Genetic diversity of barley accessions from Mongolia for greenbug resistance

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BACKGROUND

The greenbug Schizaphis graminum Rondani has caused significant damage to barley crops in the southern regions of the Russian Federation. The degree of harmfulness of the insect depends on the timing of plant colonization (the insects cause the most significant damage to winter and spring crops when migrating to the fields in the germination phase) and the abundance and duration of feeding of the phytophage [1]. The aphid’s harmfulness to barley can be significantly limited using the breeding and cultivation of resistant varieties. The specificity of the relationships between S. graminum and host plants, including barley, has been investigated previously [2]. However, the ability of S. graminum to adapt to a resistant host determines the need for a constant search for new resistance donors for breeding.

To date, only four alleles of two genes have been identified that control barley’s resistance to individual biotypes of the greenbug in the USA. Back in 1945, I.M. Atkins and R.G. Dahms [3] identified two Korean varieties of winter barley, Omugi and Dobaku, which showed high heritability of the resistance trait. Various commercial barley varieties have subsequently been produced using Omugi. An analysis of the inheritance of aphid resistance showed that the Omugi, Dobaku, Derbent, Kearney, and Will varieties have a common dominant resistance allele, subsequently designated Rsg1a, and then Rsg1 [4–7]. According to the results of trisomic analysis, the Will resistance gene is localized in the centromeric segment of chromosome 1 [7]. With the use of molecular markers, the Rsg1 locus was mapped on the long arm of chromosome 3H [8]. The Post variety was selected from the Harrison × Will hybrid population by individual selection [9]; however, the heterogeneity of this variety in terms of resistance to the aphid necessitated the selection of the Post 90 variety [10]. The Rsg1a gene is effective against aphid biotypes B–G, I–K, CWR, and WWG, but not against biotype H [11–14].

The dominant allele Rsg2b, which confers resistance to the same aphid biotypes as Rsg1a, was identified in a native barley accession from Pakistan, PI 426756 [11, 14, 15]. The expression of the Rsg2b gene is somewhat lower than that of Rsg1a; therefore, the Post 90 variety is preferable for breeding [16]. In subsequent experiments, PI 426756 was found to be more resistant than Post 90 to biotype E [17]. In addition, the Rsg2b gene, in contrast to Rsg1a, is effective against the aphid TX1 isolate, where a differential interaction between the insect and the host plant was noted. Based on this, new gene symbols, Rsg1 and Rsg2, have been proposed. The variety Wintermalt, which is susceptible to all other intraspecific forms of the insect, is weakly damaged by the G and J biotypes [12, 14]. Resistance to biotype G is also possessed by the Coulter and Bancroft varieties, which are recommended for breeding [18]. However, the Wintermalt and Coulter varieties are severely damaged by the TX1 aphid biotype [17].

The complexity of the Rsg1 locus has now been identified, where an accession of Hordeum vulgare ssp. spontaneum WBDC336 (PI 682028) had the Rsg1.a3 allele, which provides resistance to biotypes C, E, H, I, WY81, WY12 MC, and WY86 of the greenbug [19]. It was also established that the accession of H. vulgare ssp. spontaneum WBDC053 (PI 681777) carried the Rsg2.a3 allele either closely linked or allelic to Rsg2. WBDC053 is resistant to biotypes B, C, E, I, TX1, WY4A, WY4B, WY81, WY12MC, and WY86, but is severely damaged by aphid biotypes F, H, WY10MC, and WY10B [20].

Accessions of local barley represent a rich source of replenishment of the bank of effective resistance genes to S. graminum. Thus, among the accessions from East and South Asian countries, heterogeneous forms were identified that differed in the level of resistance to the Krasnodar population of the insect. The high resistance of 98 accessions is controlled by alleles that are not identical to Rsg1. As a result of the testing of 150 accessions from Mongolia, three heterogeneous forms were identified, among which, two accessions contained plants with high resistance and one accession contained plants with moderate resistance to the aphid [21].

The experiments mentioned above were performed in 2002–2004 using the Krasnodar aphid population. The level of resistance of Mongolian barley accessions to new populations of S. graminum almost 20 years later is an interesting question. In this work, we study the efficiency and genetic control of aphid resistance in barley accessions isolated in previous experiments.

