Genetic evaluation of F2 and F3 interspecific hybrids of mung bean ( Vigna radiata L. Wilczek) using retrotransposon‐based insertion polymorphism and sequence‐related amplified polymorphism markers

Mung bean ( Vigna radiata L. Wilczek) is a self‐pollinating and indispensable pulse crop in Indonesia. While low yield productivity is a major concern, genetic improvement is possible through interspecific hybridization. However, interspecific hybridization is relatively infrequent and produces low recombination exchanges, significantly limiting crop breeding efficiency. Thus, a comprehensive study is needed of the selection and genetic diversity evaluation of progenies in advanced generations derived from interspecific hybridization using a specific molecular marker. This study aims to confirm the heterozygosity in the F2 population and assess the genetic diversity in F3 mung bean populations resulting from interspecific hybridization between the mung bean and common bean. We designed the retrotransposon‐based insertion polymorphism (RBIP) marker by identifying the syntenic regions in the flanking sequences of retrotransposon insertion in common bean and mung bean. The RBIP marker can be applied to distinguish the heterozygote progenies from the homozygote progenies. Six combinations of sequence‐related amplified polymorphism (SRAP) primers were used in the genotyping of F3 mung bean progenies. The SRAP marker showed a high degree of polymorphism of up to 100%, while high genetic variation was observed within the population (71%) of mung bean progenies. The F3.4 population had the greatest number of genotypes and displayed the highest number of effective alleles, private alleles, and percentage of polymorphic loci, suggesting the existence of high genetic diversity within this population. These genetic diversity data are exceptionally critical for future genetic research since it has potentially high yield production. The genomic and marker‐assisted selection studies will support the major goals of the mung bean breeding program.


