Inbred Lines Research Articles

Assessing genotype-by-environment interactions for maydis leaf blight disease in maize (Zea mays L.) germplasm and identification of stable donors for breeding resistant hybrid varieties

Abstract This study investigates the impact of environmental factors and genotype-by-environment interactions (GEI) on the expression of maydis leaf blight (MLB) resistance in a diverse maize germplasm comprising 359 genotypes. Extensive field trials were conducted, involving artificial inoculations and disease scoring across two locations over two years. Using genotype and genotype–environment (GGE) biplot analysis based on the site regression model (SREG), we identified stable MLB-resistant 10 donors with consistent genotypic responses. These inbred lines, which consistently exhibited disease scores of ⩽3 across locations, are recommended as potential parents for breeding MLB-resistant varieties. Furthermore, the identification of a non-crossover interaction and high correlations among testing locations allowed us to define a single mega-environment for the initial screening of MLB resistance in a large set of maize germplasm. This study suggests that initial screenings can be efficiently conducted in one representative location, with validation of resistant lines at multiple sites during advanced breeding stages. This approach optimizes the use of land, labour and resources in MLB resistance testing.

Solanum pimpinellifolium exhibits complex genetic resistance to Pseudomonas syringae pv. tomato.

Pseudomonas syringae pv. tomato (Pst) is the causal agent of bacterial speck disease in tomatoes. The Pto/Prf gene cluster from Solanum pimpinellifolium was introgressed into several modern tomato cultivars and provided protection against Pst race 0 strains for many decades. However, virulent Pst race 1 strains that evade Pto-mediated immunity now predominate in tomato-growing regions worldwide. Here we report the identification of resistance to a Pst race 1 strain (Pst19) in the wild tomato accession S. pimpinellifolium LA1589 (hereafter LA1589), using our rapid high-throughput seedling screen. LA1589 supports less bacterial growth than cultivars, and does not exhibit a hypersensitive response to Pst19. We tested an existing set of 87 Inbred Backcross Lines (IBLs) derived from a cross between susceptible Solanum lycopersicum E-6203 and Solanum pimpinellifolium LA1589 for resistance to Pst19. Using single-marker analysis, we identified three genomic regions associated with resistance. Bacterial growth assays on IBLs confirmed that these regions contribute to resistance in planta. We also mapped candidate genes associated with resistance in a cross between the Solanum lycopersicum var. lycopersicum cultivar Heinz BG-1706 and S. pimpinellifolium LA1589. By comparing candidates from the two mapping approaches, we were able to identify 3 QTL and 5 candidate genes in LA1589 for a role in resistance to Pst19. This work will assist in molecular marker-assisted breeding to protect tomato from bacterial speck disease.

EQTL mapping in transgenic alpha-synuclein carrying Caenorhabditis elegans recombinant inbred lines.

Protein aggregation of α-synuclein (αS) is a genetic and neuropathological hallmark of Parkinson's disease (PD). Studies in the model nematode Caenorhabditis elegans suggested that variation of αS aggregation depends on the genetic background. However, which genes and genetic modifiers underlie individual differences in αS pathology remains unknown. To study the genotypic-phenotypic relationship of αS aggregation, we constructed a Recombinant Inbred Line (RIL) panel derived from a cross between genetically divergent strains C. elegans NL5901 and SCH4856, both harboring the human αS gene. As a first step to discover genetic modifiers 70 αS-RILs were measured for whole-genome gene expression and expression quantitative locus analysis (eQTL) were mapped. We detected multiple eQTL hot-spots, many of which were located on Chromosome V. To confirm a causal locus, we developed Introgression Lines (ILs) that contain SCH4856 introgressions on Chromosome V in an NL5901 background. We detected 74 genes with an interactive effect between αS and the genetic background, including the human p38 MAPK homologue pmk-1 that has previously been associated with PD. Together, we present a unique αS-RIL panel for defining effects of natural genetic variation on αS pathology, which contributes to finding genetic modifiers of PD.

