Acta agriculturae Slovenica, 118/3, 1–7, Ljubljana 2022 doi:10.14720/aas.2022.118.3.2344 Original research article / izvirni znanstveni članek Genetic characterization of maize (Zea mays L.) landraces grown in Kosovo assessed by MITE-Hbr markers Barbara PIPAN 1, Sali ALIU 2, 3, Dukagjin ZEKA 2, Vladimir MEGLIČ 1 Received September 15, 2021; accepted June 29, 2022. Delo je prispelo 15. septembra 2021, sprejeto 29. junija 2022 1 Crop Science Department, Agricultural Institute of Slovenia, Hacquetova ulica 17, SI-1000 Ljubljana, Slovenia 2 Department of Crop Science, Faculty of Agriculture and Veterinary, Prishtina, University of Prishtina, Kosovo 3 Corresponding author,e-mail: sali.aliu@uni-pr.edu Genetic characterization of maize (Zea mays L.) landraces grown in Kosovo assessed by MITE-Hbr markers Abstract: The aim of this study was to examine and de- scribe genetic structure on autochthonous maize germplasm (flint types) from different localities in Kosovo using Hbr mark- ers. The genetic characterization of 6-8 individual seedlings per each of 20 landraces was conducted by Hbr display calculated per selective base, the most efficient genetic diversity estimator to distinguish between landraces was primer combination Hbr- Int5-F/MseI+T. The strongest genetic relatedness (r = 55.57) had landrace ACC4 having orange colored seeds, showing the highest genetic uniformity when compared to other accessions. Clustering analysis using the Bayesian approach generated two genetic clusters for observed landraces. As a measure of population structure influenced by genetic drift and migration, Fst values for each genetic cluster were obtained. Higher Fst (0.4027) was calculated within the first genetic group compar- ing to the second one (0.2001), reflecting a higher levels of out-crossing and conservation between landraces from the first genetic cluster. A similar distribution of genetic linkages was observed from dendrogram, constructed using Dice coefficient and neighbour-joining (NJ) algorithm with minor deviations for landraces ACC6 and ACC28. Genotypes of ACCmk land- race reveal the highest genetic distinction compared to other genotypes, reflecting the highest number of bands (241) and the highest number of private bands (10) as the number of bands unique to a single population, respectively. Key words: genetic variability; heartbreaker family mark- ers; maize Genetska karakterizacija lokalnih sort koruze (Zea mays L.) gojenih na Kosovu ovrednotena z MITE-Hbr označevalci Izvleček: Namen raziskave je bil preučiti in opisati genet- sko strukturo avtohtone dednine trdinke iz različnih lokalitet Kosova s Hbr označevalci. Genetska karakterizacija 6-8 sejank od vsake lokalne sorte je bila izvedena s prikazom nabora Hbr profilov, izračunanega na osnovi selektivne baze. Najučinko- vitejši določitelj genetske raznovrstnosti za razločevanje med lokalnimi sortami je bila kombinacija primerjev Hbr-Int5-F/ MseI+T. Najmočnejšo genetsko povezanost med genotipi (r = 55.57) je imela lokalna sorta ACC4 z oranžnimi zrni, ki je iz- kazovala največjo genetsko izenačenost v primerjavi z drugimi akcesijami. Klasterska analiza z uporabo modela aposteriorne verjetnosti (Bayesian approach) je za vključene lokalne sorte oblikovala dve genetski skupini. Kot merilo za analizo popu- lacijske structure, na katero vplivata genetski zdrs in migracija, so bile izračunane vrednosti Fst za obe genetski skupini. Večja vrednost Fst (0,4027) je bila izračunana znotraj prve genetske skupine v primerjavi z drugo (0,2001), kar kaže na večji delež navskrižnega križanja in ohranjanja raznolikosti med lokalnimi sortami prve genetske skupine grozda. Podobna porazdelitev genetskih povezav je bila določena na dendrogramu, izdelanem z uporabo Dice-ovega koeficienta podobnosti in algoritma raz- vrščanja po metodi združevanja najbližjih sosedov (NJ) z