Haploid Kernels Research Articles

A new approach to Fourier transform Raman: Identification of haploids in maize (Zea mays)

AbstractThe objective of this work was to adapt the FT Raman spectroscopy analysis in the differentiation of haploid and diploid kernels in maize, developing a new efficient, agile, precise, and nondestructive methodology. The main difference observed in FT Raman readings was a peak in the region between 1600 and 1700 cm−1 in possibly haploid kernels. It was possible to correlate the characteristics of the kernels with the presence of the R1‐nj gene and the readings obtained in the Raman spectrometry technique. Most of the kernels previously classified as haploid showed positive values for principal component analysis (PCAs), indicating a correlation in the identification of haploids by the techniques adopted. The identification of haploids by R‐Navajo was superior to FT Raman. However, FT Raman spectroscopy is an agile analysis technique that enables the development of non‐invasive and non‐destructive analytical methods in maize kernels, in addition to providing relevant information about the chemical structures present in the composition of the samples.

Cytological study on haploid male fertility in maize

Doubled haploid (DH) breeding technology, which relies on haploid genome doubling, is widely used in commercial maize breeding. Spontaneous haploid genome doubling (SHGD), a more simplified and straightforward method, is gaining popularity among maize breeders. However, the cytological mechanism of SHGD remains unclear. This study crossed inbred lines RL36 and RL7, which have differing SHGD abilities, with inducer line YHI-1 to obtain haploid kernels. The meiotic processes of pollen mother cells (PMCs) in the haploid plants were compared with diploid controls. The results suggested that three main pathways, the early doubling of haploid PMCs, the first meiotic metaphase chromosomal segregation distortion, and anomaly of the second meiosis, are responsible for SHGD. Furthermore, flow cytometry analysis of ploidy levels in leaves and PMCs from haploids and diploid controls revealed that somatic cell chromosome doubling and germ cell chromosome doubling are independent processes. These findings provide a foundation for further studies on the underlying mechanism of SHGD, aiding the application of DH technology in maize breeding practices.

Improving the sorting efficiency of maize haploid kernels using an NMR-based method with oil content double thresholds

BackgroundMaize haploid breeding technology can be used to rapidly develop homozygous lines, significantly shorten the breeding cycle and improve breeding efficiency. Rapid and accurate sorting haploid kernels is a prerequisite for the large-scale application of this technology. At present, the automatic haploid sorting based on nuclear magnetic resonance (NMR) using a single threshold method has been realized. However, embryo-aborted (EmA) kernels are usually produced during in vivo haploid induction, and both haploids and EmA kernels have lower oil content and are separated together using a single threshold method based on NMR. This leads to a higher haploid false discrimination rate (FDR) and requires secondary manual sorting to select the haploid kernels from the mixtures, which increases the sorting cost and decreases the haploid sorting efficiency. In order to improve the correct discrimination rate (CDR) in sorting haploids, a method to distinguish EmA kernels is required.ResultsSingle kernel weight and oil content were measured for the diploid, haploid, and EmA kernels derived from three maize hybrids and nine inbred lines by in vivo induction. The results showed that the distribution of oil content showed defined boundaries between the three types of kernels, while the single kernel weight didn't. According to the distribution of oil content in the three types of kernels, a double-threshold method was proposed to distinguish the embryo-aborted kernels, haploid and diploid kernels based on NMR and their oil content. The double thresholds were set based on the minimum oil content of diploid kernels and the maximum content of EmA kernels as the upper and lower boundary values, respectively. The CDR of EmA kernels in different maize materials was > 97.8%, and the average FDR was reduced by 27.9 percent.ConclusionsThe oil content is an appropriate indicator to discriminate diploid, haploid and EmA kernels. An oil content double-threshold method based on NMR was first developed in this study to identify the three types of kernels. This methodology could reduce the FDR of haploids and improve the sorting efficiency of automated sorting system. Thus, this technique represents a potentially efficient method for haploid sorting and provides a reference for the process of automated sorting of haploid kernels with high efficiency using NMR.

