Determination of the Allelic Composition of the sdw1/denso (HvGA20ox2), uzu1 (HvBRI1) and ari-e (HvDep1) Genes in Spring Barley Accessions from the VIR Collection

The lodging of barley significantly limits its potential yield, leads to the deterioration of grain quality, and complicates mechanized harvesting. More than 30 dwarfness and semi-dwarfness genes and loci are known for barley, and their involvement in breeding can solve the problem of lodging. The most common dwarfing alleles are of the genes sdw1/denso (HvGA20ox2), uzu1 (HvBRI1), and ari-e (HvDep1). The aim of this study was the design of dCAPS markers for the sdw1.c and ari-e.GP alleles and the molecular screening of barley accessions from the VIR collection for identifying these and other dwarfing alleles commonly used in breeding. Two dCAPS markers have been developed to identify the sdw1.c allele of the HvGA20ox2 gene and ari-e.GP of HvDep1. These dCAPS markers and two known from the literature CAPS and dCAPS markers of the alleles sdw1.a/sdw1.e, sdw1.c, sdw1.d, and uzu1.a were used in the molecular screening of 32 height-contrasting barley accessions. This made it possible to identify the accessions with alleles sdw1.a/sdw1.e, sdw1.c, and sdw1.d of the HvGA20ox2 gene, as well as accessions with a combination of sdw1.c and uzu1.a alleles of the genes HvGA20ox2 and HvBRI1. A comparison of the results of genotyping and phenotyping showed that the presence of dwarfing alleles in all genotypes determines high or medium lodging resistance regardless of the influence of weather conditions. Twelve accessions were found to contain the new allele sdw1.ins of the HvGA20ox2 gene, which differs from the known allele sdw1.c by a larger size of PCR products. It is characterized by the Thalos_2 transposon insertion; the subsequent GTTA insertion, common with the sdw1.c allele; and by a single-nucleotide G→A substitution at the 165 position.