manj- šim odstopanjem za akcesiji ACC6 in ACC28. Genotipi akcesije ACCmk so se genetsko najbolj razlikovali od drugih na osnovi največjega števila prisotnih namnožkov (241) in največjega šte- vila prisotnih “privatnih namnožkov” (10) in v številu namnož- kov, ki so bili omejeni samo na eno populacijo. Ključne besede: genetska raznolikost; družina “Heart- breaker” označevalcev; koruza Acta agriculturae Slovenica, 118/3 – 20222 B. PIPAN et al. 1 INTRODUCTION Maize (Zea mays L.) is one of the most genetical- ly diverse and widespread crops in South-East Europe (Đalović et al., 2015; Ignjatović-Micić et al., 2015). Maize landraces have been largely replaced by commercial maize hybrids in Kosovo. The share of hybrids was 4 % in the 1960’s and already at 90 % in the early 2000’s due to their high yield potential. Studies of specific combining abilities of inbred lines and physiological traits of some hybrids were already performed in agroecological condi- tions of Kosovo (Aliu et al., 2008, 2010). Maize became widely investigated plant species regarding different ap- plications; its water use efficiency (Wang et al., 2013), morphological, physiological and biochemical response to SiO2 nanoparticles (Sharifi-Rad et al., 2016), different irrigation regimes and planting methods (Singh Brar et al., 2016). There are also different marker systems, ap- plied to assess genetic characterization of maize germ- plasm, including RAPD (Random Amplified Polymor- phic DNA) (Srdić et al., 2007), SSR (Simple Sequence Repeats) (Ignjatović-Micić et al., 2015) and MITE-Hbr (Miniature Inverted Repeat Transposable Element from the family Heartbreaker) (Casa et al. (2000; 2002) and Kavar et al. (2007)). MITE-Hbr markers have been firstly exploited and developed as a new marker system (modification of the AFLP-Amplified Fragment Length Polymorphism procedure) for evaluation of the maize genome (Casa et al. (2000; 2002)). Kavar et al. (2007) used Hbr markers to evaluate the genetic diversity of Slo- venian maize germplasm, originating from Western Bal- kan (former Yugoslavia). Related to those three reports, MITE-Hbr markers were proven to be stable, highly polymorphic, cost-effective, easily mapped and evenly distributed throughout the maize genome. SSRs are highly applicable markers in our genetic studies of different plant species, among other grape- vines (Rusjan etal., 2012, 2015) sweet potato (Pipan et al., 2016), brassicas (Pipan et al., 2011, 2013), and beans (Maras et al., 2015). In a study by Ignjatović-Micić et al. (2015), using SSR markers, they reported that higher genetic variation was observed among flint genotypes, comparing to dent ones. They also suggest that landraces from Western Balkans are highly adapted to specific en- vironmental conditions and uses and therefore could be a valuable source of genetic variability. The adaptation to diverse agro-ecological conditions is a result of natural and selection by farmers. The aim of this study was to examine and describe genetic structure of autochthonous maize germplasm from Kosovo using Hbr marker system and to employ Hbr display to show genetic differences and associations between and within observed flint landraces of maize collected in different localities of Kosovo, with a possibil- ity to detect conservation of gene flow into the maize ge- nome. The knowledge of genetic characteristics and lan- drace-specific background of maize germplasm would be of a benefit for future breeding and germplasm improve- ment programmes in Kosovo. 2 MATERIALS AND METHODS Twenty maize landraces, collected from differ- ent locations in Kosovo (Table 1), were screened using MITE-Hbr markers. DNA from 6 - 8 individuals of each landrace was extracted from each individual seedling us- ing BioSprint 15 DNA Plant Kit (Qiagen) and MagMax Express Magnetic Particle Processor (Life Technologies, Grand Island, NY) following manufacturer’s instruc- tions. Hbr display with some modifications was per- formed as described by Casa et al. (2000, 2002) and Ka- var et al. (2007). 