The prospects for using haploinducers in maize breeding

The discovery of spontaneous haploid plants and the development of ways to produce them inin vitroculture have set a new direction important for breeding and for theoretical research in reproductive biology. The frequency of spontaneous haploidy in cultivated plants is extremely low and does not exceed 0.01-0.1%, therefore, the search for sources and donors capable of stimulating haploidy in hybrid combinations is of current interest. Expansion of the search for new sources and donors of the haploinduction trait, the creation of new, more effective haploinducers contribute to the accumulation of scientific information and genetic sources, characterized by a high resource potential for selection and genetic research. The causеs of haploidy are not well understood yet. According to the available information, the genes localized in theqhir1,qhir11,qhir12regions of chromosome 1 in maize are responsible for this process. The use of genes that stimulate haploinduction in maize in combination with the marker geneR1-njresponsible for anthocyanin coloration of the caryopsis and embryo, as well as genesA1andB1, which are in control of the entire plant coloration, allowed the creation of haploinducer lines with a frequency of haploid stimulation up to 15%. Phenotypic expression of dominant alleles of the marker anthocyanin coloration genes in different parts of a hybrid plant, as well as in the caryopsis and embryo, contributes to the high-quality selection of haploid kernels in the cob due to the manifestation of recessive alleles of these genes at the haploid level. The presence of anthocyanin synthesis suppressor genes in siliceous maize (C1-I,C2-Idf,In1-D) restricts the use of theR1-njgene in other representatives of siliceous maize. In order to overcome this problem, studies are underway to create other genotypes of haploinducers, which are not associated with the anthocyanin coloration of the caryopsis, but instead have other marker traits, such as the oil content in the kernel, the absence of ligules in the leaves, and root coloration in seedlings. The use of matroclinous and androclinous types of haploinduction allows breeders to obtain highly homozygous dihaploid maize lines, with both the maternal and paternal genomes. These achievements made it possible to cut five or more times the material and time inputs into the creation of inbred lines and their sterile analogs, accelerate the breeding of new maize hybrids, and signifi cantly improve the quality of seed production in terms of typicality and uniformity. The materials presented in the article should help breeders and geneticists to learn more about the innovative directions and problems of hybrid maize breeding.

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

AbstractDoubled 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.

Screening of maize haploid kernels based on near infrared spectroscopy quantitative analysis

Doubled haploid breeding (DH breeding) is one of the major maize breeding techniques. The screening of haploid seeds is an essential link of DH breeding. Oil marking method is more stable than other haploid marking methods, which makes the oil content of haploid and diploid seeds significantly different. Currently, it is the most mature way to measure the oil content of maize seeds by nuclear magnetic resonance (NMR) spectrometer. However, the slow speed, high cost, and large volume have restricted the wide applications of NMR spectrometer. Similar to NMR, near infrared spectroscopy (NIRS) can also be used for the non-destructive screening of seeds. Moreover, compared with NMR spectrometer, NIRS spectrometer has a fast speed, low cost, and small volume. This study attempts to explore a maize haploid seed screening solution based on NIRS quantitative analysis to meet the demand of large-scale DH breeding. In this study, a screening solution for Maize Haploid seeds based on the quantitative analysis of NIRS is proposed, and tests of the solution by two representative varieties show a consistent average accuracy above 90%. A model monitoring and calibration solution based on small sample set is proposed to guarantee the stability and efficiency of the screening solution. The reason for the poor separability of the non-embryo side diffuse reflectance spectra is analyzed, and the feasibility of the non-embryo side spectra is verified by analyzing the spectral information of hyperspectral data. In summary, this research provides a more economical and efficient solution for maize haploid screening.