MATERIALS AND RESEARCH METHODS

We studied the resistance to the Krasnodar (branch of the N.I. Vavilov All-Russian Institute of Plant Genetic Resources, Kuban experimental station (KES VIR, Gulkevichsky district) population of the greenbug in 175 barley accessions from Mongolia. In addition, the resistance of the Post cultivar (k-31204, USA), containing the previously identified Rsg1 resistance gene, and lines selected from heterogeneous Mongolian accessions (k-3885, k-3904, and k-4080) were evaluated. Segregation in resistance to S. graminum of F₂ hybrids resulting from crossing the selected forms with the susceptible variety Belogorskiy (k-22089, Russia, Leningrad region) was also analyzed.

The work was performed in a light room, where the air temperature was maintained at 20°C–25°C. Insects for laboratory experiments were collected on sorghum crops from KES VIR in July (the period of maximum pest abundance). A susceptible accession of sorghum was used [Sorghum bicolor (L.) Moench] SLV-2, as well as the varieties Efremovskoe beloe and Kubanskoe krasnoe 1677, which are moderately resistant to aphids.

In the laboratory, the collected samples of the aphid were cloned. Sprouted barley seeds of the Belogorskiy variety were laid out on cotton wool moistened with water, placed in halves of Petri dishes, then one female was placed on each seedling, and the dish was covered with a glass cage, while the upper part was covered with silk bolting cloth. Cages with aphid clones were placed on light units equipped with fluorescent lamps. Thirty-five clones were collected from the SLV-2 accession, 29 clones were collected from Kubanskoe krasnoe 1677, and 22 clones were collected from Efremovskoe beloe.

To assess the resistance of the barley accessions, the seeds were sown in rows in plastic flats filled with a non-sterile soil mixture. Two rows of susceptible control (variety Belogorskiy), 10 rows of tested forms, and 1 row of cultivar Post were sown. Juvenile plants were infested with a mixture of clones, and aphids of different ages were shaken off (4–5 ind./plant) on the barley accessions tested. At the feeding site of S. graminum, plant tissues become necrotic, which enables us to test the plant damage. When the control died (usually on day 10–14 after infestation), the resistance was assessed on a scale from 0 (no damage) to 10. Plants with scores of 1–4 (damaged up to 30% of the leaf surface) were classified as resistant, while those with scores of 9–10 were classified as susceptible [22]. The samples identified as resistant were then tested again.

Damage to the Post variety and three resistant barley lines from Mongolia were assessed using 86 aphid clones. For this purpose, experimental accessions and the susceptible control were sown in vessels with soil in a circular order and covered with glass cages. Seedlings were infested with aphids of the same clone at a rate of five individuals per plant, and when the control died, the resistance of plants was assessed according to the scale described above. If the results were not clear, the experiment was repeated.

To determine the number and nature of the interactions of the resistance-controlling genes, the resistant lines k-3885, k-3904, and k-4080 (maternal forms) and the non-resistant variety Belogorskiy (paternal form) were crossed. Segregation of F₂ hybrids for aphid resistance was analyzed in a laboratory. Seeds were sown in rows in plastic flats containing soil. One row each of Р₁, P₂, and F₁, and 7–8 rows of F₂ were sown. The F₂ seeds were the offspring of a single F₁ plant. Weak or late-sprouting plants were removed 2–3 days after germination. When the second leaf appeared, the hybrids were infested by the aphid (a clone avirulent to resistant lines) at a rate of four individuals per plant. The flats were examined after 2 days and, if necessary, additional infestation was performed. During the period of death of a susceptible parent, the damage to hybrids was assessed. To reduce classification errors of phenotypes, two counts were usually performed: one when approximately 70% of the plants of a susceptible form had died off and one after 2–3 days, when only single plants of the Belogorskiy variety were characterized by a damage score of 9. Plants similar to the paternal form (scores of 9 or 10) were considered homozygous non-resistant (S). The resistant class (R) included plants with similar degrees of damage as the maternal form. To determine the correspondence between the obtained (observed) and theoretically expected data, the χ² test was used.

RESULTS AND DISCUSSION

The results of the assessment of resistance of the collection of barley accessions from Mongolia to the “old” and “new” populations of S. graminum generally coincided (Table 1). In 2021, a larger number of accessions was noted, where plants with high (3 points) resistance to the pest were identified. Apparently, these accessions were not isolated in 2004 due to the low frequency of this phenotypic class. In 2021, 28 heterogeneous accessions were identified, in which the manifestation of the resistant component varied within 5–7 points (31%–60% of the leaf surface was damaged).

