h just after the tracking dye (bromophenol blue) had migrated off the gel. After electrophoresis, waxy proteins were detected by silver staining. www.nature.com/scientificreports/ Scanning electron microscopy analysis. The granular morphology of starch was examined by scanning electron microscopy (SEM). Wheat seeds were transversely cut with a knife and fixed on circular aluminum stubs with double-sided sticky tape. Samples were sputter-coated with gold particles using an ionic sprayer (Eiko E-1020, Hitachi) and observed under a scanning electron microscope (S-570, Hitachi) with an accelerating voltage of 20 kV.

Data availability
All data generated or analyzed during this study are included in this published article and its Supplementary data files.
Received: 29 January 2020; Accepted: 3 July 2020 www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/.