Genomic traces of Japanese malting barley breeding in two modern high-quality cultivars, ‘Sukai Golden’ and ‘Sachiho Golden’

Abstract

Two modern high-quality Japanese malting barley cultivars, ‘Sukai Golden’ and ‘Sachiho Golden’, were subjected to RNA-sequencing of transcripts extracted from 20-day-old immature seeds. Despite their close relation, 2,419 Sukai Golden-specific and 3,058 Sachiho Golden-specific SNPs were detected in comparison to the genome sequences of two reference cultivars: ‘Morex’ and ‘Haruna Nijo’. Two single nucleotide polymorphism (SNP) clusters respectively showing the incorporation of (1) the barley yellow mosaic virus (BaYMV) resistance gene rym5 from six-row non-malting Chinese landrace Mokusekko 3 on the long arm of 3H, and (2) the anthocyanin-less ant2 gene from a two-row Dutch cultivar on the long arm of 2H were detected specifically in ‘Sukai Golden’. Using 221 recombinant inbred lines of a cross between ‘Ishukushirazu’ and ‘Nishinochikara’, another BaYMV resistance rym3 gene derived from six-row non-malting Japanese cultivar ‘Haganemugi’ was mapped to a 0.4-cM interval on the proximal region of 5H. Haplotype analysis of progenitor accessions of the two modern malting cultivars revealed that rym3 of ‘Haganemugi’ was independently introduced into ‘Sukai Golden’ and ‘Sachiho Golden’. Residual chromosome 5H segments of ‘Haganemugi’ surrounding rym3 were larger in ‘Sukai Golden’. Available results suggest possibilities for malting quality improvement by minimizing residual segments surrounding rym3.

Introduction

Barley (Hordum vulgare L.), currently the fourth major cereal crop worldwide, is used for livestock feed, brewing malts, and human food. Barleys indigenous to Japan were disseminated from China and the Korean Peninsula about 2,000 years ago and were subsequently spread widely. Japanese barleys were all six-row for food use until two-row barley was introduced from the West in the early 1880s for production of raw materials to support the emerging beer industries in a new era (Takahashi 1980). Two-row barley tends to have plumper grains and more uniform germination than six-row barley. Moreover, it is better suited to malting. Western two-row cultivars have been predominantly late, tall, and non-adaptive to hot and humid climates in Japan, but ‘Golden Melon’ introduced from Europe showed adequate agronomic and malting characteristics in wide areas (Kawaguchi 1976). Initially Japanese two-row barley was improved by pure line selection and cross-breeding method, particularly ‘Golden Melon’, to satisfy requirements for acceptable malting quality. Consequently, ‘Golden Melon’ became the framework of Japanese malting barley. However, moderately late maturing ‘Golden Melon’ and its derivatives did not fit the barley-rice double cropping system. The harvest period overlaps the rainy season. Consequently, rain damage exacerbated infection of barley grains with Fusarium head blight or scab disease. Gradually, further improvement of Japanese two-row malting barley became necessary to compete with the imported malts, which were inexpensive and of high quality. Early maturity and lodging resistance were acquired by crossing with Japanese six-row non-malting cultivars, but balancing early maturity and high yield within a restricted grain filling period persists as an important challenge.

A major breakthrough in Japanese malting barley breeding was the adoption of Co-operative Malting Barley Variety Tests in 1971 (Kawaguchi 1973). Under this system, candidate malting cultivars were subjected to three-year evaluation for agronomic and quality characteristics based on strict criteria. Successful cultivars are approved as contract cultivars. In Japan, selected two-row cultivars are grown on a contract basis between farmers and users. Only crops satisfying the requirements are purchased by the users as malting barley at the premium price. As the fruits of such aligned efforts, two-row malting cultivars with excellent quality and acceptable yield, ‘Amagi Nijo’ (Tsuda et al. 1979) and ‘Haruna Nijo’ (Aida 1979), were released respectively in 1978 and 1979.