600 ng of genomic DNA was digested for 3 h at 65 °C in 20 µl of 10x Tango buffer containing MseI (Fermentas). Adaptors (5’gacgatgagtcctgag and 5’tactcag- gactcat) to the digested DNAs and aliquots of the restric- tion/ligation reactions were visualized on 0.9 % agarose gels to check the quality of DNA digestion. Pre-selective amplification was performed using primers Hbr-Int5-E (5’gattctccccacagccagattc) and MseI+0 (5’gacgatgagtcct- gagtaa). Selective amplification was performed with each of the three selective primer combinations (MseI+C, MseI+G and MseI+T) with a fluorescently labeled Hbr internal primer (5’-6FAM-agccagattttcagaaaagctg). Frag- ment analysis was performed on the 3130XL Genetic Analyzer (Applied Biosystems), and sizing of fluorescent fragments/bands was determined by comparison with size standard GeneScan-500 ROX (Applied Biosystems) using GeneMapper 4.0 (Applied Biosystems). A binary matrix was constructed by scoring fragments as either present (1) or absent (0) in each DNA sample. Principal Coordinate Analysis (PCoA), Analysis of Molecular Variance (AMOVA), number of different alleles (Na), number of effective alleles (Ne) and Shan- non’s information index (I) across landraces for each selective base was calculated in GenAlEx v.6.4 (Peakal and Smousse, 2006). Genetic similarities were calculated on the basis of a binary matrix using the Dice similar- ity index (Dice, 1945). These coefficients were used to construct the clustering using the neighbur-joining (NJ) algorithm by 100 bootstraps in FreeTree (Pavliček et al., 1999) and visualized using TreeView (Page, 1996) soft- ware. Genetic diversity parameters between and within landraces including AMOVA, band patterns (num- ber of bands, number of private bands, number of lo- cally common bands alleles occurring in 50 % or fewer Acta agriculturae Slovenica, 118/3 – 2022 3 Genetic characterization of maize (Zea mays L.) landraces grown in Kosovo assessed by MITE-Hbr markers landraces, expected heterozygosity) and mean within landrace pairwise values (r) were conducted using Ge- nAlEx v.6.4 (Peakal and Smousse, 2006). Structure 2.3.3 software (Pritchard et al., 2009) was employed for infer- ring landrace structure using a Bayesian approach. Ten independent runs for each K (from1 to 7) in the case of admixture model were performed and burning period of 10,000 followed by 100,000 Markov Chain Monte Carlo repeats was used. The ideal K-value was selected based on the increases in likelihood ratios between runs us- ing Evanno’s delta K statistic (Evanno et al., 2005) im- plemented in a Structure Harvester (Earl and von Holdt, 2011). The estimation of flowering time was made in the same year at different locations, which are presented in Table 1. 3 RESULTS Kosovo landraces included in our study are all of a flint type with white kernel (fruit) color, except for a lan- drace ACC4, which kernels are orange (Table 1). Genetic characterization of 6-8 individual seedlings per each landrace was conducted using Hbr display. A total of 498 markers, ranging in size from 60-500 bp, were generated using three primer combinations: Hbr- Int5-F/MseI+T, Hbr-Int5-F/MseI+C, and Hbr-Int5-F/ MseI+G. Regarding genetic diversity estimators (PCoA, AMOVA, Na, Ne and I) calculated per selective base (T, C, G), the most efficient primer combination to distin- guish between landraces from Kosovo, was Hbr-Int5-F/ MseI+T, and Hbr-Int5-F/MseI+C respectively (Table 2). First three axes in PCoA (via covariance distance ma- trix) cumulatively explained 70 % of genetic variability for Hbr-Int5-F/MseI+T; 67 % for Hbr-Int5-F/MseI+C and 59 % for Hbr-Int5-F/MseI+G (Table 2). Percent of molecular