Maize smart-canopy architecture enhances yield at high densities.

Increasing planting density is a key strategy for enhancing maize yields1-3. An ideotype for dense planting requires a 'smart canopy' with leaf angles at different canopy layers differentially optimized to maximize light interception and photosynthesis4-6, among other features. Here we identified leaf angle architecture of smart canopy 1 (lac1), a natural mutant with upright upper leaves, less erect middle leaves and relatively flat lower leaves. lac1 has improved photosynthetic capacity and attenuated responses to shade under dense planting. lac1 encodes a brassinosteroid C-22 hydroxylase that predominantly regulates upper leaf angle. Phytochrome A photoreceptors accumulate in shade and interact with the transcription factor RAVL1 to promote its degradation via the 26S proteasome, thereby inhibiting activation of lac1 by RAVL1 and decreasing brassinosteroid levels. This ultimately decreases upper leaf angle in dense fields. Large-scale field trials demonstrate that lac1 boosts maize yields under high planting densities. To quickly introduce lac1 into breeding germplasm, we transformed a haploid inducer and recovered homozygous lac1 edits from 20 diverse inbred lines. The tested doubled haploids uniformly acquired smart-canopy-like plant architecture. We provide an important target and an accelerated strategy for developing high-density-tolerant cultivars, with lac1 serving as a genetic chassis for further engineering of a smart canopy in maize.

BABY BOOM regulates early embryo and endosperm development.

The BABY BOOM (BBM) AINTEGUMENTA-LIKE (AIL) AP2/ERF domain transcription factor is a major regulator of plant cell totipotency, as it induces asexual embryo formation when ectopically expressed. Surprisingly, only limited information is available on the role of BBM during zygotic embryogenesis. Here we reexamined BBM expression and function in the model plant Arabidopsis thaliana (Arabidopsis) using reporter analysis and newly developed CRISPR mutants. BBM was expressed in the embryo from the zygote stage and also in the maternal (nucellus) and filial (endosperm) seed tissues. Analysis of CRISPR mutant alleles for BBM (bbm-cr) and the redundantly acting AIL gene PLETHORA2 (PLT2) (plt2-cr) uncovered individual roles for these genes in the timing of embryo progression. We also identified redundant roles for BBM and PLT2 in endosperm proliferation and cellularization and the maintenance of zygotic embryo development. Finally, we show that ectopic BBM expression in the egg cell of Arabidopsis and the dicot crops Brassica napus and Solanum lycopersicon is sufficient to bypass the fertilization requirement for embryo development. Together these results highlight roles for BBM and PLT2 in seed development and demonstrate the utility of BBM genes for engineering asexual embryo development in dicot species.

Co-expression of transcription factors ZmC1 and ZmR2 establishes an efficient and accurate haploid embryo identification system in maize.

Because of their high efficiency during chromosome doubling, immature haploid maize (Zea mays L.) embryos are useful for doubled haploid production. The R1-nj marker is commonly used in doubled haploid breeding and has improved the efficiency of haploid identification. However, its effectiveness is limited by genetic background and environmental factors. We addressed this technical challenge by developing an efficient and accurate haploid embryo identification marker through co-expression of two transcription factor genes (ZmC1 and ZmR2) driven by the embryo-aleurone-specific bidirectional promoter PZmBD1 ; these factors can activate anthocyanin biosynthesis in the embryo and aleurone layer during early seed development. We developed a new haploid inducer, Maize Anthocyanin Gene InduCer 1 (MAGIC1), by introducing the transgenes into the haploid inducer line CAU6. MAGIC1 could identify haploids at 12 days after pollination, which is nine days earlier than CAU6. Importantly, MAGIC1 increased haploid identification accuracy to 99.1%, compared with 88.3% for CAU6. In addition, MAGIC1 could effectively overcome the inhibition of anthocyanin synthesis in some germplasms. Furthermore, an upgraded anthocyanin marker was developed from ZmC1 and ZmR2 to generate MAGIC2, which could identify haploids from diploids due to differential anthocyanin accumulation in immature embryos, coleoptiles, sheaths, roots, leaves, and dry seeds. This haploid identification system is more efficient and accurate than the conventional R1-nj-based method, and it simplifies the haploid identification process. Therefore, this system provides technical support for large-scale doubled haploid line production.

