By using diallel crossing of Griffing method 4,7 maize inbred lines which were different in tassel characters,were evaluated for main axis length,branch number,mean length of branch,spikelet density,spikelet density per tassel as well as normal combining ability,special combing ability and corresponding genetic parameters of the five characters.And at the same time analysis was made on the correlation of the normal combing ability among the characters. The results showed the normal combining ability of the five characters were obviously higher than special combining ability and additive effect for the genes were dominant.h_B~2(%) for the five characters was 80.72,82.96,85.13,84.211 and 61.10 respectively and h_N~2 was 60.92,66.11,44.62 ,67.20 and 38.96 respectively.It was found by correlative analysis that branch number and spikelet density had highly significant positive correlation and significant positive correlation with normal combine ability of spikelet density per tassel respectively (r=0.934 7~(**),r=0.834 5~*).When selecting tassel,branch number and spikelet density should be considered in earlier generation.
Inter-hybrid heterosis is an important part of maize heterosis. Based on the obvious xenia effects of high oil and heterosis, a technique called TEU(three-effect-utilization) was developed to pyramid the cytoplasm male sterile effect, heterosis and xenia of oil content into a production system, which has patented in China. The system has been used in practical production successfully. For better use of TEU, the author suggested that a high efficient partner-hybrid production system should be developed by screening and matching normal hybrid and high oil pollinator based on their heterosis, xenia and other traits. Further more, high-oilized normal corn could also be used as seed in high oil corn production.
High oil inbred line BY815 and two normal inbred lines 1145 and F349 treated with spaceflight were used for variability analysis.Results showed that the mutation rate of BY815 was 21.61% in SP1,while the mutation rates of 1145 and F349 were 2.57% and 3.13% respectively.Only six mutants were found from these three materials in SP2,of which two mutants,HT-3 from BY815 exhibiting albino leaf color and HT-5 from 1145 exhibiting stripe-like spots leaves,were worthy of further study.Genetic analysis of the two mutants showed that the segregation ratio of normal and mutant phenotypes was 3 ∶ 1,which was in accordance with Mendel’s single gene inheritance law.Cytological observation of all the six mutants showed no chromosome abnormalities.By using SSR(simple sequence repeat) method,130 pairs of primers were employed and only one mutant originated from inbred line 1145 showed polymorphic and the mutated loci rate of the genome in this mutant was 8.46%.
Doubled haploid (DH) technology substantially accelerates crop breeding process. Wheat haploid production through interspecific hybridization requires embryo rescue and is dependent on genetic background. In vivo haploid induction (HI) in maize has been widely used and demonstrated to be independent of genetic background and to produce haploids efficiently. Recent studies revealed that loss-of-function of the gene MTL/ZmPLA1/NLD triggers HI (Gilles et al., 2017; Kelliher et al., 2017; Liu et al., 2017). In addition to producing homozygous DH lines, HI system has also been used for gene editing in different genetic backgrounds without introducing the genome of the male parent (Kelliher et al., 2019; Wang et al., 2019). Importantly, HI system had been successfully applied to rice (Yao et al., 2018), making this method more promising. However, little is known whether it can be applied to polyploids. Extension of the maize HI to wheat would create a novel approach in producing haploids in both wheat and other polyploid crop species. In this study, full-length amino acid sequence encoded by MTL/ZmPLA1/NLD was used to do BLAST search for homologues genes in different crop species (www.gramene.com). Results showed that the gene is highly conserved among 19 species of Liliopsida. Wheat homologues genes sharing ~70%-80% amino acid sequence identity with maize. Wheat genome contains three homologues genes: TraesCS4A02G018100 (TaPLA-A), TraesCS4B02G286000 (TaPLA-B) and TraesCS4D02G284700 (TaPLA-D), located on chromosomes 4A, 4B and 4D, respectively. All three includes four exons (Figure 1a). The DNA sequence identity between each of the three homologues genes and MTL/ZmPLA1/NLD is 75%, representing a high level of