Introduction
Barley (Hordeum vulgare L.) is an important food, feed, and brewing crop, characterized by its good adaptability to various growing conditions.However, the insufficient strength of the straw limits the potential yield of barley; due to this reason, plants often become lodged even in the years favorable for growth and development.Lodging leads to a deterioration of grain quality due to the development of diseases, causes sprouting in the ear, and also complicates mechanized harvesting and reduces the overall yield of plants.Therefore, the creation of lodging resistant cultivars is of particular importance in modern breeding programs [1].
The solution of the problem of lodging resistance in barley by breeding methods is associated, first of all, with a decrease in plant height.Semi-dwarfing genes played a crucial role in the Green Revolution in the 1960s, and their use in cereal crops significantly increased global food production [2].More than 30 dwarfing and semi-dwarfing genes and loci have been identified for barley, including brachytic (brh), semibrachytic (uzu), slender 1 (sln1), breviaristatum (ari), erectoides (ert), semi-dwarf (sdw), slender dwarf (sld), and dense spike (dsp) [3,4].However, only a few of them are used in barley breeding due to their negative Plants 2024, 13, 376 2 of 14 pleiotropic effect [5].The dwarfing alleles of the genes sdw1/denso (HvGA20ox2), uzu1 (HvBRI1), and ari-e (HvDep1) are most commonly used in breeding programs.
The sdw1/denso gene (HvGA20ox2) is one of the first ones cloned and characterized in barley.It is located on the long arm of chromosome 3H and is an orthologue of the sd-1 (semi-dwarf ) gene in rice.The HvGA20ox2 gene encodes gibberellin 20-oxidase, which is involved in the penultimate step of gibberellin biosynthesis [4,6].Dwarf mutants with mutations in the HvGA20ox2 gene are sensitive to treatment with gibberellic acid.Currently, five alleles of the sdw1/denso gene have been identified in cultivated barley: (1) sdw1.c(originally denso), found in the Danish cv.Abed Denso, is the only allele with a spontaneous mutation [7].The allele has a mononucleotide deletion and a 4 bp insertion in the 5 non-coding region of the first exon, as well as a number of other mutations in the coding region [4].(2) sdw1.aallele was induced in the six-row cv.Jotun by X-ray irradiation and is characterized by a complete deletion of the HvGA20ox2 gene [4,8].This allele is mainly used for creating forage barley cultivars [6].(3) The sdw1.e allele was identified in the cultivar Risø no.9265, which was created in a breeding program by treating cv.Bomi with partly moderated fission neutrons.However, there are no data on the introduction of this allele into other modern cultivars.Similar to the situation with the sdw1.aallele, a complete deletion of the HvGA20ox2 gene was also discovered in the case of the sdw1.e allele [7,9].(4) The X-ray irradiation of cv.Valticky in 1965 resulted in obtaining cv.Diamant with the sdw1.dallele (sdw1/denso), which is characterized by a functional 7 bp deletion in the first exon, leading to a frameshift and a dwarf phenotype, as well as to an G→A substitution in the second intron [4].This allele was actively used for breeding European cultivars, especially those for brewing [10].(5) The sdw1.Lockyer allele in cv.Lockyer was identified by sequencing the sdw1/denso (HvGA20ox2) gene in different cultivars.It is characterized by a single nucleotide G→A substitution at the 70 bp position from the end of the second exon and has a similar effect on plant growth and development as the sdw1.dallele [11].To identify the above-described alleles of the sdw1/denso gene the different CAPS-, both KASP and InDel markers were developed [4,6,9,11].
The sdw1/denso gene has a pleiotropic effect; that is, it affects many traits, including general adaptability, grain productivity, and grain quality [12,13].The main characteristic is a reduction in the length of culm internodes and an increase in lodging resistance.Semidwarf barley cultivars with mutations in the sdw1/denso gene take part in the pedigree of most modern cultivars and are widespread in the Western United States, Canada, and Australia, as well as among European brewing cultivars [6].
The uzu1 (HvBRI1) gene, a homolog of the OsBRI1 gene in rice, is located on the long arm of chromosome 3H and encodes a protein kinase involved in the reception of brassinosteroids, the growth phytohormones [14].The barley accessions with mutations in HvBRI1 are insensitive to brassinosteroids treatment.At least several alleles have been described for the HvBRI1 gene in cultivated barley: uzu1.a,uzu1.b,uzu1.c,uzu1.256, and uzu1.297[15].The dwarfing allele uzu1.a, the most studied and represented in East Asian cultivars, carries an A→G missense mutation at the 2612 bp position in the coding region [14].To identify the uzu1.aallele of the uzu1 gene, the dCAPS and KASP markers were developed [11,16].This allele has multiple effects on various parts of the plant, resulting in a shortened coleoptile; a small hook or projection at the coleoptile tip; shortened, wide, dark green leaves; shortened stem, spike, awns, and flower scales; and small caryopses [16,17].Such features allow denser sowing and the application of high doses of fertilizers.Cultivars with the dwarfing allele uzu1.aoccupy more than 70% of areas under barley in Japan and more than 30% of the sown area in the Korean Peninsula.Currently, over half of the naked barley forms grown in Southern Japan carry the uzu1.aallele [16].Other alleles of the HvBRI1 gene, namely uzu1.b,uzu1.c,uzu1.256, and uzu1.297, were identified in mutant barley lines [15].At the moment, there are no reliable data on their distribution in modern barley varieties.
Another source of semi-dwarfness in spring barley among the most commonly used ones is the ari-e.GP allele of the ari-e gene located on chromosome 5H [18].It has been shown that the HvDep1 gene, a homolog of Dep1 (Dense and erect panicle 1) in rice, which encodes Plants 2024, 13, 376 3 of 14 the γ-subunit of the heterotrimeric G-protein involved in the regulation of organ and seed growth, is a candidate gene that determines the effect of the ari-e locus [19,20].At least 10 alleles have been described for the ari-e gene (HvDep1), which have indels/nucleotide substitutions in the first and third exons: ari-e.GP, ari-e.1,ari-e.30,ari-e.39,ari-e.119,ari-e.156,ari-e.166,ari-e.178,ari-e.222,and ari-e.228.All of the mutations identified in the HvDep1 gene of the different ari-e mutant lines represent loss-of-function alleles [19].For the HvDep1 gene, cases of polymorphism in the 5 UTR region were also found, and the InDel markers were developed [21].The ari-e.GP allele is characterized by a single A insertion at the 1508 position in the second exon of the HvDep1 gene [20].To identify the ari-e.GP allele of the ari-e gene, KASP markers are developed [11].Carriers of the ari-e.GP allele have a semi-dwarf phenotype with short awns and exhibit salt tolerance [22].The source of ari-e.GP is the Golden Promise semi-dwarf cultivar that was obtained as a gamma mutant of cv.Maythorpe in 1956 in Great Britain [23].The cv. Golden Promise and its derivatives are still widely used for whiskey production.Other alleles of the HvDep1 gene are used only in genetic research, but their active introduction into breeding material has been increasing in recent years.
To identify the above-described alleles of the sdw1/denso, uzu1 and ari-e genes, wildly used at the breeding, KASP markers were developed [11], as well as the InDel marker for the sdw1.callele of the sdw1/denso gene [4].However, the KASP analysis is not always made available to small practice-oriented laboratories, and the identification of the sdw1.callele using the InDel marker during electrophoresis in agarose gels requires long-term separation.An alternative method is the use of markers based on the cleaved amplified polymorphic sequences (CAPSs) and derived CAPSs (dCAPSs).They are successfully used for identifying the sdw1.dand uzu1.aalleles [16,24]; however, there is no information about this type of marker for identifying sdw1.c and ari-e.GP.
Unique barley accessions with a shortened culm and identified dwarfing alleles are preserved and studied in various gene banks, including the Global Collection of the N.I.Vavilov All-Russian Institute of Plant Genetic Resources (VIR).The present study was aimed at (1) developing and testing dCAPS markers for the detection of sdw1.c and arie.GP alleles; and (2) conducting molecular screening of the global collection of the barley accessions from the VIR collection for determining the allelic composition of the sdw1/denso (HvGA20ox2), uzu1 (HvBRI1), and ari-e (HvDep1) genes.