ZmCOI2a and ZmCOI2b redundantly regulate anther dehiscence and gametophytic male fertility in maize.

In higher plants, the generation and release of viable pollen from anthers is vital for double fertilization and the initiation of seed development. Thus, the characterization of genes related to pollen development and anther dehiscence in plants is of great significance. The F-box protein COI1 plays a crucial role in the jasmonate (JA) signaling pathway and interacts with many JAZ family proteins in the presence of jasmonoyl-isoleucine (JA-Ile) or coronatine (COR). The mutation of AtCOI1 in Arabidopsis leads to defective anther dehiscence and male sterility (MS), although COI has not been shown to affect fertility in Zea mays (maize). Here we identified two genes, ZmCOI2a and ZmCOI2b, that redundantly regulate gametophytic male fertility. Both ZmCOI2a and ZmCOI2b are highly homologous and constitutively expressed in all tissues tested. Subcellular localization revealed that ZmCOI2a and ZmCOI2b were located in the nucleus. The coi2a coi2b double mutant, generated by CRISPR/Cas9, had non-dehiscent anthers, delayed anther development and MS. In addition, coi2a coi2b male gametes could not be transmitted to the next generation because of severe defects in pollen germination. The JA content of coi2a coi2b anthers was unaltered compared with those of the wild type, and the exogenous application of JA could not rescue the fertility defects of coi2a coi2b. Transcriptome analysis showed that the expression of genes involving the JA signaling transduction pathway, including ZmJAZ3, ZmJAZ4, ZmJAZ5 and ZmJAZ15, was affected in coi2a coi2b. However, yeast two-hybrid assays showed that ZmJAZs interacted with ZmCOI1s, but not with ZmCOI2s. In conclusion, ZmCOI2a and ZmCOI2b redundantly regulate anther dehiscence and gametophytic male fertility in maize.

The RUBY reporter enables efficient haploid identification in maize and tomato.

In vivo haploid induction has been extended from maize to monocotyledonous plants like rice, wheat, millet and dicotyledonous plants such as tomato, rapeseed, tobacco and cabbage. Accurate identification of haploids is a crucial step of doubled haploid technology, where a useful identification marker is very pivotal. R1-nj is an extensively used visual marker for haploid identification in maize. RFP and eGFP have been shown to be feasible in identifying haploid. However, these methods are either limited to specific species, or require specific equipment. It still lacks an efficient visual marker that is practical across different crop species. In this study, we introduced the RUBY reporter, a betalain biosynthesis system, into maize and tomato haploid inducers as a new marker for haploid identification. Results showed that expression of RUBY could result in deep betalain pigmentation in maize embryos as early as 10 days after pollination, and enabled 100% accuracy of immature haploid embryo identification. Further investigation in tomato revealed that the new marker led to deep red pigmentation in radicles and haploids can be identified easily and accurately. The results demonstrated that the RUBY reporter is a background-independent and efficient marker for haploid identification and would be promising in doubled haploid breeding across different crop species.

Maize Interveinal Chlorosis 1 links the Yang Cycle and Fe homeostasis through Nicotianamine biosynthesis.

