Improved pearl millet genomes representing the global heterotic pool offer a framework for molecular breeding applications.

High-quality reference genome assemblies, representative of global heterotic patterns, offer an ideal platform to accurately characterize and utilize genetic variation in the primary gene pool of hybrid crops. Here we report three platinum grade de-novo, near gap-free, chromosome-level reference genome assemblies from the active breeding germplasm in pearl millet with a high degree of contiguity, completeness, and accuracy. An improved Tift genome (Tift23D2B1-P1-P5) assembly has a contig N50 ~ 7,000-fold (126 Mb) compared to the previous version and better alignment in centromeric regions. Comparative genome analyses of these three lines clearly demonstrate a high level of collinearity and multiple structural variations, including inversions greater than 1 Mb. Differential genes in improved Tift genome are enriched for serine O-acetyltransferase and glycerol-3-phosphate metabolic process which play an important role in improving the nutritional quality of seed protein and disease resistance in plants, respectively. Multiple marker-trait associations are identified for a range of agronomic traits, including grain yield through genome-wide association study. Improved genome assemblies and marker resources developed in this study provide a comprehensive framework/platform for future applications such as marker-assisted selection of mono/oligogenic traits as well as whole-genome prediction and haplotype-based breeding of complex traits.

GBS-Based SNP Map Pinpoints the QTL Associated With Sorghum Downy Mildew Resistance in Maize (Zea mays L.).

Sorghum downy mildew (SDM), caused by the biotrophic fungi Peronosclerospora sorghi , threatens maize production worldwide, including India. To identify quantitative trait loci (QTL) associated with resistance to SDM, we used a recombinant inbred line (RIL) population derived from a cross between resistant inbred line UMI936 (w) and susceptible inbred line UMI79. The RIL population was phenotyped for SDM resistance in three environments [E1-field (Coimbatore), E2-greenhouse (Coimbatore), and E3-field (Mandya)] and also utilized to construct the genetic linkage map by genotyping by sequencing (GBS) approach. The map comprises 1516 SNP markers in 10 linkage groups (LGs) with a total length of 6924.7 cM and an average marker distance of 4.57 cM. The QTL analysis with the phenotype and marker data detected nine QTL on chromosome 1, 2, 3, 5, 6, and 7 across three environments. Of these, QTL namely qDMR1.2, qDMR3.1, qDMR5.1, and qDMR6.1 were notable due to their high phenotypic variance. qDMR3.1 from chromosome 3 was detected in more than one environment (E1 and E2), explaining the 10.3% and 13.1% phenotypic variance. Three QTL, qDMR1.2, qDMR5.1, and qDMR6.1 from chromosomes 1, 5, and 6 were identified in either E1 or E3, explaining 15.2%-18% phenotypic variance. Moreover, genome mining on three QTL (qDMR3.1, qDMR5.1, and qDMR6.1) reveals the putative candidate genes related to SDM resistance. The information generated in this study will be helpful for map-based cloning and marker-assisted selection in maize breeding programs.

Genomic regions associated with heat stress tolerance in tropical maize (Zea mays L.).

With progressive climate change and the associated increase in mean temperature, heat stress tolerance has emerged as one of the key traits in the product profile of the maize breeding pipeline for lowland tropics. The present study aims to identify the genomic regions associated with heat stress tolerance in tropical maize. An association mapping panel, called the heat tolerant association mapping (HTAM) panel, was constituted by involving a total of 543 tropical maize inbred lines from diverse genetic backgrounds, test-crossed and phenotyped across nine locations in South Asia under natural heat stress. The panel was genotyped using a genotyping-by-sequencing (GBS) platform. Considering the large variations in vapor pressure deficit (VPD) at high temperature (Tmax) across different phenotyping locations, genome-wide association study (GWAS) was conducted separately for each location. The individual location GWAS identified a total of 269 novel significant single nucleotide polymorphisms (SNPs) for grain yield under heat stress at a p value of < 10-5. A total of 175 SNPs were found in 140 unique gene models implicated in various biological pathway responses to different abiotic stresses. Haplotype trend regression (HTR) analysis of the significant SNPs identified 26 haplotype blocks and 96 single SNP variants significant across one to five locations. The genomic regions identified based on GWAS and HTR analysis considering genomic region x environment interactions are useful for breeding efforts aimed at developing heat stress resilient maize cultivars for current and future climatic conditions through marker-assisted introgression into elite genetic backgrounds and/or genome-wide selection.

