Artificial intelligence model system for bone age assessment of preschool children.

BACKGROUD: Our study aimed to assess the impact of inter- and intra-observer variations when utilizing an artificial intelligence (AI) system for bone age assessment (BAA) of preschool children. METHODS: A retrospective study was conducted involving a total sample of 53 female individuals and 41 male individuals aged 3-6 years in China. Radiographs were assessed by four mid-level radiology reviewers using the TW3 and RUS-CHN methods. Bone age (BA) was analyzed in two separate situations, with/without the assistance of AI. Following a 4-week wash-out period, radiographs were reevaluated in the same manner. Accuracy metrics, the correlation coefficient (ICC)and Bland-Altman plots were employed. RESULTS: The accuracy of BAA by the reviewers was significantly improved with AI. The results of RMSE and MAE decreased in both methods (p < 0.001). When comparing inter-observer agreement in both methods and intra-observer reproducibility in two interpretations, the ICC results were improved with AI. The ICC values increased in both two interpretations for both methods and exceeded 0.99 with AI. CONCLUSION: In the assessment of BA for preschool children, AI was found to be capable of reducing inter-observer variability and enhancing intra-observer reproducibility, which can be considered an important tool for clinical work by radiologists. IMPACT: The RUS-CHN method is a special bone age method devised to be suitable for Chinese children. The preschool stage is a critical phase for children, marked by a high degree of variability that renders BA prediction challenging. The accuracy of BAA by the reviewers can be significantly improved with the aid of an AI model system. This study is the first to assess the impact of inter- and intra-observer variations when utilizing an AI model system for BAA of preschool children using both the TW3 and RUS-CHN methods.

Conductive catalysis by subsurface transition metals.

The nature of catalysis has been hotly pursued for over a century, and current research is focused on understanding active centers and their electronic structures. Herein, the concept of conductive catalysis is proposed and verified by theoretical simulations and experimental observations. Metallic systems containing buried catalytically active transitional metals and exposed catalytically inert main group metals are constructed, and the electronic interaction between them via metallic bonding is disclosed. Through the electronic interaction, the catalytic properties of subsurface transitional metals (Pd or Rh) can be transferred to outermost main group metals (Al or Mg) for several important transformations like semi-hydrogenation, Suzuki-coupling and hydroformylation. The catalytic force is conductive, in analogy with the magnetic force and electrostatic force. The traditional definition of active centers is challenged by the concept of conductive catalysis and the electronic nature of catalysis is more easily understood. It might provide new opportunities for shielding traditional active centers against poisoning or leaching and allow for precise regulation of their catalytic properties by the conductive layer.

Genome-Wide Identification, Evolution, and Expression Analysis of the WD40 Subfamily in Oryza Genus.

The WD40 superfamily is widely found in eukaryotes and has essential subunits that serve as scaffolds for protein complexes. WD40 proteins play important regulatory roles in plant development and physiological processes, such as transcription regulation and signal transduction; it is also involved in anthocyanin biosynthesis. In rice, only OsTTG1 was found to be associated with anthocyanin biosynthesis, and evolutionary analysis of the WD40 gene family in multiple species is less studied. Here, a genome-wide analysis of the subfamily belonging to WD40-TTG1 was performed in nine AA genome species: Oryza sativa ssp. japonica, Oryza sativa ssp. indica, Oryza rufipogon, Oryza glaberrima, Oryza meridionalis, Oryza barthii, Oryza glumaepatula, Oryza nivara, and Oryza longistaminata. In this study, 383 WD40 genes in the Oryza genus were identified, and they were classified into four groups by phylogenetic analysis, with most members in group C and group D. They were found to be unevenly distributed across 12 chromosomes. A total of 39 collinear gene pairs were identified in the Oryza genus, and all were segmental duplications. WD40s had similar expansion patterns in the Oryza genus. Ka/Ks analyses indicated that they had undergone mainly purifying selection during evolution. Furthermore, WD40s in the Oryza genus have similar evolutionary patterns, so Oryza sativa ssp. indica was used as a model species for further analysis. The cis-acting elements analysis showed that many genes were related to jasmonic acid and light response. Among them, OsiWD40-26/37/42 contained elements of flavonoid synthesis, and OsiWD40-15 had MYB binding sites, indicating that they might be related to anthocyanin synthesis. The expression profile analysis at different stages revealed that most OsiWD40s were expressed in leaves, roots, and panicles. The expression of OsiWD40s was further analyzed by qRT-PCR in 9311 (indica) under various hormone treatments and abiotic stresses. OsiWD40-24 was found to be responsive to both phytohormones and abiotic stresses, suggesting that it might play an important role in plant stress resistance. And many OsiWD40s might be more involved in cold stress tolerance. These findings contribute to a better understanding of the evolution of the WD40 subfamily. The analyzed candidate genes can be used for the exploration of practical applications in rice, such as cultivar culture for colored rice, stress tolerance varieties, and morphological marker development.

