Identification of diagnostic KASP-SNP markers for routine breeding activities in yam (Dioscorea spp.).

Maintaining genetic purity and true-to-type clone identification are important action steps in breeding programs. This study aimed to develop a universal set of kompetitive allele-specific polymerase chain reaction (KASP)-based single nucleotide polymorphism (SNP) markers for routine breeding activities. Ultra-low-density SNP markers were created using an initial set of 173,675 SNPs that were obtained from whole-genome resequencing of 333 diverse white Guinea yam (Dioscorea rotundata Poir) genotypes. From whole-genome resequencing data, 99 putative SNP markers were found and successfully converted to high-throughput KASP genotyping assays. The markers set was validated on 374 genotypes representing six yam species. Out of the 99 markers, 50 were highly polymorphic across the species and could distinguish different yam species and pedigree origins. The selected SNP markers classified the validation population based on the different yam species and identified potential duplicates within yam species. Through penalized analysis, the male parent of progenies involved in polycrosses was successfully predicted and validated. Our research was a trailblazer in validating KASP-based SNP assays for species identification, parental fingerprinting, and quality control (QC) and quality assurance (QA) in yam breeding programs.

Agronomic and hormonal approaches for enhancing flowering intensity in white Guinea yam (Dioscorea rotundata Poir.).

Developing novel white Guinea yam (Dioscorea rotundata) varieties is constrained by the sparse, erratic, and irregular flowering behavior of most genotypes. We tested the effectiveness of nine agronomic and hormonal treatments to enhance flowering on D. rotundata under field conditions. Genotypes responded differently to flower-inducing treatments (p<0.001). Of the test treatments, pruning and silver thiosulfate (STS) were effective in increasing the number of spikes per plant and the flowering intensity on both sparse flowering and monoecious cultivars. STS and tuber removal treatments promoted female flowers on the monoecious variety while pruning and most treatments involving pruning favored male flowers. None of the treatments induced flowering on Danacha, a non-flowering yam landrace. Flower-enhancing treatments had no significant effect on flower fertility translated by the fruit set, since most treatments recorded fruit sets above the species' average crossability rate. Flower-enhancing techniques significantly influenced number of tubers per plant (p = 0.024) and tuber dry matter content (DMC, p = 0.0018) but did not significantly affect plant tuber yield. Nevertheless, treatments that could enhance substantially flowering intensity, such as pruning and STS, reduced tuber yield. DMC had negative associations with all flowering-related traits. This study provided insights into white yam flower induction and suggests promising treatments that can be optimized and used routinely to increase flowering in yam crop, without significantly affecting flower fertility and tuber yield.

Association mapping in multiple yam species (Dioscorea spp.) of quantitative trait loci for yield-related traits.

BACKGROUND: Yam (Dioscorea spp.) is multiple species with various ploidy level and considered as cash crop in many producing areas. Selection based phenotyping for yield and its related traits such as mosaic virus and anthracnose diseases resistance and plant vigor in multiple species of yam is lengthy however, marker information has proven to enhance selection efficiency. METHODOLOGY: In this study, a panel of 182 yam accessions distributed across six yam species were assessed for diversity and marker-traits association study using SNP markers generated from Diversity Array Technology platform. For the traits association analysis, the relation matrix alongside the population structure were used as co-factor to avoid false discovery using Multiple random Mixed Linear Model (MrMLM) followed by gene annotation. RESULTS: Accessions performance were significantly different (p < 0.001) across all the traits with high broad-sense heritability (H2). Phenotypic and genotypic correlations showed positive relationships between yield and vigor but negative for yield and yam mosaic disease severity. Population structure revealed k = 6 as optimal clusters-based species. A total of 22 SNP markers were identified to be associated with yield, vigor, mosaic and anthracnose diseases resistance. Gene annotation for the significant SNP loci identified some putative genes associated with primary metabolism, pest and resistance to anthracnose disease, maintenance of NADPH in biosynthetic reaction especially those involving nitro-oxidative stress for resistance to mosaic virus, and seed development, photosynthesis, nutrition use efficiency, stress tolerance, vegetative and reproductive development for tuber yield. CONCLUSION: This study provides valuable insights into the genetic control of plant vigor, anthracnose, mosaic virus resistance, and tuber yield in yam and thus, opens an avenue for developing additional genomic resources for markers-assisted selection focusing on multiple yam species.

