Investigation of mutation load and rate in androgenic mutant lines of rapeseed in early generations evaluated by high-density

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Heliyon 9 (2023) e14065

Available online 2 March 2023

Research article

Investigation of mutation load and rate in androgenic mutant lines of rapeseed in early generations evaluated by high-density

SNP genotyping

Dilyara Gritsenko

^a

, Ainash Daurova

^b

, Alexandr Pozharskiy

^a

, Gulnaz Nizamdinova

^a

, Marina Khusnitdinova

^a

, Zagipa Sapakhova

^b

, Dias Daurov

^b

, Kuanysh Zhapar

^b

, Malika Shamekova

^b

, Ruslan Kalendar

^b

, Kabyl Zhambakin

^b^,^*

aDept. of Molecular Biology, Institute of Plant Biology and Biotechnology, Almaty, 050040, Kazakhstan

bDept. of Breeding and Biotechnology, Institute of Plant Biology and Biotechnology, Almaty, 050040, Kazakhstan

A R T I C L E I N F O Keywords:

Rapeseed

Doubled haploid mutant line SNP genotyping

Genetic pool Biological impact

A B S T R A C T

Oilseed rape (Brassica napus) is an important oil crop distributed worldwide with a broad adaptation to different climate zones. The cultivation of rapeseed is one of the most commercially viable areas in crop production. Altogether 269,093 ha of rapeseed are cultivated in Kazakhstan.

However, all rapeseed cultivars and lines cultivated in Kazakhstan on an industrial scale predominantly belong to the foreign breeding system. Therefore, the formation of a diverse genetic pool for breeding new, highly productive cultivars adopted to the environmental conditions of Kazakhstan is the most important goal in country selection programs. In this work, we have developed ethyl methanesulfonate (EMS) doubled haploid mutant lines from plant material of cultivars ‘Galant’ and ‘Kris’ to broad diversity of rapeseed in Kazakhstan. The development of mutant lines was performed via embryo callusogenesis or embryo secondary callusogenesis.

Mutants were investigated by Brassica90k SNP array, and we were able to locate 24,657 SNPs from 26,256 SNPs filtered by quality control on the genome assembly (Bra_napus_v2.0). Only 18,831 SNPs were assigned to the available annotated genomic features. The most frequent combination of mutations according to reference controls was adenine with guanine (70%), followed by adenine with cytosine (28.8%), and only minor fractions were cytosine with guanine (0.54%) and adenine with thymine (0.59%). We revealed 5606.27 markers for ‘Kris’ and 4893.01 markers for ‘Galant’ by mutation occurrence. Most mutation occurrences were occupied by double mutations where progenitors and offspring were homozygous by different alleles, enabling the selection of appropriate genotypes in a short period of time. Regarding the biological impact of mutations, 861 variants were reported as having a low predicted impact, with 1042 as moderate and 121 as high; all others were reported as belonging to non-coding sequences, intergenic regions, and other features with the effect of modifiers. Protein encoding genes, such as wall- associated receptor kinase-like protein 5, TAO1-like disease resistance protein, receptor-like protein 12, and At5g42460-like F-box protein, contained more than two variable positions, with an impact on their biological activities. Nevertheless, the obtained mutant lines were able to

Abbreviations: EMS, Ethyl methanesulfonate; GWAS, Genome-wide association study; SNP, Single nucleotide polymorphism; WAKL, Wall- associated kinase-like.

* Corresponding author.

E-mail address: [email protected] (K. Zhambakin).

Contents lists available at ScienceDirect

Heliyon

journal homepage: www.cell.com/heliyon

https://doi.org/10.1016/j.heliyon.2023.e14065

Received 21 July 2022; Received in revised form 6 February 2023; Accepted 20 February 2023

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survive and reproduce. Mutant lines, which include moderate and high impact mutations in encoding genes, are a perfect pool not only for MAS but also for the investigation of the fundamental basis of protein functions. For the first time, a collection of mutant lines was developed in our country to improve the selection of local rapeseed cultivars.

1. Introduction

Rapeseed is a valuable source of edible and industrial oils, fodder protein, and diesel fuel. Global experience shows that the cultivation of rapeseed (Brassica napus oleifera Metzg.) is one of the most commercially viable areas in crop production. Accordingly, in Kazakhstan, 269,093 ha are cultivated with rapeseed [1]. At the same time, most farmland is located in high-risk environmental zones, where abiotic and biotic stress factors play an important role in low crop yields [2].

Therefore, increased breeding efficiencies are urgently required, based on creating new cultivars of rapeseed. However, the breeding process in Kazakhstan generally follows traditional methods, which are complex and require considerable time.

Mutagenesis is an effective tool for inducing novel genetic combinations in crops with limited genetic diversity, such as rapeseed [3]. Mutagenesis has also been successfully used to improve several desirable traits, such as early ripeness, dwarfism, resistance to biotic and abiotic stress, seed harvest, and oil quality [4,5]. The production of canola cultivars with no erucic acid and low glucosinolate content allowed the use of rapeseed in the food industry [6].

There are several ways to obtain mutants on a large scale, such as T-DNA insertion and physical- and chemical-induced mutagenesis, which offer great convenience for crop research and breeding. The most widely used physical mutagens include electro- magnetic ionizing radiation, such as gamma rays and X-rays [7]. For example, gamma ray ⁶⁰Co was used to irradiate a popular cultivar of Brassica juncea, resulting in two high-yielding mutants that were eventually developed into commercial cultivars [8]. Ethyl methanesulfonate (EMS) is a widely used chemical mutagen that alkylates the guanine base, which leads mainly to transitions of C/G to T/A [9].

