Molecular characterization and assessment of genetic diversity in maize parental genotypes using RAPD markers and its relationship with hybrid performance

Introduction

Maize (Zea mays L.) is one of the world's most important cereal crops and underpins food, feed, and broader agricultural productivity, sustaining continued demand for high-yielding cultivars and improved hybrids [1][2][3][4] . In modern maize breeding, heterosis exploitation remains a central route for increasing grain yield, and hybrid development is widely used to improve productivity across diverse agro-ecologies [1,2,4,5] . The efficiency of heterosis breeding depends strongly on parental choice because parental genetic divergence and allelic interactions contribute to hybrid performance, combining ability, and the extent to which heterosis is expressed [1,[7][8][9] . Accordingly, characterization of genetic diversity among candidate inbred lines is an important component of hybrid breeding and helps identify parental combinations with a greater probability of producing superior F₁ hybrids [9][10][11] . Combining ability analysis further assists breeders in identifying superior general and specific combiners and narrowing the number of crosses requiring detailed field evaluation [1,7,6] .

Diversity Assessment

Genetic divergence in maize has traditionally been assessed through morphological, biometrical, and multivariate procedures, including Mahalanobis D² clustering of agronomic traits [9,10,12,13] . These phenotypic approaches remain informative for classifying germplasm, but molecular and phenotypic groupings may differ because phenotypic expression is partly influenced by environmental and developmental factors [9,10,14] . DNA-based markers therefore provide a complementary means of estimating genetic relationships at the genome level [9,15,16,14] .

Molecular markers can improve discrimination among parental genotypes before extensive field testing [9] .SSR and other marker systems may provide robust estimates of genetic relationships [14,17] . Integration of molecular and conventional information can support more informed hybridization planning [9,15] .

RAPD Utility

Among PCR-based marker systems, RAPD markers have been widely used in maize diversity studies because they can detect polymorphism across the genome without prior sequence information and can generate multiple fragments from a single primer [16,17] . RAPD has been applied to genotype identification, purity assessment, phylogenetic grouping, and heterotic classification, making it a practical low-cost diversity tool, although newer marker systems can provide greater resolution in some applications [16,18,19] .

RAPD has detected high polymorphism in maize lines and cultivars [16] . RAPD has been used to separate elite maize lines into distinct genetic groups [8] . RAPD can support fingerprinting and purity assessment of hybrids and inbreds [16] .

Genetic Distance and Heterosis

An important objective of molecular diversity analysis is to determine whether parental genetic distance predicts hybrid performance, heterosis, or combining ability [11,15,7] . Evidence is not uniform. Some studies have reported positive relationships between molecular genetic distance and hybrid productivity, F₁ performance, or SCA, whereas other studies have found weak or non-significant relationships with grain-yield heterosis itself [7,8,11,17] . Thus, the predictive value of molecular distance depends on the germplasm, marker system, trait, and environment. Positive, significant relationship reported [8] SNP distance vs F₁ mean and SCA Positive, significant for grain yield [7] SNP distance vs grain-yield heterosis Non-significant in one maize study [7] SSR distance vs most traits Often weak or inconsistent RFLP distance in tropical maize Positive with F₁, SCA, MPH and HPH [11] The literature therefore supports molecular diversity estimates, including RAPD-based measures, as a useful complement rather than a standalone substitute for conventional combining ability and heterosis analyses in maize parent selection [7,1,8,15] . Therefore, the present investigation was undertaken to characterize twelve maize lines and five testers at the molecular level using RAPD markers, with the following objectives:

To estimate DNA-level polymorphism and genetic diversity among parental genotypes using arbitrary decamer primers.

To group the parental lines and testers into molecular clusters using UPGMA hierarchical analysis.

To examine the relationship between RAPD-based genetic similarity and hybrid performance, combining ability, and heterotic response for grain yield.

Materials and Methods

Plant Material and Genomic DNA Extraction

The experimental material comprised the seventeen parental genotypes used in the Line × Tester crossing programme: twelve inbred lines (BMI-45, CML-161, CML-163, CML-165, CML-224, CML-465, CML-468, HKI-163, HKI-323-B, K25, LM-14 and POP-61) and five male testers (DKC 9144, DKC7044, DMH 1301, HARITA_VMH 180 and PAC 751). Fresh, healthy leaf samples were collected from young seedlings. Genomic DNA was isolated using a modified CTAB extraction protocol. DNA quality and integrity were assessed by 0.8% agarose gel electrophoresis and spectrophotometric analysis before RAPD amplification.