Identification of Diverse Maize Inbred Lines using Molecular and Phenotypic Genetic Divergence Analysis

Identification of Diverse Maize Inbred Lines using Molecular and Phenotypic Genetic Divergence Analysis

Gene co-expression analysis reveals conserved and distinct gene networks between resistant and susceptible Lens ervoides challenged by hemibiotrophic and necrotrophic pathogens

As field crops are likely to be challenged by multiple pathogens during their development, the investigation of broad-spectrum resistance in the host is of great interest for crop genetic enhancement. In this study, we attempted to address this question by adopting a weighed gene co-expression approach to study the temporal transcriptome dynamics of resistant and susceptible recombinant inbred lines (RILs) derived from an intraspecific Len ervoides cross during the infection process with either the necrotrophic pathogens Ascochyta lentis or Stemphylium botryosum, or the hemibiotrophic pathogen Colletotrichum lentis. By comparing networks of resistant and susceptible RILs, seven network module pairs were found to possess high correlation coefficients (R > 0.70) and large number of overlapping genes (n > 100). The conserved co-regulation of genes in these network module pairs were involved in plant cell wall synthesis, cell division, cytoskeleton organization, and protein ubiquitin related processes and appeared to be common disease responses against these pathogens. On the other hand, we also identified eight modules with low correlation between resistance and susceptibility networks. Among those, a stronger gene co-expression in R-genes and small RNA processes in the resistant hosts may be enhancing L. ervoides resistance against A. lentis, C. lentis, and S. botryosum, whereas the higher level of synergistic regulation in the synthesis of arginine and glutamine and phospholipid and glycerophospholipids in the susceptible hosts may contribute to increased susceptibility in L. ervoides.

Hybrid grain production in wheat benefits from synchronized flowering and high female flower receptivity.

The performance of plant hybrids relative to line breeding types is generally associated with higher yields, better adaptation, and improved yield stability. In bread wheat (Triticum aestivum L.), however, a broad commercial success for hybrids has not been accomplished until now largely due to the low efficiency of hybrid grain production, which is highly attributable to its self-pollinating nature. To better understand how hybrid wheat grains can be produced more effectively, we investigated the influence of synchronized flowering between female, i.e. male-sterile, lines and their male cross-pollinator lines as well as of the duration of flowering on hybrid grain production. We found that synchronization of flowering in combination with the longest possible temporal overlap had the largest positive effect on hybrid grain production. However, despite sufficient spatial and temporal synchronization of flowering, we also found that some female lines had lower hybrid grain set than others, suggesting genetic differences in female floral receptivity. To better assess female receptivity, we established a new phenotyping scale of male-sterile wheat flowers that provides the floral basics for effective cross-pollination. Applying this scale in our field and greenhouse trials revealed that better performing female lines remained longer in the pollen-receptive phase.

Validation of QTLs associated with corn borer resistance and grain yield: implications in maize breeding.