The creation of mutant materials by EMS produces stable genetic variability at the genome level with a high density of point mutations. EMS-induced mutagenesis has been widely used in different crops, such as soybean [10,11], maize [12], rice [13,14], wheat [15], rapeseed [16–18], and tomato [19]. EMS-induced mutagenesis in microspore cultures has been shown to be an effective method for obtaining rapeseed cultivars with high oleic acid and low linolenic acid [20]. Mutagenesis in isolated microspore cultures has several benefits over traditional breeding techniques. For instance, mutagenic treatment of microspores creates homozygous lines with valuable traits, such as improved fatty acid content and no erucic acid [21]. Benefits of haploid cell mutagenesis include: (1) better ability to avoid chimerism; (2) quick mutant detection; (3) the possibility of identifying recessive mutants in the first generation; and (4) less time required to produce homozygous mutants. In particular, breeding is possible at the in vitro stage of cultivation [22,23].

The performance of mutagenesis in isolated microspore cultures has been previously shown in several reports [23–25]. Previously, we investigated the optimization of isolated rapeseed microspore cultivation [20]. In further studies, we also applied an isolated microspore culture to produce mutants.

Mutants/variants with economically valuable traits can be selected after combined genetic and phenotypic investigations in a genome-wide association study (GWAS) analysis. Determining markers associated with valuable traits results in the detection of promising variants/mutants for directed breeding [26]. SNP genotyping with deep genome coverage allows the detection of important markers located in the qualitative trait loci (QTL) regions of the genome [27]. Numerous studies on hybridization and mutagenesis have been conducted to obtain rapeseed cultivars with improved traits for oil composition, yield, biotic/abiotic stress tolerance, and fast flowering [28]. The improved lines/mutants were investigated for the spread of mutations in QTL. The most economically valuable rapeseed trait is oil composition. Recently, a number of studies have been done on obtaining mutants with improved fatty acid parameters of seed oil [29]. Obtaining lines with improved oil parameters is difficult due to the polygenicity of the trait. In addition, oil parameters vary greatly depending on the physical parameters of the environment. Currently, 18 QTL are known to be associated with rapeseed oil content [30]. In particular, chromosomes A09, C07, and C09 contain QTL associated with glucosinolate content [31,32].

The development of a mutant collection as a genetic pool for MAS is the most important goal in the diversity of lines with economically valuable traits. Additionally, different types of mutants reveal genetic plasticity for species, cultivars, and genotypes. Mutant lines with high-impact mutations in encoding genes will explain their functions and indirect involvement in metabolic processes of plants. In this work, we developed EMS doubled haploid mutant lines from plant material of cultivars ‘Galant’ and ‘Kris’ for broad diversity of rapeseed in Kazakhstan. SNP genotyping of the M2 generation revealed mutation load, the mutation rate in different regions of the genome, the distribution of mutations in the genome, and low-, medium-, and high-impact mutations. A recently developed 90 K Illumina Infinium array was used for genotyping. This study demonstrated that plants obtained from haploid microspores via callusogenesis contain a substantially lower mutation load and can be selected for useful traits in the first backcross generations. It is important to note, that the double haploid mutants are perspective genotypes for rapid and efficient selection of cultivars with new traits. But on the other hand, the double haploid mutants could introduce unwanted effects on desirable traits via pleiotropy of genes and non-additive genetic variations, as it was shown in previous works [33–37]. Thereby, mutant lines must be under careful investigation evaluating expression of traits.

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2. Results

2.1. Development and agronomic traits of doubled haploid mutant lines

Mutant lines were developed via embryo callusogenesis or embryo secondary callusogenesis. At high concentrations of the EMS mutagen (12 mM), some embryonic callus browned and died (Fig. 1D). Meanwhile, a higher percentage of the plantlets were obtained at 8 mM EMS, with 98 plantlets from ‘Galant’ and 56 plantlets from ‘Kris.’ A 12 mM concentration of EMS had a high impact on the regeneration process. Thirty-two plantlets of ‘Galant’ and twelve of ‘Kris’ were obtained after treatment with 12 mM EMS. In total, 130 plantlets were obtained for ‘Galant’ and 68 plantlets for ‘Kris.’ All plantlets were cloned for further growth and treated with colchicine before being transferred to the soil. Before treatment with colchicine, the haploid status of every plantlet was confirmed by chloroplast

Fig. 1.The process of obtaining doubled haploid mutant lines in cultures of isolated microspores. A Microspores that had been cultivated for one week, B microspore-derived embryos that were EMS-treated, C secondary embryos production, D brown and dead embryo, E and F regenerated plantlets from embryos treated with EMS. Scale bars for a and b are 100 mm (×100 magnification). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

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counting in stomatal guard cells. The results showed that the number of chloroplasts differed significantly between the haploids and doubled haploids. The average number of chloroplasts in the stomatal guard cells of haploid mutants ranged from 10 to 12 and in doubled haploids from 20 to 24 (Fig. 2A–C). The number of chloroplasts for diploid plants, which, in this case, were considered as controls, was identical to the number of doubled haploids, while a decrease was observed in haploid plants. Thus, fewer chloroplasts were found in the stomatal guard cells of haploid stomata, equivalent to half that of the diploid and doubled haploids.

The obtained doubled haploid mutants were evaluated for their agronomic traits, such as plant height, number of pods, number of seeds per pod, weight of 1000 seeds, and weight of seeds per plant. For further amplification, the mutant plantlets of M1 were selected with parameters of agronomic traits, in the range of +20% and − 20% according to their control cultivars. The color of seeds was also an important parameter; we selected seeds with colors near yellow and black. Only 4 of 17 ‘Kris’ mutant lines showed brown, light brown, or light red color. In the case of ‘Galant’ mutant lines, 9 of 20 lines contained seeds with a light brown or light red color. The other mutant lines had black seeds. We investigated the M2 plants in the same way and selected 18 samples of ‘Kris’ and 20 samples of

‘Galant’ for genetic analysis (Table 2).

2.2. SNP genotyping and genetic investigation of mutant lines

Using the Brassica90k SNP array, 20 mutants of ‘Galant’ and 18 mutants of ‘Kris’ were genotyped along with the original specimens. The genotype call rate among samples varied from 83.43 to 89.14%, with mean and median values of 86.4%; the average median genotype calling score (GC 50%) in samples varied between 0.5001 and 0.5146. For the selected quality threshold for SNPs, 63,599 of 77,970 (81.57%) markers had a call rate ≥0.95, 36,952 (47.39%) had a median GC score ≥0.5, and 38,994 markers (50.01%) were polymorphic (MAF >0). In total, 26,256 (33.67%) SNPs satisfied all filtering criteria.

