Development and Characterization of a New TILLING Population for Forward and Reverse Genetics in Barley (Hordeum vulgare L.)

Mutagenesis is an important tool in crop improvement and free of the regulatory restrictions imposed on genetically modified organisms. Barley (Hordeum vulgare L.) is a diploid species with a genome smaller than those of other members of the Triticeae crops, making it an attractive model for genetic studies in Triticeae crops. In this study, we report an ethyl methane sulfonate (EMS)-mutagenized population in the Chinese barley landrace TX9425, which is tolerant to both abiotic and biotic stress. A TILLING (Targeting Induced Locus Lesion in Genomes) population consisting of 2000 M2 lines was also constructed based on the CEL I enzyme with subsequent polyacrylamide electrophoresis, which decreased the cost and labor investment. The mutant phenotypes of the M2 and M3 generations were scored and revealed the presence of a wide spectrum of morphological diversity. The population was evaluated by screening for induced mutations in five genes of interest. A detailed analysis was performed for the HvGLR3.5 gene and three mutations were identified by screening in 2000 M2 lines. Two of three mutations displayed tuft and yellow striped leaves compared to the wild type. Altogether, our study shows the efficiency of screening and the great potential of the new TILLING population for genetic studies in the barley crop model system.


Introduction
The genus Hordeum displays a high degree of adaptation to different stressful environments [1], and cultivated barley is grown in subtropical, temperate, and subarctic areas, from sea level to the Andes and the Himalayas [2].With such a great diffusion as well as abundant genetic stocks with over 485,000 germplasm accessions [3], the barley gene pool contains characters for wide environmental adaptability and strong stress resistance [4].
Mutation is the ultimate source of all genetic variations which provides the raw materials for natural selection and a driving force in evolution.Mutations arising spontaneously are random events and many of the mutations may confer no immediate advantage at the beginning but may help to generate a wide range of useful recombinant genotypes through the subsequent process of independent segregation and crossing over of genes [5].Historically, the first natural mutant plant in cereals was found in China about 2317 years ago [6].By now, extensive use of mutation breeding has already made significant contributions to crop production.Over the past 85 years, more than 3000 varieties were derived from the mutagenesis program, with 873 rice lines, 307 barley lines, Plants 2024, 13, 2490 2 of 11 and 265 wheat lines as the top crops listed in the IAEA/FAO mutant variety database (https://nucleus.iaea.org/sites/mvd/SitePages/Home.aspx,accessed on 18 August 2024).Various effective methods are available to induce mutations, which are categorized as physical or chemical mutagens, such as gamma rays, ethyl methane sulfonate (EMS), and sodium azide (NaN 3 ) [7].With the recent advances in genomics, the use of high-throughput platforms such as TILLING (Targeting Induced Local Lesions in Genomes) in the highly efficient evaluation of mutant pools for specific genomic sequence alterations is crucial in studying the genetic variability at a molecular level [8].The TILLING method is economical and efficient for screening mutants, which is based on conventional PCR.The subsequent mutation detection is followed by any suitable method including denaturing high-pressure liquid chromatography, fluorescence-based gel electrophoresis (e.g., LI-COR), or unlabeled capillary electrophoresis of heteroduplexes digested with the endonuclease CEL I [9,10].
Up to now, several barley TILLING populations have been developed, including the cultivar Golden Promise population as the reference genotype for barley transformation, with 3072 M 2 mutants [11].Malting cultivar Barke was used to generate a TILLING resource comprising 10,279 M 2 mutants [12], and leaf material and seeds from approximately 20,000 M 2 plants in cultivar Optic were individually harvested to develop its mutant population [10].Recently, a landrace "Hatiexi" mutagenic population consisting of 8525 M 3 lines was complemented with a high-quality de novo assembly of a reference genome for this genotype [13].These resources combined with a high-quality genome reference sequence have enabled the functional validation of a list of candidate genes resulting from forward genetics and mapping [14][15][16].In this study, we developed an EMS-induced mutant population in a Chinese barley landrace "TX9425" (TX) and generated a TILLING population with 2000 M 2 mutants.Here, we evaluated and described various features of our TX mutant population in the M 2 and M 3 generations, including its development, mutation genotypes, and a detailed analysis that was performed for the glutamate receptor-like (GLR) gene HvGLR3.5.Our results suggested that the new TILLING resource of the barley cultivar TX had potential for use in fundamental research as well as for applied breeding.

