Exploiting genomic tools for genetic dissection and improving the resistance to Fusarium stalk rot in tropical maize.

KEY MESSAGE: A stable genomic region conferring FSR resistance at ~250 Mb on chromosome 1 was identified by GWAS. Genomic prediction has the potential to improve FSR resistance. Fusarium stalk rot (FSR) is a global destructive disease in maize; the efficiency of phenotypic selection for improving FSR resistance was low. Novel genomic tools of genome-wide association study (GWAS) and genomic prediction (GP) provide an opportunity for genetic dissection and improving FSR resistance. In this study, GWAS and GP analyses were performed on 562 tropical maize inbred lines consisting of two populations. In total, 15 SNPs significantly associated with FSR resistance were identified across two populations and the combinedPOP consisting of all 562 inbred lines, with the P-values ranging from 1.99 × 10-7 to 8.27 × 10-13, and the phenotypic variance explained (PVE) values ranging from 0.94 to 8.30%. The genetic effects of the 15 favorable alleles ranged from -4.29 to -14.21% of the FSR severity. One stable genomic region at ~ 250 Mb on chromosome 1 was detected across all populations, and the PVE values of the SNPs detected in this region ranged from 2.16 to 5.18%. Prediction accuracies of FSR severity estimated with the genome-wide SNPs were moderate and ranged from 0.29 to 0.51. By incorporating genotype-by-environment interaction, prediction accuracies were improved between 0.36 and 0.55 in different breeding scenarios. Considering both the genome coverage and the threshold of the P-value of SNPs to select a subset of molecular markers further improved the prediction accuracies. These findings extend the knowledge of exploiting genomic tools for genetic dissection and improving FSR resistance in tropical maize.

Low-Density Reference Fingerprinting SNP Dataset of CIMMYT Maize Lines for Quality Control and Genetic Diversity Analyses.

CIMMYT maize lines (CMLs), which represent the tropical maize germplasm, are freely available worldwide. All currently released 615 CMLs and fourteen temperate maize inbred lines were genotyped with 180 kompetitive allele-specific PCR single nucleotide polymorphisms to develop a reference fingerprinting SNP dataset that can be used to perform quality control (QC) and genetic diversity analyses. The QC analysis identified 25 CMLs with purity, identity, or mislabeling issues. Further field observation, purification, and re-genotyping of these CMLs are required. The reference fingerprinting SNP dataset was developed for all of the currently released CMLs with 152 high-quality SNPs. The results of principal component analysis and average genetic distances between subgroups showed a clear genetic divergence between temperate and tropical maize, whereas the three tropical subgroups partially overlapped with one another. More than 99% of the pairs of CMLs had genetic distances greater than 0.30, showing their high genetic diversity, and most CMLs are distantly related. The heterotic patterns, estimated with the molecular markers, are consistent with those estimated using pedigree information in two major maize breeding programs at CIMMYT. These research findings are helpful for ensuring the regeneration and distribution of the true CMLs, via QC analysis, and for facilitating the effective utilization of the CMLs, globally.

Genetic trends in CIMMYT's tropical maize breeding pipelines.

Fostering a culture of continuous improvement through regular monitoring of genetic trends in breeding pipelines is essential to improve efficiency and increase accountability. This is the first global study to estimate genetic trends across the International Maize and Wheat Improvement Center (CIMMYT) tropical maize breeding pipelines in eastern and southern Africa (ESA), South Asia, and Latin America over the past decade. Data from a total of 4152 advanced breeding trials and 34,813 entries, conducted at 1331 locations in 28 countries globally, were used for this study. Genetic trends for grain yield reached up to 138 kg ha-1 yr-1 in ESA, 118 kg ha-1 yr-1 South Asia and 143 kg ha-1 yr-1 in Latin America. Genetic trend was, in part, related to the extent of deployment of new breeding tools in each pipeline, strength of an extensive phenotyping network, and funding stability. Over the past decade, CIMMYT's breeding pipelines have significantly evolved, incorporating new tools/technologies to increase selection accuracy and intensity, while reducing cycle time. The first pipeline, Eastern Africa Product Profile 1a (EA-PP1a), to implement marker-assisted forward-breeding for resistance to key diseases, coupled with rapid-cycle genomic selection for drought, recorded a genetic trend of 2.46% per year highlighting the potential for deploying new tools/technologies to increase genetic gain.

Identification and fine mapping of a major QTL (qRtsc8-1) conferring resistance to maize tar spot complex and validation of production markers in breeding lines.

