Identification and validation of a key genomic region on chromosome 6 for resistance to Fusarium stalk rot in tropical maize.

KEY MESSAGE: A key genomic region was identified for resistance to FSR at 168 Mb on chromosome 6 in GWAS and haplotype regression analysis, which was validated by QTL mapping in two populations. Fusarium stalk rot (FSR) of maize is an economically important post-flowering stalk rot (PFSR) disease caused by Fusarium verticillioides. The pathogen invades the plant individually, or in combination with other stalk rot pathogens or secondary colonizers, thereby making it difficult to make accurate selection for resistance. For identification and validation of genomic regions associated with FSR resistance, a genome-wide association study (GWAS) was conducted with 342 maize lines. The panel was screened for FSR in three environments using standard artificial inoculation methodology. GWAS using the mixed linear model corrected for population structure and kinship was done, in which 290,626 SNPs from genotyping-by-sequencing were used. A total of 7 SNPs, five on chromosome 6 showing strong LD at 168 Mb, were identified to be associated with FSR. Haplotype regression analysis identified 32 haplotypes with a significant effect on the trait. In a QTL mapping experiment in two populations for validating the identified variants, QTLs were identified with confidence intervals having overlapped physical coordinates in both the populations on chromosome 6, which was closely located to the GWAS-identified variants on chromosome 6. It makes this genomic region a crucial one to further investigate the possibility of developing trait markers for deployment in breeding pipelines. It was noted that previously reported QTLs for other stalk rots in maize mapped within the same physical intervals of several haplotypes identified for FSR resistance in this study. The possibility of QTLs controlling broad-spectrum resistance for PFSR in general requires further investigation.

Identification and Validation of Genomic Regions Associated With Charcoal Rot Resistance in Tropical Maize by Genome-Wide Association and Linkage Mapping.

Charcoal rot is a post-flowering stalk rot (PFSR) disease of maize caused by the fungal pathogen, Macrophomina phaseolina. It is a serious concern for smallholder maize cultivation, due to significant yield loss and plant lodging at harvest, and this disease is expected to surge with climate change effects like drought and high soil temperature. For identification and validation of genomic variants associated with charcoal rot resistance, a genome-wide association study (GWAS) was conducted on CIMMYT Asia association mapping panel comprising 396 tropical-adapted lines, especially to Asian environments. The panel was phenotyped for disease severity across two locations with high disease prevalence in India. A subset of 296,497 high-quality SNPs filtered from genotyping by sequencing was correcting for population structure and kinship matrices for single locus mixed linear model (MLM) of GWAS analysis. A total of 19 SNPs were identified to be associated with charcoal rot resistance with P-value ranging from 5.88 × 10-06 to 4.80 × 10-05. Haplotype regression analysis identified 21 significant haplotypes for the trait with Bonferroni corrected P ≤ 0.05. For validating the associated variants and identifying novel QTLs, QTL mapping was conducted using two F2:3 populations. Two QTLs with overlapping physical intervals, qMSR6 and qFMSR6 on chromosome 6, identified from two different mapping populations and contributed by two different resistant parents, were co-located with the SNPs and haplotypes identified at 103.51 Mb on chromosome 6. Similarly, several SNPs/haplotypes identified on chromosomes 3, 6 and 8 were also found to be physically co-located within QTL intervals detected in one of the two mapping populations. The study also noted that several SNPs/haplotypes for resistance to charcoal rot were located within physical intervals of previously reported QTLs for Gibberella stalk rot resistance, which opens up a new possibility for common disease resistance mechanisms for multiple stalk rots.

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.

Genome-wide association studies in tropical maize germplasm reveal novel and known genomic regions for resistance to Northern corn leaf blight.

Northern Corn Leaf Blight (NCLB) caused by Setosphaeria turcica, is one of the most important diseases of maize world-wide, and one of the major reasons behind yield losses in maize crop in Asia. In the present investigation, a high-resolution genome wide association study (GWAS) was conducted for NCLB resistance in three association mapping panels, predominantly consisting of tropical lines adapted to different agro-ecologies. These panels were phenotyped for disease severity across three locations with high disease prevalence in India. High density SNPs from Genotyping-by-sequencing were used in GWAS, after controlling for population structure and kinship matrices, based on single locus mixed linear model (MLM). Twenty-two SNPs were identified, that revealed a significant association with NCLB in the three mapping panels. Haplotype regression analysis revealed association of 17 significant haplotypes at FDR ≤ 0.05, with two common haplotypes across three maize panels. Several of the significantly associated SNPs/haplotypes were found to be co-located in chromosomal bins previously reported for major genes like Ht2, Ht3 and Htn1 and QTL for NCLB resistance and multiple foliar disease resistance. Phenotypic variance explained by these significant SNPs/haplotypes ranged from low to moderate, suggesting a breeding strategy of combining multiple resistance alleles towards resistance for NCLB.

Identification and validation of genomic regions influencing kernel zinc and iron in maize.