Plant Materials
TX9425 (TX), which was one of the Chinese two-rowed landraces, was used to generate the ethyl methane sulfonate (EMS)-induced population.TX was proved to be resistant to salt and waterlogging stress but had a low malting quality.In contrast, a Japanese two-rowed malting barley, NasoNijo (NN), with good agronomic traits and sensitivities to salt and waterlogging stress, was also studied in this experiment.Barley grains of TX and NN were harvested in the field of Yangzhou University, and the grain length, width, and thousand-grain weight were detected by the WSeen SC-G automatic seed selection and analysis system (Model SC-G, Wanshen Ltd., Hangzhou, China).The total nitrogen content of the grain was measured by a semi-automatic Kjeldahl nitrogen analyzer (FOSSKjeltec8100, FOSS, Hillerød, Denmark) according to the Kjeldahl method [17].

Generation of EMS-Mutagenized Population
A total of 0.5 kg of barley seeds (approximately 10,000) was soaked in distilled water for one night and then mutagenized in 0.5% EMS (liquid, Sigma, St. Louis, MO, USA) for 8 h at room temperature.The seeds were rinsed continuously under running water for 3 h to wash off excess EMS and then laid flat on a plate and covered with moist filter paper.Seed germination was recorded as the proportion of germinated seeds per total number of seeds and germinated seeds (M 0 ) that were sown in the field in Yangzhou with an interval of 5 cm between seeds.M 1 seeds were collected from a single plant and grown in the field.The two 4 cm length leaf segments of each individual M 2 seedling were sampled: one was for genomic DNA extraction with the CTAB method and another one was kept frozen as a backup.The phenotypes of M 2 seedlings totaled to 29 traits that were recorded during the whole growth period, which include seedling habit (such as albino, etiolated), Plants 2024, 13, 2490 3 of 11 leaf shape (such as wide, thin, curled), leaf color (such as light leaf color, yellow), tillering (such as non-tiller, multiple tillers, less tiller), plant height (such as dwarf), and spikes (such as six rows, spike anomaly, white glume).The mutation frequency observed in the M 2 population was calculated by the number of M 2 mutants/total M 2 seedlings × 100%.The M 3 generation was grown for genotyping and the phenotypes recorded in the field and the seeds from the sampled M 2 seedlings were collected, indexed, and archived.The phenotypes of M 3 seedlings including plant height, spike length, spacer number, spike number, and thousand-kernel weight were recorded.

Establishment of the CEL I Enzyme Digestion System
The extraction of the CEL I enzyme was based on the method of Till et al. [9].Then, the activity of the CEL I and enzyme digestion system were verified by the barley gene HvWRKY62.In the CEL I enzyme activity experiment, two plasmids (encoding the HvWRKY62 gene) which contained a single base difference were used as PCR amplification templates, and the PCR products were separated by CEL I enzyme digestion and gel electrophoresis (agarose).In the enzyme digestion system experiment, the extracted enzyme and the DNA of M 2 samples were diluted to different concentrations to verify the best enzyme digestion system.Genomic DNA of M 2 individual samples was extracted and pooled with 5 samples.