Phenotyping
The phenotyping of accessions with respect to plant height and lodging resistance was carried out for 3 years in the fields of the Research and Practical Base "Pushkin and Pavlovsk Laboratories of VIR" (Table 1 and Supplementary Figures S1 and S2).In 2021-2023, weather conditions differed between the years and affected the growth and development of barley.During the 2021 growing season, weather conditions differed significantly from the longterm average.Temperatures of the growing season were above the average long-term values, and precipitation was rare, which led to a decrease in plant height and an almost complete absence of lodging.In 2022-2023, weather conditions were close to the long-term average; however, heavy rains and strong winds occurred during the growing season and created a favorable background for identifying lodging-resistant forms.Plants 2024, 13,

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The plant height of accessions ranged from 22 to 68 cm in 2021, from 34 to 92 cm in 2022, and from 34 to 85 cm in 2023.According to the three-year data, 24 sources of dwarfness with a straw shortened to less than 60-70 cm were identified.In 11 accessions, the plant height did not exceed 55 cm.The accessions k-30284 Namoi and k-5210 Makbo from Australia, k-28645 S-274 from Mexico, and k-28184 Local from Yemen practically did not change in height in the years with different weather conditions, thus suggesting their ecological adaptability.On the contrary, the height of accessions k-8952 and k-8953 (local from Turkey), k-30016 C.I.11077 (Peru), k-27765 Dolma (India), k-25801 Laschkes Korona, and k-25794 Rimpaus Nackta (Germany) varied greatly in different years of the study (Table 1).
According The remaining accessions were non-resistant to lodging in different years (Table 1).

Markers Development
The sequence of the wild-type allele of the HvGA20ox2 gene (GenBank: LOC123445118) was extracted from the whole genome assembly of the barley cv.Morex (MorexV3).To localize mutations characteristic of the sdw1.cand sdw1.dalleles, the data from the work by Y. Xu et al. (2017) were used [4].This way, a consensus diagram of the HvGA20ox2 gene was constructed which reflects the localization of mutations associated with the sdw1.cand sdw1.dalleles (Figure 1).The plant height of accessions ranged from 22 to 68 cm in 2021, from 34 to 92 cm in 2022, and from 34 to 85 cm in 2023.According to the three-year data, 24 sources of dwarfness with a straw shortened to less than 60-70 cm were identified.In 11 accessions, the plant height did not exceed 55 cm.The accessions k-30284 Namoi and k-5210 Makbo from Australia, k-28645 S-274 from Mexico, and k-28184 Local from Yemen practically did not change in height in the years with different weather conditions, thus suggesting their ecological adaptability.On the contrary, the height of accessions k-8952 and k-8953 (local from Turkey), k-30016 C.I.11077 (Peru), k-27765 Dolma (India), k-25801 Lasсhkes Korona, and k-25794 Rimpaus Nackta (Germany) varied greatly in different years of the study (Table 1).

Markers Development
The sequence of the wild-type allele of the HvGA20ox2 gene (GenBank: LOC123445118) was extracted from the whole genome assembly of the barley cv.Morex  To search for the HvDep1 gene sequence in the NCBI databases, we used the published sequence of its mRNA (GenBank: FJ039903.1).The BLAST search in the full genome assemblies of MorexV3 and Golden Promise (GPv1) resulted in our finding full-length gene sequence exons that were identical to the mRNA.The alignment of the HvDep1 gene sequences from cvs.Morex and Golden Promise (mutant allele ari-e.GP carrier) confirmed To search for the HvDep1 gene sequence in the NCBI databases, we used the published sequence of its mRNA (GenBank: FJ039903.1).The BLAST search in the full genome assemblies of MorexV3 and Golden Promise (GPv1) resulted in our finding full-length gene sequence exons that were identical to the mRNA.The alignment of the HvDep1 gene sequences from cvs.Morex and Golden Promise (mutant allele ari-e.GP carrier) confirmed the presence of an A-insertion at the 1508 position in the second exon of the HvDep1 gene [20].
To develop a DNA marker for the sdw1.callele of the HvGA20ox2 gene, a 4 bp insertion located in the first exon at the 64 position (+GTTA) was selected.Because the region with the insertion was found to contain no restriction sites, dCAPS primers were created for the identification of this allele.The penultimate nucleotide in the reverse primer dCsdw1.c-Rwas modified in such a way that the PCR products of all alleles, except sdw1.c,formed a restriction site, CCGG, characteristic of HpaII (Figure 2).
To develop a DNA marker for the sdw1.callele of the HvGA20ox2 gene, a 4 bp insertion located in the first exon at the 64 position (+GTTA) was selected.Because the region with the insertion was found to contain no restriction sites, dCAPS primers were created for the identification of this allele.The penultimate nucleotide in the reverse primer dCsdw1.c-Rwas modified in such a way that the PCR products of all alleles, except sdw1.c,formed a restriction site, CCGG, characteristic of HpaII (Figure 2).In a similar way, a dCAPS marker was developed for the insertion 1508 (+A) in the ari-e.GP allele of the HvDep1 gene.Due to a modification of the reverse primer sequence, the PCR product contained the restriction site CATCC (instead of the original CAACC), which allowed this mutation to be distinguished from all other alleles by FokI restriction (Figure 3 and Supplementary Table S1).The mechanism of the dCAPS marker for ari-e.GP allele action is similar: a restriction site is formed in the wild type during amplification, while the cutting of PCR products does not occur in the mutant allele ari-e.GP.In a similar way, a dCAPS marker was developed for the insertion 1508 (+A) in the ari-e.GP allele of the HvDep1 gene.Due to a modification of the reverse primer sequence, the PCR product contained the restriction site CATCC (instead of the original CAACC), which allowed this mutation to be distinguished from all other alleles by FokI restriction (Figure 3 and Supplementary Table S1).The mechanism of the dCAPS marker for ari-e.GP allele action is similar: a restriction site is formed in the wild type during amplification, while the cutting of PCR products does not occur in the mutant allele ari-e.GP.