Integrated Transcriptomic and Metabolomic Analyses Uncover the Differential Mechanism in Saline-Alkaline Tolerance between Indica and Japonica Rice at the Seedling Stage.

Saline-alkaline stress is one of the major damages that severely affects rice (Oryza sativa L.) growth and grain yield; however, the mechanism of the tolerance remains largely unknown in rice. Herein, we comparatively investigated the transcriptome and metabolome of two contrasting rice subspecies genotypes, Luohui 9 (abbreviation for Chao2R under study, O. sativa ssp. indica, saline-alkaline-sensitive) and RPY geng (O. sativa ssp. japonica, saline-alkaline-tolerant), to identify the main pathways and important factors related to saline-alkaline tolerance. Transcriptome analysis showed that 68 genes involved in fatty acid, amino acid (such as phenylalanine and tryptophan), phenylpropanoid biosynthesis, energy metabolism (such as Glycolysis and TCA cycle), as well as signal transduction (such as hormone and MAPK signaling) were identified to be specifically upregulated in RPY geng under saline-alkaline conditions, implying that a series of cascade changes from these genes promotes saline-alkaline stress tolerance. The transcriptome changes observed in RPY geng were in high accordance with the specifically accumulation of metabolites, consisting mainly of 14 phenolic acids, 8 alkaloids, and 19 lipids based on the combination analysis of transcriptome and metabolome. Moreover, some genes involved in signal transduction as hub genes, such as PR5, FLS2, BRI1, and NAC, may participate in the saline-alkaline stress response of RPY geng by modulating key genes involved in fatty acid, phenylpropanoid biosynthesis, amino acid metabolism, and glycolysis metabolic pathways based on the gene co-expression network analysis. The present research results not only provide important insights for understanding the mechanism underlying of rice saline-alkaline tolerance at the transcriptome and metabolome levels but also provide key candidate target genes for further enhancing rice saline-alkaline stress tolerance.

Genome-Wide Identification and Evolution Analysis of the CYP76 Subfamily in Rice (Oryza sativa).

The CYP76 subfamily, a member of the CYP superfamily, plays crucial roles in the biosynthesis of phytohormones in plants, involving biosynthesis of secondary metabolites, hormone signaling, and response to environmental stresses. Here, we conducted a genome-wide analysis of the CYP76 subfamily in seven AA genome species: Oryza sativa ssp. japonica, Oryza sativa ssp. indica, Oryza rufipogon, Oryza glaberrima, Oryza meridionalis, Oryza barthii, and Oryza glumaepatula. These were identified and classified into three groups, and it was found that Group 1 contained the largest number of members. Analysis of cis-acting elements revealed a large number of elements related to jasmonic acid and light response. The gene duplication analysis revealed that the CYP76 subfamily expanded mainly in SD/WGD and tandem forms and underwent strong purifying selection during evolution. Expression pattern analysis of OsCYP76s in various developmental stages revealed that the majority of OsCYP76s exhibit relatively restricted expression patterns in leaves and roots. We further analyzed the expression of CYP76s in O. sativa, japonica, and O. sativa, indica under cold, flooding, drought, and salt abiotic stresses by qRT-PCR. We found that OsCYP76-11 showed a huge increase in relative expression after drought and salt stresses. After flooding stress, OsiCYP76-4 showed a greater increase in expression compared to other genes. CYP76 in japonica and indica showed different response patterns to the same abiotic stresses, revealing functional divergence in the gene family during evolution; these may be the key genes responsible for the differences in tolerance to indica japonica. Our results provide valuable insights into the functional diversity and evolutionary history of the CYP76 subfamily and pave the way for the development of new strategies for improving stress tolerance and agronomic traits in rice.