Anuric Acute Kidney Injury Requiring Dialysis Following Acetazolamide Use for Cataract Surgery.

BACKGROUND Acetazolamide (ACTZ) is commonly used in the prevention and treatment of various clinical conditions, and anuric acute kidney injury (AKI) is one of its known life-threatening complications. CASE REPORT We hereby report the case of a middle-aged man known to have compensated heart failure and hypertension with previously normal kidney function, who received a total dose of 2250mg of ACTZ over 3 days after cataract surgery. One week after the operation, he presented with anuria and severe bilateral renal colic, as well as progressively worsening kidney function and metabolic profile, which eventually required hemodialysis prior to recovery. CONCLUSIONS The cause of the AKI was attributed to intra-tubular obstruction by ACTZ-induced crystalluria, which required discontinuing the offending agent and dialysis to correct the kidney functions.

Genome wide association study and genomic prediction for stover quality traits in tropical maize (Zea mays L.).

Maize is rapidly replacing traditionally cultivated dual purpose crops of South Asia, primarily due to the better economic remuneration. This has created an impetus for improving maize for both grain productivity and stover traits. Molecular techniques can largely assist breeders in determining approaches for effectively integrating stover trait improvement in their existing breeding pipeline. In the current study we identified a suite of potential genomic regions associated to the two major stover quality traits-in-vitro organic matter digestibility (IVOMD) and metabolizable energy (ME) through genome wide association study. However, considering the fact that the loci identified for these complex traits all had smaller effects and accounted only a small portion of phenotypic variation, the effectiveness of following a genomic selection approach for these traits was evaluated. The testing set consists of breeding lines recently developed within the program and the training set consists of a panel of lines from the working germplasm comprising the founder lines of the newly developed breeding lines and also an unrelated diversity set. The prediction accuracy as determined by the Pearson's correlation coefficient between observed and predicted values of these breeding lines were high even at lower marker density (200 random SNPs), when the training and testing set were related. However, the accuracies were dismal, when there was no relationship between the training and the testing set.

Genetic gains with rapid-cycle genomic selection for combined drought and waterlogging tolerance in tropical maize (Zea mays L.).

Rapid cycle genomic selection (RC-GS) helps to shorten the breeding cycle and reduce the costs of phenotyping, thereby increasing genetic gains in terms of both cost and time. We implemented RC-GS on two multi-parent yellow synthetic (MYS) populations constituted by intermating ten elite lines involved in each population, including four each of drought and waterlogging tolerant donors and two commercial lines, with proven commercial value. Cycle 1 (C1 ) was constituted based on phenotypic selection and intermating of the top 5% of 500 S2 families derived from each MYS population, test-crossed and evaluated across moisture regimes. C1 was advanced to the next two cycles (C2 and C3 ) by intermating the top 5% selected individuals with high genomic estimated breeding values (GEBVs) for grain yield under drought and waterlogging stress. To estimate genetic gains, population bulks from each cycle were test-crossed and evaluated across locations under different moisture regimes. Results indicated that the realised genetic gain under drought stress was 0.110 t ha-1 yr-1 and 0.135 t ha-1 yr-1 , respectively, for MYS-1 and MYS-2. The gain was less under waterlogging stress, where MYS-1 showed 0.038 t ha-1 yr-1 and MYS-2 reached 0.113 t ha-1 yr-1 . Genomic selection for drought and waterlogging tolerance resulted in no yield penalty under optimal moisture conditions. The genetic diversity of the two populations did not change significantly after two cycles of GS, suggesting that RC-GS can be an effective breeding strategy to achieve high genetic gains without losing genetic diversity.

Identification of donors for low-nitrogen stress with maize lethal necrosis (MLN) tolerance for maize breeding in sub-Saharan Africa.

After drought, a major challenge to smallholder farmers in sub-Saharan Africa is low-fertility soils with poor nitrogen (N)-supplying capacity. Many challenges in this region need to be overcome to create a viable fertilizer market. An intermediate solution is the development of maize varieties with an enhanced ability to take up or utilize N in severely depleted soils, and to more efficiently use the small amounts of N that farmers can supply to their crops. Over 400 elite inbred lines from seven maize breeding programs were screened to identify new sources of tolerance to low-N stress and maize lethal necrosis (MLN) for introgression into Africa-adapted elite germplasm. Lines with high levels of tolerance to both stresses were identified. Lines previously considered to be tolerant to low-N stress ranked in the bottom 10% under low-N confirming the need to replace these lines with new donors identified in this study. The lines that performed best under low-N yielded about 0. 5 Mg ha-1 (20%) more in testcross combinations than some widely used commercial parent lines such as CML442 and CML395. This is the first large scale study to identify maize inbred lines with tolerance to low-N stress and MLN in eastern and southern Africa.