The Yang cycle is involved in many essential metabolic pathways in plant growth and development. As extended products of the Yang cycle, the function and regulation network of ethylene and polyamines are well characterized. Nicotianamine (NA) is also a product of this cycle and works as a key metal chelator for iron (Fe) homeostasis in plants. However, interactions between the Yang cycle and NA biosynthesis remain unclear. Here, we cloned maize interveinal chlorosis 1 (mic1), encoding a 5'-methylthioadenosine nucleosidase (MTN), that is essential for 5'-methylthioadenosine (MTA) salvage and NA biosynthesis in maize (Zea mays). A single base G-A transition in the fourth exon of mic1 causes a Gly to Asp change, resulting in increased MTA, reduced Fe distribution, and growth retardation of seedlings. Knockout of ZmMIC1 but not its paralog ZmMTN2 by CRISPR/Cas9 causes interveinal chlorosis, indicating ZmMIC1 is mainly responsible for MTN activity in maize. Transcriptome analysis showed a typical response of Fe deficiency. However, metabolic analysis revealed dramatically reduced NA content in mic1, suggesting NA biosynthesis was impaired in the mutant. Exogenous application of NA transiently reversed the interveinal chlorosis phenotype of mic1 seedlings. Moreover, the mic1 mutant overexpressing a NA synthase gene not only recovered from interveinal chlorosis and growth retardation but was also fertile. These findings provide a link between the Yang cycle and NA biosynthesis, which highlights an aspect of Fe homeostasis regulation in maize.

Influential factors and transcriptome analyses of immature diploid embryo anthocyanin accumulation in maize.

BACKGROUND: Anthocyanins are widely applied as a marker for haploid identification after haploid induction in maize. However, the factors affecting anthocyanin biosynthesis in immature embryos and the genes regulating this process remain unclear. RESULTS: In this study, we analyzed the influence of genetic background of the male and female parents, embryo age and light exposure on anthocyanin accumulation in embryos. The results showed that light exposure was the most crucial factor enhancing the pigmentation of immature embryos. The identification accuracy of haploid embryos reached 96.4% after light exposure, but was only 11.0% following dark treatment. The total anthocyanin content was 7-fold higher in immature embryos cultured for 24 h under light conditions compared to embryos cultured in the dark. Transcriptome analysis revealed that the differentially expressed genes between immature embryos cultured for 24 h in dark and light chambers were significantly enriched in the pathways of flavonoid, flavone, flavonol and anthocyanin biosynthesis. Among the genes involved in anthocyanin biosynthesis, five up-regulated genes were identified: F3H, DFR, ANS, F3'H and the MYB transcription factor-encoding gene C1. The expression patterns of 14 selected genes were confirmed using quantitative reverse transcription-polymerase chain reaction. CONCLUSION: Light is the most important factor facilitating anthocyanin accumulation in immature embryos. After 24 h of exposure to light, the expression levels of the structural genes F3H, DFR, ANS, F3'H and transcription factor gene C1 were significantly up-regulated. This study provides new insight into the factors and key genes regulating anthocyanin biosynthesis in immature embryos, and supports improved efficiency of immature haploid embryo selection during doubled haploid breeding of maize.

Multi-Trait Genomic Prediction Improves Accuracy of Selection among Doubled Haploid Lines in Maize.

Recent advances in maize doubled haploid (DH) technology have enabled the development of large numbers of DH lines quickly and efficiently. However, testing all possible hybrid crosses among DH lines is a challenge. Phenotyping haploid progenitors created during the DH process could accelerate the selection of DH lines. Based on phenotypic and genotypic data of a DH population and its corresponding haploids, we compared phenotypes and estimated genetic correlations between the two populations, compared genomic prediction accuracy of multi-trait models against conventional univariate models within the DH population, and evaluated whether incorporating phenotypic data from haploid lines into a multi-trait model could better predict performance of DH lines. We found significant phenotypic differences between DH and haploid lines for nearly all traits; however, their genetic correlations between populations were moderate to strong. Furthermore, a multi-trait model taking into account genetic correlations between traits in the single-environment trial or genetic covariances in multi-environment trials can significantly increase genomic prediction accuracy. However, integrating information of haploid lines did not further improve our prediction. Our findings highlight the superiority of multi-trait models in predicting performance of DH lines in maize breeding, but do not support the routine phenotyping and selection on haploid progenitors of DH lines.