Introduction
Mung bean (Vigna radiata L. Wilczek) is an annual and pulse crop, selfpollinated, and important legume crop in Asia (Lin et al. 2020).Mung bean are rich in vitamins, fibers, minerals, carbohydrates, proteins, and can be used as potential crop for mitigation of malnutrition (Ganesan and Xu 2018), and commonly used as supplemental dishes and healthy food in Indonesia (Novidiyanto et al. 2019).Mung bean is fairly resistant to abiotic stresses, but pro ductivity has remained low due to biotic challenges as well as a lack of variation with significant yield potential, big seed size, and high weight per seed.The production po tential of an Indonesian mung bean is up to 2.5 ton/ha, with an average productivity of about 0.9 ton/ha (Taufiq and Kristiono 2016).Cultivated mung bean had a narrow genetic basis represent low genetic variability in primary gene pool (Noble et al. 2018; Fatmawati et al. 2021).
To accelerate the genetic improvement of mung bean, interspecific hybridization approach can be utilized for creating interspecific recombinant and produce superior genotypes through mung bean breeding project.Interspe cific hybridization is indispensable method for breeding program (Zhan et al. 2017).Interspecific hybridization has been reported for genetic speciation, genome evolu tion, genetic diversity, introgression novel gene as well as improve adaptability to new environment, increasing yield, and essential nutrition to support biofortified breed ing program (Abbas et al. 2015; Zhang et al. 2016; Yu et al. 2021).The common bean (Phaseolus vulgaris L.) and mung bean, belonged to the Fabaceae family, separated Fatmawati et al. Indonesian Journal of Biotechnology 28(3), 2023, 143-152 from a common ancestor between 4.9 and 8 million years ago (Lavin et al. 2005).Thus, the mung beancommon bean linkage group is generally preserved, and synteny analysis of mung bean unigene sequences indicated gene function similarities with common bean (McClean et al. 2010).
Interspecific hybridization in mung bean has been re ported to be effective (Abbas et al. 2015; Pandiyan et al. 2020), but no studies of interspecific hybridization with common bean have been reported.Although segregation distortion has been reported in the majority of F2 progenies derived from interspecific (Toyomoto et al. 2019; Shehzad et al. 2021), we were able to successfully evaluate the F2 interspecific progenies from this crossing and maintain the elite genotypes through pedigree selection for F3 genera tion.To validate the genetic constitution of interspecific hybrids in mung bean, DNA marker is preferable to be used for characterization of the genetic background on ma terial tested, compared to morphological characterization since it is laborious task and often affected by environ mental condition (Sormin et al. 2021).In addition, DNA marker can be applied to assess genetic diversity, evolu tion, and phylogeny, investigate heterosis, identify hap loid/diploid plants and cultivar genotyping, and marker as sisted selection (Nadeem et al. 2018).
Transposable element in particular retrotransposon is relatively high abundant in mung bean genome (Kang et al. 2014).This mobile element is well organized in chromosomes and inserted into multiple gene loci (Se tiawan et al. 2020).Mung bean genome comprise of repetitive sequences (50.1%), in which 36.5% consist of long terminal repeat (LTR) retrotransposon (Kang et al. 2014).Interspecific hybridization may influence the ac tivation of mobile elements in hybrids, potentially lead ing to plant speciation and insertion of specific gene of interest (Glombik et al. 2020).Thus, transposable el ements are useful to be utilized as molecular marker due to their abundance in plant genome.Transposable elementbased markers such as interretrotransposon am plified polymorphism (IRAP), miniatureinverted repeat transposable element (MITE), interSINE amplified poly morphism (ISAP), retrotransposonbased insertion poly morphism (RBIP) has been used in plant genotyping of melon (Sormin et al. 2021), identification of somaclonal variation in date palm (Mirani et al. 2020), and genetic di versity assessment in mango (Nashima et al. 2017).
Our previous works confirmed that the F2 population of mung bean derived from interspecific hybrids was veri fied as genuine hybrids by employing a dominant IRAP marker (Fatmawati et al. 2021).However, this marker could not identify heterozygote genotypes, and a codomi nant marker is prefered for analyzing the genetic constitu tion of these hybrids.In addition, interspecific hybridiza tion is typically followed by wholegenome or fragmented DNA duplication to ensure the stability of the genuine hy brids (Glombik et al. 2020).Therefore, the use of codom inant markers is critical for the identification of genotypes containing the novel genetic recombination from both par ents in the advanced generation.Even though single se quence repeat (SSR) is a codominant marker that has been used to assess genetic diversity in mung bean accessions (Kaur et al. 2018) and the hybrid vigor of mung bean F1 hybrids (Sorajjapinun et al. 2012), but it required many primersets and was only appropriate for progenies derived from intraspecific hybridization in which their parental lines shared similar genomic constitutions.The retrotrans poson based codominant marker can be designed by de termining different allelic states at certain loci from both parental lines utilized for interspecific hybridization by identifying the flanking sequence of retrotransposon in sertion.The RBIP is a PCRbased marker that can de tect transposable element insertions in plant genomes at a specific locus and provide an accurate DNA profile.The genomic DNA can be amplified using LTR and flanking regionspecific primer sets.In addition, RBIP has been used in plant genetic studies (Kim et al. 2012; Schulman et al. 2012; Nashima et al. 2017).
After confirming and selecting the F2 was genuine hy brids, we developed an F3 population by selecting elite genotypes from F2 population through pedigree selection.Genetic variation in the F3 population is critical for the mung bean breeding effort, particularly when determin ing advanced elite genotypes and/or choosing future par ents (Baenziger et al. 2011).The genetic diversity of the F3 population can be analyzed using sequencerelated am plified polymorphism (SRAP) (Purwantoro et al. 2023).SRAP is a PCRbased marker, consist of 17 or 18 nu cleotides that amplified the open reading frames (Li and Quiros 2001).SRAP is dominant marker which has been successfully applied to investigate the genetic diversity in Indian garlic (Benke et al. 2021) and mung bean (Aneja et al. 2013).This study aimed to identify the flanking ge nomic sequences from both the mung bean and common bean genomes and design the RBIP marker from a highly conserved region to investigate heterozygosity in the F2 population.This work also aims to assess genetic diversity in F3 mung bean populations resulting from interspecific hybridization between the mung bean and common bean using SRAP marker.

Plant materials
The F2 population of interspecific hybrids that used by Fatmawati et al. (2021) was utilized to study their genetic constitutions using the RBIP marker.Four elite genotypes of mung bean selected from F2 generation were used for F3 main population.Each elite genotype consisted of 16 plants.These 64 genotypes were derived from interspe cific hybridization [mung bean landrace ′lokal malang′ (Vi gna radiata L. Wilczek) × common bean cultivar ′Lebat 3′ (Phaseolus vulgaris L.)].The plants were cultivated in Research Station of Banguntapan, Faculty of Agriculture, Universitas Gadjah Mada, Yogyakarta from October 2020 until April 2021.Remark: TPL = Total polymorphic loci; TAL = Total amplified loci; DP = Degree of polymorphism.