Variability and genetic merits of white Guinea yam landraces in Nigeria.

Introduction: Landraces represent a significant gene pool of African cultivated white Guinea yam diversity. They could, therefore, serve as a potential donor of important traits such as resilience to stresses as well as food quality attributes that may be useful in modern yam breeding. This study assessed the pattern of genetic variability, quantitative trait loci (QTLs), alleles, and genetic merits of landraces, which could be exploited in breeding for more sustainable yam production in Africa. Methods: A total of 86 white Guinea yam landraces representing the popular landraces in Nigeria alongside 16 elite clones were used for this study. The yam landraces were genotyped using 4,819 DArTseq SNP markers and profiled using key productivity and food quality traits. Results and discussion: Genetic population structure through admixture and hierarchical clustering methods revealed the presence of three major genetic groups. Genome-wide association scan identified thirteen SNP markers associated with five key traits, suggesting that landraces constitute a source of valuable genes for productivity and food quality traits. Further dissection of their genetic merits in yam breeding using the Genomic Prediction of Cross Performance (GPCP) allowed identifying several landraces with high crossing merit for multiple traits. Thirteen landraces were identified as potential genitors to develop segregating progenies to improve multiple traits simultaneously for desired gains in yam breeding. Results of this study provide valuable insights into the patterns and the merits of local genetic diversity which can be utilized for identifying desirable genes and alleles of interest in yam breeding for Africa.

Multiple-Traits Selection in White Guinea Yam (Dioscorea rotundata) Genotypes.

Choosing superior parents with complementary trait values for hybridization and selecting variants with desired product profiles to release as a new cultivar are important breeding activities to progress genetic improvement in crops. This study assessed the genetic potential of 36 parental lines of white Guinea yam (Dioscorea rotundata) genotypes using multi-trait index-based factor analysis and ideotype design (FAI-BLUP). The experiment utilized 36 white yam genotypes laid out in a 6 × 6 triple lattice design with three replications and phenotyped for 18 agronomic and food quality traits. Findings showed significant differences among genotypes for all assessed traits. Fifteen traits had desired genetic gains, whereas stem diameter (-1.34%), and two starch property traits ((holding strength (-26.31%) and final paste viscosity (-3.33%)) had undesired selection gain. The FAI-BLUP index provided total genetic gains of 148.91% for traits desired for increase and -29.26% for those desired for decrease. Genotypes TDr08-21-2, TDr9518544, TDr9501932, TDr8902665 and Pampars were identified as top best candidate for simultaneous improvement of the measured traits in white yam breeding. The findings indicate the effectiveness of the FAI-BLUP index in identifying and selecting genotypes.

White Guinea yam (Dioscorea rotundata Poir.) landraces trait profiling and setting benchmark for breeding programs in the Republic of Benin.

To meet the high demand for white Guinea yam, there is a need to develop and release improved varieties to farmers. Unfortunately, low rate of adoption of most of the improved yam varieties by both producers and consumers was observed. Information regarding agronomic characteristics and food qualities of popular white Guinea yam landraces with high market value are not available to establish minimum standards to be considered by breeding programs. To fill this gap, surveys using rural appraisal tools were carried out in 20 villages and 16 markets throughout Benin. Data on the agronomic performance suggested that for an improved variety to be adopted by Beninese farmers it should have a minimum yield of 4.16 ± 0.15 kg per mound, and average number of marketable tubers of 1.23 ± 0.05, a mean tuber length of 36.41 ± 1.22 cm, and a minimum diameter of 25.44 ± 1.16 cm. The sensorial attributes for boiled and pounded tubers of this improved variety should have minimum score of 3.16 for texture, 0.75 for softness, 3.75 for elasticity, and 1.34 for colour during the sensory evaluation. The improved variety must also have a minimum average severity score of 1.1 for yam mosaic virus disease, 1.33 for anthracnose and 1 for nematodes. Landraces Amoula, Laboko, and Djilaadja should be considered as the standard for yield, sensory attributes, and tolerance to pest and diseases while landraces Danwari, Kodjewe, Mondji, and Gnidou should be characterized as possessing good flowering and fruit setting capacities for breeding programs.

Optimum time for hand pollination in yam (Dioscorea spp.).