Validations of previously detected quantitative trait loci (QTLs) to assess their reliability are crucial before implementing breeding programs. The objective of this study was to determine the reliability and practical usefulness of previously reported QTLs for resistance to stem tunneling by the Mediterranean stem borer (MSB) and yield. These authors used approximately 600 recombinant inbred lines (RILs) from a multiparent advanced generation intercross (MAGIC) population to map QTL using a genome-wide association study (GWAS) approach. We identified RILs situated at the extremes of resistance and yield distributions within the whole MAGIC, and those QTLs were evaluated per se and crossed to a tester (A638) using lattice designs. In each set, a significant single-nucleotide polymorphism (SNP) was considered validated if (1) the same SNP was associated with the trait with a p-value < 0.02, or (2) within a ±2-Mbp interval, an SNP associated with the trait exhibited a p-value < 0.02 and demonstrated linkage disequilibrium (r2 > 0.2) with the SNPs previously reported. The novel QTL validation approach was implemented using improved experimental designs that led to higher heritability estimates for both traits compared to those estimated with the whole MAGIC population. The procedure used allowed us to jointly validate several QTL and to ascertain their possible contribution to hybrid improvement. Specifically, nearly three-quarters of the QTLs for tunnel length were confirmed. Notably, QTLs located in the genomic region 6.05-6.07 were consistently validated across different sets and have been previously linked to resistance against stalk tunneling in various mapping populations. For grain yield, approximately 10 out of 16 QTLs were validated. The validation rate for yield was lower than for tunnel length, likely due to the influence of dominance and/or epistatic effects. Overall, 9 out of 21 QTLs for tunnel length and 6 out of 17 QTLs for grain yield identified in our previous research were validated across both validation sets, indicating a moderate genetic correlation between per se and testcross performance of inbred lines. These findings offer insights into the reliability of QTL and genomic predictions, both derived from assessments conducted on the entire MAGIC population. Genomic predictions for tunnel length based on inbred line evaluations could be useful to develop more resistant hybrids; meanwhile, genomic prediction for yield could only be valid in a homozygous background.

Serological Detection of Cucumber Mosaic Virus and Capsicum Chlorosis Virus in Pepper (Capsicum annuum L.) Germplasm

A survey was conducted on the basis of symptomatology for the screening of germplasm under insect proof glasshouse conditions in the Department of Vegetable Science, Dr. YSPUHF, Nauni, Solan to identify the sources of resistance in pepper crop. The most prominent symptoms were mosaic, mottling, leaf narrowing, stunted growth, mosaic with chlorotic lesions, formation of rings with necrotic lesions and mosaic with mottling. Forty-eight varieties or breeding lines were screened for the presence or absence of virus through DAS-ELISA. The study revealed that nineteen varieties or breeding lines tested positive and twenty-nine tested negative for virus infection. Sweet Banana variety recorded the least O.D. value followed by SB and UHF-Cap-30 whereas, UHF-Cap-22 was most susceptible variety with maximum O.D. value. Mixed infection of CMV and capsicum chlorosis orthotospovirus (CaCV) was recorded with six varieties or breeding lines during germplasm screening. This study signifies the presence of mixed infection with chlorotic lesions like symptoms and importance of DAS-ELISA in screening of pepper germplasm against CMV and CaCV.

Development of infectious clones of mungbean yellow mosaic India virus (MYMIV, Begomovirus vignaradiataindiaense) infecting mungbean [Vigna radiata (L.) R. Wilczek] and evaluation of a RIL population for MYMIV resistance.

Yellow mosaic disease (YMD) is a major constraint for the low productivity of mungbean, mainly in South Asia. Addressing this issue requires a comprehensive approach, integrating field and challenge inoculation evaluations to identify effective solutions. In this study, an infectious clone of Begomovirus vignaradiataindiaense (MYMIV) was developed to obtain a pure culture of the virus and to confirm resistance in mungbean plants exhibiting resistance under natural field conditions. The infectivity and efficiency of three Agrobacterium tumefaciens strains (EHA105, LBA4404, and GV3101) were evaluated using the susceptible mungbean genotype PS16. Additionally, a recombinant inbred line (RIL) population comprising 175 lines derived from Pusa Baisakhi (MYMIV susceptible) and PMR-1 (MYMIV resistant) cross was developed and assessed for YMD response. Among the tested Agrobacterium tumefaciens strains, EHA105 exhibited the highest infectivity (84.7%), followed by LBA4404 (54.7%) and GV3101 (9.80%). Field resistance was evaluated using the coefficient of infection (CI) and area under disease progress curve (AUDPC), identifying seven RILs with consistent resistant reactions (CI≤9) and low AUDPC (≤190). Upon challenge inoculation, six RILs exhibited resistance, while RIL92 displayed a resistance response, with infection occurring in less than 10% of plants after 24 to 29 days post inoculation (dpi). Despite some plants remaining asymptomatic, MYMIV presence was confirmed through specific PCR amplification of the MYMIV coat protein (AV1) gene. Quantitative PCR revealed a very low relative viral load (0.1-5.1% relative fold change) in asymptomatic RILs and the MYMIV resistant parent (PMR1) compared to the susceptible parent (Pusa Baisakhi). These findings highlight the potential utility of the developed infectious clone and the identified MYMIV-resistant RILs in future mungbean breeding programs aimed at cultivating MYMIV-resistant varieties.