Genetic Dissection of Phosphorus Use Efficiency and Genotype-by-Environment Interaction in Maize.

Genotype-by-environment interaction (G-by-E) is a common but potentially problematic phenomenon in plant breeding. In this study, we investigated the genotypic performance and two measures of plasticity on a phenotypic and genetic level by assessing 234 maize doubled haploid lines from six populations for 15 traits in seven macro-environments with a focus on varying soil phosphorus levels. It was found intergenic regions contributed the most to the variation of phenotypic linear plasticity. For 15 traits, 124 and 31 quantitative trait loci (QTL) were identified for genotypic performance and phenotypic plasticity, respectively. Further, some genes associated with phosphorus use efficiency, such as Zm00001eb117170, Zm00001eb258520, and Zm00001eb265410, encode small ubiquitin-like modifier E3 ligase were identified. By significantly testing the main effect and G-by-E effect, 38 main QTL and 17 interaction QTL were identified, respectively, in which MQTL38 contained the gene Zm00001eb374120, and its effect was related to phosphorus concentration in the soil, the lower the concentration, the greater the effect. Differences in the size and sign of the QTL effect in multiple environments could account for G-by-E. At last, the superiority of G-by-E in genomic selection was observed. In summary, our findings will provide theoretical guidance for breeding P-efficient and broadly adaptable varieties.

Establishment of a dmp based maternal haploid induction system for polyploid Brassica napus and Nicotiana tabacum.

Doubled haploid (DH) technology is used to obtain homozygous lines in a single generation, a technique that significantly accelerates the crop breeding trajectory. Traditionally, in vitro culture is used to generate DHs, but this technique is limited by species and genotype recalcitrance. In vivo haploid induction (HI) through seed is widely and efficiently used in maize and was recently extended to several other crops. Here we show that in vivo HI can be triggered by mutation of DMP maternal haploid inducer genes in allopolyploid (allotetraploid) Brassica napus and Nicotiana tabacum. We developed a pipeline for selection of DMP orthologs for clustered regularly interspaced palindromic repeats mutagenesis and demonstrated average amphihaploid induction rates of 2.4% and 1.2% in multiple B. napus and N. tabacum genotypes, respectively. These results further confirmed the HI ability of DMP gene in polyploid dicot crops. The DMP-HI system offers a novel DH technology to facilitate breeding in these crops. The success of this approach and the conservation of DMP genes in dicots suggest the broad applicability of this technique in other dicot crops.

Haploid induction and its application in maize breeding.

Maize is a heterosis-utilizing crop species, and the application of maize hybrids has significantly improved total maize yields worldwide. Breeding pure lines is the most important part of heterosis utilization. The double haploid (DH) breeding technology is the approach rising recently in breeding pure lines; compared to the conventional recurrent-selfing method, it can significantly accelerate the crop breeding process. Similar to molecular breeding and transgenic techniques, maize DH breeding has been playing an increasingly important role in commercial breeding and is becoming the core technique in modern maize breeding. In this review, we summarize recent progress in maize DH breeding and put forth our opinions on the future development of double haploid techniques in modern maize breeding.

Genetic Dissection of Phosphorus Use Efficiency in a Maize Association Population under Two P Levels in the Field.

Phosphorus (P) deficiency is an important challenge the world faces while having to increase crop yields. It is therefore necessary to select maize (Zea may L.) genotypes with high phosphorus use efficiency (PUE). Here, we extensively analyzed the biomass, grain yield, and PUE-related traits of 359 maize inbred lines grown under both low-P and normal-P conditions. A significant decrease in grain yield per plant and biomass, an increase in PUE under low-P condition, as well as significant correlations between the two treatments were observed. In a genome-wide association study, 49, 53, and 48 candidate genes were identified for eleven traits under low-P, normal-P conditions, and in low-P tolerance index (phenotype under low-P divided by phenotype under normal-P condition) datasets, respectively. Several gene ontology pathways were enriched for the genes identified under low-P condition. In addition, seven key genes related to phosphate transporter or stress response were molecularly characterized. Further analyses uncovered the favorable haplotype for several core genes, which is less prevalent in modern lines but often enriched in a specific subpopulation. Collectively, our research provides progress in the genetic dissection and molecular characterization of PUE in maize.