sequence conservation. The amino acid sequence identity among the three TaPLA genes is 96%. Next, we designed two guide RNA sequences, one targeted TaPLA-B and TaPLA-D (gRNA1), and another targeted TaPLA-A and TaPLA-D (gRNA2) to create knockout lines using CRISPR/Cas9 (Figure 1a). After Agrobacterium tumefaciens-mediated transformation into CB037, four transgenic events were obtained with mutations on both TaPLA-A and TaPLA-D. None of them had a mutation on TaPLA-B. Sequencing results of four transgenic events are shown in Figure 1a. Although different in sequence, all these mutations led to frameshifts and loss of function for both TaPLA-A and TaPLA-D (Figure 1a). These T0 transgenic and control plants were grown in greenhouse. No obvious phenotypic difference was found between wild-type and transgenic plants, except for the seed setting rate (SSR), which ranged from ~30% to ~60% in transgenic lines, significantly lower than the average value of 92.6% in wild type (Figure 1b), implying that TaPLAs may be involved in sexual reproduction. Importantly, putative haploid plants were found in self-pollinated progenies of all four transgenic lines according to their growth characteristics. Average HI rate (HIR) ranged from 5.88% to 15.66% (Figure 1e). In the progeny of T1 × Chinese Spring and T1 × Liaochun10, 3 and 2 putative haploids were found in 29 and 19 individuals, respectively (Figure 1f). To verify real ploidy level of putative haploids, flow cytometry was used. Results showed that compared with hexaploid controls which had FL2A peaks approximately 100, all putative haploids had FL2A peaks approximately 50 (Figure 1d), suggesting that putative haploids identified by phenotypic characteristics were true haploids. These haploids had shorter plant height, narrower leaves, shorter spikes and male sterility (Figure 1c). No haploid plant was identified in a control group with 267 progenies from wild type individuals (Figure 1e). Therefore, we concluded that knockout of wheat homologues genes of MTL/ZmPLA1/NLD could trigger wheat haploid induction. The HIR reported here was higher than that in previous preprint version, this was because more haploids were identified later. Since the inducer lines (T0) still contained active Cas9, there was the possibility that floral organ genotype might be different from leaf and had homozygous mutations. In addition to haploids, 7 aneuploids were found among crossed progeny, including 1 plant with ploidy level between haploid and hexaploid and 6 plants with ploidy level higher than hexaploid. This phenomenon may provide some clues on mechanism of wheat HI. To characterize the expression pattern of the TaPLAs, subcellular localization was performed using tobacco epidermal cells. As shown in Figure 1g, all three genes showed strong signals in plasma membrane and merged well with the endoplasmic reticulum marker. This result is consistent with NLD in maize (Gilles et al., 2017). Chromosome elimination and single fertilization were two leading hypotheses in explaining HI in maize (Kelliher et al., 2019; Tian et al., 2018). While other issues like low pollen viability which may also contribute to haploid induction, had not been fully ruled out. Here, we performed fluorescein diacetate (FDA) staining to examine pollen viability in wild type and mutants. There was no difference in the proportion of pollen viability classes between mutants and wild type (Figure 1h). Therefore, loss of function of both TaPLA-A and TaPLA-D does not influence pollen viability. Several technical problems require further investigation before HI can be applied efficiently in wheat, including how to further improve wheat HI efficiency and the difficulties in haploid kernel identification. Considering the potential redundancy among TaPLA-A, TaPLA-B and TaPLA-D, wild type TaPLA-B may functionally complement tapla-A and tapla-D double mutant. Nevertheless, the complement effect is not enough to rescue the phenotype of HI and reduced SSR. On the other hand, further improvement of HI efficiency in wheat may be achieved by creating triple mutants. In addition, the gene ZmDMP contributing to HIR has been identified (Liu et al., 2015; Zhong et al., 2019), and the efficiency of wheat HI may be further improved by knockout ZmDMP homologues genes in wheat. On the other hand, recent studies have simplified the identification of haploids using enhanced green fluorescent proteins and DsRed signals specifically expressed in the embryo and endosperm, respectively (Dong et al., 