Genomic Selection Outperforms Marker Assisted Selection for Grain Yield and Physiological Traits in a Maize Doubled Haploid Population Across Water Treatments.

To increase genetic gain for tolerance to drought, we aimed to identify environmentally stable QTL in per se and testcross combination under well-watered (WW) and drought stressed (DS) conditions and evaluate the possible deployment of QTL using marker assisted and/or genomic selection (QTL/GS-MAS). A total of 169 doubled haploid lines derived from the cross between CML495 and LPSC7F64 and 190 testcrosses (tester CML494) were evaluated in a total of 11 treatment-by-population combinations under WW and DS conditions. In response to DS, grain yield (GY) and plant height (PHT) were reduced while time to anthesis and the anthesis silking interval (ASI) increased for both lines and hybrids. Forty-eight QTL were detected for a total of nine traits. The allele derived from CML495 generally increased trait values for anthesis, ASI, PHT, the normalized difference vegetative index (NDVI) and the green leaf area duration (GLAD; a composite trait of NDVI, PHT and senescence) while it reduced trait values for leaf rolling and senescence. The LOD scores for all detected QTL ranged from 2.0 to 7.2 explaining 4.4 to 19.4% of the observed phenotypic variance with R2 ranging from 0 (GY, DS, lines) to 37.3% (PHT, WW, lines). Prediction accuracy of the model used for genomic selection was generally higher than phenotypic variance explained by the sum of QTL for individual traits indicative of the polygenic control of traits evaluated here. We therefore propose to use QTL-MAS in forward breeding to enrich the allelic frequency for a few desired traits with strong additive QTL in early selection cycles while GS-MAS could be used in more mature breeding programs to additionally capture alleles with smaller additive effects.

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 Mapping and Genomic Prediction Analyses Reveal the Genetic Architecture of Grain Yield and Flowering Time Under Drought and Heat Stress Conditions in Maize.

Drought stress (DS) is a major constraint to maize yield production. Heat stress (HS) alone and in combination with DS are likely to become the increasing constraints. Association mapping and genomic prediction (GP) analyses were conducted in a collection of 300 tropical and subtropical maize inbred lines to reveal the genetic architecture of grain yield and flowering time under well-watered (WW), DS, HS, and combined DS and HS conditions. Out of the 381,165 genotyping-by-sequencing SNPs, 1549 SNPs were significantly associated with all the 12 trait-environment combinations, the average PVE (phenotypic variation explained) by these SNPs was 4.33%, and 541 of them had a PVE value greater than 5%. These significant associations were clustered into 446 genomic regions with a window size of 20 Mb per region, and 673 candidate genes containing the significantly associated SNPs were identified. In addition, 33 hotspots were identified for 12 trait-environment combinations and most were located on chromosomes 1 and 8. Compared with single SNP-based association mapping, the haplotype-based associated mapping detected fewer number of significant associations and candidate genes with higher PVE values. All the 688 candidate genes were enriched into 15 gene ontology terms, and 46 candidate genes showed significant differential expression under the WW and DS conditions. Association mapping results identified few overlapped significant markers and candidate genes for the same traits evaluated under different managements, indicating the genetic divergence between the individual stress tolerance and the combined drought and HS tolerance. The GP accuracies obtained from the marker-trait associated SNPs were relatively higher than those obtained from the genome-wide SNPs for most of the target traits. The genetic architecture information of the grain yield and flowering time revealed in this study, and the genomic regions identified for the different trait-environment combinations are useful in accelerating the efforts on rapid development of the stress-tolerant maize germplasm through marker-assisted selection and/or genomic selection.

Genomic Regions Associated with Root Traits under Drought Stress in Tropical Maize (Zea mays L.).

