Introduction

The Brassicaceae family, also known as Cruciferae, encompasses approximately 350 genera and nearly 3.500 species, with Brassica standing out as the most crucial genus among these (Nawaz et al. 2018). Within this family, Brassica includes economically significant plants. The members of Brassica genus include very rich carbohydrates, lipids, proteins, vitamins, minerals and phytochemical components all of which hold medicinal value. Turnip, scientifically known as Brassica rapa subsp. rapa, stands out as a globally significant leaf and root crop (Paul et al. 2019). Brassica rapa (2n:20) plays a significant role as both an essential oil and vegetable crop across various regions worldwide. Its seeds are extracted for oil, while its leaves, flowers, stems, and roots are utilized as vegetables (Zhao et al. 2005). This species thrives especially in colder regions and can be stored for an extended period after the harvest (Fernandes et al. 2007). While young turnip roots are commonly enjoyed in salads in their raw form, areal parts have distinctive bitter taste and typically prepared by cooking (Francisco et al. 2009). In the Malatya region, this plant is known as Çelem and its roots are used as food. However, the leaves and roots of turnip are also a good forage plant that increases milk yield in cattle. In Anatolia, turnip is produced as a third product after wheat and barley and can yield a good quality silage product (Geren 2002; Parlak-Özaslan and Sevimay 2005). Turnip, which we considered locally within the scope of this study, is consumed at the table and differs morphologically and phenotypically from the species used as a forage crop. While the tuber diameter of the turnip genotypes used in this research varies ranged from 4 to 10 cm, the tuber length was flattened and ranged from 1 to 2.5 cm. The reason locally grown turnips are morphologically different from forage turnips is that these genotypes have this flatter form. All parts of forage turnips, especially the tuber parts, are used in animal nutrition. The tuber diameters of forage turnips are between 4 and 9 cm and the tuber lengths are between 9 and 18 cm (Ayan et al. 2006, Türk et al. 2009). In addition to the widespread cultivation of forage turnip, the existence and diversity of new table genotypes contribute to biodiversity. To facilitate the optimal utilization of genetic resources, it is crucial to understand genetic diversity at both morphological and molecular levels (Kumar et al. 2016). Molecular marker techniques are useful tools for genetic diversity analyses (Wu et al. 2012). A molecular marker is a gene or DNA sequence whose specific location on a chromosome is known and is linked to a particular gene or trait. In recent years, there has been a significant surge of interest in molecular markers, uncovering polymorphisms at the DNA level (Al-Samarai and Al-Kazaz 2015). Microsatellites, also known as simple sequence repeats (SSRs), are genomic regions composed of short DNA motifs repeated consecutively. Inter simple sequence repeats (ISSRs) are DNA segments bordered at both ends by these microsatellite sequences (Ng and Tan 2015). There are plenty of publications in the literature about examining the genetic diversity of Brassica rapa genotypes grown in different regions, e.g. Zhao et al. (2005) detected genetic diversity between different B. rapa genotypes using amplified fragment length polymorphism (AFLP) markers; while for same purpose Das et al. (1999), Fujimoto and Yamagishi (1996), Pino Del Carpio et al. (2011), Takuno et al. (2007) and Zhao et al. (2005) used AFLP and random amplified polymorphic DNA (RAPD) markers; Snowdon and Friedt (2004) and Ofori et al. (2008) used SSR markers. In this study, we aimed to use ISSR markers for characterizing and detecting polymorphisms among 18 Brassica rapa subsp. rapa genotypes grown in the Malatya province of Türkiye and determined the genetic variation among genotypes.