Barley yellow mosaic virus (BaYMV), the most severe disease of autumn sowing barley in Japan, was described first in Japan (Ikata and Kawai 1940), but it has now extended its infection regions to include China, South Korea, and Europe. Most Japanese six-row barley has been resistant or moderately resistant to the virus, but Japanese two-row cultivars descended from the Western introduction are highly susceptible to the disease. Japanese BaYMV resistance researchers have identified resistance gene sources such as resistance to yellow mosaic 5 (rym5) and rym3 in East Asian six-row non-malting accessions (Kawada 1991, Konishi et al. 1997, Oozeki et al. 2017, Perovic et al. 2014, Saeki et al. 1999, Takahashi et al. 1973). For BaYMV-resistance breeding, a non-malting resistant line was used as a donor, followed by repeated crossing with high-quality two-row cultivars to eliminate persisting deleterious genes. Rigorous selection developed BaYMV-resistant two-row lines that combined acceptable levels of agronomic and malting quality characteristics. The first BaYMV-resistant and high-quality malting barley, ‘Misato Golden’, was released in 1985 with the rym5 gene derived from Chinese six-row non-malting landrace Mokusekko 3 (Seko 1987). However, barley cultivars with rym5 soon became susceptible to the resistance-breaking BaYMV variant strain III (Iida et al. 1992). Then, the rym3 gene from Japanese six-row non-malting cultivar ‘Haganemugi’ was exploited (Kawada 1991). Thereafter, two Japanese malting barley cultivars with high BaYMV-resistance and acceptable malting quality were released: ‘Sukai Golden’ in 2000 (Taniguchi et al. 2001) and ‘Sachiho Golden’ in 2005 (Kato et al. 2006). ‘Sukai Golden’ additionally carries rym5 (Kato et al. 2006). ‘Sukai Golden’ and ‘Sachiho Golden’ are estimated as sharing about 50% genetic similarity with BaYMV-susceptible but high-quality ‘Haruna Nijo’ based on pedigree analysis (Sotome et al. 2009). Consequently, concern has arisen that high-quality and BaYMV-resistance breeding efforts might have narrowed genetic diversity and might therefore have caused genetic erosion in Japanese two-row malting barley.

Malting quality in barley is an economically important but genetically complicated trait. In terms of grain quality characteristics, adequate protein content (10–11%) and low (1:3; 1:4)-beta-glucan content are the main breeding objectives (Nagamine and Kato 2008). Malt quality characteristics such as (1) malt extract, (2) malt nitrogen content, and (3) diastatic power (DP), are regarded as the major limiting factors (Kawaguchi 1973, Nonaka 1973). Here, DP represents the enzymatic ability of malted grain to convert starches into sugar during mashing. Water sensitivity of grains during malting and degradation of the malt protein, both characteristics strongly exhibited in ‘Sukai Golden’, began to be evaluated critically (Nagamine and Kato 2008). Water sensitivity, which is the failure of grains to germinate in conditions of excessive moisture, is an unfavorable trait because such grains require intricate control of steeping and germination during malting (Crabb and Kirsop 1970). Genetic analysis of malting quality was performed using QTL analysis (Hayes et al. 1993, Igartua et al. 2000, Kochevenko et al. 2018), proteome phenotyping (Bahmani et al. 2022), genome-wide association studies (GWAS) (Looseley et al. 2020, Mohammadi et al. 2015), and RNA-sequencing (RNA-seq) (Muñoz-Amatriaín et al. 2010). Single nucleotide polymorphism (SNP) markers were developed by genotyping-by-sequencing (GBS) analysis (Milner et al. 2019) or the 50k iSelect SNP array (Bayer et al. 2017). These studies detected many candidate genic regions, but their causal genes remain elusive.

An invaluable method to obtain comprehensive gene expression profiles rapidly and to acquire genome-scale datasets of transcribed sequence polymorphisms in barley is RNA-seq (Takahagi et al. 2016, Tanaka et al. 2019). The objectives of this study were to assess the diversity of two modern Japanese malting barley cultivars, ‘Sukai Golden’ and ‘Sachiho Golden’, in gene expression and SNPs using RNA-seq of transcripts from 20-day-old immature seeds. By particularly examining 20-day-old immature seed transcriptomes, we anticipated the detection of seed quality related genes effectively. Our RNA-seq analyses of immature seed-transcriptomes demonstrated that the two Japanese modern elite cultivars carried unique and contrasting features related to malting quality and BaYMV resistance gene(s).

Materials and Methods

Plant material

Two Japanese malting barley cultivars, ‘Sukai Golden’ and ‘Sachiho Golden’, were grown in pots under a glass roof. Plants grew without BaYMV infection.