To make SNP coordinates consistent with the reference rapeseed genome assembly (Bra_napus_v2.0), all markers were physically mapped against the reference sequence using the BLAT pairwise alignment tool. Information on the genomic contexts of all SNPs was retrieved from the Brassica90k SNP manifest file as the left and right flanking sequences. Both flanks were aligned independently against the reference genomic sequences and compared to exclude possible nonspecific matches and ambiguous sites. Some filtering criteria were applied. First, the chromosome IDs of the subject and the query were matched. Second, the interval between the left and right flanking sequences was selected as one base pair, with the account of the assignment of SNPs to the DNA strand. Finally, only matches with at least 95% matching positions and with no more than one insertion or deletion of length five or fewer positions were selected as valid. As a result, genomic coordinates in the Bra_napus_v2.0 assembly were assigned to 24,657 SNPs, and these variants were used to predict possible biological effects. These physically positioned SNPs were used to annotate and predict possible biological consequences using SnpEff software. In total, 18,831 SNPs were assigned to the available annotated genomic features of rapeseed.

The SnpEff program considers all annotated features available for the target genome, so multiple features are reported for each SNP. The protein coding features contained 13,943 variants, 3086 SNPs were present in pseudogenes, and 3185 were identified as belonging to the intergenic regions. For example, the variant in one coding locus can also be reported as the upstream or downstream variant for other features located nearby. In these three main categories, 9253 and 679 were assigned exclusively to protein coding sequences and pseudogenes, respectively, without overlapping feature biotypes; no variants belonging exclusively to the intergenic regions were identified. All annotation data were sorted and filtered so that only a single annotation with the highest potential impact would be assigned for each SNP. After filtering, 13,347 variants were assigned to coding sequences, 1374 to pseudogenes, and 4110 to intergenic regions and other non-coding features. The prevailing numbers of variants were in intergenic regions (3866, 20.5%;

sequence ontology SO:0000605) and upstream (7643, 40.6%; SO:0001631) and downstream gene features (3696, 19.6%;

SO:0001632; Fig. 3; Table 3). Among the other functional classes, the most frequent were missense (1042, 5.53%; SO:0001583),

Fig. 2.Stomatal guard cells with chloroplasts in mutant and control plants of B. napus with different ploidy: A—haploid (number of chloroplasts, 10 pcs.), B—diploid (number of chloroplasts, 22 pcs.), C—doubled haploid (number of chloroplasts, 24 pcs.). Scale bars—100 mm ( × 40 magnification).

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synonymous (633, 3.36%; SO:0001819), and intron variants (595, 3.16%; SO:0001627). The other types of variants, including nonsense variants (SO:0001587), had minor frequencies below 3%.

Of the 18,831 SNPs annotated with SnpEff, 861 variants were reported as having a low predicted impact, 1042 as moderate, and 121 as high; all others were reported as belonging to non-coding sequences, intergenic regions, and other features with the effect of modifiers. Most variants with high impact were annotated as stop gain or nonsense (97; SO:0001587), and 13 and 7 were predicted to potentially affect splicing as splice acceptors (SO:0001574) and donor variants (SO:0001575), respectively. Two SNPs were assigned to potential losses of start (SO:0002012) and stop codons (SO:0001578) (Table 3). All SNPs with a predicted moderate effect were missense variants (SO:0001583). Among the variants with low impact, the most frequent were synonymous changes (633;

SO:0001819), splice region variants (168; SO:0001630), and gained premature start codons (56; SO:0001988).

The information on genes potentially affected by annotated SNPs is provided in Supplementary file 4. In total, 2024 SNPs predicted to have high, moderate, or low impact were distributed among 1806 unique loci, and 391 were uncharacterized. Across all loci, one Table 1

Plant regeneration from microspore-derived embryos of rapeseed (Brassica napus) treated with EMS.

Genotype EMS concentration, mM Number of EMS-treated embryos Number of mutant plantlets

Galant 0 108 96

8 108 98

12 108 32

Kris 0 90 85

8 90 56

12 90 12

Table 2

Agronomic traits of doubled haploid mutant lines of M2.

