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.
Abstract Lung cancer is a highly prevalent malignancy characterized by significant metabolic alterations. Understanding the metabolic rewiring in lung cancer is crucial for the development of effective therapeutic strategies. The hexosamine biosynthesis pathway (HBP) is a metabolic pathway that plays a vital role in cellular metabolism and has been implicated in various cancers, including lung cancer. Abnormal activation of HBP is involved in the proliferation, progression, metastasis, and drug resistance of tumor cells. In this review, we will discuss the function and regulation of metabolic enzymes related to HBP in lung cancer. Furthermore, the implications of targeting the HBP for lung cancer treatment are also discussed, along with the challenges and future directions in this field. This review provides a comprehensive understanding of the role and intervention of HBP in lung cancer. Future research focusing on the HBP in lung cancer is essential to uncover novel treatment strategies and improve patient outcomes.
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.
Although several studies have proved the relationship between the prognostic value of miRNA-15a and different types of cancer, the result remains controversial. Thus, a meta-analysis was conducted to clarify the prognostic value of miRNA-15a expression level in human cancers.We enrolled appropriate literature by searching the databases of PubMed, Embase, and Web of Science. Subsequently, we extracted HRs and their 95% CIs and calculated pooled results of miRNA-15a for overall survival (OS) and disease-free survival (DFS). Besides, subgroup analysis, sensitivity analysis, and publication bias were also revealed in this study. We also further validated this meta-analysis using the Kaplan-Meier plotter database.10 studies, including 1616 patients, were embraced in our meta-analysis. The result showed the lower expression of miRNA-15a significantly predicted adverse OS (HR=2.17, 95% CI: 1.41-3.34), but there is no significant association between the expressing level and DFS in cancer patient (HR=2.04, 95% CI: 0.60-6.88). Based on Kaplan-Meier plotter database, we found the same results in bladder Carcinoma, head-neck squamous cell carcinoma, liver hepatocellular carcinoma, lung squamous cell carcinoma, pancreatic ductal adenocarcinoma, rectum adenocarcinoma, stomach adenocarcinoma, and uterine corpus endometrial carcinoma, but opposite results were found in cervical squamous cell carcinoma and esophageal carcinoma.Low expressing levels of miRNA-15a indicated poor OS, while miRNA-15a can be used as a prediction biomarker in different cancer types.
Summary 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.
Doubled haploid (DH) technology is an important tool in crop breeding because it can significantly accelerate the breeding process. ZmPLA1/MATL/NLD and ZmDMP are two key genes controlling haploid induction (HI) in maize, exhibiting a synergistic effect. However, it is unknown whether knock out of ZmDMP orthologs can stimulate HI in rice. In this study, a ZmPLA1 ortholog (OsPLA1) and two ZmDMP orthologs (OsDMP3 and OsDMP6) were identified in rice. All three genes encode plasma membrane-localized proteins and were highly expressed in mature anthers. Knockout of OsPLA1 in both Minghui 63 and Nipponbare resulted in reduced seed setting rate (SSR) and caused HI. The osdmp3, osdmp6 and the double mutant failed to trigger HI independently, nor increased the haploid induction rate (HIR) when combined with ospla1. Repeated pollinations operations of QX654A with the ospla1 mutant significantly improve SSR, while reducing HIR. RNA-seq profiling of mature ospla1 mutant anthers indicated that a large number of differentially expressed genes (DEGs) were enriched in redox homeostasis and lipid metabolic GO terms, plant hormone signal transduction, and MAPK signaling pathways. These findings provide important insights towards construction of an efficient DH breeding technology and study of the molecular mechanism of HI in rice.
Abstract Doubled haploid breeding technology has been one of the most important techniques for accelerating crop breeding. In compare to in vivo haploid induction in maize, which is efficient and background independent, wheat haploid production by interspecific hybridization pollinated with maize is influenced by genetic background and requires rescue of young embryos. Here, we analyzed the homologues of maize haploid induction gene MTL / ZmPLA1 / NLD in several crop species systematically, the homologues are highly conserved in sorghum, millet and wheat etc. Since wheat is a very important polyploidy crop, as a proof of concept, we demonstrated that the in vivo haploid induction method could be extended from diploid maize to hexaploid wheat by knocking out the wheat homologues ( TaPLAs ). Result showed that double knock-out mutation could trigger wheat haploid induction at ~ 2%-3%, accompanied by 30% - 60% seed setting rate. The performance of haploid wheat individual showed shorter plant, narrower leaves and male sterile. Our results also revealed that knockout of TaPLA -A and TaPLA -D do not affect pollen viability. This study not only confirmed the function of the induction gene and explored a new approach for haploid production in wheat, but also provided an example that the in vivo haploid induction could be applied in more crop species with different ploidy levels. Furthermore, by combining with gene editing, it would be a fast and powerful platform for traits improvement in polyploidy crops breeding.
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