Hand pollination success rate is low in yam (Dioscorea spp.), due partly to suboptimal weather conditions. Thus, determining the most suitable time for pollination could improve the pollination success in yam breeding programs. We performed continuous hand pollination within flowering windows of D. rotundata and D. alata for two consecutive years to determine the most appropriate month, week, and hours of the day allowing maximum pollination success. In D. alata crossing block, we observed significant differences among crossing hours for pollination success (p = 0.003); morning hours (8-12 a.m.) being more conducive than afternoons (12-5 p.m.). No significant differences existed between crossing hours in D. rotundata, though the mid-day seemed optimal. For both species, the time interval 11-12 a.m. was more appropriate for crossing while 4-5 p.m. was the poorest. However, in vitro pollen germination tests showed that mid-day pollen collection (12 noon-2 p.m.) had better results than both extremes, though there were strong genotypic effects on outcomes. Pollination success rates differed significantly among months for D. alata (p < 0.001) but not for D. rotundata (p > 0.05). Differences in pollination success existed across weeks within flowering windows of both D. alata (p < 0.001) and D. rotundata (p = 0.004). The seed production efficiency (SPE) had a similar trend as the pollination success rate. No clear pattern existed between the pollination time and the seed setting rate (SSR) or seed viability (SV), though their dynamics varied with weeks and months. This study provided an insight on the dynamics of pollination outcomes under the influence of pollination times and allows detecting months, weeks, and hours of the day when hybridization activities should be focused for better results.

Optimizing use of U.S. Ex-PVP inbred lines for enhancing agronomic performance of tropical Striga resistant maize inbred lines.

BACKGROUND: Temperate maize inbred lines with expired Plant Variety Protection Act certificates (Ex-PVP) are potential sources of desirable alleles for tropical germplasm improvement. Up to now, the usefulness of the Ex-PVP inbred lines as a potential source of novel beneficial alleles for Striga hermonthica resistance breeding to enhance genetic gain in tropical maize has not been reported. RESULTS: This study was thus conducted to characterize the combining ability of 24 Ex-PVP inbred lines in crosses with two tropical Striga resistant inbred testers under Striga-infested and non-infested conditions and across three locations for 2 years. Many testcrosses between Ex-PVP inbred lines and the first tester (T1) produced competitive or significantly higher grain yields compared to the hybrid between the two resistant testers under Striga infested and non-infested conditions and across multiple test locations. Also, most of the testcrosses with positive heterosis for grain yield and negative heterosis for Striga damage and emerged Striga count involved T1 as a tester. Our study identified six Ex-PVP inbred lines with positive GCA effects for grain yield under Striga infested and non-infested conditions and across multiple test locations. Amongst these, inbred lines HB8229-1 and WIL900-1 also displayed negative GCA effects for emerged Striga count and Striga damage rating. The inbred line HB8229-1 showed positive SCA effects for grain yield with T2, whereas WIL900-1 had positive SCA effects for grain yield with T1. Over 70% of the Ex-PVP inbred lines were consistently assigned to specific heterotic groups using yield-based classifying methods (mean grain yield and SCA effects). CONCLUSIONS: These results could facilitate systematic introgression of the Ex-PVP inbred lines into the existing Striga resistant heterotic groups in IITA. The Ex-PVP inbred lines with positive GCA effects and producing high grain yields in hybrid combinations could be useful parents for enhancing Striga resistance and agronomic performance of tropical maize hybrids.

Association mapping of plant sex and cross-compatibility related traits in white Guinea yam (Dioscorea rotundata Poir.) clones.

BACKGROUND: White Guinea yam (Dioscorea rotundata) is primarily a dioecious species with distinct male and female plants. Its breeding is constrained by sexual reproduction abnormalities, resulting in low success rates in cross-pollination. An accurate method for early detection of this plant's sex and compatible fertile parents at the seedling stage would improve levels of cross-pollination success in breeding. We used the genome-wide association studies (GWAS) to dissect the molecular basis of plant sex and cross-compatibility-related traits in a panel of 112 parental clones used in D. rotundata crossing blocks from 2010 to 2020. RESULTS: Population structure and phylogeny analyses using 8326 single nucleotide polymorphism (SNP) markers grouped the 112 white yam clones into three subpopulations. Using Multi-locus random-SNP-effect Mixed Linear Model, we identified three, one, and three SNP markers that were significantly associated with the average crossability rate (ACR), the percentage of high crossability (PHC), and the plant sex, respectively. In addition, five genes considered to be directly linked to sexual reproduction or regulating the balance of sex hormones were annotated from chromosomal regions controlling the assessed traits. This study confirmed the female heterogametic sex determination (ZZ/ZW) system proposed for D. rotundata. CONCLUSIONS: This study provides valuable insights on the genomic control of sex identity and cross-pollination success in D. rotundata. It, therefore, opens an avenue for developing molecular markers for predicting plant sex and cross-pollination success at the early growth stage before field sex expression in this crop.