Name of doubled haploid

line Plant height

(cm) Number of

pods Number of seeds in

pods Weight of seeds from the 1st

plant Weight of 1000

seeds Color of

seeds

Kris (Control) 129.4 ± 9.6 114.4 ± 21.3 15.0 ± 2.8 6.5±1.6 3.1±0.6 black

DH2K12-3 99.8 ±18.3 175.6 ±59.8 12.0 ±5.2 17.4 ±5.7 4.0 ±1.8 black

DH2K12-5 129.3 ±21.3 207.6 ±98.7 16.0 ±3.7 5.3 ±2.7 3.8 ±1.7 light brown

DH2K12-6 123.0 ±18.0 307.8 ±127.6 19.0 ±6.8 13.3 ±9.3 3.8 ±1.6 black

DH2K12-7 125.8 ±17.3 225.6 ±121.8 17.0 ±4.1 9.1 ±4.3 4.5 ±2.0 black

DH2K12-8 121.2 ±24.3 178.3 ±86.8 20.0 ±4.8 11.1 ±6.3 4.2 ±0.9 red brown

DH2K12-10 95.8 ±19.3 152.3 ±97.2 19.0 ±7.3 5.6 ±2.8 3.6 ±1.0 black

DH2K12-11 93.2 ±23.6 162.3 ±89.2 18.0 ±7.1 7.3 ±5.3 3.6 ±0.8 black

DH2K12-12 88.7 ±19.5 149.2 ±99.2 22.0 ±9.2 7.3 ±4.3 3.6 ±1.3 black

DH2K12-13 128.2 ±24.3 214.2 ±116.2 21.0 ±7.5 11.0 ±6.1 3.7 ±1.9 black

DH2K12-14 125.1 ±19.7 212.6 ±115.1 17.0 ±5.9 6.2 ±3.0 4.9 ±2.0 black

DH2K8-1 121.5 ±21.3 163.8 ±82.1 18.0 ±9.8 8.3 ±4.5 4.3 ±1.5 red brown

DH2K8-2 126.4 ±18.6 108.6 ±58.2 16.0 ±6.0 13.4 ±6.1 4.7 ±1.3 black

DH2K8-3 112.9 ±14.6 158.7 ±84.7 17.0 ±8.9 7.2 ±3.9 3.6 ±1.2 black

DH2K8-4 128.2 ±18.2 113.8 ±74.6 16.0 ±6.7 5.3 ±1.3 3.4 ±1.4 black

DH2K8-5 106.8 ±16.8 100.8 ±62.7 14.0 ±8.6 13.1 ±6.9 4.0 ±2.1 brown

DH2K8-6 120.9 ±20.5 92.3 ±54.2 20.0 ±8.3 4.3 ±2.8 3.8 ±1.7 black

DH2K8-7 111.5 ±19.1 154.3 ±41.7 16.0 ±4.8 7.0 ±4.1 4.4 ±1.9 black

DH2K4-1 120.3 ±10.1 121.5 ±71.3 13.0 ±5.2 3.9 ±1.7 3.3 ±1.2 black

Galant (Control) 132.5 ± 5.3 137.5 ± 15.6 17.0 ± 3.2 6.3±1.5 3.4±0.6 black

DH2G12-1 128.5 ±31.2 157.8 ±23.6 18.0 ±3.7 20.0 ±11.3 4.3 ±1.9 black

DH2G12-2 125.3 ±25.8 130.6 ±24.6 16.0 ±5.1 5.9 ±3.9 3.8 ±1.0 red brown

DH2G12-3 119.2 ±21.3 168.9 ±75.3 19.0 ±5.8 9.2 ±3.1 4.4 ±1.9 red brown

DH2G12-5 128.7 ±12.3 142.8 ±55.3 15.0 ±4.1 6.9 ±4.1 3.1 ±1.2 black

DH2G12-6 124.3 ±19.3 185.6 ±102.4 16.0 ±3.0 7.4 ±4.3 3.7 ±1.0 black

DH2G12-7 103.8 ±16.8 177.0 ±52.1 23.0 ±3.7 6.2 ±3.8 3.9 ±1.6 red brown

DH2G12-8 123.9 ±15.3 163.6 ±41.0 17.0 ±5.2 6.1 ±2.2 3.8 ±1.3 red brown

DH2G12-9 127.9 ±14.2 201.5 ±62.8 19.0 ±2.8 12.1 ±7.3 3.8 ±1.8 red brown

DH2G12-10 117.6 ±14.5 188.1 ±65.3 18.0 ±3.7 17.4 ±9.8 4.4 ±2.1 black

DH2G12-13 108.9 ±11.5 142.8 ±52.3 19.0 ±3.3 6.8 ±3.1 3.2 ±1.8 black

DH2G12-14 96.7 ±13.8 150.3 ±47.6 21.0 ±5.5 7.4 ±4.0 3.8 ±1.6 black

DH2G12-15 126.3 ±15.7 145.1 ±70.2 18.0 ±2.5 9.5 ±5.6 3.5 ±1.0 light brown

DH2G12-16 102.8 ±13.6 157.0 ±40.9 19.0 ±3.6 10.4 ±6.1 3.8 ±1.7 black

DH2G12-17 109.7 ±13.4 152.3 ±68.0 16.0 ±2.7 7.8 ±3.0 2.9 ±1.5 black

DH2G12-18 129.4 ±15.8 163.0 ±49.1 20.0 ±2.7 5.4 ±2.9 5.4 ±2.0 light brown

DH2G12-19 127.6 ±12.4 158.2 ±67.1 25.0 ±4.2 5.5 ±3.1 2.9 ±1.0 black

DH2G8-1 115.9 ±15.8 130.7 ±60.4 17.0 ±3.9 6.7 ±3.3 3.5 ±1.0 black

DH2G8-2 108.0 ±19.5 142.6 ±45.8 19.0 ±4.2 10.3 ±4.9 3.9 ±1.9 red brown

DH2G8-4 128.3 ±15.7 138.8 ±46.2 21.0 ±6.5 5.5 ±2.9 3.4 ±1.6 light brown

DH2G8-5 124.3 ±18.6 152.0 ±76.8 19.0 ±4.0 7.3 ±5.1 3.6 ±1.0 black

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had nine annotated variants, including 1 with seven variants, 3 with six, 2 with five, 6 with four, 16 with three, 121 with two, and 1646 with only a single annotated variant. The highest number of variants was reported for the uncharacterized locus LOC106369673, containing four potential positions of missense (impact moderate), four synonymous variants (impact low), and a single position with a splice donor variant (impact high). An uncharacterized locus LOC106393131 contained seven potential variants, including five synonymous and two missense variants. In the latter, SNPs Bn-scaff_16197_1-p2880558 and Bn-scaff_16197_1-p2872919 had heterozygous genotypes in ‘Galant’ and homozygous in all corresponding mutants; thus, the mutation status was unclear. The ‘Kris’

specimen had homozygous genotypes on these two markers (A/A and G/G, respectively), and four mutants (DH2k12-10, DH2k4-1, DH2k8-2, and DH2k8-3) had simultaneous double mutations (A » C, G » A). Uncharacterized loci LOC106373355, LOC106416957, and LOC111212099 had six variants, each with low or moderate impact. Among the loci with predicted functions, the highest number of significant variants, four, was reported for LOC106395522, encoding wall-associated receptor kinase-like protein 5; it contained three missense and a single synonymous variant. However, no mutations were found in ‘Kris’ doubled haploids, and the genotypes of

‘Galant’ plants were unclear. Another locus, LOC111200312, encoding the TAO1-like disease resistance protein, had two missense and two synonymous variants; these variants were homozygous in both ‘Kris’ and ‘Galant’ and mutated in different combinations in their

Fig. 3. Distribution of predicted effects across 18,831 annotated SNPs.