Markers Approbation
tion located in the first exon at the 64 position (+GTTA) was selected.Because the region with the insertion was found to contain no restriction sites, dCAPS primers were created for the identification of this allele.The penultimate nucleotide in the reverse primer dCsdw1.c-Rwas modified in such a way that the PCR products of all alleles, except sdw1.c,formed a restriction site, CCGG, characteristic of HpaII (Figure 2).In a similar way, a dCAPS marker was developed for the insertion 1508 (+A) in the ari-e.GP allele of the HvDep1 gene.Due to a modification of the reverse primer sequence, the PCR product contained the restriction site CATCC (instead of the original CAACC), which allowed this mutation to be distinguished from all other alleles by FokI restriction (Figure 3 and Supplementary Table S1).The mechanism of the dCAPS marker for ari-e.GP allele action is similar: a restriction site is formed in the wild type during amplification, while the cutting of PCR products does not occur in the mutant allele ari-e.GP.

Markers Approbation
The testing of all CAPS and dCAPS markers for the detection of the sdw1.d,sdw1.c,uzu1.a, and ari-e.GP alleles was carried out on a sample of 12 barley accessions from the VIR collection with the known alleles of dwarfing genes.
To identify the sdw1.dallele (SNP G→A) of the HvGA20ox2 gene, the corresponding CAPS marker was used (Supplementary Table S1).The presence of the sdw1.dallele was determined from the absence of PCR products' restriction with the HvGA20ox2F/R primers, while all other alleles, including the wild type, had a site for the HaeIII restriction enzyme in the SNP region (Figure 4a).The sdw1.d allele was detected in control accessions k-19691 Diamant (Czechoslovakia), k-30293 Franklin (Australia), k-31247 Tipple (Great Britain), and k-31355 Braemar (Germany).Accessions with the sdw1.aallele had no amplification products with the HvGA20ox2F/R primers because they are characterized by a complete Plants 2024, 13, 376 7 of 14 deletion of the gene, which was confirmed in accessions k-30291 Yerong (Australia) and k-19037 Jotun (Sweden) carrying sdw1.a(Figure 4a) [6,24].
To identify the sdw1.dallele (SNP G→A) of the HvGA20ox2 gene, the corresponding CAPS marker was used (Supplementary Table S1).The presence of the sdw1.dallele was determined from the absence of PCR products' restriction with the HvGA20ox2F/R primers, while all other alleles, including the wild type, had a site for the HaeIII restriction enzyme in the SNP region (Figure 4a).The sdw1.d allele was detected in control accessions k-19691 Diamant (Czechoslovakia), k-30293 Franklin (Australia), k-31247 Tipple (Great Britain), and k-31355 Braemar (Germany).Accessions with the sdw1.aallele had no amplification products with the HvGA20ox2F/R primers because they are characterized by a complete deletion of the gene, which was confirmed in accessions k-30291 Yerong (Australia) and k-19037 Jotun (Sweden) carrying sdw1.a(Figure 4a) [6,24].To detect the sdw1.callele of the HvGA20ox2 gene, we used the dCAPS marker developed in this study, which detects a 4 bp insertion at the 64 position (+GTTA) (Supplementary Table S1).The sdw1.c allele was detected in the controls k-19308 Dayton (USA) and k- To detect the sdw1.callele of the HvGA20ox2 gene, we used the dCAPS marker developed in this study, which detects a 4 bp insertion at the 64 position (+GTTA) (Supplementary Table S1).The sdw1.c allele was detected in the controls k-19308 Dayton (USA) and k-19808 Deba Abed (Denmark); there was no cutting of PCR products in their case (Figure 4b).
The uzu1.a mutant allele of the HvBRI1 gene was determined using the dCAPS marker proposed by Saisho et al. (2004).The marker identifies the SNP A→G substitution at the 2612 position from the start codon [16].The presence of uzu1.a was determined by the restriction of PCR products (BstHHI restriction enzyme) (Supplementary Table S1).The used sample contained no controls with the uzu1.aallele from literary sources.However, several accessions, which presumably carry this allele, were selected based on the phenotypic assessment of seedlings that have a small hook or protrusion at the coleoptile tip, as is characteristic of uzu1.a.Indeed, the dCAPS marker confirmed the presence of uzu1.a in k-21338 Ehimehadaka N4, k-21341 Jamatohadaka, k-21363 Shikokuhadaka N3, and k-21378 Shinjinryoku N1 (Figure 5).
the restriction of PCR products (BstHHI restriction enzyme) (Supplementary Table S1).The used sample contained no controls with the uzu1.aallele from literary sources.However, several accessions, which presumably carry this allele, were selected based on the phenotypic assessment of seedlings that have a small hook or protrusion at the coleoptile tip, as is characteristic of uzu1.a.Indeed, the dCAPS marker confirmed the presence of uzu1.a in k-21338 Ehimehadaka N4, k-21341 Jamatohadaka, k-21363 Shikokuhadaka N3, and k-21378 Shinjinryoku N1 (Figure 5).Also, the dCAPS marker was used to differentiate the ari-e.GP allele of the HvDep1 gene.We developed primers that flank the region with the A insertion at the 1508 position in the start codon (Supplementary Table S1).During amplification, a CATCC restriction site, the recognition site for FokI restriction endonuclease, is formed in wild-type alleles; no cutting of 154 bp PCR products in the mutant allele occurs (Figure 4d).Testing of the dCAPS marker for the identification of ari-e.GP confirmed its presence in the control accession k-20491 Golden Promise (Great Britain).