QTL mapping of spontaneous haploid genome doubling using genotyping-by-sequencing in maize (Zea mays L.).

KEY MESSAGE: A major QTL for SHGD was identified on chromosome 5 with stable expression across environments. The introgression this QTL can overcome the need of colchicine in DH lines development. Genome doubling of haploids is one of the major constraints of large-scale doubled haploid (DH) technology. Improving spontaneous haploid genome doubling (SHGD) is an alternative to overcome this limitation. In this study, we aimed to construct a high-density linkage map based on genotyping by sequencing of single nucleotide polymorphism, to detect QTL and QTL by environment (Q by E) interactions affecting SHGD and to identify the best trait for mapping and selection of haploid male fertility (HMF). To this end, a biparental population of 220 F2:3 families was developed from a cross between A427 (high HMF) and CR1Ht (moderate HMF) to be used as donor. A high-density linkage map was constructed containing 4171 SNP markers distributed over 10 chromosomes with an average distance between adjacent markers of 0.51 cM. QTL mapping for haploid fertile anther emergence, pollen production, tassel size, and HMF, identified 27 QTL across three environments, and Q by E interactions were significant. A major QTL was identified on chromosome 5. This QTL explained over 45% of the observed variance for all traits across all environments. The introgression of this major QTL, using marker-assisted backcrossing, has great potential to overcome the need of using colchicine in DH line development.

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

BACKGROUND: In 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. RESULTS: We 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. CONCLUSION: These 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.

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.

Novel technologies in doubled haploid line development.

haploid inducer line can be transferred (DH) technology can not only shorten the breeding process but also increase genetic gain. Haploid induction and subsequent genome doubling are the two main steps required for DH technology. Haploids have been generated through the culture of immature male and female gametophytes, and through inter- and intraspecific via chromosome elimination. Here, we focus on haploidization via chromosome elimination, especially the recent advances in centromere-mediated haploidization. Once haploids have been induced, genome doubling is needed to produce DH lines. This study has proposed a new strategy to improve haploid genome doubling by combing haploids and minichromosome technology. With the progress in haploid induction and genome doubling methods, DH technology can facilitate reverse breeding, cytoplasmic male sterile (CMS) line production, gene stacking and a variety of other genetic analysis.

QTL mapping for haploid male fertility by a segregation distortion method and fine mapping of a key QTL qhmf4 in maize.

KEY MESSAGE: Four QTL related to haploid male fertility were detected by a segregation distortion method and the key QTL qhmf4 was fine mapped to an interval of ~800 kb. Doubled haploid (DH) technology enables rapid development of homozygous lines in maize breeding programs. However, haploid genome doubling is a bottleneck for the commercialization of DH technology and is limited by haploid male fertility (HMF). This is the first study reporting the quantitative trait locus (QTL) analysis of HMF in maize. Four QTL, qhmf1, qhmf2, qhmf3, and qhmf4, controlling HMF have been identified by segregation distortion (SD) loci detection in the selected haploid population derived from 'Yu87-1/Zheng58'. Three loci, qhmf1, qhmf2, and qhmf4, were also detected in the selected haploid population derived from '4F1/Zheng58'. The QTL qhmf4 showed the strongest SD in both haploid populations. Based on the sequence information of 'Yu87-1' and 'Zheng58', thirteen markers being polymorphic between the two lines were developed to saturate the qhmf4 region. A total of 8168 H1BC2 (haploid backcross generation) plants produced from 'Yu87-1' and 'Zheng58' were screened for recombinants. All the 48 recombinants were backcrossed to 'Zheng58' to develop H1BC3 progeny. The heterozygous H1BC3 individuals were crossed with CAU5 to induce haploids. In each H1BC3 progeny, haploids were genotyped and evaluated for anther emergence score (AES). Significant (or no significant) difference (P < 0.05) between haploids with or without 'Yu87-1' donor segment indicated presence or absence of qhmf4 in the donor segment. The analysis of the 48 recombinants narrowed the qhmf4 locus down to an ~800 kb interval flanked by markers IND166 and IND1668.