An association mapping panel, named as CIMMYT Asia association mapping (CAAM) panel, involving 396 diverse tropical maize lines were phenotyped for various structural and functional traits of roots under drought and well-watered conditions. The experiment was conducted during Kharif (summer-rainy) season of 2012 and 2013 in root phenotyping facility at CIMMYT-Hyderabad, India. The CAAM panel was genotyped to generate 955, 690 SNPs through GBS v2.7 using Illumina Hi-seq 2000/2500 at Institute for Genomic Diversity, Cornell University, Ithaca, NY, USA. GWAS analysis was carried out using 331,390 SNPs filtered from the entire set of SNPs revealed a total of 50 and 67 SNPs significantly associated for root functional (transpiration efficiency, flowering period water use) and structural traits (rooting depth, root dry weight, root length, root volume, root surface area and root length density), respectively. In addition to this, 37 SNPs were identified for grain yield and shoot biomass under well-watered and drought stress. Though many SNPs were found to have significant association with the traits under study, SNPs that were common for more than one trait were discussed in detail. A total 18 SNPs were found to have common association with more than one trait, out of which 12 SNPs were found within or near the various gene functional regions. In this study we attempted to identify the trait specific maize lines based on the presence of favorable alleles for the SNPs associated with multiple traits. Two SNPs S3_128533512 and S7_151238865 were associated with transpiration efficiency, shoot biomass and grain yield under well-watered condition. Based on favorable allele for these SNPs seven inbred lines were identified. Similarly, four lines were identified for transpiration efficiency and shoot biomass under drought stress based on the presence of favorable allele for the common SNPs S1_211520521, S2_20017716, S3_57210184 and S7_130878458 and three lines were identified for flowering period water-use, transpiration efficiency, root dry weight and root volume based on the presence of favorable allele for the common SNPs S3_162065732 and S3_225760139.

Correction: Identification of QTL for Early Vigor and Stay-Green Conferring Tolerance to Drought in Two Connected Advanced Backcross Populations in Tropical Maize (Zea mays L.).

[This corrects the article DOI: 10.1371/journal.pone.0149636.].

Identification of QTL for Early Vigor and Stay-Green Conferring Tolerance to Drought in Two Connected Advanced Backcross Populations in Tropical Maize (Zea mays L.).

We aimed to identify quantitative trait loci (QTL) for secondary traits related to grain yield (GY) in two BC1F2:3 backcross populations (LPSpop and DTPpop) under well-watered (4 environments; WW) and drought stressed (6; DS) conditions to facilitate breeding efforts towards drought tolerant maize. GY reached 5.6 and 5.8 t/ha under WW in the LPSpop and the DTPpop, respectively. Under DS, grain yield was reduced by 65% (LPSpop) to 59% (DTPpop) relative to WW. GY was strongly associated with the normalized vegetative index (NDVI; r ranging from 0.61 to 0.96) across environmental conditions and with an early flowering under drought stressed conditions (r ranging from -0.18 to -0.25) indicative of the importance of early vigor and drought escape for GY. Out of the 105 detected QTL, 53 were overdominant indicative of strong heterosis. For 14 out of 18 detected vigor QTL, as well as for eight flowering time QTL the trait increasing allele was derived from CML491. Collocations of early vigor QTL with QTL for stay green (bin 2.02, WW, LPSpop; 2.07, DS, DTPpop), the number of ears per plant (bins 2.02, 2.05, WW, LPSpop; 5.02, DS, LPSpop) and GY (bin 2.07, WW, DTPpop; 5.04, WW, LPSpop), reinforce the importance of the observed correlations. LOD scores for early vigor QTL in these bins ranged from 2.2 to 11.25 explaining 4.6 (additivity: +0.28) to 19.9% (additivity: +0.49) of the observed phenotypic variance. A strong flowering QTL was detected in bin 2.06 across populations and environmental conditions explaining 26-31.3% of the observed phenotypic variation (LOD: 13-17; additivity: 0.1-0.6d). Improving drought tolerance while at the same time maintaining yield potential could be achieved by combining alleles conferring early vigor from the recurrent parent with alleles advancing flowering from the donor. Additionally bin 8.06 (DTPpop) harbored a QTL for GY under WW (additivity: 0.27 t/ha) and DS (additivity: 0.58 t/ha). R2 ranged from 0 (DTPpop, WW) to 26.54% (LPSpop, DS) for NDVI, 18.6 (LPSpop, WW) to 42.45% (LPSpop, DS) for anthesis and from 0 (DTPpop, DS) to 24.83% (LPSpop, WW) for GY. Lines out-yielding the best check by 32.5% (DTPpop, WW) to 60% (DTPpop, DS) for all population-by-irrigation treatment combination (except LPSpop, WW) identified are immediately available for the use by breeders.