Isolation and quantitation of genomic DNA
Total DNA was isolated from mung bean leaves using a modified CTAB (hexadecyltrimethylammonium bromide) as described in Dharajiya et al. (2017).NanoDrop (2000c Spectrometer, Thermo Scientific) was used to quantify DNA samples.Then, DNA were diluted using nuclease free water (NFW) into working solution 25 (ng/µL).

Synteny analysis of mung bean-common bean and RBIP marker design
The bacteria artificial chromosome (BAC) clone of Phase olus vulgaris PVGBa_61E16, the accession of genbank ID GU215957.1 was retrieved from the National Center for Biotechnology Information (NCBI).The LTR sequence and Ty1/Copia retrotransposon and the motif of the se quence was detected and confirmed by LTR finder (Xu and Wang 2007) and Conserved Domain Database (CDD) of NCBI (Lu et al. 2020), respectively.The synteny anal ysis of the flanking genomic sequence of retrotransposon between mung bean and common bean was conducted us ing a dotlet (Junier and Pagni 2000).The flanking se quence of 3′ LTR retrotransposon from P. vulgaris was subjected to Dotlet JS (https://dotlet.vitalit.ch)against V. radiata genome sequence (LJIH01000004.1:c168862160236) to identify the synteny region.Multiple sequence alignment (MSA) of syntenic region from the flanking genomic sequence was generated using ClustalW em bedded in BioEdit.The RBIP primers were designed from the highly conserved region to obtain PCR prod ucts with different size between P. vulgaris and V. ra diata using FastPCR (Kalendar et al. 2017).The flank ing region was amplified by PCR using primer pairs 5′ ACCATTTAAGCCCAAGGTTCAACCTCA3′ and 5′ GAGACTTTCCTCTGCATATGAAC3′.

PCR amplification
The amplification of DNA was carried out using T100™ thermal cycler (BioRad, USA).The PCR reaction of RBIP and SRAP was consisted of 50 ng of gDNA, 0.2 mM dNTPs, 0.2 µM primer, 1X GoTaq® Green Master Mix (Promega, USA), 1.25 U/µL GoTaq® polymerase, and added with NFW into final volume 12.5 µL.The ampli fication of RBIP condition consisted of predenaturation at 95 °C for 2 min, 35 cycles of denaturation at 95 °C for 1 min, annealing at 60 °C for 1 min, with an extension at 72 °C for 2 min, and the final extension at 72 °C for 10 min.The DNA amplification using IRAP marker was con ducted in accordance to Fatmawati et al. (2021).The PCR condition of SRAP were conducted in accordance with Li and Quiros (2001).In brief, predenaturation at 94 °C for 2 min, annealing at 35 °C for 1 min, and extension at 72 °C, 1 min are the first five cycles.In additional 35 cycles, the annealing temperature was raised to 50 °C for 1 min, fol lowed by an 8min extension at 72 °C.The SRAP primer sequences are listed in Table 1 as described in Uzun et al. (2009).

Data analysis
The amplified bands were scored as 1 if they were present and 0 if they were not to generated binary data.NTSYS PC software was used to do the cluster analysis (Rohlf 2009).The binary data were subjected to genetic simi larity matrix using simple matching on the similarity of quantitative data (SIMQUAL) program.The mean of the unweighted pair group method with arithmetic average (UPGMA) technique was used to create a dendrogram.Number of distinct alleles (Na), number of effective al leles (Ne), expected heterozygosity (He), number of loci with private allele (Pa), and percentage of polymorphic loci (PPL) were used to compute genetic indices.The ge netic distance between mung bean genotypes was used to perform principal coordinate analysis (PCoA).The analy sis of molecular variance (AMOVA) and PCoA were car ried out using the GenAIEx software version 6.5 (Peakall and Smouse 2012).