Integrated multi-locus genome-wide association studies and transcriptome analysis for seed yield and yield-related traits in Brassica napus.

Rapeseed (Brassica napus L.), the third largest oil crop, is an important source of vegetable oil and biofuel for the world. Although the breeding and yield has been improved, rapeseed still has the lowest yield compared with other major crops. Thus, increasing rapeseed yield is essential for the high demand of vegetable oil and high-quality protein for live stocks. Silique number per plant (SN), seed per pod (SP), and 1000-seed weight (SW) are the three important factors for seed yield in rapeseed. Some yield-related traits, including plant height (PH), flowering time (FT), primary branch number (BN) and silique number per inflorescence (SI) also affect the yield per plant (YP). Using six multi-locus genome-wide association study (ML-GWAS) approaches, a total of 908 yield-related quantitative trait nucleotides (QTNs) were identified in a panel consisting of 403 rapeseed core accessions based on whole-genome sequencing. Integration of ML-GWAS with transcriptome analysis, 79 candidate genes, including BnaA09g39790D (RNA helicase), BnaA09g39950D (Lipase) and BnaC09g25980D (SWEET7), were further identified and twelve genes were validated by qRT-PCRs to affect the SW or SP in rapeseed. The distribution of superior alleles from nineteen stable QTNs in 20 elite rapeseed accessions suggested that the high-yielding accessions contained more superior alleles. These results would contribute to a further understanding of the genetic basis of yield-related traits and could be used for crop improvement in B. napus.

The Pyramiding of Elite Allelic Genes Related to Grain Number Increases Grain Number per Panicle Using the Recombinant Lines Derived from Indica-japonica Cross in Rice.

Indica(xian)-japonica(geng) hybrid rice has many heterosis traits that can improve rice yield. However, the traditional hybrid technology will struggle to meet future needs for the development of higher-yield rice. Available genomics resources can be used to efficiently understand the gene-trait association trait for rice breeding. Based on the previously constructed high-density genetic map of 272 high-generation recombinant inbred lines (RILs) originating from the cross of Luohui 9 (indica, as female) and RPY geng (japonica, as male) and high-quality genomes of parents, here, we further explore the genetic basis for an important complex trait: possible causes of grain number per panicle (GNPP). A total of 20 genes related to grains number per panicle (GNPP) with the differences of protein amino acid between LH9 and RPY were used to analyze genotype combinations, and PCA results showed a combination of PLY1, LAX1, DTH8 and OSH1 from the RPY geng with PYL4, SP1, DST and GNP1 from Luohui 9 increases GNPP. In addition, we also found that the combination of LAX1-T2 and GNP1-T3 had the most significant increase in GNPP. Notably, Molecular Breeding Knowledgebase (MBK) showed a few aggregated rice cultivars, LAX1-T2 and GNP1-T3, which may be a result of the natural geographic isolation between the two gene haplotypes. Therefore, we speculate that the pyramiding of japonica-type LAX-T2 with indica-type GNP1-T3 via hybridization can significantly improve rice yield by increasing GNPP.

Chloroplast inner envelope protein FtsH11 is involved in the adjustment of assembly of chloroplast ATP synthase under heat stress.

The maintenance of a proton gradient across the thylakoid membrane is an integral aspect of photosynthesis that is mainly established by the splitting of water molecules in photosystem II and plastoquinol oxidation at the cytochrome complex, and it is necessary for the generation of ATP in the last step of photophosphorylation. Although environmental stresses, such as high temperatures, are known to disrupt this fundamental process, only a few studies have explored the molecular mechanisms underlying proton gradient regulation during stress. The present study identified a heat-sensitive mutant that displays aberrant photosynthesis at high temperatures. This mutation was mapped to AtFtsH11, which encodes an ATP-dependent AAA-family metalloprotease. We showed that AtFtsH11 localizes to the chloroplast inner envelope membrane and is capable of degrading the ATP synthase assembly factor BFA3 under heat stress. We posit that this function limits the amount of ATP synthase integrated into the thylakoid membrane to regulate proton efflux from the lumen to the stroma. Our data also suggest that AtFtsH11 is critical in stabilizing photosystem II and cytochrome complexes at high temperatures, and additional studies can further elucidate the specific molecular functions of this critical regulator of photosynthetic thermotolerance.

