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.

Loss-of-function alleles of ZmPLD3 cause haploid induction in maize.

Doubled haploid technology has been widely applied to multiple plant species and is recognized as one of the most important technologies for improving crop breeding efficiency. Although mutations in MATRILINEAL/Zea mays PHOSPHOLIPASE A1/NOT LIKE DAD (MTL/ZmPLA1/NLD) and Zea mays DOMAIN OF UNKNOWN FUNCTION 679 MEMBRANE PROTEIN (ZmDMP) have been shown to generate haploids in maize, knowledge of the genetic basis of haploid induction (HI) remains incomplete. Therefore, cloning of new genes underlying HI is important for further elucidating its genetic architecture. Here, we found that loss-of-function mutations of Zea mays PHOSPHOLIPASE D3 (ZmPLD3), one of the members from the phospholipase D subfamily, could trigger maternal HI in maize. ZmPLD3 was identified through a reverse genetic strategy based on analysis of pollen-specifically expressed phospholipases, followed by validation through the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR-Cas9) system. Mutations of ZmPLD3 resulted in a haploid induction rate (HIR) similar to that of mtl/zmpla1/nld and showed synergistic effects rather than functional redundancy on tripling the HIR (from 1.19% to 4.13%) in the presence of mtl/zmpla1/nld. RNA-seq profiling of mature pollen indicated that a large number of pollen-specific differentially expressed genes were enriched in processes related to gametogenesis development, such as pollen tube development and cell communication, during the double-fertilization process. In addition, ZmPLD3 is highly conserved among cereals, highlighting the potential application of these in vivo haploid-inducer lines for other important crop plant species. Collectively, our discovery identifies a novel gene underlying in vivo maternal HI and provides possibility of breeding haploid inducers with further improved HIR.

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.

Single nucleus sequencing reveals spermatid chromosome fragmentation as a possible cause of maize haploid induction.

Production of maternal haploids using a conspecific haploid inducer is routine and highly efficient in maize. However, the underlying mechanism of haploid induction (HI) is unclear. We develop a method to isolate three nuclei from a pollen grain and four microspores from a tetrad for whole-genome sequencing. A high rate of aneuploidy is observed at the three-nucleus stage (6/22 pollens) rather than at the tetrad stage (1/72 microspores) in one HI line CAU5. Frequent aneuploidy is also observed in another two inducer lines, but not in two regular lines, which implies that HI may be associated with pollen aneuploidy. We further sequence the individual embryos and endosperms of 88 maize kernels crossing between regular and inducer lines. Genome-wide elimination of the CAU5-derived chromosome is identified in eight of 81 embryos. Together, these results suggest that continuous chromosome fragmentation occurring post meiosis in the gametophyte may cause haploidy of the embryo.

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.

Rapid and accurate identification of in vivo-induced haploid seeds based on oil content in maize.

The needs of a growing human population require rapid and efficient development of improved cultivars by plant breeders. The doubled haploid (DH) technology enables generating completely homozygous lines in a single step and, thus, is central to modern genetics and breeding approaches. Rapid and reliable identification of seeds with a haploid embryo after in vivo haploid induction is elementary in the method utilized in maize but current systems have severe shortcomings preventing their use in many germplasm types. Here, we describe an alternative method for discrimination of haploid from diploid seeds based on differences in their oil content stemming from pollination with high oil inducers. After presenting some fundamental theory, we provide a proof-of-concept with experimental results, demonstrating acceptable error rates across different germplasm. Our approach represents a breakthrough in DH technology in maize, because it is amenable to automated high-throughput screening and applicable to any maize germplasm worldwide.

Gametophytic and zygotic selection leads to segregation distortion through in vivo induction of a maternal haploid in maize.

Production of maternal haploids via a male inducer can greatly accelerate maize breeding and is an interesting biological phenomenon in double fertilization. However, the mechanism behind haploid induction remains elusive. Segregation distortion, which is increasingly recognized as a potentially powerful evolutionary force, has recently been observed during maternal haploid induction in maize. The results present here showed that both male gametophytic and zygotic selection contributed to severe segregation distortion of a locus, named segregation distortion 1 (sed1), during maternal haploid induction in maize. Interestingly, analysis of reciprocal crosses showed that sed1 is expressed in the male gametophyte. A novel mapping strategy based on segregation distortion has been used to fine-map this locus. Strong selection for the presence of the sed1 haplotype from inducers in kernels with haploid formation and defects could be detected in the segregating population. Dual-pollination experiments showed that viable pollen grains from inducers had poor pollen competitive ability against pollen from normal genotypes. Although defective kernels and haploids have different phenotypes, they are most probably caused by the sed1 locus, and possible mechanisms for production of maternal haploids and the associated segregation distortion are discussed. This research also provides new insights into the process of double fertilization.