Screening of the Mutation Lines
The primers of target genes were designed using the NCBI primer-BLAST website (https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi,accessed on 10 January 2024).The PCR reactions were conducted using a thermal cycler (T100 Thermal Cycler, Bio-rad, Hercules, CA, USA) as follows: heat denaturation at 95 • C for 3 min, followed by 35 cycles (94 • C for 30 s, 60 • C for 60 s, 72 • C for 1 min), 72 • C for 5 min, and 95 • C for 5 min; 95 The PCR product was subjected to CEL I enzyme digestion at 45 • C for 45 min and then added to the termination solution to terminate the reaction.The CEL I enzyme digestion system included the following: 10 µL of the PCR product, 1 µL of the CEL I enzyme, 2 µL of the CEL I digestion buffer, and 7 µL of ddH 2 O.The enzyme digestion product was then detected by 8% polyacrylamide gel.For confirmation of presumed mutated loci, amplicons of the respective target gene were generated from putative mutants by utilizing the same PCR conditions as established for CEL I analysis and sent to the company for sequencing.

Phenotype Experiment
The seeds of TX, mutant line FM10587, and FM10811 were grown in pots (8 cm × 12 cm) in a greenhouse with a day/night temperature of 22 ± 3 • C in a 16 h/8 h day/night regime.The plant height (from the base of the plant to the tip of the flag leaf), root length (from the base of the plant to the tip of the longest root), and relative chlorophyll content (SPAD value) were measured after four weeks.

Generation of a TILLING Population in the Barley Cultivar "TX9425"
The two-rowed barley cultivar TX9425 (TX) was used to develop a mutant population.TX is characterized by various traits including salinity tolerance [18,19], waterlogging tolerance [18,20,21], disease resistance [22,23], and dwarf genes [24].However, this variety also showed some unfavorable traits, including thicker husk, short and twisty spike length (Figure 1A,B), and high grain density [25,26].Compared with NasoNijo (NN), which has good agronomic traits but is sensitive to salt and waterlogging stress, the thousand-kernel weight of TX is higher than NN (Figure 1C) and the grain of TX is fuller than NN with a shorter grain length and much wider grain width (Figure 1C,E,F).The nitrogen content in the grain of TX was lower than NN (Figure 1G).length (Figure 1A,B), and high grain density [25,26].Compared with NasoNijo (NN), which has good agronomic traits but is sensitive to salt and waterlogging stress, the thousand-kernel weight of TX is higher than NN (Figure 1C) and the grain of TX is fuller than NN with a shorter grain length and much wider grain width (Figure 1C,E,F).The nitrogen content in the grain of TX was lower than NN (Figure 1G).Based on previous experience with EMS mutagenesis in a different barley cultivar [10,12], we established a population of mutants in the TX background by treating seeds with 0.5% v/v EMS for 8 h.The germination rate of M1 seedlings was around 70%, and the leaves of some M1 seedlings grown in the field were albino and etiolated, which decreased the survival rate of M1 seedlings to around 60%. Finally, 1022 M1 plants advanced to the M2 generation.We sowed all the M2 lines and collected leaf samples from a single seedling for DNA extraction and thereby obtained a library with 2000 high-quality M2 DNA samples.

Mutant Types and Mutation Frequency Observed in the M2 Population
In total, 7224 M2 individuals were scored for their phenotypes during the field cultivation by visual assessment of 29 traits for plant seedlings, leaves, stems, and spikes.The results showed that TX produced abundant phenotypic variations after EMS mutagenesis, and the seedlings were divided into mutated individual lines in terms of seedling growth habits, leaf shape, leaf color, tiller number, plant height, spike type, and row type (Figure 2 and Table 1).A total of 319 mutant lines were found in the M2 generation, with a mutation frequency of 4.42%.At seedling stage, a total of three types of mutation were found in the seedling habits of 48 strains (Table 1).Among them, there were 12 lines with etiolated leaves in the whole plant or growth period of seedlings with a mutation frequency of 0.17%, and 34 mutant lines with a mutation frequency of 0.47% showed a seedling tuft phenotype (Table 1).In leaf phenotypes, this population in the M2 generation displayed abundant phenotypes which included leaf yellow stripes, light leaf color, yellow leaf, Based on previous experience with EMS mutagenesis in a different barley cultivar [10,12], we established a population of mutants in the TX background by treating seeds with 0.5% v/v EMS for 8 h.The germination rate of M 1 seedlings was around 70%, and the leaves of some M 1 seedlings grown in the field were albino and etiolated, which decreased the survival rate of M 1 seedlings to around 60%. Finally, 1022 M 1 plants advanced to the M 2 generation.We sowed all the M 2 lines and collected leaf samples from a single seedling for DNA extraction and thereby obtained a library with 2000 high-quality M 2 DNA samples.