KEY MESSAGE: A major QTL of qRtsc8-1 conferring TSC resistance was identified and fine mapped to a 721 kb region on chromosome 8 at 81 Mb, and production markers were validated in breeding lines. Tar spot complex (TSC) is a major foliar disease of maize in many Central and Latin American countries and leads to severe yield loss. To dissect the genetic architecture of TSC resistance, a genome-wide association study (GWAS) panel and a bi-parental doubled haploid population were used for GWAS and selective genotyping analysis, respectively. A total of 115 SNPs in bin 8.03 were detected by GWAS and three QTL in bins 6.05, 6.07, and 8.03 were detected by selective genotyping. The major QTL qRtsc8-1 located in bin 8.03 was detected by both analyses, and it explained 14.97% of the phenotypic variance. To fine map qRtsc8-1, the recombinant-derived progeny test was implemented. Recombinations in each generation were backcrossed, and the backcross progenies were genotyped with Kompetitive Allele Specific PCR (KASP) markers and phenotyped for TSC resistance individually. The significant tests for comparing the TSC resistance between the two classes of progenies with and without resistant alleles were used for fine mapping. In BC5 generation, qRtsc8-1 was fine mapped in an interval of ~ 721 kb flanked by markers of KASP81160138 and KASP81881276. In this interval, the candidate genes GRMZM2G063511 and GRMZM2G073884 were identified, which encode an integral membrane protein-like and a leucine-rich repeat receptor-like protein kinase, respectively. Both genes are involved in maize disease resistance responses. Two production markers KASP81160138 and KASP81160155 were verified in 471 breeding lines. This study provides valuable information for cloning the resistance gene, and it will also facilitate the routine implementation of marker-assisted selection in the breeding pipeline for improving TSC resistance.

Genetic Dissection of Quantitative Resistance to Common Rust (Puccinia sorghi) in Tropical Maize (Zea mays L.) by Combined Genome-Wide Association Study, Linkage Mapping, and Genomic Prediction.

Common rust is one of the major foliar diseases in maize, leading to significant grain yield losses and poor grain quality. To dissect the genetic architecture of common rust resistance, a genome-wide association study (GWAS) panel and a bi-parental doubled haploid (DH) population, DH1, were used to perform GWAS and linkage mapping analyses. The GWAS results revealed six single-nucleotide polymorphisms (SNPs) significantly associated with quantitative resistance of common rust at a very stringent threshold of P-value 3.70 × 10-6 at bins 1.05, 1.10, 3.04, 3.05, 4.08, and 10.04. Linkage mapping identified five quantitative trait loci (QTL) at bins 1.03, 2.06, 4.08, 7.03, and 9.00. The phenotypic variation explained (PVE) value of each QTL ranged from 5.40 to 12.45%, accounting for the total PVE value of 40.67%. Joint GWAS and linkage mapping analyses identified a stable genomic region located at bin 4.08. Five significant SNPs were only identified by GWAS, and four QTL were only detected by linkage mapping. The significantly associated SNP of S10_95231291 detected in the GWAS analysis was first reported. The linkage mapping analysis detected two new QTL on chromosomes 7 and 10. The major QTL on chromosome 7 in the region between 144,567,253 and 149,717,562 bp had the largest PVE value of 12.45%. Four candidate genes of GRMZM2G328500, GRMZM2G162250, GRMZM2G114893, and GRMZM2G138949 were identified, which played important roles in the response of stress resilience and the regulation of plant growth and development. Genomic prediction (GP) accuracies observed in the GWAS panel and DH1 population were 0.61 and 0.51, respectively. This study provided new insight into the genetic architecture of quantitative resistance of common rust. In tropical maize, common rust could be improved by pyramiding the new sources of quantitative resistance through marker-assisted selection (MAS) or genomic selection (GS), rather than the implementation of MAS for the single dominant race-specific resistance gene.

Beat the stress: breeding for climate resilience in maize for the tropical rainfed environments.

KEY MESSAGE: Intensive public sector breeding efforts and public-private partnerships have led to the increase in genetic gains, and deployment of elite climate-resilient maize cultivars for the stress-prone environments in the tropics. Maize (Zea mays L.) plays a critical role in ensuring food and nutritional security, and livelihoods of millions of resource-constrained smallholders. However, maize yields in the tropical rainfed environments are now increasingly vulnerable to various climate-induced stresses, especially drought, heat, waterlogging, salinity, cold, diseases, and insect pests, which often come in combinations to severely impact maize crops. The International Maize and Wheat Improvement Center (CIMMYT), in partnership with several public and private sector institutions, has been intensively engaged over the last four decades in breeding elite tropical maize germplasm with tolerance to key abiotic and biotic stresses, using an extensive managed stress screening network and on-farm testing system. This has led to the successful development and deployment of an array of elite stress-tolerant maize cultivars across sub-Saharan Africa, Asia, and Latin America. Further increasing genetic gains in the tropical maize breeding programs demands judicious integration of doubled haploidy, high-throughput and precise phenotyping, genomics-assisted breeding, breeding data management, and more effective decision support tools. Multi-institutional efforts, especially public-private alliances, are key to ensure that the improved maize varieties effectively reach the climate-vulnerable farming communities in the tropics, including accelerated replacement of old/obsolete varieties.