RNA extraction and NGS-aided transcriptome analysis (RNA-seq)

Barley caryopses (including hulls) 20 days after flowering were subjected to RNA-seq analysis with three biological replications for each cultivar. Plant total RNA was extracted using RNA-suisui-S (Rizo Inc.), with subsequent on-column DNase treatment with an After Tri-Reagent RNA Clean-Up Kit (Favorgen Biotech Corp.). The harvested RNA quality was assessed using a bioanalyzer (Agilent Bioanalyzer 2100; Agilent Technologies Inc.) with a kit (Agilent RNA 6000 Nano Kit; Agilent Technologies Inc.).

Sequencing libraries were prepared according to a kit (TruSeq RNA Sample Preparation Kit v2; Illumina Inc.), except for one ‘Sukai Golden’ RNA sample because of poor quality. Paired-end sequencing was performed using an Illumina HiSeq 2500 system, which generated an average of 26.6 million reads (2 × 100 nucleotides) per library.

Data analysis

The NGS reads were quality-checked and trimmed (Trimmomatic ver. 0.36; Bolger et al. 2014) with the default parameters. The cleaned reads were aligned to the barley cv. ‘Morex’ reference genome (IBSC_Ver.2) (Mascher et al. 2017) with the help of STAR two-pass mode ver. 2.7.9a (Dobin et al. 2013). ‘Morex’ Ver.3 genome was not used because its gene annotation showed a significantly lower mapping ratio. Only properly paired and unique alignments were retrieved for subsequent analyses. For gene expression analysis, the digital expression value TPM (transcripts per million) was estimated for each transcript using RSEM ver. 1.3.1 (Li and Dewey 2011). Then differentially expressed genes were evaluated with the help of DESeq2 ver. 1.38.1 (Love et al. 2014) and tximport ver. 1.26.0 (Soneson et al. 2015) in R ver. 4.2.2 (R Core Team 2022). For genetic polymorphism analysis, the mpilup command of BCFtools ver. 1.9 (Danecek et al. 2021) was used to call the genetic variations of each alignment dataset. Only biallelic, non-missing, and replicate-common SNPs supported by more than four reads were retained for analysis. The genetic variations of ‘Haruna Nijo’ were obtained using the NGS read data of its genomic DNA from a published study (Sakkour et al. 2022). The procedure was mostly the same, except for the use of BWA-MEM version 0.7.17-r1188 (https://doi.org/10.48550/arXiv.1303.3997) with default parameters for aligning the reads to the genome reference.

Data and code availability

Raw sequence data of barley RNA-seq were deposited at the DNA Databank of Japan (DDBJ) Sequence Read Archive (DRA) under accession number PRJDB14939. The raw sequence data of ‘Haruna Nijo’ genome are available at the DDBJ DRA under accession number DRR045273. In-house R and Python scripts used for this study are available through a GitHub repository (https://github.com/junesk9).

Validation of the six SNPs in the BaYMV resistance rym5 locus by PCR markers

RNA-seq of seed RNA detected six SNPs in the rym5 locus (HORVU3Hr1G113940; HORVU.MOREX.r3.3HG0327270 in V3) between ‘Sukai Golden’ and ‘Sachiho Golden’, and three of them were specific to ‘Sukai Golden’. Previous primers (Nagamine unpublished data, Stein et al. 2005) and newly designed primers were used to develop polymorphic CAPS and dCAPS markers to validate the SNPs of the rym5 locus. For this experiment, the two cultivars plus ‘Morex’, Mokusekko 3 and ‘Haruna Nijo’, were included. We retrieved the complete Hv-eIF4E CDS sequences of ‘Morex’ (AY661558), Mokusekko 3 (LC037398), and ‘Haruna Nijo’ (AK365250) from the public database. Polymorphic CAPS or dCAPS markers were designed.

Mapping of the rym3 gene using RIL population and haplotype analysis

Because rym3 has not been cloned yet, we mapped the rym3 resistance gene using F₁₁ generation of the 221 recombinant inbred lines (RIL) derived from an ‘Ishukushirazu’ and ‘Nishinochikara’ cross. ‘Ishukushirazu’ is a carrier of the rym3 gene from ‘Haganemugi’; ‘Nishinochikara’ is a non-carrier. Resistance and susceptible phenotypes of rym3 were accessed by visual observation when the RIL materials were grown in the BaYMV type III infested field of the Tochigi Branch, Tochigi Prefectural Agricultural Experiment Station. Fifteen seeds each were early planted in late October according to two replications and were assessed individually for the mosaic symptom of the leaves in early March of the following year. Equivocal lines were tested repeatedly in multiple years and were also subjected to the ELISA test (Usugi et al. 1984).