Mutant Types and Mutation Frequency Observed in the M 2 Population
In total, 7224 M 2 individuals were scored for their phenotypes during the field cultivation by visual assessment of 29 traits for plant seedlings, leaves, stems, and spikes.The results showed that TX produced abundant phenotypic variations after EMS mutagenesis, and the seedlings were divided into mutated individual lines in terms of seedling growth habits, leaf shape, leaf color, tiller number, plant height, spike type, and row type (Figure 2 and Table 1).A total of 319 mutant lines were found in the M 2 generation, with a mutation frequency of 4.42%.At seedling stage, a total of three types of mutation were found in the seedling habits of 48 strains (Table 1).Among them, there were 12 lines with etiolated leaves in the whole plant or growth period of seedlings with a mutation frequency of 0.17%, and 34 mutant lines with a mutation frequency of 0.47% showed a seedling tuft phenotype (Table 1).In leaf phenotypes, this population in the M 2 generation displayed abundant phenotypes which included leaf yellow stripes, light leaf color, yellow leaf, striata, flag leaf deformation, leaf spot, and wide, thin, curled and non-midribs (Figure 2 and Table 1).Among these mutation types, the mutation frequency of yellow leaf and leaf yellow stripes were higher than other mutation types with 0.57% and 0.50%, respectively (Table 1).The mutation of yellow leaf stripes occurred at the tillering stage, and the chlorophyll contents at the edges or middle of some leaves were significantly reduced.Another mutation type which is similar to yellow leaf stripes was white stripes with a low mutation frequency of 0.06% (Table 1).In the M 2 generation, there were 75 lines related to stem mutation type, with a total mutation frequency of 1.03% (Table 1), and the less-tillering phenotype showed Plants 2024, 13, 2490 5 of 11 the highest mutation frequency compared with other phenotypes (Table 1).Phenotypes related to spike mutations were investigated, which included six rows, spike anomaly, white glume, red glume, long awn, short awn, awnless, and sterile (Figure 2 and Table 1).Among these phenotypes, the mutation frequency of spike anomaly and white glume was relatively higher than other phenotypes (Table 1).
and Table 1).Among these mutation types, the mutation frequency of yellow leaf and leaf yellow stripes were higher than other mutation types with 0.57% and 0.50%, respectively (Table 1).The mutation of yellow leaf stripes occurred at the tillering stage, and the chlorophyll contents at the edges or middle of some leaves were significantly reduced.Another mutation type which is similar to yellow leaf stripes was white stripes with a low mutation frequency of 0.06% (Table 1).In the M2 generation, there were 75 lines related to stem mutation type, with a total mutation frequency of 1.03% (Table 1), and the less-tillering phenotype showed the highest mutation frequency compared with other phenotypes (Table 1).Phenotypes related to spike mutations were investigated, which included six rows, spike anomaly, white glume, red glume, long awn, short awn, awnless, and sterile (Figure 2 and Table 1).Among these phenotypes, the mutation frequency of spike anomaly and white glume was relatively higher than other phenotypes (Table 1).