variability among landraces in Hbr screen- ing varied from 10 (Hbr-Int5-F/MseI+C) to 18 (Hbr- Int5-F/MseI+T), depending on selective primer applied (Table 2). Additionally, the highest values of Na (7.400), Ne (1.067) and I (0.074) were calculated for Hbr-Int5-F/ MseI+T (Table 2). Landrace-specific genetic diversity was estimated by applying different algorithms to compare a genetic composition among and within autochthonous landrac- es from Kosovo. The summary of mean within landrace pairwise values, based on genetic distance, is presented in Figure 1. The lowest mean r value was calculated Landrace label Locality Latitude [° ‘ ‘’] Longitude [° ‘ ‘’] Altitude [m] Vernacular name Landscape Kernel type Kernel Color Kernel shape Flowering time [days] ACC2 Ferizaj 42.25.21 21.09.06 555 Bardhosh Flat Flint White Oval 69 ACC4 Shtime 42.26.40 21.42.96 642 Kolomboq Mountain Flint Orange Oval 72 ACC6 Skenderaj 42.44.39 20.48.04 603 Miser Valley Flint White Oval 71 ACC8 Skenderaj 42.44.39 20.47.39 597 Miser Valley Flint White Oval 75 ACC12 Skenderaj 42.45.00 20.48.23 623 Miser Flat Flint White Oval 76 ACC14 Drenas 42.39.30 20.42.46 565 Kolomboq Flat Flint White Oval 76 ACC16 Drenas 42.39.21 20.42.32 586 Kolomboq Flat Flint White Oval 75 ACC26 Vushtrri 42.48.38 20.58.30 518 Kolomboq Flat Flint White Oval 72 ACC28 Suharekë 42.21.45 20.49.02 388 Miser Valley Flint White Oval 77 ACC30 Vushtrri 42.50.46 20.59.26 557 Kolomboq Flat Flint White Oval 71 ACC32 Drenas 42.34.50 20.54.06 585 Kolomboq Flat Flint White Oval 71 ACC34 Podujevë 42.53.39 21.12.12 598 Kolomboq Mountain Flint White Oval 75 ACCmk Lipjan 42.31.45 21.07.20 551 Miser Flat Flint White Oval 69 ACC38 Kamenicë 42.33.56 21.31.32 812 Kolomboq Mountain Flint White Longi 67 ACC40 Kamenicë 42.34.16 21.31.32 766 Kolomboq Mountain Flint White Oval 72 ACC42 Prishtinë 42.35.35 21.20.40 824 Kolomboq Mountain Flint White Oval 65 ACC44 Drenas 42.41.21 20.45.31 691 Kolomboq Flat Flint White Oval 71 ACC46 Malisheve 42.27.56 20.43.22 576 Miser Mountain Flint White Oval 72 ACC48 Malisheve 42.28.01 20.44.04 562 Miser Mountain Flint White Oval 70 ACC50 Drenas 42.41.50 20.44.43 567 Kolomboq Flat Flint White Oval 76 Table 1: Characteristics of maize landraces from Kosovo Acta agriculturae Slovenica, 118/3 – 20224 B. PIPAN et al. across ACC50 (39.03) where r was also outside U and L limits (Figure 1) reflecting the weakest genetic related- ness of genotypes within ACC50 landrace. The strongest genetic relatedness (r = 55.57) was reached within orange colored seeds of ACC4 landrace (Figure 1) showing the highest genetic uniformity of included genotypes within ACC4 compared to other landraces. The genetic structure of observed landraces, described by Bayesian clustering approach in Figure 2 shows higher uniformity of ACC4, with 98.1 % probability that landrace ACC4 belongs to the first (red) genetic cluster and lower genetic uniform- ity within ACC50 with 79.9 % probability that genotypes Cumulative % in PCoA (first 3 axes) AMOVA (% among landraces) Na Ne I Hbr-Int5-F/MseI+T 70 18 7.400 1.067 0.074 Hbr-Int5-F/MseI+C 67 10 6.850 1.066 0.072 Hbr-Int5-F/MseI+G 59 16 7.100 1.065 0.069 Table 2: Analysis of genetic diversity among landraces by selective bases from ACC50 belong to the second genetic cluster (green) which is also confirmed by r value (Figure 1). In general, clustering analysis using the Bayesian method generated two genetic clusters (ideal K, con- ducted using Structure Harvester) for observed landraces (Figure 2). Similary colorored segments represents the estimat- ed membership to the genetic cluster. The first genetic cluster (red) posses 0.0778 of expected heterozygosity between genotypes and 0.0925 was calculated for the second one (green), respectively. Regarding their genetic structure, landraces ACC6, ACC12, ACC14, ACC34, Notes: Na-The number of alleles; Ne- The