Application of SPARK teaching in acute abdomen radiography teaching for undergraduate medical students.

BACKGROUND: Acute abdomen is a series of acute and severe abdominal diseases commonly encountered in clinic. It is important to strengthen the image teaching of acute abdomen for undergraduates. AIM: This study aimed to explore the application effect of SPARK[sub-speciality (S), problem-based learning (P), assessment (A), report (R) and reading skill (K)] teaching mode in the experimental teaching of acute abdomen for undergraduate medical students. METHODS: We selected a total of 58 third year medical students for observation. The students were divided into experimental group and control group. Among them, 29 students in the experimental group studied in SPARK teaching mode, 29 students in the control group studied in traditional teaching mode. The two groups of students were tested after the theory class, before and after the experimental class, and one week after the experimental class, to compare the application effects of the two teaching modes. After the test one week after the experimental class, the two groups of students jointly adopted SPARK mode to learn, and were tested again one month after the experimental class to compare whether the two groups of students achieved the same results. The total score of all tests was 150. RESULTS: The average scores of the experimental group and the control group after theory class were (69.0 ± 26.4) and (72.1 ± 24.1) respectively, with no statistical difference (t = 0.468, P = 0.642). The average scores of the experimental group before, after and one week after the experimental class were higher than those of the control group. The experimental group was (84.5 ± 23.1), (109.7 ± 23.8), (105.5 ± 31.0) respectively, and the control group was (52.8 ± 15.1), (93.8 ± 17.0), (80.0 ± 22.8) respectively. The differences were statistically significant (t = -6.195, P = 0.00; t = -2.919, P = 0.05; t = -3.569, P = 0.01). The average scores of the experimental group and the control group after one month were (99.0 ± 31.0) and (95.5 ± 25.6) respectively, and there was no significant difference between the two groups (t = -0.462, P = 0.646). CONCLUSIONS: The SPARK teaching mode was helpful for undergraduate medical students to consolidate image foundation, improve image reading skills.

Genome-Wide Identification of the AGC Protein Kinase Gene Family Related to Photosynthesis in Rice (Oryza sativa).

The cAMP-dependent protein kinase A, cGMP-dependent protein kinase G and phospholipid-dependent protein kinase C (AGC) perform various functions in plants, involving growth, immunity, apoptosis and stress response. AGC gene family is well described in Arabidopsis, however, limited information is provided about AGC genes in rice, an important cereal crop. This research studied the AGC gene family in the AA genome species: Oryza sativa ssp. japonica, Oryza sativa ssp. indica, Oryza nivara, Oryza rufipogon, Oryza glaberrima, Oryza meridionalis, Oryza barthii, Oryza glumaepatula and Oryza longistaminata were searched and classified into six subfamilies, and it was found that these species have similar numbers of members. The analysis of gene duplication and selection pressure indicated that the AGC gene family expanded mainly by segmental or whole genome duplication (WGD), with purifying selection during the long evolutionary period. RNA-seq analysis revealed that OsAGCs of subfamily V were specifically highly expressed in leaves, and the expression patterns of these genes were compared with that of photosynthesis-related genes using qRT-PCR, discovered that OsAGC9, OsAGC20, and OsAGC22 might participate in photosynthesis. These results provide an informative perspective for exploring the evolutionary of AGC gene family and its practical application in rice.

A comparative study of three bone age assessment methods on Chinese preschool-aged children.