 

Table 1. Barley accessions from Mongolia, distinguished by their resistance to the greenbug, S. graminum

VIR catalogue number	Accession	Variety	Resistance, score
			2004	2021
3885	Landrace	Pallidum, coeleste, violaceum, himalayense	4–6, 8, 9	7–9
3904	«	Coeleste	4–6, 8, 9	5, 7, 8
3926	«	Pallidum	10	3, 7, 8
4080	«	Coeleste, revelatum	5, 7–10	4–10
21731	«	Revelatum, coeleste	10	3, 8, 10
21737	«	Coeleste	6, 7	3, 7, 8
22089	Belogorskiy (control)		9, 10	9, 10

 

The significant variability of the trait may be due to the manifestation of genes with low expressivity and/or the presence of clones with different virulence to the studied forms in the S. graminum population. The infestation of pure lines isolated from heterogeneous accessions (k-3885, k-3904, and k-4080) with a mixture of clones (population) of aphids caused plant damage with scores of 2–8 points. When these lines were infested with insect clones, three phenotypic classes were noted: resistant (1–4 points, manifestation of genes with high expressivity in this accession to this clone), moderately resistant (5–8 points, the action of genes with a weak phenotypic manifestation), and susceptible (9–10 points, virulence of this clone to both major and minor resistance genes). Consequently, both hypotheses were confirmed, where lines protected by aphid resistance genes with a distinct and poor phenotypic manifestation, which differentially interact with insect genotypes, were identified.

The frequency of the detection of aphid clones virulent to barley accessions with clearly manifested resistance genes on sorghum varieties differed significantly, as when feeding in the field on moderately resistant sorghum varieties, there was a clear accumulation in comparison with the susceptible accession SLV-2 (an excess of 1.6–2.2 times) of S. graminum clones that were virulent to the barley accessions (Table 2). The efficiency of the resistance genes was low, as 32%–45% of the clones in the Krasnodar insect population are virulent to accessions from Mongolia and to the Post cultivar.

 

Table 2. Frequency of virulent to barley accessions S. graminum clones collected on three sorghum varieties (%)

Barley accession	Sorghum variety
	SLV-2	Efremovskoe beloe	Kubanskoe krasnoe 1677
Post	22.9	40.9	41.4
k-3904	20.0	31.8	44.8
k-4080	25.7	54.5	55.2
k-3885	28.6	45.5	48.3

 

As a result of the assessment of resistance of four accessions of barley to 86 clones of S. graminum, 15 virulence phenotypes were identified (12 each among clones collected on SLV-2 and Kubanskoe krasnoe 1677, and 10 on Efremovskoe beloe). The phenotype that was avirulent to all barley accessions dominated (Table 3). Two virulence phenotypes (10 and 14) were noted only among the collections from Kubanskoe krasnoe 1677, and phenotype 7 was unique for the SLV-2 accession.

 

Table 3. Phenotypic diversity of the Krasnodar population of S. graminum

Virulence phenotype	Phenotype frequency, %	Barley accessions
		Post	k-3904	k-4080	k-3885
1	34.8	R	R	R	R
2	7.0	S	R	R	R
3	2.3	R	S	R	R
4	4.6	R	R	S	R
5	7.0	R	R	R	S
6	7.0	R	R	S	S
7	1.2	S	S	R	R
8	3.5	R	S	S	R
9	3.5	S	R	R	S
10	1.2	S	R	S	R
11	7.0	R	S	S	S
12	3.5	S	R	S	S
13	1.2	S	S	R	S
14	4.6	S	S	S	R
15	11.6	S	S	S	S
Note. R — resistance, S — accession susceptibility.

 

Greenbug clones with different virulence phenotypes (“test clones”) enable the identification of resistance genes in isolated barley forms. If at least one clone that is avirulent to the tester of a given resistance gene damages the variety under study, this means that the variety does not have a functional allele of this gene. When the experimental accessions were infested with S. graminum with virulence phenotype 2, the Post variety was found to be susceptible, while the accessions from Mongolia were resistant to the pest. This means that accessions k-3904, k-4080, and k388 have resistance gene alleles that differ from the previously identified Rsg1 gene. Comparison of the interaction of experimental accessions with aphids with virulence phenotypes 3, 4, and 5 indicated a difference in the genetic control of the trait in all landraces from Mongolia. These conclusions were supported by a pairwise comparison of the resistance of the accessions to aphids with virulence phenotypes 6–14.