RBIP marker design and genetic confirmation of F2 hybrids
An LTR retrotransposon was detected from BAC clone of Phaseolus vulgaris (GU215957.1)using LTR Finder (Xu and Wang 2007).The sequence length of this retrotranspo son was 4200 bp, and the lengths of its 5 and 3 LTRs were 320 and 317 bp, respectively.This retrotransposon shared 97.20% of LTR similarity and target site duplication was marked by CATTC at both the 5′ and 3′ end of LTR.This LTR retrotransposon was classified as Ty1/Copia retro transposon based on Conserved Domain Database (CDD) analysis (Figure 1).This Ty1/Copia sequence was made up of GAG, Integrase, Reverse Transcriptase, and RNAse The MSA of flanking genomic region was analyzed using Bioedit and the primers were designed from highly conserved syntenic region to obtain the PCR product with different size from both plant species (Figure 3).The PCR amplification from both of ′lokal malang′ (female parent) and ′Lebat3′ (male parent) with RBIP primers generated distinct amplicon size around 1300 bp and 900 bp, re spectively (Figure 4b).The heterozygote progenies can be clearly recognized from the homozygote progenies be cause they inherit alleles from both parents (Figure 4b).The homozygous progenies only had a single band that was identical to the female parent, such as genotypes of 43, 49, and 50.In contrast, IRAP, a dominant marker, was not unable to distinguish the heterozygotes (Figure 4a).
The successful hybridization of two parental lines re sulted in a novel genetic recombination in their progeny.Genetic recombination determines population diversity and generates unique allele combinations (Fernandes et al. 2018).Recombination rates consider to be different among species, populations, individuals, sex, chromo somes, and intrachromosomal locations (Dreissig et al. 2019).The successful genetic recombination determines by the successful hybridization and fertilization.How ever, the interspecific hybridization is relatively infre quent, produces low recombination exchanges, signifi cantly limiting crop breeding efficiency (Shen et al. 2021).Therefore, comprehensive identification of progenies re sulting from interspecific hybridization should be carried out using genetic markers.In this results, the RBIP marker used in this study was confirmed and supported that the progenies are genuine hybrids derived from interspecific hybridization between mung bean and common bean that has been validate by Fatmawati et al. (2021) using IRAP marker and the progeny had a distinct morphological char acter in seed size and seed coat color (Fatmawati et al. 2021; Fatmawati 2022).

SRAP marker analysis of F3 interspecific hybrid of mung bean
In this study, we demonstrated the application of SRAP marker to characterize genetic diversity of F3 mung bean population.High level polymorphism was observed when SRAP marker applied in mung bean genotyping.Six com bination SRAP primers has been successfully amplified PCR products to characterize genetic diversity in all mung bean genotypes.All primers produced 122 of total ampli fied loci out of which 120 loci were polymorphic (Table 1).
The amplicon sizes and degree of polymorphism were var ied from 100 to 2000 bp and 77.78% to 100.00%, respec tively.These results imply that all these SRAP primers were highly effective for mung bean genotyping.The use of molecular markers is essential, especially when assess ing the genetic diversity of a population produced via inter specific hybridization.Mung bean lacks a barier in which to cross with closely related species (Pandiyan et al. 2020).Furthermore, rigorous research should be carried out uti lizing molecular markers to determine genetic variety in advanced genotypes.Thus, using SRAP marker, we con firmed that this F3 population of was genetically varied and exhibited high degree of polymorphism.

Genetic diversity of F3 mung bean population
The values of Na and Ne ranged from 0.967 to 1.336 and 1.127 to 1.148, respectively (Table 2).When compared to other populations, the F3.2 population has the most dis tinct alleles.The He value ranged from 0.089 to 0.117, while the PPL value ranged from 45.08 to 63.11 percent.
The F3.2 population has the highest He and PPL values.The Pa value for the entire population ranged from 2 to 14 bands.Both the F3.2 and F3.4 populations have a high Pa value, with 13 and 14 bands, respectively.These findings indicate that the genotypes in the F3.2 and F3.4 popula tions have a significant genetic diversity when compared to other populations.This finding is corroborated by the AMOVA, which shows that genetic variation is prevalent within populations (Table 3).According to the AMOVA data, there was 71% variance within the population and 29% variation among populations (Table 3).The variance was calculated using 999 permutations.With a moderate degree of genetic diversity, population variation is consid erably different (29%).This finding is confirmed by the large number of total private alleles produced in all pop ulations (34), of which 13 and 14 private alleles occurred in F3.2 (38.23%) and F3.4 (41.17%), respectively (Table 2).The selection of characteristics linked to yield compo nents and seed coat colors in F2 population of mung bean may have resulted in high diversity within the population.
The F3.4 population presented remarkable genetic dif ferentiation in the number of private allele (Table 2).The reason for the increased genetic diversity among geno types of the F3.4 population might be because it is free of segregation distortion in F2 and its progenies inherit the alleles from their male parent (common bean).The private allele denotes that the allele exists solely in one population.The F3.4 population comprised 14 private al leles compared to F3.1 and F3.3, which had 5 and 2 private alleles, respectively, showing the considerable genetic di versity that occurred among the F3.4 genotypes.Private allele data provide valuable information on the unique ge netic variety at specific loci, as well as identifying highly diverse genotypes that might be used as parental lines in plant breeding programs to enhance allele diversity in the population (Salem and Sallam 2016).The allelic pattern and genetic diversity indices were beneficial in determin ing genetic variation in each population.Even though the three populations had distinct diversity, the F3.4 had the