2018). This method may provide a potential solution for haploid kernel identification in wheat. In summary, our study provided the proof that HI is not limited to diploid crop species but can be extended to polyploid species. Meanwhile, this study also provided a promising platform for wheat haploid gene editing and mechanism studies of HI. Considering the conservation of gene sequence and function, the system could potentially be extended to a wider variety of crop species. We thank Prof. Pu Wang for providing greenhouse, Prof. Zhongfu Ni for providing CB037 seeds, Prof. Xingguo Ye for wheat transformation and Dr. Qiguo Yu for carefully reading the manuscript. This research was supported by the National Key Research and Development Program of China (2016YFD0101200), the Modern Maize Industry Technology System (CARS-02-04) and China Postdoctoral Science Foundation (2018M631634). The authors declare no conflicts of interest. S.C. and C.L. conceived and designed the project. C.L. and Y.Z. constructed plasmid. X.Q., Y.Z., M.C. and Z.L. planted transgenic plants in greenhouse and performed haploid identification and verification and phenotype investigations. Y.Z., C.L., X.Q., M.L. and W.L. performed data analysis. C.L., Y.Z., X.Q., M.X. and S.C. wrote the paper with inputs from all authors.
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.
Southern corn rust (SCR), caused by Puccinia polysora Underw, is a destructive disease that can severely reduce grain yield in maize ( Zea mays L.). Owing to P. polysora being multi-racial, it is very important to explore more resistance genes and develop more efficient selection approaches in maize breeding programs. Here, four Doubled Haploid (DH) populations with 384 accessions originated from selected parents and their 903 testcross hybrids were used to perform genome-wide association (GWAS). Three GWAS processes included the additive model in the DH panel, additive and dominant models in the hybrid panel. As a result, five loci were detected on chromosomes 1, 7, 8, 8, and 10, with P -values ranging from 4.83×10 -7 to 2.46×10 -41 . In all association analyses, a highly significant locus on chromosome 10 was detected, which was tight chained with the known SCR resistance gene RPPC and RPPK . Genomic prediction (GP), has been proven to be effective in plant breeding. In our study, several models were performed to explore predictive ability in hybrid populations for SCR resistance, including extended GBLUP with different genetic matrices, maker based prediction models, and mixed models with QTL as fixed factors. For GBLUP models, the prediction accuracies ranged from 0.56-0.60. Compared with traditional prediction only with additive effect, prediction ability was significantly improved by adding additive-by-additive effect ( P -value< 0.05). For maker based models, the accuracy of BayesA and BayesB was 0.65, 8% higher than other models (i.e., RRBLUP, BRR, BL, BayesC). Finally, by adding QTL into the mixed linear prediction model, the accuracy can be further improved to 0.67, especially for the G_A model, the prediction performance can be increased by 11.67%. The prediction accuracy of the BayesB model can be further improved significantly by adding QTL information ( P -value< 0.05). This study will provide important valuable information for understanding the genetic architecture and the application of GP for SCR in maize breeding.
Efficient haploid induction is the prerequisites in maize doubled haploid breeding.In this study,the results showed that the haploid induction rates differed significantly among the 20 single hybrids,and the highest rate was about five times higher than the lowest.From the multi-year haploid induction experiment using single hybrid ZD958 as female and haploid inducer CAUHOI-1 as male,the average induction rate in winter season in Hainan(3.39%) was higher than that of Beijing in summer season(1.86%).Haploid rates differed between different seed positions on the ear,and the higher haploid frequency was found on the top of the ear.Haploid induction rate was also affected by the silk age,and the younger silk could yield more haploids.The results above supported that maternal genotype,maternal silk age and inducing location could be important in haploid induction.To increase haploid induction rate and optimize the haploid induction,induction should be conducted in locations with stable environment and pollination in early time after ear silking.
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