Transgressive segregation and generation mean analysis reveal the gene action underlying the inheritance of drought tolerance in rice

Climate change, an effective driver of unprecedented seasonal droughts, is greatly affecting rice production in Africa by threatening food security and safety. Rice, one of the major staple crops on the continent, can save the situation through the development of drought-tolerant cultivars, presenting a major challenge for future rice improvement programs as drought is regarded as a critical limitation in rain-fed ecosystems. This study sought to understand the genetic basis and inheritance behind the expression of tolerance of rice breeding lines to drought-stress through generation mean analysis. To achieve these objectives, two drought-sensitive genotypes (Jasmine 85 and CRI-Agrarice) were crossed with a drought-tolerant genotype (APO) to develop six populations (F1, F2, BC1, BC2, P1 and P2) under screenhouse drought-stress and non-stress evaluation. Data were collected on grain yield and yield-related traits among which the generation mean analysis was conducted. At least one transgressive phenotype was produced in the F2 population for each trait whether there is a significant difference or not among the parental lines under drought-stress. Under non-stress conditions, there was a significance for all six types of gene action for days to flowering in both crosses. Among both crosses and water-regimes, additive x additive gene interaction was significant for most of the traits even though the scaling tests were not significant indicating the effectiveness of selection in early generations. Therefore, either forward breeding or backcross breeding can be adopted as breeding strategies for rapid improvement for these lines to drought tolerance.

Early Generation Selection of Potato Breeding Lines

Potato is the third most important food crop in the world in terms of adaptability, yield potential, and nutritional advantages. This study aimed to conduct potato breeding work for cultivation in Kazakhstan; potato breeding for further processing into chips with cultivation in the northern regions; and the selection of potatoes for processing into frozen French fries with cultivation in the southern and south-eastern regions. Potato varieties (Fontane, Santana, and Punchy) were used as reference varieties to check molecular markers linked to maturity, tuber shape, and flesh color. A total of 42 potato breeding lines crossed from Yagodnyi 19 and CIP clone 397079-6 were used in this study to identify prospective breeding lines. The research was carried out between 2023 and 2024 and under greenhouse conditions. According to the results of molecular analysis and phenotypic data, 21 breeding lines were identified as prospective potato breeding lines. The majority of these potato breeding lines had a round tuber shape and were recommended for chip processing. Three breeding lines had a long oval tuber shape, making them suitable for French fries. Six breeding lines with short-oval and oval tuber shapes were found for consumer potato processing. It is recommended that the breeding process and studies of biochemical properties are continued in all of these identified potato breeding lines.

Identification of genes governing YMD resistance in soybean cultivar SL 958.

Yellow mosaic disease (YMD) is the most devastating disease of soybean under north Indian conditions. A recombinant inbred line population derived from SL 958 (R) × AGS 456 (S) was used to map the genes imparting YMD resistance. Genotyping by sequencing (GBS) of 148 individuals and the parents was used to identify polymorphic SNPs in the population. These were used to generate linkage maps and subsequently map the QTL. A QTL, MYMIV 1-1.1, was mapped onto chromosome 6. The QTL was mapped at a genetic distance of 114.31 cM, with likelihood ratio of 81.34 (LOD 17.68), explaining 33.80% of the variation for the trait, with an additive effect of 1.22. MYMIV 1-1.1 was flanked by SNPs Gm06_12614920 explaining 33.80% of variation and Gm06_12887789 explaining 31.04% of the variation. The QTL region spanned for a total of 2.798 Mbp. The QTL was mapped in an approximately 4 cM region. Flanking SNPs can be developed into linked markers for effective marker-assisted selection. Out of the genes in the QTL region, Glyma.06G161700 (Suppressor of Gene Silencing/SGS3) is the strongest contender for the resistance gene. This is the first study documenting GBS-based mapping of YMD resistance in soybean. © 2024 Society of Chemical Industry.

