Harnessing Heterogeneity: Clustering Kazakh Spring Rapeseed for Breeding Value

The Republic of Kazakhstan (RK) offers optimal climatic conditions for rapeseed cultivation, a pivotal oilseed crop. Addressing the demand for raw materials in the processing industry, edible vegetable oil, and livestock feedstock necessitates the development and implementation of high-yielding rapeseed varieties. Rapeseed, as noted by Suleimenova et al. [1], contains high proportions of crude fat, crude protein, starch, and structural carbohydrates. It is exploited across diverse industries, including food, metallurgy, paint and varnish, soap, chemical, textile, and, more recently, biofuels. Post-processing, cakes and meals rich in easily digestible protein are produced, serving as valuable feedstock [2-5].

This study aims to generate breeding genotypes of spring rapeseed and categorize them into clusters based on yield level and ecological adaptability. Commercial varieties and hybrids of oilseed spring rapeseed are subjected to various abiotic stresses, including temperature fluctuations, light intensity variations, water distribution, soil pH, heavy metal concentrations, salinity, nutrient deficiency, and gaseous pollutants. The selection of cultivars and hybrids typically prioritizes the biological benefits of the crop, with hybrids often yielding higher than varieties, particularly under non-extreme conditions [6-8].

Rapeseed is a leading oilseed crop in the European Union (EU) and Canada, with yields averaging 27-35 c/ha [9]. In RK, low erucic and low glucosinolate varieties of spring rapeseed are cultivated extensively across different soil and climatic zones for edible oil production and as high-protein components in animal diets. Oilseed rape (Brassica napus L.), a promising crop for temperate climates, is grown for food, feed products, and biodiesel production. As Khan et al. [10] report, rapeseed is the primary oil source for biodiesel production in EU countries, requiring significant chemical fertilizer inputs to sustain an alternative crop.

The North Kazakhstan and East Kazakhstan regions of RK concentrate the majority of oilseed production. According to the Bureau of National Statistics of the Republic of Kazakhstan, as of 2022, oilseed crops and gross yields have increased by 67% and 1.5 times, respectively. Thirteen primary agricultural producers are engaged in rapeseed cultivation in RK. Early maturing, oil-bearing, and drought-resistant varieties of spring rape are particularly well-adapted to RK's soil and climatic conditions, providing high seed and green mass yields [11]. German-bred varieties also exhibit adaptive traits suitable for cultivation in RK.

The present study is nested within the broader context of spring rapeseed breeding research. Significant genetic diversity in spring rapeseed offers potential for trait enhancement, such as yield, oil content, and disease resistance. However, effectively leveraging this variability remains an elusive goal. With the growing demand for vegetable oils and biofuels, enhancing rapeseed yield is a critical focus of breeding programs. Developing varieties with high and stable yields under diverse environmental conditions is essential. Rapeseed cultivation often confronts various abiotic stresses, including drought, salinity, and temperature variations [12, 13]. Breeding for improved stress tolerance is crucial for sustainable production. The use of advanced genomic tools can expedite breeding progress by facilitating efficient identification of desirable traits.

To address these research gaps, this study undertakes a comprehensive analysis of spring rapeseed samples using Hierarchical Cluster Analysis (HCA) within the Statistical Package for the Social Sciences (SPSS) software. Through this, the study endeavors to elucidate the genetic variability, stress resistance, and other economically valuable traits of spring rapeseed, contributing to ongoing efforts to enhance this crop for agricultural and environmental sustainability.

The following methods were used in the experimental study: laboratory methods included monitoring and assessing the quality of plant material; field – parameters of a comprehensive analysis of phenotypic environmental conditions; statistical – analysis of information, construction of a dendrogram for a cluster assessment of varieties of spring oilseed rape.

Growing technology – generally accepted for conditions in the northern region of RK. Variety Maykudyk, approved for use in the Akmola region, was sown as a standard. The research was carried out in the field by the laboratory for the selection of legumes and oilseeds in the period 2021-2022. based on A.I. Barayev Research and Production Centre for Grain Farming, Shortandinsky district, Akmola region. Breeding lines of spring oilseed rape were studied during a two-year competitive variety testing.

Indicators of breeding value, genotypic effect, stress resistance and genetic flexibility were determined by quantitative traits of yield, by the number of grains per ear and the weight of 1000 grains. Under laboratory conditions, the seed suitability of rapeseed breeding material was determined: seed germination averaged 87-96%, the weight of 1000 seeds ranged from 5.2 to 6.1 g. Seeds were harvested in the same way as standard seed. The laying and conducting of experiments, the selection of plant samples, their preparation for analysis, was carried out according to observations and accounting for yields according to the methods of Eberhardt and Thomas [24].

The material for the study is the best basic and some new unknown 36 varieties of spring rapeseed collected in various ecological and geographical zones. The studied spring rapeseed genotypes, grown in 11 countries, are presented in Figure 1.

1.png

Figure 1. The ecological and geographical diversity of spring rapeseed genotypes

Source: Study [25].