In the analysis of the F₂ hybrids, the damage to lines isolated from accessions k-3904, k-4080, and k3885 varied between 1 and 5 points. In the F₁ hybrid, k-4080 × Belogorskiy, resistance dominated, with a plant damage score of 2–3 points. In two other cases, the level of resistance of heterozygotes was somewhat lower (2–7 points), indicating incomplete dominance of the trait. In the F₂ populations, the resulting phenotype ratio corresponded to the theoretically expected 3R:1S (Table 4), indicating that barley landraces from Mongolia carry by one dominant resistance gene.

 

Table 4. Segregation for resistance to S. graminum of F₂ hybrids from crossing resistant barley accessions with a susceptible tester

Cross combination	Total number of plants	The ratio of phenotypes R : S		χ2	р
		observed	expected
k-3885 × Belogorskiy	125	93 : 32	3 : 1	0.024	0.8–0.9
k-3904 × Belogorskiy	207	147 : 60	3 : 1	1.754	0.1–0.2
k-04080 × Belogorskiy	294	225 : 69	3 : 1	0.367	0.5–0.75
Note. χ20,05 = 3,84. R — resistance, S — susceptibility.

 

CONCLUSION

The genetic diversity of barley accessions from Mongolia in terms of resistance to the greenbug is low. Six heterogeneous accessions were identified, in which plants with high resistance to the pest were detected. Among 28 accessions, damage to the leaf surface of the resistant component varied from 31% to 60%. As a result of assessment of the resistance of pure lines selected from the heterogeneous accessions, k-3885, k-3904, and k-4080, as well as the Post variety (carrier of the previously identified Rsg1 gene) to 86 aphid clones, 15 virulence phenotypes were identified. Accessions k-3885, k-3904, and k-4080 each have one dominant resistance allele, which differ from each other and also differ from Rsg1. S. graminum genotypes were revealed to interact differentially not only with the main genes but also with weakly expressed resistance genes of barley lines.

ADDITIONAL INFORMATION

Author contribution. Thereby, all authors made equal contribution to the conception of the work, acquisition, analysis, interpretation of data for the work, drafting and revising the work, final approval of the version to be published and agree to be accountable for all aspects of the work. Contribution of each author: E.E. Radchenko — concept and design of the study, analysis of the data obtained, writing the text; R.A. Abdullaev — analysis of the data obtained, writing the text; D.E. Akimova — collection and processing of materials, writing text, literature review; I.Yu. Zaitseva — collection and processing of materials, writing text.

Competing interests. The authors declare that they have no competing interests.

Funding source. This work was supported by the Research Foundation Flanders (project No. 21-76-00018, https://rscf.ru/project/21-76-00018/).

About the authors

Evgeny E. Radchenko

N.I. Vavilov All-Russian Institute of Plant Genetic Resources

Author for correspondence.
Email: eugene_radchenko@rambler.ru
ORCID iD: 0000-0002-3019-0306
SPIN-code: 1667-0530
Scopus Author ID: 7005353107

Dr. Sci. (Biol.), Main Research Associate

Russian Federation, Saint Petersburg

Renat A. Abdullaev

N.I. Vavilov All-Russian Institute of Plant Genetic Resources

Email: abdullaev.1988@list.ru
ORCID iD: 0000-0003-1021-7951
Scopus Author ID: 57211915135

Cand. Sci. (Biol.), Senior Research Associate

Russian Federation, Saint Petersburg

Daria E. Akimova

N.I. Vavilov All-Russian Institute of Plant Genetic Resources

Email: nezabutkabitter@yandex.ru
ORCID iD: 0000-0002-0657-1715

Postgraduate Student

Russian Federation, Saint Petersburg

Irina Yu. Zajtseva

Saint Petersburg State Agrarian University

Email: irzajtseva@yandex.ru
ORCID iD: 0000-0001-8283-9747

Student

Russian Federation, Saint Petersburg

References

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