We screened public markers for polymorphism. Monomorphic public primers were sequenced to convert them to polymorphic markers. Additionally, we originally designed primers from selected barley ESTs that were syntenic to rice chromosome 12. Mapping was conducted using 4 public (Haruyama et al. 2011, Sato et al. 2009) and 14 originally developed PCR markers (Supplemental Tables 1, 2). The map distance was calculated using Map Maker Ver.2 with the recombinant inbred setting according to an earlier report by Taketa et al. (2006, 2021). Wheat–barley ditelosomic addition lines for 5HS and 5HL (Islam 1983) were used to ascertain their chromosome arm locations. Furthermore, haplotype analysis of 29 barley accessions in the pedigrees of ‘Sukai Golden’ and ’Sachiho Golden’ were performed using the same 18 markers to trace the introduction route of rym3 and to deduce the residual chromosome segment sizes surrounding rym3.

Results

Morphological and BaYMV resistance genes

‘Sukai Golden’ and ‘Sachiho Golden’ both had compact spikes, but ‘Sukai Golden’ had about 20% shorter awns than ‘Sachiho Golden’ had (Fig. 1A, Supplemental Fig. 1). ‘Sukai Golden’ lacked anthocyanin pigmentation in the awn tips, auricle, culm node, and basal leaf sheath, but ‘Sachiho Golden’ had purple pigmentation in all those tissues (Fig. 1B–1E). We inferred that this pigmentation difference resulted from the ANT2 locus, which encodes the anthocyanin pathway gene HvbHLH1 (HORVU2Hr1G096810, HORVU.MOREX.r3.2HG0188710) on the 2HL (Cockram et al. 2010). Genotyping with the published HvbHLH1 PCR primers showed that ‘Sukai Golden’ had a 16-bp deletion allele (ant2), but that ‘Sachiho Golden’ had a WT allele (Supplemental Fig. 2). This observation explained the lack of pigmentation in the vegetative tissues of ‘Sukai Golden’. Pedigree analyses indicated that ant2 of ‘Sukai Golden’ was derived from the Dutch cultivar ‘Cambrinus’ (Russel et al. 2000) through ‘Tsuyushirazu’ and Kanto Nijo 25 (Fig. 2). Mature grains were phenotypically similar between the two cultivars (Fig. 1F).

Fig. 1.

Morphology of ‘Sukai Golden’ (left) and ‘Sachiho Golden’ (right), in about two-week-old spikes (A), awn tips (B), auricle (C), culm node (D), leaf sheath base (E), and mature grains, left is dorsal side and right is ventral side (F). Bars are 1 cm in A, and 5 mm in B, C, D, E, and F.

Fig. 2.

Pedigrees of ‘Sukai Golden’ (A) and ‘Sachiho Golden’ (B) bred in Japan with improved BaYMV resistance and malting quality. Orange box shows the rym5 donor, Mokusekko 3, blue boxes show ‘Haganemugi’, the rym3 donor. The integration route of anthocyanin-less2 (ant2) is shown in italic magenta in A. For rym3, different haplotypes are distinguished by blue letters with a dash followed by the cultivar/accession abbreviations, where simple rym3 shows the intact ‘Haganemugi’ type. In ‘Sukai Golden’s pedigree, a rym3-T216 haplotype was detected, whereas in ‘Sachiho Golden’s pedigrees, two haplotypes, rym3-SK32 in Saikai Kawa 32, and rym30-KN29 in Kanto Nijo 29 (highlighted in magenta in B), were distinguished. Details of rym3 haplotypes are summarized in Table 1.

Regarding the BaYMV resistance genes, ‘Sukai Golden’ reportedly carries both rym3 and rym5, but ‘Sachiho Golden’ harbors only rym3. Their pedigrees (Fig. 2) showed six-row non-malting Mokusekko 3 and ‘Haganemugi’ as donors of rym5 and rym3 BaYMV resistance genes, respectively (Kato et al. 2006, Taniguchi et al. 2001). Spikes at about three weeks old of the resistance donors are shown along with those of the recipient malting cultivars (Supplemental Fig. 1).