Identifying quantitative trait loci and determining closely related stalk traits for rind penetrometer resistance in a high-oil maize population.

Stalk lodging in maize causes annual yield losses between 5 and 20% worldwide. Many studies have indicated that maize stalk strength significantly negatively correlates with lodging observed in the field. Rind penetrometer resistance (RPR) measurements can be used to effectively evaluate maize stalk strength, but little is known about the genetic basis of this parameter. The objective of this study was to explore a genetic model and detect quantitative trait loci (QTL) of RPR and determine relationships between RPR and other stalk traits, especially cell wall chemical components. RPR is quantitative trait in nature, and both additive and non-additive effects may be important to consider for the improvement of RPR. Nine additive-effect QTLs covering nine chromosomes, except chromosome 5, and one pair of epistatic QTLs were detected for RPR. CeSA11 involved in cellulose synthesis and colorless2 involved in lignin synthesis were identified as possible candidate genes for RPR. Internode diameter (InD), fresh weight of internode (FreW), dry weight of internode (DryW), fresh weight and dry weight as well as cell wall components per unit volume significantly positively correlated with RPR. The internode water content (InW) significantly negatively correlated with RPR. Notably, these traits significantly correlated with RPR, and the QTLs of these traits co-localized with those of RPR. The corresponding results obtained from correlation analysis and QTL mapping suggested the presence of pleitropism or linkage between genes and indicated that these different approaches may be used for cross authentication of relationships between different traits.

[Nondestructive identification of different oil content maize kernels by near-infrared spectra].

Using 220 maize single kernels, containing 75 common maize single kernels, 72 high-oil maize single kernels and 73 super high-oil maize single kernels as study materials, BPANN identification model was set up for maize single kernel with different oil content based on principal components of near infrared (NIR) spectra. Four fifths of the samples were randomly selected as training set and the other samples as prediction set. Fourteen principal components from the second to the fifteenth were selected as nets input and -1, 0, 1 as nets output. Ten models were set up like this and the accurate identification rate of all the training sets can reach 100%. For prediction sets, fifteen common corn grain samples had an average accurate identification rate of 99.33%, fourteen high-oil corn grain samples had an average accurate identification rate of 97.88%, fourteen super high-oil corn grain samples had an average accurate identification rate of 91.43%, and total maize grains in prediction set had an average accurate identification rate of over 95%. Results showed that NIR spectroscopy combined with BP-ANN technology could identify maize kernels fast and nondestructively according to oil content, which offered a very useful classification method for maize seed breeding. The effect of different principal component on BPANN models was also studied. Results told us that the first principal component with over 99% of variance contribution had negative effect on the identification model. The predictive ability of identification models set up by different principal component was discriminatory, although the learning accurate identification rates were all 100%. So it is necessary to choose correlative principal component to set up identification model.

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.

Isolation and partial characterization of rennet-like proteases from Australian cardoon (Cynara cardunculus L.).

The yield, protein content, proteolytic activity, and substrate specificity of crude and partially purified extracts from dried and fresh Australian cardoon (Cynara cardunculus L.) flowers were determined. Crude water extracts had high yield but low protein content and proteolytic activity, whereas citric acid extracts had low yield but high protein content and proteolytic activity. Fresh flower extracts gave higher yield and proteolytic activity but lower protein content in comparison with dried flower extracts. Purification with ammonium sulfate resulted in significantly increased proteolytic activity for water extracts from both fresh and dried cardoon flowers, whereas the proteolytic activity of citric acid extracts did not change significantly after purification. Irrespective of extraction method, all extracts had higher proteolytic activity against ovine whole and kappa-caseins compared to their bovine counterparts, showing optimal activity at 37 degrees C and pH 6.0. Separation of purified extracts by ion-exchange liquid chromatography yielded three active fractions, each of which when assayed with sodium dodecyl sulfate capillary electrophoresis revealed two subunits with molecular masses of 15.5 and 33.1 kDa, respectively.