Genome-Wide Association Analysis Reveals Genetic Architecture and Candidate Genes Associated with Grain Yield and Other Traits under Low Soil Nitrogen in Early-Maturing White Quality Protein Maize Inbred Lines.

Maize production in the savannas of sub-Saharan Africa (SSA) is constrained by the low nitrogen in the soils. The identification of quantitative trait loci (QTL) conferring tolerance to low soil nitrogen (low-N) is crucial for the successful breeding of high-yielding QPM maize genotypes under low-N conditions. The objective of this study was to identify QTLs significantly associated with grain yield and other low-N tolerance-related traits under low-N. The phenotypic data of 140 early-maturing white quality protein maize (QPM) inbred lines were evaluated under low-N. The inbred lines were genotyped using 49,185 DArTseq markers, from which 7599 markers were filtered for population structure analysis and genome-wide association study (GWAS). The inbred lines were grouped into two major clusters based on the population structure analysis. The GWAS identified 24, 3, 10, and 3 significant SNPs respectively associated with grain yield, stay-green characteristic, and plant and ear aspects, under low-N. Sixteen SNP markers were physically located in proximity to 32 putative genes associated with grain yield, stay-green characteristic, and plant and ear aspects. The putative genes GRMZM2G127139, GRMZM5G848945, GRMZM2G031331, GRMZM2G003493, GRMZM2G067964, GRMZM2G180254, on chromosomes 1, 2, 8, and 10 were involved in cellular nitrogen assimilation and biosynthesis, normal plant growth and development, nitrogen assimilation, and disease resistance. Following the validation of the markers, the putative candidate genes and SNPs could be used as genomic markers for marker-assisted selection, to facilitate genetic gains for low-N tolerance in maize production.

Cross compatibility in intraspecific and interspecific hybridization in yam (Dioscorea spp.).

Yam (Dioscorea spp.) is a staple crop for millions of people in the tropics and subtropics. Its genetic improvement through breeding is being challenged by pre-zygotic and post-zygotic cross-compatibility barriers within and among species. Studies dissecting hybridization barriers on yam for improving the crossability rates are limited. This study aimed to assess the cross-compatibility, which yielded fruit set, viable seeds and progeny plants in an extensive intraspecific and interspecific crossing combinations in a yam genetic improvement effort to understand the internal and exogenous factors influencing pollination success. Cross-compatability was analyzed at the individual genotype or family level using historical data from crossing blocks and seedling nurseries from 2010 to 2020 at the International Institute of Tropical Agriculture (IITA). The average crossability rate (ACR) was lower in interspecific crossing combinations (6.1%) than intraspecific ones (27.6%). The seed production efficiency (SPE) values were 1.1 and 9.3% for interspecific and intraspecific crosses, respectively. Weather conditions and pollinator's skills are the main contributors to the low success rate in the intraspecific cross combinations in yam breeding. At the same time, genetic distance and heterozygosity played little role. Interspecific cross barriers were both pre-zygotic and post-zygotic, resulting from the evolutionary divergence among the yam species. Dioscorea rotundata had higher interspecific cross-compatibility indices than D. alata. Distant parents produced intraspecific crossbred seeds with higher germination rates compared to closest parents (r = 0.21, p = 0.033). This work provided important insights into interspecific and intraspecific cross-compatibility in yam and suggested actions for improving hybridization practices in yam breeding programs.

Diversity, trait preferences, management and utilization of yams landraces (Dioscorea species): an orphan crop in DR Congo.