In vivo haploid induction leads to increased frequency of twin-embryo and abnormal fertilization in maize

BackgroundIn vivo haploid induction (HI) based on Stock6-derived inducer lines has been the most prevalent means of producing haploids. Nevertheless, the biological mechanism of HI is not fully understood, the twin-embryo kernels had been found during haploid induction, which may provide potential evidence for the abnormal double fertilization during HI.ResultsWe investigated twin-embryo frequency in progenies of different haploid inducers. Results reveal that increasing the HI potential significantly improved the frequency of twin-embryo kernels. Compared with the average twin-embryo kernel frequency (average frequency = 0.07%) among progenies pollinated by the haploid inducer line CAUHOI, the frequency of twin-embryo was improved to 0.16% in progenies pollinated by the haploid inducer line CAU5. This result was further confirmed by pollinating single hybrid ND5598 with four haploid inducers possessing differentiated HIRs, where twin-embryo frequency was highly correlated with HIR. Among 237 twin-embryo kernels, we identified 30 haploid twin-embryo kernels (12.66%), a frequency which was much greater than the average HI rate for three other inducer lines (frequency range 2–10%). In addition, aneuploids, occurred at high frequency (8 in 41 twin plants). This level of aneuploidy provides new insight into the abnormal double fertilization during HI. Moreover, we observed differences in growth rate between twin plants in the field, as 4.22% of the twin plants grew at a significantly different rate. Both simple sequence repeats markers (SSR) and 3072 SNP-chip genotyping results revealed that > 90% of the twin plants shared the same origin, and the growth difference could be attributed to aneuploidy, competition for nutrients, and possible hormone regulation.ConclusionThese results demonstrate that an enhanced HI ability can increase twin-embryo kernel frequency, and high frequency of both haploid twin-embryo kernels and aneuploidy observed in this research give us new insights to understand the mechanism of both HI and abnormal embryogenesis.

Identification of maize haploid kernels based on hyperspectral imaging technology

Haploid breeding is a significant technology of maize breeding. Rapid and accurate haploid kernel identification method has great significance to accelerating the efficiency of haploid breeding. At present, detecting genetic markers on the embryo of kernels by machine vision and determining oil content of maize kernels by nuclear magnetic resonance (NMR) are widely used to automatically identify haploid maize kernel. However, the machine vision method can only identify the haploid through embryo side of kernels, and the NMR method cannot distinguish them when haploid and diploid have the overlap oil content. The study was aimed exploring a rapid and accurate method to identify haploid maize kernel using near-infrared hyperspectral imaging technology to overcome the limitations of current automated haploid identification and to achieve more accurate screening of haploid. In terms of two representative varieties of maize (Zhengdan 958 and Nongda 616), the study adopted spectral features of hyperspectral imaging to discuss the influence of embryonic orientation (embryo faces to or against light source) on haploid identification model. Meanwhile, the separability of embryo and non-embryo and identification accuracy of joint modeling of embryo and non-embryo were analyzed. The study showed that the greater difference between embryo and non-embryo of haploid and diploid, but hyperspectral imaging method could effectively distinguish haploid and diploid through embryo or non-embryo. At the same time, with the qualitative analysis method, two maize varieties could accurately distinguished haploid and diploid with overlapping oil content based on joint modeling. In this case, the test set of haploid and diploid achieved yielded higher correct acceptance rate (CAR) of 99% and the false acceptance rate (FAR) were both below 1%, with a high accuracy rate. The study showed that it is feasible to recognize maize haploid using hyperspectral imaging technology, which can provide a reference for the later haploid sorting systems.

Hetero-fertilization together with failed egg–sperm cell fusion supports single fertilization involved in in vivo haploid induction in maize

In vivo doubled-haploid technology is widely applied in commercial maize breeding programs because of its time-saving and cost-reducing features. The production of maize haploids primarily depends on the use of Stock6-derived haploid inducer lines. Although the gene underlying haploid induction, MTL/ZmPLA1/NLD, was cloned recently, the mechanism of haploid induction is still unknown. Hetero-fertilization can occur via a single fertilization, which provides a means to investigate single-fertilization events by studying the hetero-fertilization phenomenon. In this study, we found that the hetero-fertilization rate increased significantly when female maize lines were first individually crossed with pollen from the inducer CAU5 in dual-pollination experiments 4 h before a second pollination with common lines. We also examined embryogenesis during haploid induction by confocal laser-scanning microscopy and observed single-fertilized ovules, indicating that single fertilization occurred during haploid induction. We therefore postulate that both single fertilization and chromosome elimination contribute to haploid induction in maize. We also propose a scheme for the formation of hetero-fertilized and haploid kernels. Our results provide an efficient approach to identify hetero-fertilized kernels for research on interactions between embryo and endosperm.

