Improvements to the maize (Zea mays L.) in vivo maternal doubled haploid system

The maize (Zea mays L.) in vivo maternal doubled haploid system has been widely applied to maize breeding and genetics in recent decades and is an important part of the majority of public and private maize breeding programs today. The principal advantage of the doubled haploid system is the ability to generate completely homozygous inbred lines in as little as two seasons. Other advantages to this system include more rapid integration of loci of interest and increased usefulness over traditional lines developed through self-pollination. In this dissertation, some of the major problems in the maternal doubled haploid system are addressed. Namely, improvement of maternal inducers, improved understanding of the genetics controlling inducibility, development of an automated system to sort haploid kernels, investigation and application of spontaneous chromosome doubling, and a proposal for the acceleration of the breeding cycle beyond doubled haploids through the in vitro nursery. This dissertation provides some new insight into these problems, as follows. The development and release of a new improved maternal haploid inducer for use in doubled haploid programs. Improved understanding of the quantitative nature of inducibility and the effects of misclassification are discussed. Successful automated discrimination of haploid and diploid kernels using optical and fluorescence methods is described. In an effort to make the doubled haploid system more efficient and safe, a bypass of the colchicine doubling step is proposed through the application and investigation of spontaneous chromosome doubling in haploid plants. Finally, as a proposal for what could be the next step in accelerating the breeding cycle, the in vitro nursery and its applications is discussed

Germplasm enhancement of maize: Utilization of doubled haploid breeding on adapted tropical sources

The allelic diversity (AD) project of the Germplasm Enhancement of Maize program utilized the double haploid (DH) breeding method to expedite release of lines from 300 exotic maize races. Differential effects on haploid induction rates (HIR) and chromosome doubling rates (DR) associated with the recurrent parents PHB47 and PHZ51, the elevation that the race is traditionally grown at, and the race itself were examined. Races from the AD project were grouped by elevation (high, middle, or low altitude) of their country of origin. Six races per elevation were randomly selected and backcrossed using both recurrent parents to generate 36 populations. Ten replications were randomized in a complete randomized design for two growing seasons (2011, 2012) to estimate HIR, and one growing season (2013) to estimate DR. In the HIR study, populations were pollinated with a haploid inducer line. Seed progeny were screened the following winter and the number of haploid, hybrid, and contaminate seeds were recorded and analyzed. The recurrent parent effect was significant with PHB47 having a higher HIR than PHZ51. Elevation was significant with higher HIR associated with low elevation origin, and race also proved to be significant. In the DR study, the populations were treated with colchicine as seedlings to promote chromosome doubling. Haploid plants that successfully doubled and had functional ear and tassel structures were self-pollinated. Recurrent parent, elevation, and race effects were all non-significant for DR and kernel set per ear. The doubled haploid breeding method can produce maize inbred lines more rapidly than traditional methods, but there are challenges. Sorting haploid from hybrid seed based on visual color markers is time consuming, and can be difficult due to color inhibitors that obscure pigmentation needed to distinguish between haploid, hybrid, and outcrossed seed. In this study, weight was evaluated as a method to sort haploid from hybrid seed. A preliminary experiment utilized two families for analysis. Eleven haploid and eleven hybrid kernels from each family were weighed for a total of 44 experimental units. A second experiment used six families and the same format as the previous, for 132 experimental units. Hybrid seed weighed significantly more than haploid seed in both experiments. However, the interaction between line and kernel type was significant in the second experiment. In conclusion, efficacy of sorting haploid from hybrid kernels based on weight depends on the genotypes involved

New methods for haploid selection in maize

The major aim of this study was to evaluate new methods to select haploids for maize breeding. The first method was to evaluate different inbreds for sensitivity to herbicides that could be developed as a tool for haploid selection, and the second was to evaluate automated sorting based on near-infrared reflectance spectroscopy (NIRS) and SIMCA analysis

Discrimination of haploid and diploid maize kernels via multispectral imaging

The use of doubled haploids (DHs) in maize has become ubiquitous in maize breeding programmes as it allows breeders to go from cross to evaluation in as little as 2 years. Two important aspects of the in vivo DH system used in maize are as follows: (i) the identification of haploid progeny and (ii) doubling of the haploid genome to produce fertile inbred lines. This study is focused on the first step. Currently, identification of maize haploid progeny is performed manually using the R1-nj seed colour marker. This is a labour-intensive and time-consuming process; a method for automated sorting of haploids would increase the efficiency of DH line development. In this study, six inbred lines were crossed with the maternal haploid inducer ‘RWS/RWK-76’ and a sample of seed was sorted manually for each line. Using the VideometerLab 3 system, spectral imaging techniques were applied to discriminate between haploids and hybrids. Using DNA markers to confirm the haploid/diploid state of the tested seed, for the majority of genotypes haploid identification was possible with over 50% accuracy.This is an accepted manuscript of an article published as De La Fuente, Gerald N., Jens Michael Carstensen, and Michael A. Edberg. "Discrimination of haploid and diploid maize kernels via multispectral imaging." Plant Breeding 136, no. 1 (2017): 50-60. doi:10.1111/pbr.12445/full.</p

A Diallel Analysis of a Maize Donor Population Response to In Vivo Maternal Haploid Induction I: Inducibility