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mutant offspring. LOC111215021, encoding receptor-like protein 12, had two missense and two synonymous variants; however, three could not be assessed for ‘Galant’ mutants because of missing genotypes in the parent specimen.

The gene LOC106424136, encoding the At5g42460-like F-box protein gene, was the only locus with two potential positions of premature stop gain: SNP Bn-A02-p4761483 (c.510A >G; p. Tyr170*) and Bn-A02-p4913845 (c.848G >A; p. Ser283*). The parent specimens of ‘Galant’ and ‘Kris’ had genotypes on these markers (A/G and G/A, A/A and G/G, respectively). Seven ‘Galant’ mutants, DH2g12-5, DH2g12-10, DH2g12-16, DH2g12-17, DH2g12-18, DH2g12-6, and DH2g8-4, had mutations in Bn-A02-p4913845, and one, DH2g12-8, had a mutation in Bn-A02-p4761483. The offspring of ‘Kris’ contained multiple mutants of either of these two markers.

3. Discussion

The breeding program in Kazakhstan aims only to develop new wheat, barley, rice, apple, pear, grapevine, and some vegetable cultivars for industrial purposes, which leads to complete dependency on foreign cultivars in the case of rapeseed and other crops. All rapeseed cultivars and lines cultivated in our country for industrial scale predominantly belong to the foreign breeding system [38].

However, the cultivation area for rapeseed expands annually, and factories in North Kazakhstan and Almaty regions for seed pro- cessing and oil production have been established [39]. Therefore, new highly productive cultivars regarding yield traits adopted to the environmental conditions of Kazakhstan will advance oil production and lead to a decreased oil cost.

In this research, we developed doubled haploid mutant lines to broaden the genetic pool for MAS and investigated the mutation load and rate in prospective plantlets according to agronomic traits. In previous work, M4–M6 plantlets were used to select mutant lines with high-impact mutations and a stable genome type because earlier M generations provide abnormal diversity in progenitors.

This process of developing stable mutants is time-consuming for the creation of new lines. Doubled haploid mutant lines decrease the time of the breeding process and lead to rapid evaluation of major and minor mutations in earlier generations. However, such characteristics as heat and drought tolerance, oil content are typically polygenic—regulated traits and formed by many low-effect genes with combination of different alleles [40]. The chance to obtain the right combination of known trait forming alleles by development of mutants is low, but still the double haploid mutants are very reasonable in case of revealing new additive and non-additive genetic variants with high positive impact on the traits. Further investigation of later generations will allow to reduce the number of lines with unstable phenotypic characteristics induced by possible pleiotropy effect.

In this research, only regeneration via callusogenesis and second callusogenesis were possible. Of embryos, 64% and 44% were able to provide calluses in cases of 8- and 12-mM EMS treatment, respectively. To form a genetic pool from the M2 mutant lines, we selected plants with agronomic traits that fluctuated between +20% and − 20% regarding their controls. Only 38 of the 198 mutant lines were suitable for these conditions, with 20 mutants of ‘Galant’ and 18 mutants of ‘Kris.’ Using high-density SNP genotyping, we found genetic diversity among 38 mutant lines and new mutations in encoding sequences, with different impacts on the biological activity of genes and regulation regions. Additionally, using the new Brassica90k SNP array, we located 24,657 SNPs from 26,256 SNPs filtered by quality control on the genome assembly (Bra_napus_v2.0). Only 18,831 SNPs were assigned to the available annotated genomic features. We found that all mutant lines were heterozygous at different rates, despite the doubled haploid development process of these mutants. No more than 1350 heterozygous SNPs were found from 26,256 markers in most mutant lines, however, several mutants included 2176 to 8575 heterozygous SNPs. We divided them into 2 groups: rate of heterozygosity under but higher than 1350 SNPs or above the reference controls. The control for ‘Galant’ and ‘Kris’ contained 4947 and 3160 heterozygous SNP markers, respectively. The first group included mutant lines DH2g12-17 (3247 SNPs), DH2g8-4 (4366 SNPs), and DH2g12-18 (2176 SNPs). The second group was formed by mutant lines DH2g12-8 (8083 SNPs), DH2g8-2 (5347 SNPs), DH2k12-3 (8575 SNPs), DH2k8-1 (7046 SNPs), DH2k12-11 Table 3

Distribution of annotated rapeseed SNP across functional types, according to sequence ontology.

Sequence ontology term effect SNP count Percentage

SO:0001631 upstream_gene_variant 7643 40.59

SO:0000605 intergenic_region 3866 20.53

SO:0001632 downstream_gene_variant 3696 19.63

SO:0001583 missense_variant 1042 5.53

SO:0001819 synonymous_variant 633 3.36

SO:0001627 intron_variant 595 3.16

SO:0001624 3_prime_UTR_variant 431 2.29

SO:0001623 5_prime_UTR_variant 275 1.46

SO:0002011 intragenic_variant 244 1.30

SO:0001630 splice_region_variant 168 0.89

SO:0001587 stop_gained 97 0.52

SO:0001792 non_coding_transcript_exon_variant 57 0.30

SO:0001988 5_prime_UTR_premature_start_codon_gain_variant 56 0.30

SO:0001574 splice_acceptor_variant 13 0.07

SO:0001575 splice_donor_variant 7 0.04

SO:0002012 start_lost 2 0.01

SO:0001578 stop_lost 2 0.01

SO:0001567 stop_retained_variant 2 0.01

SO:0001582 initiator_codon_variant 1 0.01

SO:0002019 start_retained_variant 1 0.01

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(5908 SNPs), and DH2k8-7 (6303 SNPs). Low or medium heterozygosity in mutant lines could be related to callusogenesis and colchicine treatment, as shown in previous studies [41–43]. Therefore, around 5% heterozygosity was observed in most mutants. A high rate of heterozygosity in mutants could be achieved because of diploidization before EMS treatment and lead to higher diversity compared to the control [44]. However, before colchicine treatment, all lines were checked to study the ploidy of the plantlets. We counted the number of chloroplasts in the closing stomata of the leaves using a standard cytological method. The accuracy of counting was 93.93% [45], indicating that around 6% may consist of diploid mutants with a high rate of heterozygosity. No particular dif- ferences were found in mutation load and rates between the 8- and 12-mM EMS treatments.