Background: Bone age assessment (BAA) is an essential tool utilized in outpatient pediatric clinics. Three major BAA methods, Greulich-Pyle (GP), Tanner-Whitehouse 3 (TW3), and China 05 RUS-CHN (RUS-CHN), were applied to comprehensively compare bone age (BA) and chronological age (CA) in a Chinese sample of preschool children. This study was designed to determine the most reliable method. Methods: The BAA sample consisted of 207 females and 183 males aged 3-6 years from the Zhejiang Province in China. The radiographs were estimated according to the GP, TW3, and RUS-CHN methods by two pediatric radiologists. The data was analyzed statistically using boxplots, the Wilcoxon rank test, and Student's t-test to explore the difference (D) between BA and CA. Results: According to the distributions of D, the boxplots showed that the median D of the TW3 method was close to zero for both male and female subjects. The TW3 and RUS-CHN methods overestimated the age of both genders. The TW3 method had the highest correct classification rate for males but a similar rate for females. The GP method did not show any significant difference between the BA and CA when applied to 3-year-old males and 4-year-old females while the TW3 method showed similar results when applied to 6-year-old females. The RUS-CHN method showed the least consistent results among the three methods. Conclusion: The TW3 method was superior to the GP and RUS-CHN methods but not reliable on its own. It should be noted that a precise age diagnosis for preschool children cannot be easily made if only one of the methods is utilized. Therefore, it is advantageous to combine multiple methods when assessing bone age.

De novo assembly of two chromosome-level rice genomes and bin-based QTL mapping reveal genetic diversity of grain weight trait in rice.

Following the "green revolution," indica and japonica hybrid breeding has been recognized as a new breakthrough in further improving rice yields. However, heterosis-related grain weight QTLs and the basis of yield advantage among subspecies has not been well elucidated. We herein de novo assembled the chromosome level genomes of an indica/xian rice (Luohui 9) and a japonica/geng rice (RPY geng) and found that gene number differences and structural variations between these two genomes contribute to the differences in agronomic traits and also provide two different favorable allele pools to produce better derived recombinant inbred lines (RILs). In addition, we generated a high-generation (> F15) population of 272 RILs from the cross between Luohui 9 and RPY geng and two testcross hybrid populations derived from the crosses of RILs and two cytoplasmic male sterile lines (YTA, indica and Z7A, japonica). Based on three derived populations, we totally identified eight 1,000-grain weight (KGW) QTLs and eight KGW heterosis loci. Of QTLs, qKGW-6.1 and qKGW-8.1 were accepted as novel KGW QTLs that have not been reported previously. Interestingly, allele genotyping results revealed that heading date related gene (Ghd8) in qKGW-8.1 and qLH-KGW-8.1, can affect grain weight in RILs and rice core accessions and may also play an important role in grain weight heterosis. Our results provided two high-quality genomes and novel gene editing targets for grain weight for future rice yield improvement project.

Epstein-Barr virus infection-associated cholangiocarcinoma: a report of one case and the review of literature.

The clinical data of a patient with Epstein-barr virus (EBV) associated with cholangiocarcinoma was reported in this paper: a case of a 36-year-old female presented with abdominal pain and systemic skin yellowing combined with skin itching. Laboratory studies showed increase in alanine aminotransferase 242 U/L, aspartate aminotransferase 404 U/L, r-glutamyltransferase 1516 U/L, total bilirubin 308.2 µmol/L and CA199 (101.0 U/ml). AFP (4.5 ng/ml) was normal. CT revealed multiple space-occupying lesions in the liver. PET-CT revealed liver malignant tumor and lymph node metastasis. Liver puncture pathology revealed infiltrative growth of significant heterocyst nests in the liver tissue, which was morphologically consistent with malignant tumors, considering poorly differentiated carcinoma. Pathology suggestion: combining liver puncture with morphology, immunohistochemistry, and EBV in situ hybridization results, it was consistent with EB virus-associated poorly differentiated carcinoma, therefore, consider EBV infection-associated poorly differentiated cholangiocarcinoma (CCA) (LELC morphology). The patient underwent liver transplantation in Hangzhou Shulan Hospital on June 8, 2021 successfully. After surgery, the patient orally took tacrolimus for anti-rejection, entecavir for antiviral therapy, gemcitabine 1.2 g + cis-platinum 30 mg for chemotherapy. After following up for more than 5 months post liver transplantation, the condition of the patient deteriorated. The patient subsequently died. Based on the case of our patient and the review of existing literature, when the patient's serum CA199 increased, AFP did not change significantly, and there was no previous history of hepatitis B. CT revealed a low-density mass in the liver, ring enhancement in the arterial phase, and heterogeneous enhancement of the tumor in the delayed phase. Ring enhancement of the liver lesion mass was observed on MRI. Consider the might possibility of hepatic CCA. When patients showed recurrent tonsillitis at an early age, EBV virus infection should be vigilant and oropharyngeal tissue should persist, diagnosis of EBV-associated liver cancer should be considered. In particular, EBV infection-related liver cancer is relatively rare, the clinician should improve the recognition of the disease to strive for early diagnosis and therapy.