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.

Genome-wide association and genomic prediction of resistance to maize lethal necrosis disease in tropical maize germplasm.

KEY MESSAGE: Genome-wide association analysis in tropical and subtropical maize germplasm revealed that MLND resistance is influenced by multiple genomic regions with small to medium effects. The maize lethal necrosis disease (MLND) caused by synergistic interaction of Maize chlorotic mottle virus and Sugarcane mosaic virus, and has emerged as a serious threat to maize production in eastern Africa since 2011. Our objective was to gain insights into the genetic architecture underlying the resistance to MLND by genome-wide association study (GWAS) and genomic selection. We used two association mapping (AM) panels comprising a total of 615 diverse tropical/subtropical maize inbred lines. All the lines were evaluated against MLND under artificial inoculation. Both the panels were genotyped using genotyping-by-sequencing. Phenotypic variation for MLND resistance was significant and heritability was moderately high in both the panels. Few promising lines with high resistance to MLND were identified to be used as potential donors. GWAS revealed 24 SNPs that were significantly associated (P < 3 × 10(-5)) with MLND resistance. These SNPs are located within or adjacent to 20 putative candidate genes that are associated with plant disease resistance. Ridge regression best linear unbiased prediction with five-fold cross-validation revealed higher prediction accuracy for IMAS-AM panel (0.56) over DTMA-AM (0.36) panel. The prediction accuracy for both within and across panels is promising; inclusion of MLND resistance associated SNPs into the prediction model further improved the accuracy. Overall, the study revealed that resistance to MLND is controlled by multiple loci with small to medium effects and the SNPs identified by GWAS can be used as potential candidates in MLND resistance breeding program.

Fine mapping of Msv1, a major QTL for resistance to Maize Streak Virus leads to development of production markers for breeding pipelines.

Msv1 , the major QTL for MSV resistance was delimited to an interval of 0.87 cM on chromosome 1 at 87 Mb and production markers with high prediction accuracy were developed. Maize streak virus (MSV) disease is a devastating disease in the Sub-Saharan Africa (SSA), which causes significant yield loss in maize. Resistance to MSV has previously been mapped to a major QTL (Msv1) on chromosome 1 that is germplasm and environment independent and to several minor loci elsewhere in the genome. In this study, Msv1 was fine-mapped through QTL isogenic recombinant strategy using a large F 2 population of CML206 × CML312 to an interval of 0.87 cM on chromosome 1. Genome-wide association study was conducted in the DTMA (Drought Tolerant Maize for Africa)-Association mapping panel with 278 tropical/sub-tropical breeding lines from CIMMYT using the high-density genotyping-by-sequencing (GBS) markers. This study identified 19 SNPs in the region between 82 and 93 Mb on chromosome 1(B73 RefGen_V2) at a P < 1.00E-04, which coincided with the fine-mapped region of Msv1. Haplotype trend regression identified a haplotype block significantly associated with response to MSV. Three SNPs in this haplotype block at 87 Mb on chromosome 1 had an accuracy of 0.94 in predicting the disease reaction in a collection of breeding lines with known responses to MSV infection. In two biparental populations, selection for resistant Msv1 haplotype demonstrated a reduction of 1.03-1.39 units on a rating scale of 1-5, compared to the susceptible haplotype. High-throughput KASP assays have been developed for these three SNPs to enable routine marker screening in the breeding pipeline for MSV resistance.

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.

Genome-wide association analysis reveals new targets for carotenoid biofortification in maize.