Yam (Dioscorea spp.) is cultivated in many villages of DR Congo as a means to sustain food security and alleviate poverty. However, the extent of the existing diversity has not been studied in details thus, considered as an orphan. A survey covering 540 farmers in 54 villages was conducted in six major yam growing territories covering three provinces in DR Congo to investigate the diversity, management and utilization of yam landraces using pre-elaborate questionnaires. Subject to synonymy, a total of 67 landraces from five different species were recorded. Farmers' challenges limiting yam production were poor tuber qualities (69%), harvest pest attack (7%), difficulty in harvesting (6%), poor soil status (6%). The overall diversity was moderate among the recorded yam germplasm maintained at the household level (1.32) and variability exist in diversity amongst the territories and provinces. Farmers' in territories of Tshopo and Mongala provinces maintained higher level of germplasm diversity (2.79 and 2.77) compared to the farmers in territories of Bas-Uélé (1.67). Some yam landraces had limited abundance and distribution due to loss of production interest in many villages attributable to poisons contained hence, resulting in possible extinction. Farmers' most preferred seed source for cultivation were backyard (43%) and exchange with neighboring farmers (31%) with the objective of meeting food security and generating income. In villages where yam production is expanding, farmers are relying on landraces with good tuber qualities and high yield even though they are late maturing. This study revealed the knowledge of yam landrace diversity, constraints to production and farmers' preferences criteria as a guide for collection and conservation of yam germplasm for yam improvement intervention.

Diversity of Water Yam (Dioscorea alata L.) Accessions from Côte d'Ivoire Based on SNP Markers and Agronomic Traits.

Dioscorea alata (L.), also referred to as water, winged, or greater yam, is one of the most economically important staple food crops in tropical and subtropical areas. In Côte d'Ivoire, it represents, along with other yam species, the largest food crop and significantly contributes to food security. However, studies focusing on better understanding the structure and extent of genetic diversity among D. alata accessions, using molecular and phenotypic traits, are limited. This study was, therefore, conducted to assess the pattern of genetic variability in a set of 188 D. alata accessions from the National Agronomic Research Centre (CNRA) genebank using 11,722 SNP markers (generated by the Diversity Arrays Technology) and nine agronomic traits. Phylogenetic analyses using hierarchical clustering, admixture, kinship, and Discriminant analysis of principal component (DAPC) all assigned the accessions into four main clusters. Genetic diversity assessment using molecular-based SNP markers showed a high proportion of polymorphic SNPs (87.81%). The analysis of molecular variance (AMOVA) showed low molecular variability within genetic groups. In addition, the agronomic traits evaluated for two years in field conditions showed a high heritability and high variability among D. alata accessions. This study provides insights into the genetic diversity among accessions in the CNRA genebank and opens an avenue for sustainable resource management and the identification of promising parental clones for water yam breeding programs in Côte d'Ivoire.

Identification of quantitative trait nucleotides and candidate genes for tuber yield and mosaic virus tolerance in an elite population of white guinea yam (Dioscorea rotundata) using genome-wide association scan.

BACKGROUND: Improvement of tuber yield and tolerance to viruses are priority objectives in white Guinea yam breeding programs. However, phenotypic selection for these traits is quite challenging due to phenotypic plasticity and cumbersome screening of phenotypic-induced variations. This study assessed quantitative trait nucleotides (QTNs) and the underlying candidate genes related to tuber yield per plant (TYP) and yam mosaic virus (YMV) tolerance in a panel of 406 white Guinea yam (Dioscorea rotundata) breeding lines using a genome-wide association study (GWAS). RESULTS: Population structure analysis using 5,581 SNPs differentiated the 406 genotypes into seven distinct sub-groups based delta K. Marker-trait association (MTA) analysis using the multi-locus linear model (mrMLM) identified seventeen QTN regions significant for TYP and five for YMV with various effects. The seveteen QTNs were detected on nine chromosomes, while the five QTNs were identified on five chromosomes. We identified variants responsible for predicting higher yield and low virus severity scores in the breeding panel through the marker-effect prediction. Gene annotation for the significant SNP loci identified several essential putative genes associated with the growth and development of tuber yield and those that code for tolerance to mosaic virus. CONCLUSION: Application of different multi-locus models of GWAS identified 22 QTNs. Our results provide valuable insight for marker validation and deployment for tuber yield and mosaic virus tolerance in white yam breeding. The information on SNP variants and genes from the present study would fast-track the application of genomics-informed selection decisions in breeding white Guinea yam for rapid introgression of the targeted traits through markers validation.