number of detected effective alleles; I-Shannon’s information index Figure 1: Mean within landrace pairwise values (r) according to genetic distance. Upper (U) and lower (L) confidence limits bound the 95 % confidence interval about the null hypothesis of ‘No difference ‘ across the landraces as determined by permuta- tion (99) Figure 2: Structure plot of maize landraces from Kosovo Acta agriculturae Slovenica, 118/3 – 2022 5 Genetic characterization of maize (Zea mays L.) landraces grown in Kosovo assessed by MITE-Hbr markers ACC28, ACC4, ACC46, ACC38, ACC16, ACC44, ACC2, and ACC8 belong to the first cluster of landraces (red); meanwhile landraces ACC30, ACC40, ACC42, ACC32, ACC48, ACCmk, ACC50, and ACC26 comprise the sec- ond genetic cluster (green) (Figure 2). A similar distribu- tion of genetic relations between landraces was observed in a dendrogram, constructed using Dice coefficient (Dice, 1945) and the NJ algorithm with minor deviations for landraces ACC6 and ACC28 (Figure 3). Figure 3: Genetic linkages of observed landraces from Kosovo using Dice coefficient and neighbour-joining (NJ) algorithm. (Green and red colours assign cluster colours from structure plot on Figure 2) Figure 4: Band patterns across maize landraces from Kosovo We have calculated landrace-specific parameters of genetic diversity (Figure 4) to compare genetic character- istics between all maize landraces collected, applying Hbr display. Genotypes of landrace ACCmk reveal the high- est genetic distinction compared to genotypes within other landraces, reflecting the highest number of bands (241) and the highest number of private bands (10) (Fig- ure 4), respectively. A number of common bands with a frequency of > 5 %, which are found in the 50 % assessed landraces, reached the highest values for ACC40 (115), ACC32 (114) and ACCmk (111), respectively (Figure 4). Evaluation of genetic differentiation within and be- tween landraces, applying Hbr markers, provided use- ful information about genetic the structure, relatedness and genetic diversity of autochthonous maize germplasm from Kosovo. Genetic uniformity of genotypes within landraces is high, regardless to low values of expected heterozygosity (max He = 0.067, data not shown) as a measure of genetic diversity within landraces. Genotypes within the second (green) cluster reveal lower genetic di- versity (Fst = 0.2001) compared to the first (red) cluster (Fst = 0.4027). 4 DISCUSSION Results presented in Table 2 indicate that Hbr- Int5-F/MseI+T is the most informative selective primer provided by Hbr display to distinguish twenty maize landraces collected from different locations in Kosovo. In the study by Kavar et al. (2007) evaluating Slovenian maize landraces, the most informative primer combi- nation was Hbr-Int5-F/MseI+G, revealing the highest number of loci (103) scored using Hbr display. On the other hand, calculated genetic diversity values for maize landraces from Kosovo are similar using Hbr display. This was as well the case evaluating Slovenian accessions by Kavar et al. (2007), where similar values of scored loci (73-103) were obtained applying different selec- tive primers. Cluster analysis using Bayesian approach revealed no genetic relatedness (regarding their genetic Acta agriculturae Slovenica, 118/3 – 20226 B. PIPAN et al. structure) between landraces from the same geographic origin (locality and landscape), a name of landrace and kernel shape (no difference for ACC38 which is oval), respectively. Structure plot also shows that individual plants from landraces ACC14 (87.3 % red, 12.7 % green), ACC50 (20.1 % red; 79.9 % green), ACC26 (28.0 % red, 72.0 % green), and ACC28 (75.0 % red, 25.0 % green) are sharing on average higher proportion of germplasm belonging to both genetic clusters (Figure 2). According to the data in Table 1, four landraces named