Material and methods

Plant materials

In the screening conducted in 2022 throughout Malatya, it was determined that Brassica rapa subsp. rapa plants are exclusively cultivated in the Pütürge district. The plants grown in this region are sent to other parts of the province. The seeds of 18 genotypes (eight Standart (Us), (B1, B2, B3, B4, B5, B6, B7 and B8), four Gündeğer Pütürge (B9, B10, B11 and B12), four Karşıyaka Pütürge (B13, B14, B15 and B16), one Centre (B17), and one Şahit (B18)), collected from the villagers in the Karşıyaka region between November 2021 and January 2022, were planted under greenhouse conditions in April 2023 and the seeds were germinated. For germinating the seeds, a 1:3 perlite + peat mixture was prepared and the seeds were planted in germination vials. The prepared vials were placed in a plastic greenhouse with an approximate temperature of 20 ± 2 °C. Leaf samples were coded and taken into transparent bags for analysis (DNA) from the turnip seeds that produced real leaves. The leaves of the turnip genotypes taken were stored at − 80 °C for analysis. The collected leaf samples were prepared for genomic DNA isolation (Gene Mark commercial kit) in the plant biotechnology laboratory.

PCR amplifications and ISSR-PCR analysis

We used nine ISSR primers for PCR. PCR components and PCR protocol are shown in Table 1. ISSR-PCR results were visualized by 1.5% agarose gel electrophoresis. Gel images of PCR results with UBC-807 primer are shown in Fig. 1. Each ISSR gel image was evaluated individually for the presence (indicated as “1”) or absence (indicated as “0”) of amplified fragments, and missing data were imputed by assigning (“9”). Genetic similarity values between genotypes were analyzed using NTSYS-pc package version 2.10. The genetic similarity coefficient was calculated using the NEI72 method (Rohlf 2000). At the same time, a similarity matrix was created using UPGMA cluster analysis and NEI72 coefficient and groups were determined by dendrogram. PCA analysis was performed using JMP Statistical Software. Additionally, major allele frequency, Nei's H and PIC values were calculated using the PowerMarker software package version 3.25 (Liu and Muse 2005).

Table 1 Primers used to amplify the inter simple sequence repeat - polymerase chain reaction (ISSR-PCR) sequences, PCR components and protocol
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Fig. 1
figure 1

Gel image of inter simple sequence repeat - polymerase chain reaction (ISSR-PCR) bands amplified with UBC-807. B1: Standart (Us), B2: Standart (Us), B3: Standart (Us), B4: Standart (Us), B5: Standart (Us), B6: Standart (Us), B7: Standart (Us), B8: Standart (Us), B9: Gündeğer Pütürge, B10: Gündeğer Pütürge, B11: Gündeğer Pütürge, B12: Gündeğer Pütürge, B13: Karşıyaka Pütürge, B14: Karşıyaka Pütürge, B15: Karşıyaka Pütürge, B16: Karşıyaka Pütürge, B17: Centre, B18: Şahit