Genome-Wide Association Studies for Sex Determination and Cross-Compatibility in Water Yam (Dioscorea alata L.).

Yam (Dioscorea spp.) species are predominantly dioecious, with male and female flowers borne on separate individuals. Cross-pollination is, therefore, essential for gene flow among and within yam species to achieve breeding objectives. Understanding genetic mechanisms underlying sex determination and cross-compatibility is crucial for planning a successful hybridization program. This study used the genome-wide association study (GWAS) approach for identifying genomic regions linked to sex and cross-compatibility in water yam (Dioscorea alata L.). We identified 54 markers linked to flower sex determination, among which 53 markers were on chromosome 6 and one on chromosome 11. Our result ascertained that D. alata is characterized by the male heterogametic sex determination system (XX/XY). The cross-compatibility indices, average crossability rate (ACR) and percentage high crossability (PHC), were controlled by loci on chromosomes 1, 6 and 17. Of the significant loci, SNPs located on chromosomes 1 and 17 were the most promising for ACR and PHC, respectively, and should be validated for use in D. alata hybridization activities to predict cross-compatibility success. A total of 61 putative gene/protein families with direct or indirect influence on plant reproduction were annotated in chromosomic regions controlling the target traits. This study provides valuable insights into the genetic control of D. alata sexual reproduction. It opens an avenue for developing genomic tools for predicting hybridization success in water yam breeding programs.

Genomic and Phenotypic Diversity of Cultivated and Wild Tomatoes with Varying Levels of Heat Tolerance.

Assessment of genetic variability in heat-tolerant tomato germplasm is a pre-requisite to improve yield and fruit quality under heat stress. We assessed the population structure and diversity in a panel of three Solanum pimpinellifolium (wild tomatoes) and 42 S. lycopersicum (cultivated tomatoes) lines and accessions with varying heat tolerance levels. The DArTseq marker was used for the sequencing and 5270 informative single nucleotide polymorphism (SNP) markers were retained for the genomic analysis. The germplasm was evaluated under two heat stress environments for five yield and flower related traits. The phenotypic evaluation revealed moderate broad-sense heritabilities for fruit weight per plant and high broad-sense heritabilities for fruit weight, number of inflorescences per plant, and number of flowers per inflorescence. The hierarchical clustering based on identity by state dissimilarity matrix and UPGMA grouped the germplasm into three clusters. The cluster analysis based on heat-tolerance traits separated the germplasm collection into five clusters. The correlation between the phenotypic and genomic-based distance matrices was low (r = 0.2, p < 0.05). The joint phenotypic and genomic-based clustering grouped the germplasm collection into five clusters well defined for their response to heat stress ranging from highly sensitive to highly tolerant groups. The heat-sensitive and heat-tolerant clusters of S. lycopersicum lines were differentiated by a specific pattern of minor allele frequency distribution on chromosome 11. The joint phenotypic and genomic analysis revealed important diversity within the germplasm collection. This study provides the basis for efficient selection of parental lines to breed heat-tolerant varieties.

Optimized Protocol for In Vitro Pollen Germination in Yam (Dioscorea spp.).

Yam (Dioscorea spp.) plants are mostly dioecious and sometimes monoecious. Low, irregular, and asynchronous flowering of the genotypes are critical problems in yam breeding. Selecting suitable pollen parents and preserving yam pollen for future use are potential means of controlling these constraints and optimizing hybridization practice in yam breeding programs. However, implementing such procedures requires a robust protocol for pollen collection and viability testing to monitor pollen quality in the field and in storage. This study, therefore, aimed at optimizing the pollen germination assessment protocol for yam. The standard medium composition was stepwisely modified, the optimal growth condition was tested, and in vivo predictions were made. This study showed that the differences in yam pollen germination percentage are primarily linked to the genotype and growing conditions (i.e., medium viscosity, incubation temperature, and time to use) rather than the medium composition. The inclusion of polyethylene glycol (PEG) in the culture medium caused 67-75% inhibition of germination in D. alata. Although the in vivo fertilization was dependent on female parents, the in vitro germination test predicted the percentage fruit set at 25.2-79.7% and 26.4-59.7% accuracy for D. rotundata and D. alata genotypes, respectively. This study provides a reliable in vitro yam pollen germination protocol to support pollen management and preservation efforts in yam breeding.