Qualitative Analysis of Maize Haploid Kernels Based on Calibration Transfer by Near-Infrared Spectroscopy

ABSTRACTHaploid breeding is one of the most important modern crop selection technologies. Near-infrared spectroscopy (NIRS) has been used to identify haploids rapidly and to non-destructively accelerate the selection process. However, the change of the external environment weakens the performance of the model, as the training and the test spectra may be collected separately from different environments. Thus, a novel calibration transfer method is proposed to calibrate the model in order to reduce the impact of the environment. The near-infrared spectra of 400 maize kernels of two varieties were collected from 9000 to 4000 cm−1. Principal component analysis was performed to construct a feature space and extract features. In the constructed feature space, the calibration transfer method was used to calibrate test sets. Finally, support vector machine was employed to establish a haploid identification model. The results show that when the spectra of the test set and the training set were collected in the same environment, the corrected acceptance of the model was above 90%. While the spectra of the test set and the training set were collected from different environments, the corrected acceptance was 77.87%. However, when the model used the calibration transfer method, the corrected acceptance increased by 12.46%. Moreover, compared with direct standardization, this calibration transfer method achieved better results without detailed sample chemical information and many standards. The results demonstrate that the calibration transfer method based on NIRS was effective for identifying maize haploid kernels in variable environments.

Design and Experiment of a Sorting System for Haploid Maize Kernel

This study designed an automatic sorting system that can separate haploid maize kernels from cross-breeding kernels that are marked with the Navajo label. This system comprises the seed feeding, image acquisition, sorting and system control units. The seed feeding unit distributes the maize kernels over the synchronous belt. The image acquisition unit acquires the images of maize kernels, as well as distinguishes the heterozygous from haploid kernels based on the color feature of the endosperm. The sorting unit contains mechanical arms and solenoid valves that can select the heterozygote kernels using air suction. Lastly, the system control unit is compatible with other units. Four maize varieties, namely, 958-CAU, 1050-37, LC0990-LN75 and LC0995-CAU, were provided by China Agricultural University, National Maize Improvement Center. These varieties were selected for the experiments conducted. The successful unloading rates of the system are 85.56%, 91.39%, 87.22% and 86.39%, respectively. (R[Formula: see text]G)[Formula: see text]G–B[Formula: see text]/B was employed to distinguish the pigmented endosperm area. The accuracy rates of identification for the heterozygous kernels are 92.91%, 95.04%, 88.82% and 92.65% for 958-CAU, 1050-37, LC0990-LN75 and LC0995-CAU, respectively.

Fully-Automated High-Throughput NMR System for Screening of Haploid Kernels of Maize (Corn) by Measurement of Oil Content.

One of the modern crop breeding techniques uses doubled haploid plants that contain an identical pair of chromosomes in order to accelerate the breeding process. Rapid haploid identification method is critical for large-scale selections of double haploids. The conventional methods based on the color of the endosperm and embryo seeds are slow, manual and prone to error. On the other hand, there exists a significant difference between diploid and haploid seeds generated by high oil inducer, which makes it possible to use oil content to identify the haploid. This paper describes a fully-automated high-throughput NMR screening system for maize haploid kernel identification. The system is comprised of a sampler unit to select a single kernel to feed for measurement of NMR and weight, and a kernel sorter to distribute the kernel according to the measurement result. Tests of the system show a consistent accuracy of 94% with an average screening time of 4 seconds per kernel. Field test result is described and the directions for future improvement are discussed.