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Results and discussion

Molecular markers are independent of environmental influences and can be easily determined in all tissues (Thakur et al. 2017). These markers reveal inter-varietal genomic variability by determining genetic variation and linkage maps (Demirel 2020), also powerful tools for plant systematics and plant breeding (Takahashi et al. 2016). The ISSR molecular marker technique was identified by Zietkiewicz et al. (1994) and this technique has been used in phylogenetic analysis, molecular characterization and genetic variation of many plant genotypes (Svetleva et al. 2006; Oğraş et al. 2017; Pınar et al. 2021; El-Esawi et al. 2022). In this study, a total of 73 bands were obtained using nine ISSR primers. Of these 67 were polymorphic and six were monomorphic. The polymorphism rate was approximately 91.78%. The lowest “PIC” value was detected in the UBC-873 primer as 0.1710, and the highest “PIC” value was detected in the UBC-834 primer as 0.3750. The mean “PIC” value was determined as 0.3319. While the lowest “H” value was found as 0.1888 in the UBC-873 primer, the highest “H” value was determined as 0.4999 in the UBC-834 primer. The mean “H” value was determined as 0.4276. Also, the lowest “major allele frequency” value was detected in the UBC-834 primer as 0.5054, and the highest “major allele frequency” value was detected in the UBC-873 primer as 0.8944. The mean “major allele frequency” value was determined as 0.6531 (Table 2). Similarity index values were determined to be between 0.0928 and 1.0924 (Table 3). The UPGMA dendrogram consist of two groups (Fig. 2). Group A was divided into two subgroups. Subgroup A, consists of Standart Us (B1, B2, B3, B4, B5, B6, B7 and B8) genotypes. In subgroup B Gündeğer Pütürge and Karşıyaka Pütürge (B9, B13, B14, B15, B10, B11 and B12) genotypes emerged together. Group B consists of Karşıyaka Pütürge, Centre, and Şahit (B16, B17 and B18) genotypes. The UPGMA dendrogram was partially compatible with the distribution of genotypes in the regions where they were collected. Based on PCA analyses, the graphic results and eigen values obtained on the two-dimensional plane are given in Fig. 3. The first four eigen values explained 65% (eigen > 1) of the total variance in the population. As a result of the analysis, the 18 genotypes were reduced to two dimensions. There are 10 genotypes in the first dimension and eight genotypes in the second dimension. The clustering in the two-dimensional graph obtained as a result of the PCA analysis was partially compatible with the UPGMA dendrogram results. The genetic relationship of eleven Brassica rapa var. rapa genotypes from Erzurum province of Türkiye was determined using RAPD-PCR by Yildirim et al. (2010). They obtained an average of 90.4% polymorphism from 32 RAPD primers and revealed that the RAPD technique would be useful in genotyping Brassica rapa var. rapa genotypes. Talebi et al (2010) studied 47 Brassica rapa genotypes from all canola and mustard producing countries worldwide to create a gene plasma collection and obtained 99 polymorphic bands from 12 primers in the ISSR-PCR analysis. Their results revealed that these can be used to make informed decisions about the selection of genotypes to cross for developing populations or to facilitate the identification of different parents to cross in hybrid combinations in order to maximize heterosis. Using the SSR technique, Soengas et al (2011) determined the genetic diversity of 80 Brassica rapa subsp. rapa populations collected in northwestern Spain and northern Portugal. They obtained a total of 83 different alleles from eighteen SSR primers and determined the polymorphic information content to be between 0.28 and 0.80. SSR research revealed that new information on the genetic diversity and genetic structure of the Brassica rapa subsp. rapa population will be useful for its use and management.

Table 2 Major allele frequency, gene diversity (H) and polymorphism information content (PIC) values
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Table 3 Pairwise genetic distance matrix obtained from inter-simple sequence repeat data
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Fig. 2
figure 2

The unweighted pair group method with arithmetic mean (UPGMA) tree-generated inter simple sequence repeat - polymerase chain reaction (ISSR-PCR) data. B1: Standart (Us), B2: Standart (Us), B3: Standart (Us), B4: Standart (Us), B5: Standart (Us), B6: Standart (Us), B7: Standart (Us), B8: Standart (Us), B9: Gündeğer Pütürge, B10: Gündeğer Pütürge, B11: Gündeğer Pütürge, B12: Gündeğer Pütürge, B13: Karşıyaka Pütürge, B14: Karşıyaka Pütürge, B15: Karşıyaka Pütürge, B16: Karşıyaka Pütürge, B17: Centre, B18: Şahit

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Fig. 3
figure 3

Two dimensional graph and eigen values created as a result of principal component analysis with inter simple sequence repeat - polymerase chain reaction (ISSR-PCR). B1: Standart (Us), B2: Standart (Us), B3: Standart (Us), B4: Standart (Us), B5: Standart (Us), B6: Standart (Us), B7: Standart (Us), B8: Standart (Us), B9: Gündeğer Pütürge, B10: Gündeğer Pütürge, B11: Gündeğer Pütürge, B12: Gündeğer Pütürge, B13: Karşıyaka Pütürge, B14: Karşıyaka Pütürge, B15: Karşıyaka Pütürge, B16: Karşıyaka Pütürge, B17: Centre, B18: Şahit

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Conclusion

In this study, 91.73% polymorphism, 0.4276 average “H”, 0.3319 average “PIC” and 0.6531 average major allele frequency were determined using nine ISSR primers. In the past, the ISSR, RAPD, and SSR techniques for detecting the genetic diversity of Brassica rapa genotypes, cultivar development and diagnosis have all proven to be suitable. In our study, the ISSR-PCR technique was revealed as a suitable marker for breeding, genetic variations and phylogenetic relationship of Brassica rapa subsp. rapa genotypes and the development of different cultivars.