Mean Performance and Heterotic Pattern of Maize (Zea mays L.) Inbred Lines Adapted to Sub-humid Central Highland of Ethiopia

In spite of abiotic and biotic stresses and low availability of high-yielding cultivars restrict potential production of maize, it is a major food crop in Africa. To overcome this production barrier, promising germplasm must be chosen, combining ability must be understood, and heterotic groups must be formed in order to generate high-yielding maize varieties. The study set out to evaluate plant height, ear height, ear diameter, thousand seed weight, kernel per row, kernel row per ear, and number of ear per plant in order to assess the performance evaluation and heterotic pattern of inbred lines. In 2019, twenty-six inbred lines and two single cross testers were crossed using a line by tester mating design to create fifty-two three way cross F1 hybrids. The experiment, which used an alpha lattice design with two replications, was conducted at the Ambo and Kulumsa Agricultural Research Centers during the 2020 cropping season. Analyses of variance revealed that among the parameters with significant mean squares arising from crosses and lines both within and across locations were grain yield, anthesis date, silking date, plant height, ear height, kernel row per ear, and number of ears per plant. Because of the lines, testers, and crossings for grain yield, anthesis date, silking date, plant height, ear height, kernel row per ear, and number of ear per plant, the line x tester ANOVA results revealed significant mean squares. Due to line x site, line x tester, tester x site, and line x tester x site, there was a noticeable difference in plant height, ear height, and the number of ears per plant. This proved that different environment have different selection criteria for materials and different performance levels. This genetic study Categorized, L1, L22, and L24 under heterotic group A (CEL08008/CEL08047), while L8, L10, L14, L17, L20, and L26 were placed under heterotic group-B (CEL08024/CML561).

Yr29 combined with QYr.nwafu-4BL.3 confers durable resistance to stripe rust in wheat cultivar Jing 411.

The combination of a QTL on chromosome arm 4BL and Yr29 provides durable resistance with no significant yield penalty. Wheat stripe rust or yellow rust (YR), caused by Puccinia striiformis f. sp. tritici (Pst), causes substantial yield reductions globally, but losses can be minimized by using resistance genes. Chinese wheat cultivar Jing 411 (J411) has continued to display an acceptable level of adult-plant resistance (APR) to YR in varied field conditions since its release in the 1990s. A recombinant inbred line (RIL) population comprising 187 lines developed from a cross of J411 and Kenong 9204 (KN9204) was evaluated in multiple environments to identify genomic regions carrying genes for YR resistance. A total of five quantitative trait loci (QTL) on chromosome arm 1BL, 3BS, 4BL, 6BS, and 7BL from J411 and two QTL on 3DS and 7DL from KN9204 were detected using inclusive composite interval mapping with the wheat 660K SNP array. QYr.nwafu-1BL.5 and QYr.nwafu-4BL.3 from J411 were robust and showed similar effects in all environments. QYr.nwafu-1BL.5 was likely the pleiotropic gene of Yr29/Lr46. QYr.nwafu-4BL.3 was located within a 1.0cM interval delimited by KASP markers AX-111609222 and AX-89755491. Based on haplotype analysis, Yr29 and QYr.nwafu-4BL.3 were identified as genetic components of quantitative resistance in a number of wheat cultivars. Moreover, RILs with Yr29 and QYr.nwafu-4BL.3 individually or when combined showed higher resistance to YR in rust nurseries compared with RILs without them, and there was no negative effect of their presence on agronomic traits under rust-free conditions. These results suggest that effective polymerization strategy is important for breeding high yielding and durable resistance cultivars.