To sum it up, the testing of CAPS and dCAPS markers for the sdw1.d,sdw1.с(HvGA20ox2 gene), uzu1.a(HvBRI1), and ari-e.GP (HvDep1) alleles using a sample of control accessions confirmed that the tested markers effectively differentiate mutant variants from the wild-type and other alleles (Figures 4 and 5).
Barley accessions with various dwarfing alleles of the sdw1/denso gene were most represented in the sample (Table 1).Four accessions, namely k-31430 AF Lucius (Czechia, var.nudum), k-27080 Belorusskij 76 (Belarus, var.nudum), k-28650 S-281 (Mexico, var.himalayense), and k-30284 Namoi (Australia, var.nudum), are characterized by the presence of the sdw1.dgene allele.Based on the absence of amplification products from primers HvGA20ox2F/R, three accessions were identified as presumably carrying the sdw1.aor sdw1.e allele.These accessions characterized by a large deletion affecting the HvGA20ox2 gene are k-28957 Karan 3 (India, var.coeleste), k-28961 Karan 19 (India, var.coeleste), and Also, the dCAPS marker was used to differentiate the ari-e.GP allele of the HvDep1 gene.We developed primers that flank the region with the A insertion at the 1508 position in the start codon (Supplementary Table S1).During amplification, a CATCC restriction site, the recognition site for FokI restriction endonuclease, is formed in wild-type alleles; no cutting of 154 bp PCR products in the mutant allele occurs (Figure 4d).Testing of the dCAPS marker for the identification of ari-e.GP confirmed its presence in the control accession k-20491 Golden Promise (Great Britain).
To sum it up, the testing of CAPS and dCAPS markers for the sdw1.d,sdw1.c(HvGA20ox2 gene), uzu1.a(HvBRI1), and ari-e.GP (HvDep1) alleles using a sample of control accessions confirmed that the tested markers effectively differentiate mutant variants from the wild-type and other alleles (Figures 4 and 5).
The sdw1.c allele was identified in two accessions: k-29730 Xima La 6 (China, var.coeleste) and k-26209 Potra (Finland, var.parallelum).Additionally, this allele was detected in four more accessions, though in combination with the dwarfing allele uzu1.a of the uzu1 gene; these accessions are k-21338 Ehimehadaka N4 (Japan, var.subnudipyramidatum), k-21341 Jamatohadaka (Japan, var.subnudipyramidatum), k-21363 Shikokuhadaka N3 (Japan, var.brevisetum), and k-21378 Shinjinryoku N1 (Japan, var.brevisetum).The mentioned accessions belong to six-row naked forms of Japanese origin.Indeed, the presence of the uzu1.aallele is typical for samples of Chinese, Japanese, and Korean origin [16].A study by Zhang J. and Zhang W. (2003) showed that 68.4% of barley cultivars bred in China originated from only six dwarf cultivars, four of which carried a mutant allele of the uzu1 gene, and the other two probably carried a mutation in the sdw1/denso gene [25].
During the screening of the sample using the dCAPS marker of the sdw1.callele designed in the present study, 12 accessions were found to differ in terms of the sdw1.callele by a larger size of PCR products (Figure 4b).This may indicate the presence of an insertion in the sdw1/denso gene sequence.
The studied sample was found to contain no barley accessions with the ari-e.GP allele (ari-e gene); a restriction of PCR products occurred in all genotypes, except for the control k-20491 cv.Golden Promise (Figure 4d).
The remaining seven barley accessions did not show the presence of the studied alleles of the dwarfing genes.
A comparison of the phenotyping and molecular screening data (Table 1 and Supplementary Figures S1 and S2) showed that all barley accessions with the sdw1.dand sdw1.a/sdw1.e alleles of the sdw1/denso gene (HvGA20ox2) are characterized by medium resistance to lodging.High resistance to lodging was demonstrated by accessions with the sdw1.callele, or with its combination with the uzu1.aallele of the HvBRI1 gene.The height of these accessions did not exceed 62 cm in the years with high humidity, and 43 cm in the driest period.The accessions with these alleles or combinations of alleles are a promising source material for their introduction into the breeding process as sources of dwarfness and lodging resistance.
Among the accessions that did not show the presence of the studied alleles of the dwarfing genes, the results of three-year observations have shown three dwarf ones to be most stable, namely k-31431 AF Cesar (Czechia), k-28868 complex hybrid (Mexico), and Kibtsel (Leningrad Province).Most likely, their resistance to lodging is due to the presence of other dwarfing genes and requires further study.