Transcriptome sequencing and transcribed region annotation

RNA-seq was performed for 20-day-old immature seeds of the two cultivars. Their genome-wide gene expression profiles were analyzed first. The 26,070 of 39,734 protein-coding loci were detected being expressed in the seeds (Fig. 3A). Differentially expressed genes (DEGs) were evaluated by comparing the expression data of the two cultivars. The ‘Sukai Golden’ data were estimated as having the 111 and 130 loci of significantly higher and lower DEGs (false-discovery rate <0.01), respectively, in comparison to the ‘Sachiho Golden’ data (Fig. 3A, Supplemental Table 3). This mere abundance of DEGs and their even distribution on the genome implies that the gene expression profiles in seeds of the two cultivars appear to be generally similar.

Fig. 3.

RNA-seq results of transcriptomes of 20-day-old caryopses of ‘Sukai Golden’ and ‘Sachiho Golden’: (A) Gene expression levels with ‘Sukai Golden’ on the x-axis, and ‘Sachiho Golden’ on the y-axis; (B) Venn-diagram of the caryopsis RNA-seq of ‘Sukai Golden’ (upper left) and ‘Sachiho Golden’ (upper right), in comparison to the genome sequences of ‘Haruna Nijo’ (lower) and ‘Morex’ (IBSC_Ver.2); (C) Venn-diagram of the number of genes with SNPs of ‘Sukai Golden’ (upper left) and ‘Sachiho Golden’ (upper right), in comparison to the genome sequences of ‘Haruna Nijo’ (lower) and ‘Morex’ (IBSC_Ver.2); (D) Chromosomal proportion of modern Japanese malting cultivar-specific SNPs. The middle bars are those of ‘Sukai Golden’. The right bars are those of ‘Sachiho Golden’, relative to the whole SNPs (65,622). (E) The physical distribution of Japanese modern barley cultivar specific SNPs sorted according to the barley chromosomes 1H to 7H and Un (unknowns). Bars above the horizontal lines show cultivar specific SNPs of ‘Sukai Golden’ (shown in black bars), whereas bars below the line show those of ‘Sachiho Golden’ (shown in gray bars). Arrowheads indicate the presumed centromere positions according to Mascher et al. (2017). Approximate physical positions of ant2 on 2HL arm and rym5 on 3HL arm are shown, and rym3 candidate interval is shown by a double-headed arrow.

Then, the genetic variations of the two cultivars against the ‘Morex’ ver.2 reference genome were analyzed using the RNA-seq data. The variations of another cultivar ‘Haruna Nijo’ (Sakkour et al. 2022) were analyzed together, because, based on pedigree analysis, it is estimated as sharing about 50% genetic similarity with the two cultivars (Kato et al. 2006, Sotome et al. 2009, Taniguchi et al. 2001). Our analysis pipeline identified the 65,622 biallelic SNPs in total, of which the 47,189 SNPs were commonly identified from the two RNA-seq data, whereas the 10,160 SNPs were identified exclusively from the ‘Haruna Nijo’ data. The 3,893 and 4,380 SNPs were identified, respectively, as different alleles in ‘Sukai Golden’ or ‘Sachiho Golden’ data relative to the ‘Morex’ reference. Among them, the 1,474 and 1,322 SNPs were also identified respectively from the ‘Haruna Nijo’ data. By subtractions, the 2,419 and 3,058 SNPs were finally identified, respectively, as ‘Sukai Golden’-specific and ‘Sachiho Golden’-specific SNP loci (Fig. 3B, 3D, Supplemental Table 4). The number of genes with SNPs in respective cultivar was presented in Fig. 3C. ‘Sukai Golden’ and ‘Sachiho Golden’ shared 959 genes with SNP(s). Next, the chromosomal distribution of cultivar specific SNPs was analyzed. The ‘Sukai Golden’-specific SNPs distributed over chromosomes in a proportion similar to the whole SNPs detected, whereas the ‘Sachiho Golden’-specific SNPs were excessive in chromosomes 3H and 7H and scarce in chromosomes 4H and 6H (Fig. 3D, Supplemental Table 4).