Pangenome Data Analysis Reveals Characteristics of Resistance Gene Analogs Associated with Sclerotinia sclerotiorum Resistance in Sunflower

The sunflower, an important oilseed crop and food source across the world, is susceptible to several pathogens, which cause severe losses in sunflower production. The utilization of genetic resistance is the most economical, effective measure to prevent infectious diseases. Based on the sunflower pangenome, in this study, we explored the variability of resistance gene analogs (RGAs) within the species. According to a comparative analysis of RGA candidates in the sunflower pangenome using the RGAugury pipeline, a total of 1344 RGAs were identified, comprising 1107 conserved, 199 varied, and 38 rare RGAs. We also identified RGAs associated with resistance against Sclerotinia sclerotiorum (S. sclerotiorum) in sunflower at the quantitative trait locus (QTL). A total of 61 RGAs were found to be located at four quantitative trait loci (QTLs). Through a detailed expression analysis of RGAs in one susceptible and two tolerant sunflower inbred lines (ILs) across various time points post inoculation, we discovered that 348 RGAs exhibited differential expression in response to Sclerotinia head rot (SHR), with 17 of these differentially expressed RGAs being situated within the QTL regions. In addition, 15 RGA candidates had gene introgression. Our data provide a better understanding of RGAs, which facilitate genomics-based improvements in disease resistance in sunflower.

Combined BSA-Seq and RNA-Seq Analysis to Identify Candidate Genes Associated with Aluminum Toxicity in Rapeseed (Brassica napus L.).

Exchangeable aluminum (Al) ions released from acidic soils with pH < 5.5 inhibit root elongation of crops, ultimately leading to yield reduced. It is necessary to identify the quantitative trait locus (QTLs) and candidate genes that confer toxicity resistance to understand the mechanism and improve tolerance of rapeseed. In this study, an F2 segregating population was derived from a cross between Al-tolerance inbred line FDH188 (R178) and -sensitive inbred line FDH152 (S169), and the F2:3 were used as materials to map QTLs associated with the relative elongation of taproot (RET) under Al toxicity stress. Based on bulked segregant analysis sequencing (BSA-seq), three QTLs (qAT-A07-1, qAT-A07-2, and qAT-A09-1) were detected as significantly associated with RET, and 656 candidate genes were screened. By combined BSA and RNA-seq analysis, 55 candidate genes showed differentially expressed, including genes encoding ABC transporter G (ABCG), zinc finger protein, NAC, ethylene-responsive transcription factor (ERF), etc. These genes were probably positive factors in coping with Al toxicity stress in rapeseed. This study provides new insight into exploring the QTLs and candidate genes' response to Al toxicity stress by combined BSA-seq and RNA-seq and is helpful to further research on the mechanism of Al resistance in rapeseed.

A major falling number locus on chromosome 4B governs resistance to pre-harvest sprouting in bread wheat line Soru#1

Pre-harvest sprouting (PHS) is a serious threat to wheat quality and occurs when maturing seeds on plants encounter substantial rain before harvest. In the present study, the genetics of PHS resistance were studied using a population of 131 recombinant inbred lines (RILs) from a cross between Soru#1 and Naxos. The population was genotyped with the Illumina 90 K and Axiom 35 K wheat SNP arrays and tested in field trials across two locations in Norway and one in China. Seed dormancy as a measure of sprouting damage was evaluated using germination index (GI) and falling number (FN). Days to heading, days to maturity, and plant height were also recorded to evaluate their potential impact on PHS. Thirteen PHS-related quantitative trait loci (QTL) were detected. The most consistent QTL for FN, contributed by Soru#1, on the long arm of chromosome 4B explained 10.0–19.2% of the phenotypic variation across trials. Other important PHS resistance QTL from Soru#1 detected on chromosome arms 2AL, 2BL, and 4BS, affected both GI and FN. Naxos carried favorable QTL alleles for PHS resistance on chromosome arm 4AS and the Phs1 allele on 4AL. The reduced height Rht-D1 and vernalization Vrn-A1 alleles were the major determinants of plant height and earliness; however, these traits had minimal impact on the genetic control of PHS resistance in this population. Thus, both parents contributed valuable alleles for improving PHS resistance in breeding programs.