Considering the 18,831 physically mapped SNPs, the mutation load of the obtained lines of ‘Kris’ and ‘Galant’ amounted to 42%

regarding the corresponding controls and was sufficient to form a genetic pool for further MAS. The following scenarios were considered for the detection of potential mutated genotypes: (0) progenitor and offspring have the same homozygous genotype - no mutation; (1) heterozygous progenitor and homozygous offspring -the difference of genotypes can be attributed to allele segregation rather than mutation, so the mutation event cannot be directly identified based on these limited data; (2) homozygous progenitor and heterozygous offspring – mutation; (3) progenitor and offspring both heterozygous - allele segregation combined with consequent mutation; and (4) progenitor and offspring homozygous by the different alleles – double mutation (Fig. 4). On average, 10,500.58 markers were unchanged, with 1733.05, 1203.00, 504.74, and 3898.53 corresponding to the four mutation scenarios, respectively, in the ‘Kris’ offspring. For the ‘Galant’ offspring, 10,071.71 markers were unchanged, with 2969.57, 886.29, 496.43, and 3510.29 demonstrating alterations corresponding to the four scenarios defined above. The frequencies of particular changes in the genotype for each scenario were in agreement with the general distribution of alleles for the annotated SNP set (Table 4). According to the developed scenarios, we revealed 5606.27 markers for ‘Kris’ and 4893.01 markers for ‘Galant,’ which was confirmed by mutation occurrence regarding the last three mutation scenarios. Additionally, 2–4 scenarios were occupied by double mutations when progenitor and offspring were homozygous by different alleles, which helps to select appropriate genotypes in a short period of time.

Scenarios 1 and 2 could not be directly confirmed by mutation occurrence. Considering allelic variant pairs annotated as the reference and alternative alleles (Table 5), the most frequent combination was adenine with guanine (70%), followed by adenine with cytosine (28.8%), and only minor fractions were cytosine with guanine (0.54%) and adenine with thymine (0.59%).

In this research, we predicted the biological impact of variants located in encoding or regulation sequences. However, due to the small number of mutation lines, we did not consider the connection between agronomic traits and genomic background by eliciting the influence of particular genes or SNPs on traits. The main aim of this study was to determine the diversity and possible impact of obtained mutations in lines and to reveal the load and rate of mutations in different genomic regions. Similar genetic pools were formed in previous studies, and most of them considered additional agronomic traits, such as oil content, flowering time, and black leg resistance to select mutant lines [18,46]. The investigation considering GWAS will continue in developing new lines based on the formed genetic pool.

Despite revealing 1042 variants with moderate predicted impact and 121 with high impact, mutant lines were able to survive and reproduce in this study. In the case of characterized encoding sequences, wall-associated receptor kinase-like protein 5, TAO1-like

Fig. 4.Frequencies of genotype changes in analyzed doubled haploid rapeseed mutants obtained from cultivars ‘Galant’ (top panel) and ‘Kris’ (bottom panel).

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disease resistance protein, receptor-like protein 12, and At5g42460-like F-box protein contained more than two variable positions, with an impact on their biological activities. Membrane-localized receptor-like kinases are well-characterized plant pattern recognition receptors involved in pathogen recognition and resistance [47]. In a previous study, the gene Rlm9, encoding a wall-associated kinase-like (WAKL) protein, was identified for resistance to race-specific blackleg, and WAKL is a newly emerging class of race-specific plant R genes [48]. The TAO1-like disease resistance protein is a TIR-NB-LRR bearing protein that contributes to disease resistance and was investigated in Arabidopsis thaliana. This protein is usually induced by the Pseudomonas syringae type III effector AvrB and has not been researched in rapeseed before [49]. The variation in resistance genes to pathogens offers possibilities to select prospective genotypes for MAS and also for acceleration of yield quantity and quality.

The At5g42460-like F-box protein gene also carries variable positions detected in the obtained mutants. F-box genes form large multigene superfamilies and control many important biological functions in plants [50]. Mutants survived with high impact variation Table 4

Summary of the number of changes of different types across the doubled haploid mutants originated from rapeseed cultivars ‘Kris’ and ‘Galant’.

Change scenario Change Galant Kris

mean SD mean SD

0 (no change) CC 1564.06 257.39 1465.70 135.47

AA 5538.72 914.55 5039.15 485.38

GG 3759.17 628.25 3446.00 325.79

TT 32.56 5.81 22.30 1.72

all 10071.71 2435.95 10500.58 2984.03

1 (segregation; not enough data to confirm mutation) A?>?C 259.61 50.14 436.60 54.74