Cultivar-specific SNPs tended to distribute in cluster with different patterns, but the wide regions around the centromeres were scarce in SNPs for all chromosomes. ‘Sukai Golden’ had readily apparent clusters of SNPs in the distal region of 2HL and the sub-terminal region of 3HL (Fig. 3E). The cluster in 2HL appears to surround the ANT2 locus with its physical location at around 677 Mb. The cluster in 3HL coincided the rym5, which encodes the eukaryotic translation elongation initiation factor 4E (Hv-eIF4E), at around 690 Mb. ‘Sachiho Golden’ had main SNP clusters in the middle region of 3HL and the middle region of 7HS, but these were not associated with any breeding history (Fig. 3E). Chromosomes 4H and 6H of ‘Sachiho Golden’ were particularly rare in SNPs, which agrees with their scarce SNP proportions (Fig. 3D)

Seed RNA-seq detected multiple SNPs in the rym5 resistance gene

The RNA-seq conduced for 20-day-old immature seeds detected the transcriptomes of the rym5 locus for Hv-eIF4E in the sub-terminal region of 3HL (Kanyuka et al. 2005, Stein et al. 2005). ‘Sukai Golden’ (rym5) and ‘Sachiho Golden’ (without rym5) differed in six SNPs of the Hv-eIF4E transcribed region, three (positions 359, 478 and 481) of which were unique to ‘Sukai Golden’ with rym5 (Fig. 4A, Supplemental Table 4). The rym5 haplotype of ‘Sukai Golden’ was identical to that of Mokusekko 3, which is the donor of the resistance allele rym5. Based on the complete Hv-eIF4E CDS sequences of Mokusekko 3 and ‘Haruna Nijo’ from the public database, we developed CAPS or dCAPS markers for distinguishing the six SNPs at the positions of minus 44, 157, 359, 478, 481, and 483 nt (Supplemental Table 2). Two markers developed at positions 359 and 478 nt were diagnostic to the rym5 allele derived from Mokusekko 3 because it unequivocally distinguished rym5 resistant accessions from susceptible ones tested (Supplemental Fig. 3). The six SNPs of Hv-eIF4E detected in the present RNA-seq were all described previously in the comprehensive polymorphism studies reported by Stracke et al. (2007) and Hofinger et al. (2011).

Fig. 4.

SNPs of the BaYMV resistance gene rym5 (HORVU3Hr1G113940) on the long arm of chromosome 3H among the five barley cultivars (A), and genetic and physical mapping of the BaYMV resistance rym3 gene on the proximal region on 5H (B). In (B), left is graphical genotype of ‘Ishukushirazu’ (abbreviated as Is, rym3 carrier), ‘Nishinochikara’ (Nic, non-rym3 carrier), ‘Sukai Golden’ (Suk) and ‘Sachiho Golden’ (Sac). Blue and yellow bars respectively denote Is-derived and Nic-derived chromosome segments. The genetic map of rym3 (in the middle) was obtained in the Is and Nic F₁₁ generation 221 RILs. Only three markers in magenta color were polymorphic between Suk and Sac. Genetic markers’ physical positions in Morex Ver.2 genome sequences are shown at the right.

Mapping of the rym3 resistance gene and haplotype analysis

Because rym3 has not been cloned yet, we first deduced its location by genetic mapping using the 211 RILs between ‘Ishukushirazu’ and ‘Nishinochikara’, which were rather polymorphic. Of 18 markers mapped, rym3 cosegregated with eight 5H markers spanning the centromere. Wheat–barley ditelosomic addition lines localized four markers each, respectively to the short and long arm of 5H. The rym3 gene was mapped to a 0.4 cM interval that was flanked by TBr3-3 and O12B5H14, suggesting its location in a centromeric region with highly suppressed recombination (Fig. 4B). In the ‘Morex’ V2 physical map, the rym3 candidate region was estimated as 268 Mb between HORVU5Hr1G020400 and HORVU5Hr1G045700 (Figs. 3E, 4B). This interval was estimated as 234 Mb in V3.