Cluster analysis of F3 mung bean population
Understanding the genetic diversity of this population re quires the identification of F3 population structure.UP GMA clustering of simple matching similarity data from all six marker combinations split 64 F3 genotypes into six different groups (Figure 5).The similarity coefficient was varied from 0.75 to 0.98.There were 39 individuals in Cluster I. On the other hand, Clusters II, III, IV, V and VI had 2 members, 2 members, 6 members, 16 members, and 1 member, respectively.This finding suggests that ISAP is a rather good marker for identifying genetic diversity in an interspecific hybrid F3 population.A PCoA biplot analysis validated the UPGMA clustering result, which di vided the 64 mung bean genotypes into four groups (Fig ure 6).All 64 genotypes of F3 mung bean population were divided into four quadrants.Quadrant I included the genotypes of the F3.1 population.The predominance of F3.2 and F3.3 genotypes were found in quadrant II.Sev eral genotypes of F3.2 and F3.4 populations were found in quadrant III.The most of F3.4 populations were found in quadrant IV.This result implies that the F3.4 popula tion was distinguished from other populations, which is corroborated by the UPGMA dendrogram result (Figure 5).The findings of the dendrogram analysis (UPGMA) agreed with the results of the PCoA biplot (Figures 5 and  6).This result implies that the F3 population structure that has been selected from F2 generation had a distinct genetic background which supported by the phenotypic variation of seed coat color and yield traits (Fatmawati 2022).Furthermore, knowing population structure is criti cal for identifying markerassociated characteristics using genomewide association studies (GWAS) (Eltaher et al. 2018).As a result, before doing GWAS to find a proper correlation between a characteristic of interest and markers that might lead to the identification of underlying genes, the first stage is to evaluate the population structure.

Conclusions
In conclusion, the RBIP marker can be used to differentiate the heterozygote progenies in F2 population of interspe cific hybrids.The F3.4 genotype population had the high est number of private alleles, polymorphic loci percentage, and effective alleles.Despite their tremendous selection, the elite genotypes were genetically diverse.This mung bean population structure and genetic diversity informa tion is critical for future genetic studies such as GWAS and markerassisted selection studies for high yield poten tial and nutritional value of mung bean.

FIGURE 1
FIGURE 1 Ty1/Copia retrotransposon structure identified in Phaseolus vulgaris containing flanked DNA sequence that high synteny to Vigna radiata.

FIGURE 2
FIGURE 2 Dot plot analysis of Phaseolus vulgaris and Vigna radiata genomic sequences synteny region that flanked at the 3′ LTR of Ty1/Copia retrotransposon.

FIGURE 3 FIGURE 4
FIGURE 3 Multiple sequence alignment of flanking genomic region of Phaseolus vulgaris and Vigna radiata.Yellow colors depict the primer position.

FIGURE 5 FIGURE 6
FIGURE 5 Dendrogram of 64 genotypes of mung bean in F3 population based on SRAP markers which separated the genotypes into 6 clusters with the genetic similarity coefficient is 0.82.

TABLE 1
Marker utility of SRAP in F3 interspecific hybrid of mung bean.

TABLE 2
The average of various genetic factors in each mung bean population.
Remark: df = degree of freedom; SS = sum of square; MS = mean of square; Est.Var = estimated variance; % Var = percentage of variation.