During the growing season, the field crop was carefully cared for, and a planned fight against weeds, diseases, and pests was carried out. Fertilizer Kemira Hydro was applied at a ratio of 10 g/10 l of water once every two weeks, containing magnesium, sulphur, and trace elements. Twice a month with an interval of 10 days, Fitoverm and Fitosporin were treated at a dosage of 1 ml/5 l and 10 ml/10 l of water, respectively. Collection and accounting of the crop was carried out in the phase of full ripeness of spring rapeseed manually from each site of the experiment by the weight method. Spring rapeseed was harvested directly on the Wintersreiger Classic combine, upon reaching full maturity and physiological ripeness at seed moisture from 8 to 12% in terms of the bulk. Seeds were dried as needed, weighed per plot and recalculated in quintal yield units: yield per hectare; herbage height, cm; the number of branches and pods, pcs.; the number of seeds in pods and per plant, pcs.; the weight of seeds per plant and weight of 1000 seeds, g.

Statistical analysis of yield data was performed using the dispersion method based on data from the study by Xiong et al. [26]. Cluster analysis of samples was carried out for a complex of economically valuable traits of spring rapeseed using the Hierarchical Cluster Analysis (HCA) module and Statistical Package for the Social Sciences (SPSS). To construct dendograms, a measure of the difference or similarity of traits in the multidimensional space of grouping selected genotypes according to adaptability to the environment, reflecting the intuitive properties of the distance between points – Euclidean metric, was used.

The number of replicates is 3, in case of this study it’s an appropriate. It, thus, depends on the number of treatments, availability of material, experimental unit and expenses. The size of individual plots where spring oilseed rape varieties were cultivated was 2.1 m². Cluster analysis was conducted based on a complex of economically valuable traits of spring rapeseed. While it specifies that the analysis was based on quantitative traits like yield, the number of grains per ear, and the weight of 1000 grains, it doesn't provide details about other variables or traits considered in the analysis.

The methods of modern breeding in the climate of Northern Kazakhstan allow the cultivation of high-quality spring rapeseed, despite the frequent manifestation of early spring drought and high humidity at low air temperatures during periods of flowering, fruiting, and intensive seed ripening. In modern diversified agriculture, when new hybrids and varieties of spring rapeseed are discovered, breeders need the means to achieve the expected results with minimal effort and time. According to Hu et al. [30], the development of spring rapeseed breeding for resistance to abiotic environmental factors of drought resistance and acid resistance is carried out using breeding, physicochemical and biotechnological methods. No tangible success has been achieved in breeding spring rapeseed in this direction, so the creation of source material with pronounced stress-resistant properties is an important breeding problem [31, 32].

In the conditions of 2021-2022, as close as possible to production, various varieties of spring rapeseed were tested, among which are Kalibr, Jeros, ANIIZiS-1, NPSR0607, Smilla and Yarvelon. A promising result was noted under the conditions of cultivation in 2022, with an average yield of 1 and 2 clusters at the level of 24.4 and 21.7 c/ha, respectively. In the work of Cherkasova and Rzaeva [33], the most promising lines of spring rapeseed with high yields were noted: Maykudyk variety – 29.2 c/ha and Bilder hybrid – 32.7 c/ha. These samples were used as seed material for the creation of new hybrids, with a seeding rate of 2.5 million viable seeds and intensive cultivation technologies in the conditions of Northern Kazakhstan.

Cluster analysis was employed to categorize rapeseed varieties into two distinct groups, primarily based on average yield indicators and physiological and biochemical analyses. The initial cluster, denoted as the first group, comprised spring rape varieties characterized by robust resistance to drought stress. Notable varieties in this cluster included 196, Tavrion, Oredej, Vanguard, Vikros, Ole, Caliber, Ability, Gladiator, NPCZR 11109, Licolli, Karoo Black, and Hight. In contrast, the second group encompassed high-yielding varieties, such as NPSR0507, Stellar, Maikudyk, OR005, RY105, 601, Rubezh, Krasnodar 3, Khidalgo, Mirko, 44C73, and Viking.

This clustering approach aligns with the findings of Fan et al. [34], which suggest a significant positive correlation between yield and specific characteristics, including the flowering period, as well as the number of branches and pods per plant. The utilization of cluster analysis to delineate relationships and distances among spring rapeseed varieties proves to be an effective strategy in the examination of crop collections. It facilitates the efficient and rational utilization of alternative germplasm for the purpose of synthesizing early-maturing, high-yielding varieties and hybrids, thereby contributing to the advancement of rapeseed breeding and cultivation practices [35, 36].