Only three (16.7%) of the 18 markers, i.e., TBr3-4, k05331GR and O12B5H1, were polymorphic between ‘Sukai Golden’ and ‘Sachiho Golden’ (Fig. 4B, Table 1). However, by referring to the haplotype data, we depicted graphical genotypes of chromosome 5H including rym3 in ‘Sukai Golden’ and ‘Sachiho Golden’ (Fig. 4B). The graphical genotypes revealed that ‘Sachiho Golden’ harbored smaller chromosome segments from the rym3 donor ‘Haganemugi’ compared to ‘Sukai Golden’. ‘Sachiho Golden’ likely had many recombination events near rym3, which probably reflect rigorous selection to eliminate persisting deleterious genes. By contrast, ‘Sukai Golden’ carried ‘Haganemugi’ segments spanning the entire the 5HS arm and the proximal 5HL region, which were able to retain genes that are deleterious for malting. Haplotypes of 5H near rym3 were examined for 29 accessions including mainly progenitor lines and a standard ‘Morex’ (Table 1). The data corroborated restricted recombination in the rym3 candidate region. The rym3-resistant lines/cultivars were classified into four types: (1) entirely ‘Haganemugi’ chromosome 5H segment, such as Hakei J-7; (2) left-most two 5HS arm markers recombined, but others were all ‘Haganemugi’ type, such as Saikai Kawa 32; (3) five 5HL markers were introduced, such as Tochikei 216 (‘Sukai Golden’); and (4) left-most two 5HS, the middle one and right five 5HL marker segments are introduced, such as Kanto Nijo 29 (a parent of ‘Sachiho Golden’).

Table 1.

Haplotypes around the rym3 locus of representative accessions in the pedigrees of ‘Sukai Golden’ and ‘Sachiho Golden’. Alleles A and B represent ‘Isyukushirazu’-type (rym3 resistant, ‘Haganemugi’ allele) and ‘Nishinochikara’-type (susceptible), respectively. Accessions in bold letters are presumed to carry the rym3 gene.

Marker	TBr3-4	k05331GR	TBr3-3	k05052GR	k03447GR	k03041GR	k03390	k09554T	TBr3-1	k00267	O12B5H1	O12B5H14	O12B5H12	TBr3-2	k09404GR	k07650GR	TBr3-8	TBr3-5
Accession	5HS	5HS	5HS	5HS	5HS	5HS	5HS	5HL	5HL	5HL	5HL	5HL	5HL	5HL	5HL	5HL	5HL	5HL
Ishukushirazu	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A
Nishinochikara	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B
Sukai Golden	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	A
Sachiho Golden	B	B	A	A	A	A	A	A	A	A	B	A	B	B	B	B	B	A
Haruna Nijo	B	B	B	B	B	B	B	B	A	A	B	A	B	B	B	B	B	A
Golden Melon	B	B	B	B	B	B	B	B	A	A	B	A	B	B	B	B	B	A
Morex	A	B	B	B	B	B	B	B	B	B	B	A	B	B	A	B	B	A
Kanto Nijo 25	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B
Nankei B1303	A	A	B	A	B	B	A	A	A	A	A	A	A	A	A	A	B	A
Seijo 17	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A
Mokusekko 3	A	A	B	A	B	B	A	A	A	A	A	A	A	A	A	A	B	A
Klages	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	B	B
Tsuyushirazu	B	B	B	B	B	B	B	B	A	A	B	A	B	B	B	B	B	A
Cambrinus	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A
Tochikei 216	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	A
Satukibare	B	B	B	B	B	B	B	B	A	A	B	A	B	B	B	B	B	A
Tochikei 133	B	B	B	B	B	B	B	B	A	A	B	A	B	B	B	B	B	A
Haganemugi	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A
Daikei R4224	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A
Nittakei 30	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A
Saikai Kawa 32	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A
Hakei J-7	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A
Asahi 5	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A
Kanto Nijo 29	B	B	A	A	A	A	A	A	A	A	B	A	B	B	B	B	B	A
Amagi Nijo	B	B	B	B	B	B	B	B	A	A	B	A	B	B	B	B	B	A
Saikai Kawa 33	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A
Kawasaigoku	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A
Tochikei 166	B	B	B	B	B	B	B	B	A	A	B	A	B	B	B	B	B	A
Mona	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A

Discussion

For this study, we compared two modern Japanese malting cultivars with contrasting brewery quality and BaYMV resistance by RNA-seq of seed trancriptomes at 20-day after flowering. Gene expression levels in the two cultivars were generally similar, but their mutual cultivar-specific SNPs distribution patterns differed. Although the two cultivars are expected to share about 50% genetic similarity with ‘Haruna Nijo’ based on the pedigree analysis (Sotome et al. 2009), a large number of specific SNPs was detected: 2,419 for ‘Sukai Golden’ and 3,058 for ‘Sachiho Golden’. In ‘Sukai Golden’, clusters of SNPs in 2HL and 3HL respectively coincided with previously identified ant2 and rym5. ‘Sachiho Golden’ had two prominent cultivar-specific SNP clusters in the middle of 3HL and in the distal region of 7HS, but their associations with any breeding traits are unknown.