The Main Agronomic Traits of Stable Mutant Lines in the M 3 Generation
The main agronomic traits of stable mutant lines in the M 3 generation were investigated under field conditions from the subsequent year, and the results showed that there were abundant mutation phenotypes in plant height, spike length, spike number, and thousand-kernel weight (Table 2).In the M 3 generation, the plant height was in the range from 27.72 to 91.50 cm; the spike length was in the range from 2.50 to 12.03 cm; the spike number was in the range from 2.78 to 15.56 cm; and the thousand-kernel weight was in the range from 15.63 to 48.39 g (Table 2).Among these agronomic traits, the coefficient of variation (CV) in spike number was the highest with 43.04% (Table 2).

Mutant Screening
Before the mutant screening from the TILLING population, the CEL I enzyme digestion system was established.In this experiment, the CEL I enzyme was extracted from fresh celery in Yangzhou, and each time, the enzymatic activity was detected after extraction to ensure that it had a good enzymatic effect during the digestion process.Two plasmids (PW1 and PW2) containing the same HvWRKY62 gene (with a single base difference) were used as PCR amplification templates, and the PCR products were separated by CEL I digestion and gel electrophoresis.The two template plasmids were amplified to 1327 bp products and digested to produce 371 bp and 956 bp fragments.The amplification primers, differential bases, and fragment sizes are listed in the Supplementary Figure S1A.After confirming the activity of the CEL I enzyme, the effect of the CEL I enzyme concentration on the enzyme digestion was analyzed, and the enzyme digestion system was optimized.In this experiment, the extracted CEL I enzyme was diluted into different concentrations (1×, 0.5×, 0.25×, 0.1×, 0.05×, 0.025×, 0.0001×, H 2 O) to explore the optimal concentration of the CEL I enzyme.Then, the two plasmids were mixed in ratio of 1:1, 1:2, 1:4, and 1:8, respectively, to determine the suitable barley DNA pool.Based on these results (Supplementary Figure S1B), a 1× concentration of the CEL I enzyme was best for the digestion and the number of mixed DNA samples was five.
A total of 400 DNA pools were constructed in groups of five M 2 DNA samples to screen for mutants.The screen revealed a total of 11 mutant strains from the EMS mutant pool, including HvRBOHB, HvRBOHE, HvDTX16, HvGLR2.8, and HvGLR3.5 genes (Table 3).In these mutant lines, there was one mutation located in the intron; six of the exon-located mutations did not induce a change in the amino acid (AA) sequence, and four induced a change in AAs in the protein sequence (Table 3).3.5.New Alleles of Functional Mutations in the Gene HvGLR3.5 A detailed analysis was performed for the HvGLR3.5 (HORVU.MOREX.r3.7HG0733700.2) gene that belongs to the glutamate receptor-like (GLR) family of ion channels [27], and GLR3.5 functions as an off switch that blocks the transmission of wound-induced electrical potentials [28].HvGLR3.5 had six exons and five introns, and three mutations were found in Exon 2 (Figure 3A).Two of three Hvglr3.5 mutants (FM10811 and FM10587) showed mutant phenotypes different to wild-type TX (Figure 3C-E).At the one-month seedling stage, phenotype measurements displayed that the relative chlorophyll content (SPAD), root length, and plant height of the FM10587 mutant were all lower than wild-type TX (Figure 3B).Compared with the wild type, these two homozygous mutants showed a significantly reduced plant height, both at the seedling stage and in the field (Figure 3B,F,G).In the field observation, the FM10811 variety showed a tuft phenotype with more tillers and lower plant height compared to TX.Meanwhile, the FM10587 mutant displayed yellow striped leaves (Figure 3F,G).