Genome-Wide Classification and Evolutionary Analysis Reveal Diverged Patterns of Chalcone Isomerase in Plants.

Flavonoids as a class of important secondary metabolites are widely present in land plants, and chalcone isomerase (CHI) is the key rate-limiting enzyme that participates in catalyzing the stereospecific isomerization of chalcones to yield their corresponding flavanones. However, the phylogenetic dynamics and functional divergence of CHI family genes during the evolutionary path of green plants remains poorly understood. Here, a total of 122 CHI genes were identified by performing a genome-wide survey of 15 representative green plants from the most ancestral basal plant chlorophyte algae to higher angiosperm plants. Phylogenetic, orthologous groups (OG) classification, and genome structure analysis showed that the CHI family genes have evolved into four distinct types (types I-IV) containing eight OGs after gene duplication, and further studies indicated type III CHIs consist of three subfamilies (FAP1, FAP2, and FAP3). The phylogeny showed FAP3 CHIs as an ancestral out-group positioned on the outer layers of the main branch, followed by type IV CHIs, which are placed in an evolutionary intermediate between FAP3 CHIs and bona fide CHIs (including type I and type II). The results imply a potential intrinsic evolutionary connection between CHIs existing in the green plants. The amino acid substitutions occurring in several residues have potentially affected the functional divergence between CHI proteins. This is supported by the analysis of transcriptional divergence and cis-acting element analysis. Evolutionary dynamics analyses revealed that the differences in the total number of CHI family genes in each plant are primarily attributed to the lineage-specific expansion by natural selective forces. The current studies provide a deeper understanding of the phylogenetic relationships and functional diversification of CHI family genes in green plants, which will guide further investigation on molecular characteristics and biological functions of CHIs.

CBL-Interacting Protein Kinase OsCIPK18 Regulates the Response of Ammonium Toxicity in Rice Roots.

Ammonium ( NH 4 + ) is one of the major nitrogen sources for plants. However, excessive ammonium can cause serious harm to the growth and development of plants, i.e., ammonium toxicity. The primary regulatory mechanisms behind ammonium toxicity are still poorly characterized. In this study, we showed that OsCIPK18, a CBL-interacting protein kinase, plays an important role in response to ammonium toxicity by comparative analysis of the physiological and whole transcriptome of the T-DNA insertion mutant (cipk18) and the wild-type (WT). Root biomass and length of cipk18 are less inhibited by excess NH 4 + compared with WT, indicating increased resistance to ammonium toxicity. Transcriptome analysis reveals that OsCIPK18 affects the NH 4 + uptake by regulating the expression of OsAMT1;2 and other NH 4 + transporters, but does not affect ammonium assimilation. Differentially expressed genes induced by excess NH 4 + in WT and cipk18 were associated with functions, such as ion transport, metabolism, cell wall formation, and phytohormones signaling, suggesting a fundamental role for OsCIPK18 in ammonium toxicity. We further identified a transcriptional regulatory network downstream of OsCIPK18 under NH 4 + stress that is centered on several core transcription factors. Moreover, OsCIPK18 might function as a transmitter in the auxin and abscisic acid (ABA) signaling pathways affected by excess ammonium. These data allowed us to define an OsCIPK18-regulated/dependent transcriptomic network for the response of ammonium toxicity and provide new insights into the mechanisms underlying ammonium toxicity.