The BaYMV resistance gene rym3, derived from an old Japanese six-row non-malting cultivar ‘Haganemugi’ bred in 1955, has been a successful resistance gene source without conferring deleterious effects on agricultural characteristics (Kato et al. 2006, Taniguchi et al. 2001). Both ‘Sukai Golden’ and ‘Sachiho Golden’ carry the rym3 resistance gene derived from ‘Haganemugi’, but their pedigrees and the present haplotype data clarified that the rym3 resistance gene was introduced through different routes. The present genetic mapping demonstrated that the residual chromosome segment sizes surrounding the rym3 resistance gene differed considerably between the two cultivars. ‘Sukai Golden’ carries large chromosome 5H segments harboring rym3 of ‘Haganemugi’-origin without nearby crossovers. Such large residual chromosome 5H segments might partly explain its excessive malt protein degradation and high water sensitivity, both of which are unfavorable traits found in ‘Sukai Golden’ (Nagamine and Kato 2008). However, ‘Sachiho Golden’ carries a smaller residual chromosome segments of ‘Haganemugi’ 5H, most likely attributable to multiple cross-overs near rym3. ‘Haganemugi’ alleles in the proximal region of chromosome 5H loci might be likely to degrade malting characteristics. Pedigrees of ‘Sachiho Golden’ implies that multiple crosses with quality but BaYMV-susceptible parents and repeated rigorous selection up to advanced F₄ generations were effective to select rare recombinants that might provide a balance of high malting quality and rym3 resistance.

Saeki et al. (1999) mapped rym3 to the proximal region of the 5HS arm. Similarly, Perovic et al. (2014) mapped rym_HOR4224 to 5HS; HOR4224 is a Japanese six-row accession and its resistance gene is likely to be allelic to rym3. The present study mapped rym3 to a 0.4 cM interval spanning the centromeric region of 5H, but due to highly suppressed recombination, its physical candidate region is still as large as 268 Mb (Fig. 3E). Genomic analyses of the Japanese rym3 resistance breeding materials may be useful to narrow down the rym3 candidate region because the reliable materials are well maintained.

The present RNA-seq analyses of immature seed transcriptomes in two elite Japanese malting barley cultivars are expected to provide important insights as deciphering mechanisms that can lead to compatible improvements in both seed quality and BaYMV disease resistance. These findings are expected to pave the way to molecular cloning of key genes carried by modern Japanese malting barley cultivars, even those with close genetic relatedness.

Author Contribution Statement

S.T., K.M. and J-S.K. designed the research; S.T., S.Y., Y.K., T.S. and T.K. performed the experiments, J-S.K., K.M., H.T. and S.T. analyzed the data, S.T. and J-S.K. wrote the paper.

Acknowledgments

This research was partly supported by a Cooperative Research Grant for Genome Research for BioResource, NODAI Genome Research Center, Tokyo University of Agriculture (13B-16), from MEXT, Japan; the Brewers Association of Japan; the Yakumo Environmental Science Promotion Foundation, Japan; and a grant in aid (no. 23H02184) from MEXT, Japan. The authors are obliged to Dr. K. Tanaka and to Dr. T. Ishige for critical comments and discussions. The authors are grateful to Mr. T. Nagamine and Mr. N. Haruyama for their generous supply of unpublished marker information for rym5 and rym3, respectively. Barley seeds including RILs for the rym3 mapping were generously supplied by Mr. N. Kawada. Some barley genetic resources were obtained from National Bioresource Project – Barley, Japan. We appreciate the skillful technical assistance of Dr. E. Himi, Mr. M. Yamaguchi, Mr. T. Sekiwa, Mr. T. Okiyama, Mr. M. Ooyama, Mr. Y. Arai, Ms. M. Oozeki, Ms. M. Shiraga, and Ms. N. Yamamoto.

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