KEY MESSAGE: Genome-wide association analysis in CIMMYT's association panel revealed new favorable native genomic variations in/nearby important genes such as hydroxylases and CCD1 that have potential for carotenoid biofortification in maize. Genome-wide association studies (GWAS) have been used extensively to identify allelic variation for genes controlling important agronomic and nutritional traits in plants. Provitamin A (proVA) enhancing alleles of lycopene epsilon cyclase (LCYE) and ß-carotene hydroxylase 1 (CRTRB1), previously identified through candidate-gene based GWAS, are currently used in CIMMYT's maize breeding program. The objective of this study was to identify genes or genomic regions controlling variation for carotenoid concentrations in grain for CIMMYT's carotenoid association mapping panel of 380 inbred maize lines, using high-density genome-wide platforms with ~476,000 SNP markers. Population structure effects were minimized by adjustments using principal components and kinship matrix with mixed models. Genome-wide linkage disequilibrium (LD) analysis indicated faster LD decay (3.9 kb; r (2) = 0.1) than commonly reported for temperate germplasm, and therefore the possibility of achieving higher mapping resolution with our mostly tropical diversity panel. GWAS for various carotenoids identified CRTRB1, LCYE and other key genes or genomic regions that govern rate-critical steps in the upstream pathway, such as DXS1, GGPS1, and GGPS2 that are known to play important roles in the accumulation of precursor isoprenoids as well as downstream genes HYD5, CCD1, and ZEP1, which are involved in hydroxylation and carotenoid degradation. SNPs at or near all of these regions were identified and may be useful target regions for carotenoid biofortification breeding efforts in maize; for example a genomic region on chromosome 2 explained ~16% of the phenotypic variance for ß-carotene independently of CRTRB1, and a variant of CCD1 that resulted in reduced ß-cryptoxanthin degradation was found in lines that have previously been observed to have low proVA degradation rates.

Analysis of effectiveness of R1-nj anthocyanin marker for in vivo haploid identification in maize and molecular markers for predicting the inhibition of R1-nj expression.

KEY MESSAGE: R1-nj anthocyanin marker inhibition is highly frequent in tropical maize germplasm considerably affecting efficiency of haploid identification. Molecular markers reliably differentiating germplasm with anthocyanin color inhibitor have been identified in this study. The R1-Navajo (R1-nj) color marker facilitates easy and quick identification of haploid kernels at the seed stage during in vivo haploid induction process in maize. However, the Navajo phenotype can be completely suppressed or poorly expressed in some germplasm, making it impossible or inefficient to identify haploids at the seed stage. In this study, we characterized the expression of R1-nj marker in a large array of tropical/subtropical inbred lines, breeding populations and landraces by crossing with the R1-nj-based tropicalized haploid inducer. There was a high frequency of inhibition of the Navajo phenotype in the maize inbred lines, which are used in tropical breeding programs. Genome-wide association mapping showed that the C1 anthocyanin regulatory locus is the most significant genetic factor influencing inhibition of the Navajo phenotype. Molecular marker assays were designed based on polymorphism in the C1 vs C1-I alleles. Analysis of a set of 714 inbred lines demonstrated that a combination of two gene-specific markers--8 bp C1-I InDel and C1-I SNP--could predict with high accuracy the presence of anthocyanin color inhibition in the germplasm analyzed. Information generated in this study aids in making informed decisions on the constitution of source populations for doubled haploid (DH) line development in tropical germplasm, particularly those derived from elite maize lines from CIMMYT. The C1-I gene-specific molecular markers identified and validated will facilitate high-throughput and cost-effective evaluation of a large pool of germplasm for the presence of the dominant color inhibitor in maize germplasm.

Molecular mapping across three populations reveals a QTL hotspot region on chromosome 3 for secondary traits associated with drought tolerance in tropical maize.

Identifying quantitative trait loci (QTL) of sizeable effects that are expressed in diverse genetic backgrounds across contrasting water regimes particularly for secondary traits can significantly complement the conventional drought tolerance breeding efforts. We evaluated three tropical maize biparental populations under water-stressed and well-watered regimes for drought-related morpho-physiological traits, such as anthesis-silking interval (ASI), ears per plant (EPP), stay-green (SG) and plant-to-ear height ratio (PEH). In general, drought stress reduced the genetic variance of grain yield (GY), while that of morpho-physiological traits remained stable or even increased under drought conditions. We detected consistent genomic regions across different genetic backgrounds that could be target regions for marker-assisted introgression for drought tolerance in maize. A total of 203 QTL for ASI, EPP, SG and PEH were identified under both the water regimes. Meta-QTL analysis across the three populations identified six constitutive genomic regions with a minimum of two overlapping traits. Clusters of QTL were observed on chromosomes 1.06, 3.06, 4.09, 5.05, 7.03 and 10.04/06. Interestingly, a ~8-Mb region delimited in 3.06 harboured QTL for most of the morpho-physiological traits considered in the current study. This region contained two important candidate genes viz., zmm16 (MADS-domain transcription factor) and psbs1 (photosystem II unit) that are responsible for reproductive organ development and photosynthate accumulation, respectively. The genomic regions identified in this study partially explained the association of secondary traits with GY. Flanking single nucleotide polymorphism markers reported herein may be useful in marker-assisted introgression of drought tolerance in tropical maize.