A?>?G 644.83 127.83 1046.65 120.55

A?>?T 8.44 2.62 16.70 3.37

C?>?A 265.67 52.70 418.20 52.88

C?>?G 8.67 1.97 19.75 3.34

G?>?A 625.50 120.17 1150.30 135.12

G?>?C 8.56 3.01 14.35 2.91

T?>?A 8.06 3.19 15.50 3.25

all 2969.57 768.32 1733.05 544.63

2 (single mutation) A >C 173.94 251.68 129.40 165.13

A >G 466.00 613.77 326.15 400.55

A >T 5.94 5.62 4.90 2.49

C >A 170.89 230.86 129.05 155.12

C >G 4.11 3.39 5.55 1.76

G >A 437.50 587.32 329.20 388.25

G >C 6.11 4.74 3.00 1.38

T >A 5.33 6.14 3.35 1.69

all 886.29 1104.86 1203.00 1678.21

3 (mutation and segregation) A<>C 78.61 44.25 72.00 41.83

A<>G 174.72 106.81 184.70 113.04

A<>T 5.83 2.55 6.40 2.66

C<>A 68.94 45.59 66.25 41.38

C<>G 6.89 2.89 4.70 1.95

G<>A 187.89 112.30 174.65 93.01

all 496.43 309.00 504.74 330.48

G<>C 4.67 2.54 6.65 3.07

T<>A 5.22 2.32 5.90 2.86

4 (double mutation) A » C 629.50 182.12 536.30 138.66

A » G 1390.22 430.03 1289.15 320.05

A » T 3.72 2.19 6.05 1.88

C » A 599.06 179.34 525.45 131.25

C » G 5.11 2.25 6.05 1.50

G » A 1476.28 461.19 1310.70 329.90

G » C 5.22 2.32 5.00 1.49

T » A 6.00 2.95 7.10 1.41

all 3510.29 1205.25 3898.53 1546.04

Table 5

Frequencies of occurrence of four nucleotides as the reference and alternative alleles across 18,831 SNPs annotated for the reference rapeseed genome (Bra_napus_v2.0).

ALT A C G T

REF Frequency (%) 48.9 14.87 35.91 0.3

A 50.54 0 14.57 35.66 0.3

C 14.46 14.22 0 0.24 0

G 34.7 34.39 0.3 0 0

T 0.29 0.29 0 0 0

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in the At5g42460-like F-box protein, specifically a premature stop gain, making it possible to conclude that this protein is not major for the life cycle and that the protein function could be restored by homologous or other proteins.

Considering the fact that rapeseed is the most important crop as an oil source, the exploration of mutant lines in the frame of fatty acid content is promising. The fatty acid desaturase 2 (FAD2) and fatty acid desaturase 3 genes encode important enzymes involved in producing precursors of oleic and/or linoleic acid in the lipid biosynthetic pathway of canola [51–53]. Rapeseed FAD2 genes are located on different chromosomes A01, A05, C01, and C05, and are involved in oleic acid regulation. Considering the obtained mutant lines in this study, 779 and 932, mutations assigned to annotated genome features were detected in A05 and A01, respectively.

Potentially, these mutations could be located in FAD2 genes and also influence oil content. In previous research, the SNPs on chromosomes A08 and C03 were confirmed to influence linoleic and linolenic acid content [54,55], in our mutant lines we have also identified 515 mutations on chromosome A08. The upstream region of gene BNAa08G06530D involved in the arachidic acid trait includes four upstream variants with a high impact on its biological activity for developed mutant lines and the location of this gene is assigned also to chromosome A08.

Additionally, the chromosomes A06, A09, A10, C02, and C07 were involved in changing erucic acid, oleic acid, and linoleic acid contents [56]. 625, 735, and 660 variants in our study were as well detected in chromosomes A06, A09, and A10, respectively, according to our mutant lines with a high possibility to influence oil contents. Regarding the C genome of Brassica napus all markers were mapped on scaffolds, wherefore considering the number of mutations in a particular chromosome was not possible. Fatty acid content is a quantitative trait controlled by multiple genes and most genes were often detected on chromosomes A03, A05, A06, A10, and C02 [57].

The oil content as the most valuable trait of rapeseed is under careful investigation. The expression of loci associated with important characteristics of oil are influenced not only by genetic background (GxG interactions) including load and rate of mutations, but also genotype-by-environment (GxE) interactions make a significant contribution [58]. Complicacy of this trait is an outcome of different reactions of genotypes on changeable environmental conditions. The further testing of the obtained lines in this research with significant mutations in oil related loci in diverse environmental conditions will revealed the most perspective genotypes for cultivation and MAS. The mutations with the impact on biological activity of proteins involved in formation of oil content could enhance or inhibit the penetrance and expressivity of trait in different regions of sharply continental climate of Kazakhstan. The parental cultivars of present mutant lines have not shown any significant deviation in quality and quantity of oil content during several cultivations on Kazakhstanian fields. The precise prediction of oil content and other valuable traits based on the data from GxG and GxE interactions using such modern techniques as artificial intellect, machine learning, neural network algorithms, allows to improve the breading process in times [37,40,59–64].

Chromosome A09 is well involved in the regulation of flowering time and plant height traits by assigned conserved regions as was shown in previous research [65]. This region contains AGL transcription factor BnaFUL and histone methylation factor BnaGRP3. In our study, chromosome A09 comprised 649 mutations with high impact and 86 with moderate or low including mutations in encoding and non-coding sequences. The upstream gene variant was also observed in BNAa08G26230D gene of the chromosome A09 in our mutant lines, the expression of this gene was significantly up-regulated in the young inflorescences of absolutely apetalous and early flowering line compared with normally petalled one, as was previously confirmed in flowering and apetalous characteristic research [66]. Most loci with the predicted variants contained only single SNPs, with a significant predicted effect. As their occurrence depends on both the initial genotype and the viability of obtained mutants, the particular genes are the subject of further individual evaluation with respect to the genotypes and phenotypes of obtained mutant doubled haploid plants. Mutant lines that include moderate- and high-impact mutations in encoding genes are a perfect pool not only for MAS but also for the investigation of the fundamental basis of protein functions.

3.1. Conclusion

Taken together, we report here the first Kazakhstanian collection of mutant rapeseed lines comprising high, moderate, and low impact variants to improve the selection of local cultivars. The mutation load and rate in the prospective lines according to agronomic traits were investigated. Further, an attempt to derive homozygous plants from most perspectives’ lines by self-pollinating and via the isolated microspore culture will be carried out.

4. Material and methods 4.1. Development of mutant lines

In our research, mutant doubled haploid lines of rapeseed cultivars ‘Galant’ and ‘Kris’ were used. All mutant lines were obtained when treated with EMS mutagen of haploid microspore-derived embryos [67]. The originator of the cultivars was V.S. Pustovoit All-Russian Research Institute of Oil Crops.