Genomic Prediction with Genotype by Environment Interaction Analysis for Kernel Zinc Concentration in Tropical Maize Germplasm.

Zinc (Zn) deficiency is a major risk factor for human health, affecting about 30% of the world's population. To study the potential of genomic selection (GS) for maize with increased Zn concentration, an association panel and two doubled haploid (DH) populations were evaluated in three environments. Three genomic prediction models, M (M1: Environment + Line, M2: Environment + Line + Genomic, and M3: Environment + Line + Genomic + Genomic x Environment) incorporating main effects (lines and genomic) and the interaction between genomic and environment (G x E) were assessed to estimate the prediction ability (rMP ) for each model. Two distinct cross-validation (CV) schemes simulating two genomic prediction breeding scenarios were used. CV1 predicts the performance of newly developed lines, whereas CV2 predicts the performance of lines tested in sparse multi-location trials. Predictions for Zn in CV1 ranged from -0.01 to 0.56 for DH1, 0.04 to 0.50 for DH2 and -0.001 to 0.47 for the association panel. For CV2, rMP values ranged from 0.67 to 0.71 for DH1, 0.40 to 0.56 for DH2 and 0.64 to 0.72 for the association panel. The genomic prediction model which included G x E had the highest average rMP for both CV1 (0.39 and 0.44) and CV2 (0.71 and 0.51) for the association panel and DH2 population, respectively. These results suggest that GS has potential to accelerate breeding for enhanced kernel Zn concentration by facilitating selection of superior genotypes.

Genomic Prediction of Kernel Zinc Concentration in Multiple Maize Populations Using Genotyping-by-Sequencing and Repeat Amplification Sequencing Markers.

Enriching of kernel zinc (Zn) concentration in maize is one of the most effective ways to solve the problem of Zn deficiency in low and middle income countries where maize is the major staple food, and 17% of the global population is affected with Zn deficiency. Genomic selection (GS) has shown to be an effective approach to accelerate genetic gains in plant breeding. In the present study, an association-mapping panel and two maize double-haploid (DH) populations, both genotyped with genotyping-by-sequencing (GBS) and repeat amplification sequencing (rAmpSeq) markers, were used to estimate the genomic prediction accuracy of kernel Zn concentration in maize. Results showed that the prediction accuracy of two DH populations was higher than that of the association mapping population using the same set of markers. The prediction accuracy estimated with the GBS markers was significantly higher than that estimated with the rAmpSeq markers in the same population. The maximum prediction accuracy with minimum standard error was observed when half of the genotypes were included in the training set and 3,000 and 500 markers were used for prediction in the association mapping panel and the DH populations, respectively. Appropriate levels of minor allele frequency and missing rate should be considered and selected to achieve good prediction accuracy and reduce the computation burden by balancing the number of markers and marker quality. Training set development with broad phenotypic variation is possible to improve prediction accuracy. The transferability of the GS models across populations was assessed, the prediction accuracies in a few pairwise populations were above or close to 0.20, which indicates the prediction accuracies across years and populations have to be assessed in a larger breeding dataset with closer relationship between the training and prediction sets in further studies. GS outperformed MAS (marker-assisted-selection) on predicting the kernel Zn concentration in maize, the decision of a breeding strategy to implement GS individually or to implement MAS and GS stepwise for improving kernel Zn concentration in maize requires further research. Results of this study provide valuable information for understanding how to implement GS for improving kernel Zn concentration in maize.

Estimation of physiological genomic estimated breeding values (PGEBV) combining full hyperspectral and marker data across environments for grain yield under combined heat and drought stress in tropical maize (Zea mays L.).