The difference in productive parameters of spring rapeseed varieties of clusters 1 and 2 was noted in terms of plant height – 133.1 and 142.3 cm, the number of seeds in the pod – 20.8 and 22.1 pcs., weight of 1000 seeds – 4.2 and 3.7 g, respectively. The oil content of spring rape seeds in terms of crude fat for clusters 1 and 2 was in the range of 45-49%, the yield was 21-24 q/ha. Given State Data Register of Selection Achievements Approved for use in the Republic of Kazakhstan [37], the spring barley cultivar Maykudyk was separately noted, which was sown as a standard sample. Among its characteristics, it is necessary to highlight the following: plant height 112-130 cm, number of branches – 1-2, weight of 1000 seeds – 3.5 g, seed oil content in terms of crude fat – 48-50%, crude protein content 21.9-27.7%, yield 35.2 c/ha.

The practical value of a culture is determined by the biochemical composition of the seeds. The oil content of wild rapeseed is a determining factor in productivity, its decrease is compensated by an increase in seed yield, hence the yield of oil from 1 ha. Studies have shown that the content of crude protein and fat together make up 68-74% of the total mass of seeds. Based on data from Raza et al. [38], rapeseed protein is characterized by a higher albumin fraction of 36-48% than in other crops, characterized by a high content of essential amino acids, including methionine and lysine. One of the main indicators of the quality of raw fat is the fatty acid composition, with the lowest content of saturated fatty acids.

When conducting research, the weather conditions of the period 2021-2022 differed, which made it possible to give an objective assessment of the studied methods of cultivation of spring rapeseed. The vegetative period of spring rapeseed in clusters 1 and 2 was 91-97 days from sowing to full maturation of seeds. Cultivation of rapeseed as a green fertilizer in the period from sowing to harvesting, the maturation of green mass is 36-38 days. According to Bognár et al. [39], the growing season of spring rapeseed is 97-114 days, but in favourable weather years, a green mass can form, which will be harvested a second time. When sown in summer at a short harvest time, the crop resists frost, which allows extending the late seasonal green conveyor for feeding animals by 1.5-2 months. According to Geddes et al. [40], in their work, the breeding of early maturing varieties can be successfully carried out if it is based on the use of source material from the global and national assortment, which was previously studied for the presence of traits selected under the conditions in which the varieties are created. The earlier maturing varieties have many advantages over regular varieties, including cold hardiness, drought tolerance, disease, and insect resistance. In spring rapeseed, varieties with a growing season of 110 days or more are called late-ripening, varieties with a growing season of 90 to 110 days are called mid-ripening, and varieties with a growing season of less than 80 days are called early-ripening.

During the initial period of growing spring rapeseed, its development is extremely slow, so it is necessary to carry out a targeted fight against weeds, since if the crops are completely drowned out by weeds, seed yield losses can reach 45-60%. In the fight against weeds, planned harrowing was carried out by seedlings with the introduction of herbicide a Buckler. Spring rapeseed is also damaged by rapeseed and cruciferous fleas, rapeseed flower beetle, cabbage mosquito, cabbage aphids, leaf-gnawing pests, therefore, spring rapeseed was treated with insecticides Sufron, which reliably protected crops from damage by pests, while maintaining its growth activity [41, 42]. In the studies of Washuck et al. [43], noted the use of alternative options for crop protection, instead of complex applications of herbicides and insecticides, leading to an increase in protein and fat content in seeds. As an alternative, instead of treating the seeds with insecticides, it is necessary to increase the seeding rates at earlier sowing dates, since the faster-formed pods and the seeds in them are less susceptible to insect attack, beneficially affecting the processes of nutrient accumulation [44].

Enhancing agronomic selection methods for oilseed rape, particularly with the integration of fertilization practices, is imperative to unlock the potential for increased overall yield under ideal growth conditions, ultimately leading to heightened seed productivity. Soil preparation preceding sowing, coupled with the judicious application of mineral fertilizers, has assumed paramount importance [45]. Furthermore, it is advisable to incorporate liming procedures into field management practices, given that soil pH levels below 7 can negatively impact nutrient uptake by plants. Empirical research conducted by Gugala et al. [46] underscores the vulnerability of spring rapeseed during its early growth stages. However, precise sowing techniques on a uniformly prepared and sufficiently compact seedbed promote consistent seeding depth and foster robust seedling development [47]. It is worth noting that specific pH benchmarks have been established, falling within the range of 6 to 7. Maintaining soil pH within this range has been associated with a notable increase in oil content, ranging from 1.6% to 2.2%. Conversely, this optimal pH range is correlated with a reduction in crude protein content by approximately 4.2% to 5%.

One limitation of our study is the relatively limited number of genotypes and growing environments assessed. While our sample provided valuable insights, it may not capture the full spectrum of genetic diversity or environmental variability present in spring rapeseed cultivation. Expanding the study to include a broader range of genotypes and diverse growing environments would enhance the generalizability of our findings. Additionally, factors such as seasonal variations and microclimatic conditions may influence trait expressions and cluster formation. Acknowledging these limitations, we recommend future studies with larger genotype sets and multi-location trials to account for environmental variations. To validate and build upon our findings, future research directions could include conducting multi-year trials to assess the stability of cluster formations across seasons and to account for interannual variability in climatic conditions.