The maize in vivo maternal doubled haploid (DH) system is an important tool used by maize breeders and geneticists around the world. The ability to rapidly produce DH lines of maize for breeding allows breeders to quickly respond to new selection criteria based on the ever changing biotic and abiotic stresses that maize is subjected to across its growing area. There are two important steps in the generation of DH lines using the in vivo maternal DH system: 1) the production and identification of haploid progeny, and 2) the doubling of genomes to create fertile, diploid inbred lines that can be used for topcross and per se evaluation. For this study, the focus is the first step, the production and identification of haploid progeny. A diallel mating between six inbred lines of maize, three highly inducible lines (CR1HT, PA91HT1, WF9) and three lines with low inducibility (NK778, A427, A637) was produced to study the genetic makeup of inducibility in temperate maize germplasm. A maximum estimated rate of inducibility was found in A427/A637 at 14.6%. Significant general combining ability (GCA) specific combining ability (SCA), reciprocal (REC), environmental (ENV), as well as GCA by ENV and SCA by ENV interactions were found. Misclassification rates ranged from 0-45.2% in the 30 hybrids considered. This study supports the use of germplasm with improved inducibility for breeding to improve rates of inducibility in germplasm which has low induction rates

Classification approaches for sorting maize (Zea mays subsp. mays) haploids using single‐kernel near‐infrared spectroscopy

Doubled haploids (DHs) are an important breeding tool for creating maize inbred lines. One bottleneck in the DH process is the manual separation of haploids from among the much larger pool of hybrid siblings in a haploid induction cross. Here, we demonstrate the ability of single‐kernel near‐infrared reflectance spectroscopy (skNIR) to identify haploid kernels. The skNIR is a high‐throughput device that acquires an NIR spectrum to predict individual kernel traits. We collected skNIR data from haploid and hybrid kernels in 15 haploid induction crosses and found significant differences in multiple traits such as percent oil, seed weight, or volume, within each cross. The two kernel classes were separated by their NIR profile using Partial Least Squares Linear Discriminant Analysis (PLS‐LDA). A general classification model, in which all induction crosses were used in the discrimination model, and a specific model, in which only kernels within a specific induction cross, were compared. Specific models outperformed the general model and were able to enrich a haploid selection pool to above 50% haploids. Applications for the instrument are discussed

Haploid identification in maize

Doubled haploid (DH) line production through in vivo maternal haploid induction is widely adopted in maize breeding programs. The established protocol for DH production includes four steps namely in vivo maternal haploid induction, haploid identification, genome doubling of haploid, and self-fertilization of doubled haploids. Since modern haploid inducers still produce relatively small portion of haploids among undesirable hybrid kernels, haploid identification is typically laborious, costly, and time-consuming, making this step the second foremost in the DH technique. This manuscript reviews numerous methods for haploid identification from different approaches including the innate differences in haploids and diploids, biomarkers integrated in haploid inducers, and automated seed sorting. The phenotypic differentiation, genetic basis, advantages, and limitations of each biomarker system are highlighted. Several approaches of automated seed sorting from different research groups are also discussed regarding the platform or instrument used, sorting time, accuracy, advantages, limitations, and challenges before they go through commercialization. The past haploid selection was focusing on finding the distinguishable marker systems with the key to effectiveness. The current haploid selection is adopting multiple reliable biomarker systems with the key to efficiency while seeking the possibility for automation. Fully automated high-throughput haploid sorting would be promising in near future with the key to robustness with retaining the feasible level of accuracy. The system that can meet between three major constraints (time, workforce, and budget) and the sorting scale would be the best option

Compatibility and Stability Analysis of Haploid Inducers under Different Source Germplasm and Seasons in Maize Using GGE Biplot

Multiple factors can affect the R1-nj purple kernel expression and seed set, reducing its efficiency in identifying haploids in maize. The complex interaction among the haploid inducer (HI), source germplasm (SG), and season (S) is inevitable in in vivo maize haploid induction but could be used through compatibility and stability tests. We tested five HI genotypes on 25 distinct source germplasm in two different seasons of tropical savanna in Thailand. The dry season was more suitable than the rainy season for haploid induction. We noticed varying degrees of R1-nj inhibition among the 25 tropical source germplasm, with some of them exhibiting significant issues with the R1-nj purple kernel expression. Therefore, using the R1-nj alone may not provide accurate ploidy identification in maize. Despite the intense R1-nj expression, haploid inducer BHI306 showed poor stability and compatibility with tropical source germplasm for pollination rate and seed set during the rainy season. The GGE biplot suggested KHI42 and KHI64 as the most compatible haploid inducers under their respective two different mega-source germplasm for the pollination rate and R1-nj seed set. These findings can guide breeders in selecting the most compatible and stable haploid inducers under varying conditions.This article is published as Dermail, A.; Lübberstedt, T.; Suwarno, W.B.; Chankaew, S.; Lertrat, K.; Ruanjaichon, V.; Suriharn, K. Compatibility and Stability Analysis of Haploid Inducers under Different Source Germplasm and Seasons in Maize Using GGE Biplot. Agronomy 2024, 14, 1505. https://doi.org/10.3390/agronomy14071505.© 2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/)