New Alleles of Functional Mutations in the Gene HvGLR3.5
A detailed analysis was performed for the HvGLR3.5 (HORVU.MOREX.r3.7HG0733700.2) gene that belongs to the glutamate receptor-like (GLR) family of ion channels [27], and GLR3.5 functions as an off switch that blocks the transmission of wound-induced electrical potentials [28].HvGLR3.5 had six exons and five introns, and three mutations were found in Exon 2 (Figure 3A).Two of three Hvglr3.5 mutants (FM10811 and FM10587) showed mutant phenotypes different to wild-type TX (Figure 3C-E).At the one-month seedling stage, phenotype measurements displayed that the relative chlorophyll content (SPAD), root length, and plant height of the FM10587 mutant were all lower than wild-type TX (Figure 3B).Compared with the wild type, these two homozygous mutants showed a significantly reduced plant height, both at the seedling stage and in the field (Figure 3B,F,G).In the field observation, the FM10811 variety showed a tuft phenotype with more tillers and lower plant height compared to TX.Meanwhile, the FM10587 mutant displayed yellow striped leaves (Figure 3F,G).

Discussion
Forward and reverse genetics approaches are two classical approaches often used to discover the gene functions underlying the phenotype.The classical forward genetic approach involves random mutagenesis and subsequent isolation of defective genetic mutants for a specific biological process or phenotype [5].Whilst the frequency of mutations occurring spontaneously in nature is too low, physical and chemical mutagens are induced for accelerated plant breeding and are applicable to all plant species.Mutagens introduce random changes throughout the plant genome, resulting in manageable population sizes [29].
With the recent advances in next-generation sequencing technologies, high-throughput genome characterization has radically changed the value of TILLING.TILLING popula-tions can be utilized either for visible developmental and morphological phenotypes or for scoring phenotypic behavior after the application of specific mutant screens [30].The initial mutation detection method for TILLING was performed using a commercial denaturing HPLC method, referred to as Denaturing High-Performance Liquid Chromatography (DHPLC) [8], which was originally developed to examine a mutant population of barley [10].The above method requires expensive equipment, but an enzymatic mismatch cleavage approach was successfully adapted for high-throughput mutant screening which employed the LI-COR system for the identification of Arabidopsis and wheat mutations [9,[31][32][33][34].Furthermore, a more economical and cost-effective substitute for the LI-COR system has been demonstrated with the use of conventional agarose or polyacrylamide electrophoresis [35,36].In this study, we implemented a mutation scanning method based on the CEL I enzyme double-stranded cutting of mismatches with subsequent polyacrylamide electrophoresis, which reduced the outlay required to purchase the DHPLC system and the fluorescent primers as well as labor investment.Therefore, this method of mutation scanning technology will both increase the throughput and decrease the cost of rapid mutation discovery in mutagenized plants and other populations.
As described above, TILLING has the benefit of genome-wide mutation discovery and broader applicability for the pursuit of point mutations in any organism.Many TILLING populations have been constructed for numerous crop species using diverse mutagens.In barley, several TILLING populations were constructed and most of them were EMS-mutagenic [10,12,13,37,38].Similar conclusions were drawn from other crop studies including rice, wheat, and maize [39][40][41].Compared to other chemical and physical mutagenesis methods, EMS mutagenesis is more convenient and efficient since the mutation frequency of EMS appears to be high in all plant species and, based on historical accounts, appears to be independent of genome size [10,42].Meanwhile, the mutant population development period is short, which makes it possible to generate a working reverse genetic population in approximately one and a half years, without having to carry out any plant transformation or selection of transformants [10].In this study, 1022 mutant materials were obtained from the barley variety TX by EMS mutagenesis, and the morphological observation of the M 2 and M 3 generations showed that the mutant library was rich in morphological variation types, and the phenotypic mutation frequency reached 4.42%, involving leaves, plant height, stems, and spikes.This mutant library can provide ideal experimental materials for the study of functional genomes and breeding of barley.
We have previously shown that ion homeostasis plays a crucial role in resistance to waterlogging stress [43][44][45].Thus, we were interested in identifying newly induced ion transporter mutants for exploration in our research program.In this study, we screened 5 gene mutants from this population and a total of 11 mutant strains were found.Compared to other gene mutants, the mutant number found in our study was low.In the study by Caldwell and his colleagues [10], there were six induced alleles of the HvFor1 gene and four induced alleles of the Hin-a gene which were confirmed in the mutant pool.For the Hvhox1 mutant, 31 alleles were identified, and the majority of these mutations (25 of 31) were G/C to A/T transitions [12].Most of the analysis of induced mutagenesis focuses on characterizing non-synonymous changes or insertions/deletions. Interestingly, in our study, most of the mutant lines we found were synonymous.However, there are instances where synonymous base changes can induce a significant phenotypic effect, such as base changes within the miRNA binding site and possibly even synonymous changes, which could have dramatic effects on phenotypes.
TILLING of the HvGLR3.5 gene in our newly developed TX cultivar TILLING population demonstrated the feasibility of generating an allelic series of a gene in barley with subtle phenotypic variation.In our study, three synonymous mutations were identified.Two of these mutations showed tuft and yellow striped leaves compared to the wild type.In Marchantia polymorpha L., Mpglr-7 and Mpglr-9 mutants were indistinguishable from the parental lines when grown in vitro or in soil, indicating that MpGLR activity is not required for normal vegetative growth and development [46].In Arabidopsis, engineer-ing the GLR3.3 channel by CRISPR impairs Ca 2+ /CaM-mediated desensitization which enhance plant systemic wound responses and anti-herbivore defense [47].Meanwhile, GLR mutations in Arabidopsis produced tip growth defects in pollen tubes [48].However, mutant phenotypes mentioned in M. polymorpha and Arabidopsis are typically the result of a single mutation; in the EMS mutant population, the mutant lines we explored carry multiple additional mutations in addition to those selected.Therefore, we observed various mutant phenotypes in the M 3 generation between different allele mutants.