Trait Association Studies to Determine Selection Indices in F5 Segregating Populations of Rice (Oryza sativa L.)

The experiment consisted of 38 promising F5 breeding lines developed and evaluated for correlation coefficient, path coefficient and selection indices studies with four check varieties viz., NVSR-2435, Sardar, Lalkada Gold and GR-18 in RBD with three replications during kharif-2023 at RRRS, Vyara, Gujarat. Genotypic correlation analysis revealed that grain yield per plant appeared to be positively and significantly correlated with productive tillers per plant, plant height, panicle length, straw yield per plant and 100 grain weight, which suggested that these characters can be improved simultaneously towards higher grain yield per plant by direct selection. Genotypic path analysis revealed that positive and direct effects on grain yield per plant were exhibited by days to maturity, productive tillers per plant, panicle length, 100 grain weight and straw yield per plant. Among these traits, panicle length, productive tillers per plant, 100 grain weight and straw yield per plant exhibited direct effect along with highly significant and positive association with grain yield per plant. Therefore, selection for such traits may be considered as most important yield attributing characters. The negligible residual effect of 0.037 was observed in path analysis, indicates that the model explained large proportion of variance. Thirty-one selection indices were constructed in all possible combinations of grain yield per plant (X1) along with four components viz., productive tiller per plant (X2), panicle length (X3), 100 grain weight (X4) and straw yield per plant (X5) through equal weight method. Among all the selection indices, the relative efficiency (%) and expected genetic advance (GA) was noted maximum with four characters selection index (I1245) compared to grain yield per plant (I1).

Doubled Haploid Technology: Opportunities and Challenges for the Rapid Generation of Maize Homozygous Lines.

Maize is used for multiple purposes, including food, feed, and energy production, and since transitioning to hybrid cultivars at around 1930, maize yield has significantly increased. This is largely due to hybrid vigor, which refers to the superior performance of the progeny from two unrelated inbred parents. Consequently, nearly all maize cultivars grown in the United States are hybrids. Hybrid breeding programs comprise two essential components; namely, inbred line development and hybrid production. Traditionally, developing inbred lines takes a long time, requiring six to 10 generations of self-pollination. The doubled haploid (DH) technology, however, accelerates this process, enabling the derivation of fully homozygous lines within two generations. DH technology is applicable in several crop species and has been most successful in maize due to in vivo maternal haploid induction. Here, we review the origins of the DH technology, and discuss advantages and challenges of the technology as well as applications of DH lines.

Doubled Haploid Technology: Generation of Doubled Haploid Maize Lines Using Haploid Inducers.

Doubled haploid (DH) technology allows for the development of completely homozygous lines from heterozygous plants in only two generations. This approach has been widely adopted in maize breeding programs, as it expedites the generation of inbred lines compared to traditional methods. The DH approach is based on the use of maize genotypes that have the ability to induce haploid seeds when used as the pollen parent. The most common method for producing maize haploid plants for the generation of DH lines is in vivo maternal haploid induction. The process involves pollination with a haploid inducer maize line to generate haploid seeds. Then, haploids are screened for and identified (typically via the expression of a particular marker gene), germinated, treated with an exogenous doubling agent to induce genome duplication, and transplanted to the field. Following successful self-pollination, seeds harvested from the ear represent fully homozygous lines. The seed set at this stage, however, is often low, necessitating one or two additional rounds of self-pollination to increase the number of fully homozygous inbred lines. Here, we describe a protocol for the generation of maize DH lines using maternal haploid-inducing maize lines. We outline the steps for setting up the donor material, performing induction crosses, selecting haploids based on two different marker alleles, treating seedlings with colchicine to double the genome, transplanting the treated seedlings to the field, and self-pollinating the treated plants.