KEY MESSAGE: Genome-wide association study (GWAS) on 923 maize lines and validation in bi-parental populations identified significant genomic regions for kernel-Zinc and-Iron in maize. Bio-fortification of maize with elevated Zinc (Zn) and Iron (Fe) holds considerable promise for alleviating under-nutrition among the world's poor. Bio-fortification through molecular breeding could be an economical strategy for developing nutritious maize, and hence in this study, we adopted GWAS to identify markers associated with high kernel-Zn and Fe in maize and subsequently validated marker-trait associations in independent bi-parental populations. For GWAS, we evaluated a diverse maize association mapping panel of 923 inbred lines across three environments and detected trait associations using high-density Single nucleotide polymorphism (SNPs) obtained through genotyping-by-sequencing. Phenotyping trials of the GWAS panel showed high heritability and moderate correlation between kernel-Zn and Fe concentrations. GWAS revealed a total of 46 SNPs (Zn-20 and Fe-26) significantly associated (P ≤ 5.03 × 10-05) with kernel-Zn and Fe concentrations with some of these associated SNPs located within previously reported QTL intervals for these traits. Three double-haploid (DH) populations were developed using lines identified from the panel that were contrasting for these micronutrients. The DH populations were phenotyped at two environments and were used for validating significant SNPs (P ≤ 1 × 10-03) based on single marker QTL analysis. Based on this analysis, 11 (Zn) and 11 (Fe) SNPs were found to have significant effect on the trait variance (P ≤ 0.01, R2 ≥ 0.05) in at least one bi-parental population. These findings are being pursued in the kernel-Zn and Fe breeding program, and could hold great value in functional analysis and possible cloning of high-value genes for these traits in maize.

Genome-wide association study in Asia-adapted tropical maize reveals novel and explored genomic regions for sorghum downy mildew resistance.

Globally, downy mildews are among the important foliar diseases of maize that cause significant yield losses. We conducted a genome-wide association study for sorghum downy mildew (SDM; Peronosclerospora sorghi) resistance in a panel of 368 inbred lines adapted to the Asian tropics. High density SNPs from Genotyping-by-sequencing were used in GWAS after controlling for population structure and kinship in the panel using a single locus mixed model. The study identified a set of 26 SNPs that were significantly associated with SDM resistance, with Bonferroni corrected P values ≤ 0.05. Among all the identified SNPs, the minor alleles were found to be favorable to SDM resistance in the mapping panel. Trend regression analysis with 16 independent genetic variants including 12 SNPs and four haplotype blocks identified SNP S2_6154311 on chromosome 2 with P value 2.61E-24 and contributing 26.7% of the phenotypic variation. Six of the SNPs/haplotypes were within the same chromosomal bins as the QTLs for SDM resistance mapped in previous studies. Apart from this, eight novel genomic regions for SDM resistance were identified in this study; they need further validation before being applied in the breeding pipeline. Ten SNPs identified in this study were co-located in reported mildew resistance genes.

QTL mapping of agronomic waterlogging tolerance using recombinant inbred lines derived from tropical maize (Zea mays L) germplasm.

Waterlogging is an important abiotic stress constraint that causes significant yield losses in maize grown throughout south and south-east Asia due to erratic rainfall patterns. The most economic option to offset the damage caused by waterlogging is to genetically incorporate tolerance in cultivars that are grown widely in the target agro-ecologies. We assessed the genetic variation in a population of recombinant inbred lines (RILs) derived from crossing a waterlogging tolerant line (CAWL-46-3-1) to an elite but sensitive line (CML311-2-1-3) and observed significant range of variation for grain yield (GY) under waterlogging stress along with a number of other secondary traits such as brace roots (BR), chlorophyll content (SPAD), % stem and root lodging (S&RL) among the RILs. Significant positive correlation of GY with BR and SPAD and negative correlation with S&RL indicated the potential use of these secondary traits in selection indices under waterlogged conditions. RILs were genotyped with 331 polymorphic single nucleotide polymorphism (SNP) markers using KASP (Kompetitive Allele Specific PCR) Platform. QTL mapping revealed five QTL on chromosomes 1, 3, 5, 7 and 10, which together explained approximately 30% of phenotypic variance for GY based on evaluation of RIL families under waterlogged conditions, with effects ranging from 520 to 640 kg/ha for individual genomic regions. 13 QTL were identified for various secondary traits associated with waterlogging tolerance, each individually explaining from 3 to 14% of phenotypic variance. Of the 22 candidate genes with known functional domains identified within the physical intervals delimited by the flanking markers of the QTL influencing GY and other secondary traits, six have previously been demonstrated to be associated with anaerobic responses in either maize or other model species. A pair of flanking SNP markers has been identified for each of the QTL and high throughput marker assays were developed to facilitate rapid introgression of waterlogging tolerance in tropical maize breeding programs.

Approaches for gene targeting and targeted gene expression in plants.

Transgenic science and technology are fundamental to state-of-the-art plant molecular genetics and crop improvement. The new generation of technology endeavors to introduce genes 'stably' into 'site-specific' locations and in 'single copy' without the integration of extraneous vector 'backbone' sequences or selectable markers and with a 'predictable and consistent' expression. Several similar strategies and technologies, which can push the development of 'smart' genetically modified plants with desirable attributes, as well as enhance their consumer acceptability, are discussed in this review.