New Allelic Variant of the sdw1/denso (HvGA20ox2) Gene
To confirm the insertion in the sdw1/denso gene, identified using dCAPS and InDel markers, we carried out the sequencing of PCR products.In the case of the MC40861P3F/R primers selected from the literature, sequencing was carried out for the following barley accessions: cv.Dolma (GenBank: PP053705), cv.Makbo (PP053704), cv.Rimpaus Nackta (PP053706), C.I.10405 (PP053707), and k-8953 (PP053708); those carrying the new identified insert; and cvs.Deba Abed (sdw1.callele, PP053703) and Morex (wild type allele, PP053702).The sequencing of PCR products from the dCsdw1.c-F/Rprimers designed in the present study was carried out for the accession k-18374 (PP053712) with an insert; for Dayton (PP053710) and Xima La 6 (PP053711), both with the sdw1.callele; and for Morex (PP053709).
Indeed, all accessions with a larger PCR product contained a 166 bp insertion (Figure 6 and Supplementary Figure S3) located at the 64 bp position from the beginning of the 5 non-coding region of the first exon of the HvGA20ox2 gene.It was the same in all the six sequenced barley accessions.It is noteworthy that the sdw1.callele has a characteristic GTTA insertion located at the position that completely coincides with the 3 end of the 166 bp insertion identified in the present study at the same position.The BLAST search for the sequence of the 166 bp insertion identified high homology (92.02%, 98% coverage) to a region annotated as transposon Thalos_2 (GenBank: KR813336.1).This transposon is 162 bp long and does not contain the GTTA sequence at its end, characteristic of the sdw1.callele.The alignment of the sequencing results also revealed a single nucleotide G→A substitution at the 135 position from the beginning of the first exon.Like the 166 bp insertion, this SNP is located in the first exon upstream of the start codon.
As a result, we identified a new allele of the sdw1/denso gene, characterized by an insertion of the Thalos_2 transposon, a subsequent GTTA insertion, common with the sdw1.callele, as well as by a single-nucleotide G→A substitution at the 165 position, which we named sdw1.ins.The same sequence with exactly the same transposon insertion and single nucleotide substitution was found in the barley pan-genome [26] in three accessions (HOR8148, HOR10350, and HOR13821).However, the allelic composition of the HvGA20ox2 gene was not described by the authors.
sequence of the 166 bp insertion identified high homology (92.02%, 98% coverage) to a region annotated as transposon Thalos_2 (GenBank: KR813336.1).This transposon is 162 bp long and does not contain the GTTA sequence at its end, characteristic of the sdw1.callele.The alignment of the sequencing results also revealed a single nucleotide G→A substitution at the 135 position from the beginning of the first exon.Like the 166 bp insertion, this SNP is located in the first exon upstream of the start codon.As a result, we identified a new allele of the sdw1/denso gene, characterized by an insertion of the Thalos_2 transposon, a subsequent GTTA insertion, common with the sdw1.callele, as well as by a single-nucleotide G→A substitution at the 165 position, which we named sdw1.ins.The same sequence with exactly the same transposon insertion and single nucleotide substitution was found in the barley pan-genome [26] in three accessions (HOR8148, HOR10350, and HOR13821).However, the allelic composition of the HvGA20ox2 gene was not described by the authors.
The allele sdw1.ins was detected in 12 spring barley accessions of different ecological and geographical origin (Table 1).The plant height in accessions with the insertion ranged from 36 to 91 cm and varied depending on weather conditions.Under different weather conditions, accessions with the sdw1.insallele did not retain high lodging resistance.According to the data from 2022 to 2023, it was medium or low.Presumably, this insertion at the beginning of the non-coding region does not affect lodging resistance.The greater part of the accessions is represented by local forms, mainly from high mountainous regions.The allele sdw1.ins was detected in 12 spring barley accessions of different ecological and geographical origin (Table 1).The plant height in accessions with the insertion ranged from 36 to 91 cm and varied depending on weather conditions.Under different weather conditions, accessions with the sdw1.insallele did not retain high lodging resistance.According to the data from 2022 to 2023, it was medium or low.Presumably, this insertion at the beginning of the non-coding region does not affect lodging resistance.The greater part of the accessions is represented by local forms, mainly from high mountainous regions.Their ecogeographic diversity suggests that the transposon insertion occurred independently.