Kolomboq (vernacular name or local name), originating from flat landscape have the longest flowering period of more than 72 days. There is one exception in this cluster, landrace ACC28 Miser (local name), originating from the valley (Suharekë) with the longest flowering period (77 days). A difference in landrace distributions for genetic cluster- ing, when comparing the structure plot (Figure 2) and dendrogram (Figure 3) could be a logical consequence of different algorithms/approaches applied for specific pur- pose, required for genetic diversity assessment. According to the landrace-specific parameters of genetic diversity (Figure 4), landrace ACCmk represents a potentially interesting source for further genetic studies and germplasm improvement. A high number of private bands actually represents unique copies of Hbr transpo- sons that ACCmk landrace harbors compared to private alleles observed in other studies using SSR markers. Pri- vate alleles in those cases could reflect accumulation and conservation of introduced genes via out-crossing along generations to the plant genome (Pipan et al., 2013). Cal- culated number of common bands with a frequency > 5 % (Figure 4) for landraces ACC40, ACC32 and even ACCmk, are sharing the highest number of scored bands with other landraces, even though that the three landrac- es belong to the second (green) genetic cluster/group (Figures 2 and 3). Evaluation of maize landraces from Kosovo was successfully assessed by a rare type of marker system, miniature inverted repeat transposable element - Hbr marker, using three selective primer combinations. To distinguish genotypes between and within different landraces, application of only one selective marker could be sufficient, as reported Kavar et al. (2007). Applica- tion of SSR markers as a codominant marker system is also used in genetic diversity studies of maize landraces (Ignjatović-Micić et al., 2015). According to the results presented, there are strong genetic relations between dif- ferent landraces, comprising two genetic groups, which could be assigned to the two general micro centers of diversity in Kosovo. As a measure of a population struc- ture influenced by genetic drift and migration, Fst values for each genetic cluster generated using Bayesian cluster analysis, were obtained. Higher Fst (0.4027) was calcu- lated within the first (red) genetic group compared to the second (green) one (0.2001), reflecting higher levels of out-crossing and conservation between landraces from the first genetic cluster. This result could be also a con- sequence of simultaneous cultivation and uncontrolled gene flow between observed maize landraces listed in Table 1 within the same production area of Kosovo in the past. 5 CONCLUSIONS Related to mean r value, there is 29.7 % of a variable genetic part, which is dispersed along included landraces from Kosovo. It is important to point out that landraces evaluated, originated from different localities with a di- verse landscape (flat, mountain, and valley) and from various production areas. According to the results, there are strong genetic relations between different landraces, comprising from two genetic groups, which could in- dicate on two micro diversification locations of flint type in Kosovo. The results provided, we can conclude that MITE-Hbr markers are highly applicable and cost- effective tool for maize genetic diversity studies and as in this case for a genetic distinction between and within landraces collected in Kosovo. 6 ACKNOWLEDGEMENT This work was in part financially supported by grant P4-0072 (Agrobiodiversity programme) from the Slo- venian Research Agency. We are thankful to the mag. Romana Rutar providing seedlings, dr. Katarina Rudolf- Pilih for an extra hand with DNA extraction and to the dr. 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