High throughput phenotyping technologies are lagging behind modern marker technology impairing the use of secondary traits to increase genetic gains in plant breeding. We aimed to assess whether the combined use of hyperspectral data with modern marker technology could be used to improve across location pre-harvest yield predictions using different statistical models. A maize bi-parental doubled haploid (DH) population derived from F1, which consisted of 97 lines was evaluated in testcross combination under heat stress as well as combined heat and drought stress during the 2014 and 2016 summer season in Ciudad Obregon, Sonora, Mexico (27°20" N, 109°54" W, 38 m asl). Full hyperspectral data, indicative of crop physiological processes at the canopy level, was repeatedly measured throughout the grain filling period and related to grain yield. Partial least squares regression (PLSR), random forest (RF), ridge regression (RR) and Bayesian ridge regression (BayesB) were used to assess prediction accuracies on grain yield within (two-fold cross-validation) and across environments (leave-one-environment-out-cross-validation) using molecular markers (M), hyperspectral data (H) and the combination of both (HM). Highest prediction accuracy for grain yield averaged across within and across location predictions (rGP) were obtained for BayesB followed by RR, RF and PLSR. The combined use of hyperspectral and molecular marker data as input factor on average had higher predictions for grain yield than hyperspectral data or molecular marker data alone. The highest prediction accuracy for grain yield across environments was measured for BayesB when molecular marker data and hyperspectral data were used as input factors, while the highest within environment prediction was obtained when BayesB was used in combination with hyperspectral data. It is discussed how the combined use of hyperspectral data with molecular marker technology could be used to introduce physiological genomic estimated breeding values (PGEBV) as a pre-harvest decision support tool to select genetically superior lines.

Molecular Breeding for Nutritionally Enriched Maize: Status and Prospects.

Maize is a major source of food security and economic development in sub-Saharan Africa (SSA), Latin America, and the Caribbean, and is among the top three cereal crops in Asia. Yet, maize is deficient in certain essential amino acids, vitamins, and minerals. Biofortified maize cultivars enriched with essential minerals and vitamins could be particularly impactful in rural areas with limited access to diversified diet, dietary supplements, and fortified foods. Significant progress has been made in developing, testing, and deploying maize cultivars biofortified with quality protein maize (QPM), provitamin A, and kernel zinc. In this review, we outline the status and prospects of developing nutritionally enriched maize by successfully harnessing conventional and molecular marker-assisted breeding, highlighting the need for intensification of efforts to create greater impacts on malnutrition in maize-consuming populations, especially in the low- and middle-income countries. Molecular marker-assisted selection methods are particularly useful for improving nutritional traits since conventional breeding methods are relatively constrained by the cost and throughput of nutritional trait phenotyping.

Molecular characterization of CIMMYT maize inbred lines with genotyping-by-sequencing SNPs.

KEY MESSAGE: Molecular characterization information on genetic diversity, population structure and genetic relationships provided by this research will help maize breeders to better understand how to utilize the current CML collection. CIMMYT maize inbred lines (CMLs) have been widely used all over the world and have contributed greatly to both tropical and temperate maize improvement. Genetic diversity and population structure of the current CML collection and of six temperate inbred lines were assessed and relationships among all lines were determined with genotyping-by-sequencing SNPs. Results indicated that: (1) wider genetic distance and low kinship coefficients among most pairs of lines reflected the uniqueness of most lines in the current CML collection; (2) the population structure and genetic divergence between the Temperate subgroup and Tropical subgroups were clear; three major environmental adaptation groups (Lowland Tropical, Subtropical/Mid-altitude and Highland Tropical subgroups) were clearly present in the current CML collection; (3) the genetic diversity of the three Tropical subgroups was similar and greater than that of the Temperate subgroup; the average genetic distance between the Temperate and Tropical subgroups was greater than among Tropical subgroups; and (4) heterotic patterns in each environmental adaptation group estimated using GBS SNPs were only partially consistent with patterns estimated based on combining ability tests and pedigree information. Combining current heterotic information based on combining ability tests and the genetic relationships inferred from molecular marker analyses may be the best strategy to define heterotic groups for future tropical maize improvement. Information resulting from this research will help breeders to better understand how to utilize all the CMLs to select parental lines, replace testers, assign heterotic groups and create a core set of breeding germplasm.

A GeneTrek analysis of the maize genome.

Analysis of the sequences of 74 randomly selected BACs demonstrated that the maize nuclear genome contains approximately 37,000 candidate genes with homologues in other plant species. An additional approximately 5,500 predicted genes are severely truncated and probably pseudogenes. The distribution of genes is uneven, with approximately 30% of BACs containing no genes. BAC gene density varies from 0 to 7.9 per 100 kb, whereas most gene islands contain only one gene. The average number of genes per gene island is 1.7. Only 72% of these genes show collinearity with the rice genome. Particular LTR retrotransposon families (e.g., Gyma) are enriched on gene-free BACs, most of which do not come from pericentromeres or other large heterochromatic regions. Gene-containing BACs are relatively enriched in different families of LTR retrotransposons (e.g., Ji). Two major bursts of LTR retrotransposon activity in the last 2 million years are responsible for the large size of the maize genome, but only the more recent of these is well represented in gene-containing BACs, suggesting that LTR retrotransposons are more efficiently removed in these domains. The results demonstrate that sample sequencing and careful annotation of a few randomly selected BACs can provide a robust description of a complex plant genome.