Ploidy effect and genetic architecture exploration of stalk traits using DH and its corresponding haploid populations in maize.

BACKGROUND: Doubled haploid (DH) lines produced via in vivo haploid induction have become indispensable in maize research and practical breeding, so it is important to understand traits characteristics in DH and its corresponding haploids which derived from each DH lines. In this study, a DH population derived from Zheng58 × Chang7-2 and a haploid population, were developed, genotyped and evaluated to investigate genetic architecture of eight stalk traits, especially rind penetrometer resistance (RPR) and in vitro dry matter digestion (IVDMD), which affecting maize stalk lodging-resistance and feeding values, respectively. RESULTS: Phenotypic correlation coefficients ranged from 0.38 to 0.69 between the two populations for eight stalk traits. Heritability values of all stalk traits ranged from 0.49 to 0.81 in the DH population, and 0.58 to 0.89 in the haploid population. Quantitative trait loci (QTL) mapping study showed that a total of 47 QTL for all traits accounting for genetic variations ranging from 1.6 to 36.5% were detected in two populations. One or more QTL sharing common region for each trait were detected between two different ploidy populations. Potential candidate genes predicated from the four QTL support intervals for RPR and IVDMD were indirectly or directly involved with cellulose and lignin biosynthesis, which participated in cell wall formation. The increased expression levels of lignin and cellulose synthesis key genes in the haploid situation illustrated that dosage compensation may account for genome dosage effect in our study. CONCLUSIONS: The current investigation extended understanding about the genetic basis of stalk traits and correlations between DH and its haploid populations, which showed consistence and difference between them in phenotype, QTL characters, and gene expression. The higher heritabilities and partly higher QTL detection power were presented in haploid population than in DH population. All of which described above could lay a preliminary foundation for genetic architecture study with haploid population and may benefit selection in haploid-stage to reduce cost in DH breeding.

Identification of Haploid Maize Kernel Using NIR Spectroscopy in Reflectance and Transmittance Modes: A Comparative Study.

The spectra measurements mode that suitable for haploid maize kernel identification was explored using MicroNIR-1700 series of miniature near infrared spectrometer by JDSU company. Based on Near Infrared Spectroscopy (NIRS) qualitative analysis techniques, we conducted a comparative study using reflectance and transmittance spectra to identify haploid maize kernels. Partial least squares-discriminant analysis (PLS-OLDA) was used to compress the pretreated spectral data, and then the identification models were built based on Support Vector Machine (SVM). The measured data were recorded in reflectance and transmittance modes and the recognition correct rates were calculated. For measurements taken in reflectance mode, the average recognition rate was less than 60% regardless of embryo side positions. In transmittance mode, however, the average recognition rate reached 93.2%. The experiment results show that diffuse reflection spectrum could only obtain corn grain surface information, so embryo side positions severely affect haploid maize kernel identification effect when reflectance measurements mode have been employed, but they have far less impact on transmittance mode. The near infrared diffuse transmittance spectra analyzes non-uniform samples can achieve the analysis of optical path depth information accumulation, all information of the sample interior can be obtained, so transmittance spectra could identify haploid maize effectively and be desensitized to kernel positions. NIRS qualitative analysis techniques with features of rapid, nondestructive could identify the haploid and Micro-NIR spectrometer scan fast and cost less, which have utility for automatically selecting haploid maize kernels from hybrid kernels.