Plant Material
The material for the study was 32 height-contrasting barley accessions from the VIR collection.They were represented by 14 botanical varieties from 19 countries.A set of 12 cultivars and almost isogenic lines, selected from literature sources, was taken as the control.

Phenotyping
Phenotyping with respect to plant height and lodging resistance was carried out in accordance with the "Methodological guidelines for studying and maintaining the global collection of barley and oat" [27] in 2021-2023 on the fields of the Research and Practical Base "Pushkin and Pavlovsk Laboratories of VIR".

Primers Design
The sequences of different alleles of the HvGA20ox2 and HvDep1 genes were aligned using the MEGA XI [28] and Unipro UGENE programs [29].
The whole genome assembly of cv.Morex (MorexV3) was used as a source of wildtype alleles of HvGA20ox2 and HvDep1 genes.The sequence of the sdw1.callele was given according to Y. Xu et al., 2017 [4].The ari-e.GP allele of the HvDep1 gene was extracted from the genome assembly of cv.Golden Promise (GPv1).
Primers for dCAPS markers were designed using the dCAPS Finder 2.0 program [30].Annealing specificity was tested via in silico PCR, using genome assemblies of Morex (MorexV3) and Golden Promise (GPv1) cultivars.The quality control of primers, i.e., their tendency to form secondary structures (hairpins and homo-and heterodimers) was assessed using the OligoAnalyzer resource (Integrated DNA Technologies, Inc., Coralville, IA, USA).

Molecular Screening
DNA was isolated from the upper leaves of individual plants (3 plants per accession) collected during the heading period in the field.The method of SDS extraction was used [31].The concentration and quality of the isolated DNA were controlled by an Implen N60 nanophotometer (IMPLEN, Munich, Germany), as well as by the 0.8% agarose gel electrophoresis.
Intragenic DNA markers selected from literature sources were chosen for molecular screening of the sdw1.cand sdw1.dalleles of the HvGA20ox2 gene and the uzu1.aallele of the HvBRI1 gene.Additionally, dCAPS markers were developed for sdw1.c, as well as for the ari-e.GP allele of the HvDep1 gene (Figures 1-3).The primers used in the study and PCR programs for them are listed in Supplementary Table S1.
PCR was carried out under standard conditions.The PCR with primers MC40861P3F/R and primers for detecting the uzu1.aallele used 20 µL of a reaction mixture that contained 40 ng of template DNA, 1× reaction buffer (Dialat, Moscow, Russia), 1.5 mM MgCl 2 , 0.3 mM of each dNTP, 200 nM of each forward and reverse primers (Evrogen, Moscow, Russia), and 1 unit of Taq polymerase (Dialat, Russia).For all other PCR primers, the mixture contained 2.5 mM MgCl 2 .
The restriction of PCR products was carried out overnight in 15 µL of the reaction mixture according to the manufacturer's protocol (SibEnzyme, Novosibirsk, Russia).
PCR products were separated via electrophoresis in 2% and 3% agarose gels at 5 V/cm of gel length for 2-3 h.The gels were stained with ethidium bromide and visualized in UV light.

Sequencing
Amplification products generated by the MC40861P3 F/R and dCsdw1.c-F/Rprimers were used for sequencing.PCR products were purified using the Cleanup Standard Kit (Evrogen, Russia).Sanger sequencing was performed on a 24-capillary 3500xL Genetic Analyzer (Applied Biosystems, Waltham, MA, USA) at the Center for Shared Use "Genomic Technologies, Proteomics and Cell Biology" of the All-Russia Research Institute for Agricultural Microbiology.The sequences were read and aligned in the programs MEGA XI [28], Unipro UGENE [29], and BioEdit Sequence Alignment Editor [32].