Upon reaching 1.5–2.5 mm in size, the embryos derived from the microspores were treated with EMS (Sigma Aldrich, US). EMS was added to the Petri dishes at two concentrations: 8 and 12 mM. The dishes were then placed on a shaker (40–50 rpm) in a temperature controller at 25 ^◦C for 1 h. After treatment, the embryos were dried on a sterile paper sheet for 5 s. Thereafter, they were transplanted onto a solid B5 medium with 0.8% agar and 2% sucrose and incubated for 24 h in a thermostat at 10 ^◦C. After incubation, the tubes with embryos were placed under light at 25 ^◦C. After two weeks of cultivation, the embryos were transplanted onto fresh B5 medium with 0.05 mg/L gibberellic acid (GA) for regeneration. The obtained plantlets were transferred to MS medium with half salts (½MS).

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The regeneration of mutant haploid plants occurred through embryo callusogenesis and, in some cases, through embryo secondary callusogenesis. In some cases, the formation of callus was followed by secondary embryos after 2–4 weeks (Fig. 1 A–C). The secondary callus from embryos was then transplanted to a nutrient medium Gamborg B5+0.05 mg/L GA for further development of plantlets.

After 28–30 days of culture, we observed different rates of regeneration from embryos depending on the different EMS treatments (Table 1; Fig. 1E and F). In ‘Galant’ and ‘Kris,’ 108 and 90 embryos, respectively, were used for each treatment with different concentrations of the mutagen.

Plantlet cloning was carried out on ½MS medium, and the reproduction coefficient was 1:3. After 4 weeks of cultivation in vitro, one-third of the plantlets were left for cloning, and two-thirds were transplanted into the ground. Before transplanting into the soil, plantlets were treated with 0.05% colchicine solution for 16 h to double the set of chromosomes at 4 ^◦C [68]. To check the ploidy of plantlets before colchicine treatment, we counted the number of chloroplasts in the closing stomata of leaves using the cytological method [45]. After treatment with colchicine, the plantlets were transplanted into plastic pots with a diameter of 10–12 cm (three plantlets per pot). Then plants were covered with plastic pots to increase humidity and gradually opened after 2–3 days. Adaptation took place under controlled conditions at a temperature of 23–26 ^◦C, light mode of 16/8 (day/night), lighting of 5000 Lux, and humidity of 50–60%. After acclimatization, the plants were transplanted into vegetative vessels (10 L), with three plants per vegetative vessel.

All mutant lines were marked DH2g (‘Galant’) or k (‘Kris’), 12 (12 mM of EMS), or 8 (8 mM of EMS). Only the DH2k 4-1 mutant line was developed with 4 mM EMS treatment.

4.2. Evaluation of agronomic traits in mutant lines

The mutant lines were cultivated in an experimental field for the analysis of their agronomic traits. One hundred seeds of each fertile mutant (M1) were selected for sowing. As a result, 35–45 plants were viable for each mutant (M1). From each mutant, 30 plants were selected for the analysis of agronomic parameters, such as plant height (cm), number of pods, number of seeds per pod, weight of 1000 seeds (g), and weight of seeds per plant (g). Statistical analysis was performed in Excel.

4.3. SNP genotyping

DNA was extracted from 100 mg of frozen rapeseed leaves using a modified CTAB protocol [69]. The yield and quality of the DNA were evaluated by electrophoresis in 1% agarose gel and a spectrophotometer NanoDrop2000 (Thermo Fisher Scientific, USA).

Genotyping was conducted using the Brassica90k SNP array [70] on an iScan platform (Illumina, USA). Array preparation and scanning were performed in accordance with the manufacturer’s protocols. Genotype calling was performed in GenomeStudio (Illu- mina), and genotyping data were exported in PLINK data format, along with a summary of genotype calling by loci and samples.

PLINK1.9 [71] was used for data format conversion and manipulation, and R [72] with the ‘adegenet’ [73] package was used for general data handling, filtering, and analysis. SNP markers with a call rate ≤0.95, median GC score ≤0.5, and zero minor allele frequency (MAF) were excluded.

4.4. SNP marker mapping in the genome

All selected SNPs were re-mapped using rapeseed genome assembly Bra_napus_v2.0 (GenBank accession GCA_000686985.2) as follows. First, flanking sequences of each marker were extracted from the manifest file for the Brassica90k array (‘SourceSeq’ field).

Second, left and right flanking sequences were aligned independently against the reference genome sequence using BLAT [74]. The following conditions were selected to determine the accuracy of matching: (1) same chromosome and strand assignment for the left and right sequences; (2) 95% of exactly matched positions, with no more than one insertion or deletion with a length ≤5; and (3) the distance between flanks equals exactly one nucleotide position. SNPs matching these requirements were selected for the annotation and prediction of biological consequences using SnpEff [75].

Author contribution statement

Dilyara Gritsenko: Conceived and designed the experiments; Analyzed and interpreted the data; Wrote the paper.

Ainash Daurova: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Wrote the paper.

Alexandr Pozharskiy: Performed the experiments; Analyzed and interpreted the data.

Gulnaz Nizamdinova, Marina Khusnitdinova: Performed the experiments.

Zagipa Sapakhova, Dias Daurov, Kuanysh Zhapar: Performed the experiments; Contributed reagents, materials, analysis tools or data.

Malika Shamekova: Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data.

Ruslan Kalendar: Analyzed and interpreted the data; Wrote the paper.

Kabyl Zhambakin: Conceived and designed the experiments; Wrote the paper.

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Funding statement

This work was supported by the Committee of Science of the Ministry of Science and Higher Education of the Republic of Kazakhstan under the program BR18574149 “Development of highly productive cultivars and lines of agricultural crops using innovative technologies” the task 03“Development of spring rapeseed cultivar for cultivation in the northern regions of Kazakhstan using mutagenesis, distant hybridization and haploid biotechnology”, as well as the Science Committee of the Ministry of Education and Science of the Republic of Kazakhstan in the in the framework grant funding project AP08856576 “Creating source material of turnip rape (Brassica rapa) to create new cultivars for Northern Kazakhstan” from the Science Committee of the Ministry of Education and Science of the Republic of Kazakhstan.

Data availability statement

Data included in article/supp. material/referenced in article.

Declaration of interest’s statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.heliyon.2023.e14065.

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