A green fluorescent protein-engineered haploid inducer line facilitates haploid mutant screens and doubled haploid breeding in maize

Doubled haploid (DH) technology is a useful tool for maize breeding and gene discovery. The production of a large number of DH lines has been possible recently with the development of various in vivo haploid inducer lines based on the discovery of the initial stock 6. Most of the haploid induction systems use the R1-navajo (R1-nj) gene whose expression in the kernel produces diploid kernels with colored aleurone crowns and scutella and haploid kernels with colored aleurone crowns but colorless scutella. However, the R1-nj gene expression depends on various genetic and environmental factors, which can mask the typical R1-nj phenotype. To solve this problem, we introduced a dominant green fluorescent protein (GFP) marker gene into a maize haploid inducer, RWS, to generate a RWS-GFP inducer. This line allows the identification of haploids in the early germination stage by visualizing the GFP expression of germinated kernels. Germinated diploid seeds will produce GFP fluorescence in emerged radicles and coleoptiles, but haploids will be GFP negative because of the lack of paternal GFP gene during hybridization with the haploid inducer. We demonstrated that this system can be used to screen haploid mutants and to produce haploids from various commercial sweet corn hybrids, which have a genetic background that prevents haploid identification by other systems. Haploid plants were treated with colchicine to produce DH plants. The GFP-engineered haploid inducer line will be a powerful tool in maize genetic studies and DH breeding.

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.

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.

Mapping of maternal QTLs for in vivo haploid induction rate in maize (Zea mays L.)

Haploid technology can significantly shorten the time required for inbred line improvement, accelerate the breeding process, and reduce breeding costs. The production of haploids is not only dependent on the genetics of the paternal haploid inducer, but it is also affected by the genetic background of the maternal donor during the process of haploid production. To address the maternal genetic contribution to haploid production, we pollinated a mapping population consisting of 186 F2:3 family lines derived from a cross between Zheng58 and Chang7-2 with the inducer line CAU5 and selected haploid kernels using R1-nj kernel markers. Two quantitative trait loci (QTLs), qmhir1 and qmhir2, which contribute to the maternal genetics of haploid induction, were detected on chromosomes 1 and 3, respectively. The qmhir1 locus is located between the flanking marker loci umc1292 and bnlg1014, and it explained 14.70 % of the phenotypic variation. The qmhir2 locus is located between marker loci umc1844 and umc2277 and it explained 8.42 % of the phenotypic variation. The genetic effect of both QTLs is partial dominance.

Morphological and molecular evidences for DNA introgression in haploid induction via a high oil inducer CAUHOI in maize

The phenomenon of maternal haploid induction in maize was first described many years ago, but the underlying mechanism is still unclear. In this study, the Stock-6-derived, haploid-inducing line CAUHOI with high kernel oil content (KOC), was used as the pollinator to produce maternal haploids from the maize hybrid ZD958 with low KOC. CAUHOI is homozygous for the dominant marker gene R1-nj. Haploids were identified by morphological and cytological investigations. The frequency of haploid induction from this cross was 2.21%. Unexpectedly, many haploid kernels had weakly pigmented purple color on the embryo, and some haploid kernels had high KOC. Simple sequence repeat (SSR) analysis showed that 43.18% of the haploids carried segments from CAUHOI, and a small proportion (average 1.84%) of the genome of CAUHOI was introgressed into haploids. Haploid kernels with high KOC had a higher frequency of segment introgression from CAUHOI (2.92%) than that in haploid kernels with low KOC (1.79%), showing that the marker gene R1-nj and high-oil genes from CAUHOI were expressed during the development of some haploid embryos, and confirmed that the DNA introgression from the inducer parent occurred during maternal haploid induction. Together, these results suggested that the chromosome elimination was probably responsible for haploid induction in maize, and late somatic elimination might occur. Several possible mechanisms underlying haploid formation are discussed.