RAPD-PCR Amplification

PCR amplification was performed using fifteen random decamer RAPD primers under standardized reaction and thermal-cycling conditions. Amplified products were separated by agarose gel electrophoresis and visualized using a gel documentation system.

Electrophoresis and Scoring

Amplified PCR products were resolved on 1.5% agarose gels and scored manually for the presence (1) or absence (0) of clear and reproducible amplification fragments across the seventeen parental genotypes.

Statistical and Cluster Analyses

Polymorphism percentage was calculated from the total and polymorphic bands generated by each primer. Binary banding matrices were used to calculate Jaccard's genetic similarity coefficients among parental genotype pairs. UPGMA hierarchical clustering was used to construct a dendrogram showing molecular relationships among lines and testers. Molecular similarity was compared with corresponding hybrid performance, SCA and heterosis information obtained from the Line × Tester evaluation.

Results and Discussion

RAPD Polymorphism and Marker Efficiency

All fifteen RAPD primers produced clear and reproducible amplification profiles across the seventeen parental genotypes. The primers generated 123 bands, of which 97 were polymorphic and 26 were monomorphic. The average number of bands per primer was 8.20, while average polymorphic bands per primer was 6.47. Mean polymorphism was 78.86%, with a range of 66.67-90.91%. OPB-05 generated the maximum number of bands (11) and the highest polymorphism (90.91%), followed by OPC-05 (90.00%) and OPB-03 (88.89%). OPA-04 and OPC-04 showed the lowest polymorphism (66.67%). These results demonstrate substantial molecular variability among the parental material. The separation of the five testers into a distinct molecular cluster indicates marked differentiation between the female line pool and tester pool. Such differentiation provides a useful molecular basis for examining the performance of crosses involving contrasting parental groups.

Relationship Between Molecular Diversity and Hybrid Performance

A major objective was to examine whether molecularly divergent parental combinations were also associated with superior hybrid performance. The results indicate that several highly divergent parental combinations identified through RAPD analysis produced hybrids with strong seed yield and combining ability. K25 × DMH 1301 had the lowest similarity among the highlighted pairs (0.55) and recorded the highest seed yield among the five top heterotic hybrids (241.63 g plant⁻¹), together with 92.64% mid-parent heterosis and 69.13% better-parent heterosis. BMI-45 × HARITA_VMH 180 also showed high molecular divergence (0.57) and a strong positive SCA effect for seed yield (+ 50.23) and biological yield (+ 56.24).

The comparison nevertheless should be interpreted as evidence of association within the present germplasm rather than proof that molecular distance alone determines heterosis. Hybrid performance is also influenced by parental combining ability and specific interactions. The result chapter itself shows that the most successful crosses combined molecular divergence with strong SCA and high heterotic expression. The top heterotic combinations identified in the result chapter further included HKI-163 × DKC7044, which recorded 237.51 g seed yield per plant and the highest better-parent heterosis among the five highlighted crosses (72.02%). The molecular similarity information therefore provides a useful additional dimension for interpreting why particular parental combinations performed well, but conventional SCA and heterosis estimates remain essential for final hybrid selection.

Conclusions

The RAPD analysis demonstrated substantial molecular diversity among the twelve maize lines and five testers used in the Line × Tester programme. Fifteen primers generated 123 bands, of which 97 were polymorphic, with an average polymorphism of 78.86%. Jaccard's similarity coefficients ranged from 0.55 to 0.86, and UPGMA analysis separated the seventeen parents into four major molecular clusters. The molecularly divergent combinations K25 × DMH 1301 and BMI-45 × HARITA_VMH 180 were also associated with strong hybrid performance and SCA, while K25 × DMH 1301 recorded particularly high mid-parent and better-parent heterosis for seed yield. These results support the use of RAPD characterization as a complementary tool for identifying divergent parental combinations in maize. However, molecular distance should be considered together with per se performance, GCA, SCA, heterosis and field evaluation before advancing hybrids for breeding or commercial testing.

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