Conclusions
The present study resulted in the development of dCAPS markers for identifying the sdw1.cdwarf alleles of the HvGA20ox2 gene and ari-e.GP of the HvDep1 gene in barley.These alleles are widespread and have been used for many years in breeding aimed at reducing plant height.The advantage of dCAPS markers is the ability to identify alleles on standard PCR equipment using agarose gel electrophoresis, as well as good reproducibility and unambiguous interpretation of the obtained results.
The testing of all CAPS and dCAPS markers on a sample of barley accessions with the known dwarfing alleles sdw1.d and sdw1.c of the HvGA20ox2 gene and uzu1.a of the HvBRI1 gene, as well as ari-e.GP and wild-type alleles, showed complete compliance with the expected results and high efficiency of mutant variant differentiation.
The molecular screening of 32 height-contrasting barley accessions from the VIR collection has found the sdw1.dallele of the sdw1/denso gene in four accessions, the sdw1.aor sdw1.e allele of the sdw1/denso gene in three accessions, the sdw1.callele of the sdw1/denso gene in two accessions, and the sdw1.callele in combination with the uzu1.aallele of the uzu1 gene in four accessions.The ari-e.GP allele was absent in the studied sample; it was identified only in control cultivars.A comparison of the results of genotyping and phenotyping has shown that the presence of dwarfing alleles in all genotypes determines high or medium lodging resistance, regardless of the influence of weather conditions.
The accessions with the identified alleles of the sdw1 (HvGA20ox2) and uzu1 (HvBRI1) genes are recommended for the use as sources of dwarfness and resistance to lodging in breeding programs.
In 12 samples, a previously undescribed allele of the sdw1/denso gene was identified which differs from the sdw1.callele because of its larger size of PCR products generated by the same primers.The presence of a 166 bp insertion was confirmed by sequencing with two pairs of primers, dCsdw1.c-F/Rand MC40861P3F/R.A new allele of the HvGA20ox2 gene that was identified in this study was named sdw1.ins.It is characterized by a Thalos_2 transposon insertion; a subsequent GTTA insertion, which is common with the sdw1.callele; and a single-nucleotide G→A substitution at the 165 position.
(MorexV3).To localize mutations characteristic of the sdw1.cand sdw1.dalleles, the data from the work by Y. Xu et al. (2017) were used [4].This way, a consensus diagram of the HvGA20ox2 gene was constructed which reflects the localization of mutations associated with the sdw1.cand sdw1.dalleles (Figure 1).

Figure 1 .
Figure 1.Diagram of the HvGA20ox2 gene with the localized mutation characteristic of the sdw1.c,sdw1.ins, and sdw1.dalleles, as well as the location of primers for their detection.The non-coding regions of exons are highlighted in light gray; the coding ones are highlighted in dark gray.

Figure 1 .
Figure 1.Diagram of the HvGA20ox2 gene with the localized mutation characteristic of the sdw1.c,sdw1.ins, and sdw1.dalleles, as well as the location of primers for their detection.The non-coding regions of exons are highlighted in light gray; the coding ones are highlighted in dark gray.

Figure 2 .
Figure 2. Sequence alignment flanked by the dCsdw1.c-F/RdCAPS primers for wild-type (from the MorexV3 genome assembly) and sdw1.c(cv.Deba Abed according to Xu et al., 2017 [4]) alleles of the HvGA20ox2 gene.The oval indicates the nucleotide that has been changed in the dCAPS-primer compared to the original gene sequence.

Figure 3 .
Figure 3. Sequence alignment flanked by the dCari-e.GP-F/R dCAPS primers for the wild-type (from the MorexV3 genome assembly) and ari-e.GP (from the GPv1 assembly) alleles of the HvDep1 gene.The oval indicates the nucleotide that was changed in the dCAPS-primer compared to the original gene sequence.

Figure 2 .
Figure 2. Sequence alignment flanked by the dCsdw1.c-F/RdCAPS primers for wild-type (from the MorexV3 genome assembly) and sdw1.c(cv.Deba Abed according to Xu et al., 2017 [4]) alleles of the HvGA20ox2 gene.The oval indicates the nucleotide that has been changed in the dCAPS-primer compared to the original gene sequence.

Figure 2 .
Figure 2. Sequence alignment flanked by the dCsdw1.c-F/RdCAPS primers for wild-type (from the MorexV3 genome assembly) and sdw1.c(cv.Deba Abed according to Xu et al., 2017 [4]) alleles of the HvGA20ox2 gene.The oval indicates the nucleotide that has been changed in the dCAPS-primer compared to the original gene sequence.

Figure 3 .
Figure 3. Sequence alignment flanked by the dCari-e.GP-F/R dCAPS primers for the wild-type (from the MorexV3 genome assembly) and ari-e.GP (from the GPv1 assembly) alleles of the HvDep1 gene.The oval indicates the nucleotide that was changed in the dCAPS-primer compared to the original gene sequence.

Figure 3 .
Figure 3. Sequence alignment flanked by the dCari-e.GP-F/R dCAPS primers for the wild-type (from the MorexV3 genome assembly) and ari-e.GP (from the GPv1 assembly) alleles of the HvDep1 gene.The oval indicates the nucleotide that was changed in the dCAPS-primer compared to the original gene sequence.

Figure 6 .
Figure 6.Sequence alignment of PCR-products flanked by the dCsdw1.c-F/Rprimers.The sequence homologous to the Thalos_2 transposon is highlighted in bold.The oval indicates the nucleotide that was changed in the dCAPS-primer compared to the original gene sequence.

Figure 6 .
Figure 6.Sequence alignment of PCR-products flanked by the dCsdw1.c-F/Rprimers.The sequence homologous to the Thalos_2 transposon is highlighted in bold.The oval indicates the nucleotide that was changed in the dCAPS-primer compared to the original gene sequence.

Table 1 .
Characteristics of barley accessions with respect to plant height and lodging resistance, Pushkin, 2021-2023.