Conclusions
EMS mutagenesis can offer useful novel insights into the gene function in a species under investigation, and the availability of TILLING resources in different germplasms can be important.We developed a TILLING population in the two-rowed Chinese landrace TX9425 based on the CEL I enzyme with subsequent polyacrylamide electrophoresis, which decreased the cost and labor investment of rapid mutation discovery in mutagenized plants.In our population, the mutant phenotype variation was abundant, and we have demonstrated the availability for reverse genetics by identifying five ion transport-related gene mutations.This population will be a resource for functional analysis of genes and for high-throughput gene discovery in large-genome cereals.

Figure 1 .
Figure 1.The phenotype of TX9425 (TX).TX is a Chinese two-rowed and hulled barley variety, which is mainly used for feeding.The image of the left panel (A) shows the whole plant after the filling period, and the top right image (B) shows the whole spike and seeds with awns.The bottom right image (C) shows the mature seeds.The thousand-kernel weight (D), relative water content (E), nitrogen content (F), and crude protein content (G) of TX and NasoNijo.NasoNijo (NN) is a Japanese two-rowed and hulled barley variety, which is mainly used for malting.

Figure 1 .
Figure 1.The phenotype of TX9425 (TX).TX is a Chinese two-rowed and hulled barley variety, which is mainly used for feeding.The image of the left panel (A) shows the whole plant after the filling period, and the top right image (B) shows the whole spike and seeds with awns.The bottom right image (C) shows the mature seeds.The thousand-kernel weight (D), relative water content (E), nitrogen content (F), and crude protein content (G) of TX and NasoNijo.NasoNijo (NN) is a Japanese two-rowed and hulled barley variety, which is mainly used for malting.

Figure 3 .
Figure 3. Hvglr3.5 mutants detected from the TILLING population.The mutation locations (red main string) were shown in HvGLR3.5 gene structure and all the mutations were synonymously (A).

Table 1 .
Mutant types and mutation frequency observed in M2 population.

Table 1 .
Mutant types and mutation frequency observed in M 2 population.

Table 2 .
Agronomic traits of some homozygous mutant lines in M 3 generation.

Table 3 .
List of TILLING targets and the number and distribution of mutation types.