Genome-Wide Identification and Evolution Analysis of the Gibberellin Oxidase Gene Family in Six Gramineae Crops.

The plant hormones gibberellins (GAs) regulate plant growth and development and are closely related to the yield of cash crops. The GA oxidases (GAoxs), including the GA2ox, GA3ox, and GA20ox subfamilies, play pivotal roles in GAs' biosynthesis and metabolism, but their classification and evolutionary pattern in Gramineae crops remain unclear. We thus conducted a comparative genomic study of GAox genes in six Gramineae representative crops, namely, Setaria italica (Si), Zea mays (Zm), Sorghum bicolor (Sb), Hordeum vulgare (Hv), Brachypodium distachyon (Bd), and Oryza sativa (Os). A total of 105 GAox genes were identified in these six crop genomes, belonging to the C19-GA2ox, C20-GA2ox, GA3ox, and GA20ox subfamilies. Based on orthogroup (OG) analysis, GAox genes were divided into nine OGs and the number of GAox genes in each of the OGs was similar among all tested crops, which indicated that GAox genes may have completed their family differentiations before the species differentiations of the tested species. The motif composition of GAox proteins showed that motifs 1, 2, 4, and 5, forming the 2OG-FeII_Oxy domain, were conserved in all identified GAox protein sequences, while motifs 11, 14, and 15 existed specifically in the GA20ox, C19-GA2ox, and C20-GA2ox protein sequences. Subsequently, the results of gene duplication events suggested that GAox genes mainly expanded in the form of WGD/SD and underwent purification selection and that maize had more GAox genes than other species due to its recent duplication events. The cis-acting elements analysis indicated that GAox genes may respond to growth and development, stress, hormones, and light signals. Moreover, the expression profiles of rice and maize showed that GAox genes were predominantly expressed in the panicles of the above two plants and the expression of several GAox genes was significantly induced by salt or cold stresses. In conclusion, our results provided further insight into GAox genes' evolutionary differences among six representative Gramineae and highlighted GAox genes that may play a role in abiotic stress.

Genome-Wide Identification and Expression Profiles of 13 Key Structural Gene Families Involved in the Biosynthesis of Rice Flavonoid Scaffolds.

Flavonoids are a class of key polyphenolic secondary metabolites with broad functions in plants, including stress defense, growth, development and reproduction. Oryza sativa L. (rice) is a well-known model plant for monocots, with a wide range of flavonoids, but the key flavonoid biosynthesis-related genes and their molecular features in rice have not been comprehensively and systematically characterized. Here, we identified 85 key structural gene candidates associated with flavonoid biosynthesis in the rice genome. They belong to 13 families potentially encoding chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), flavonol synthase (FLS), leucoanthocyanidin dioxygenase (LDOX), anthocyanidin synthase (ANS), flavone synthase II (FNSII), flavanone 2-hydroxylase (F2H), flavonoid 3'-hydroxylase (F3'H), flavonoid 3',5'-hydroxylase (F3'5'H), dihydroflavonol 4-reductase (DFR), anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR). Through structural features, motif analyses and phylogenetic relationships, these gene families were further grouped into five distinct lineages and were examined for conservation and divergence. Subsequently, 22 duplication events were identified out of a total of 85 genes, among which seven pairs were derived from segmental duplication events and 15 pairs were from tandem duplications, demonstrating that segmental and tandem duplication events play important roles in the expansion of key flavonoid biosynthesis-related genes in rice. Furthermore, these 85 genes showed spatial and temporal regulation in a tissue-specific manner and differentially responded to abiotic stress (including six hormones and cold and salt treatments). RNA-Seq, microarray analysis and qRT-PCR indicated that these genes might be involved in abiotic stress response, plant growth and development. Our results provide a valuable basis for further functional analysis of the genes involved in the flavonoid biosynthesis pathway in rice.

High-resolution bin-based linkage mapping uncovers the genetic architecture and heterosis-related loci of plant height in indica-japonica derived populations.

Plant height (PH) is an important trait affecting the plant architecture, seed yield, and harvest index. However, the molecular mechanisms underlying PH heterosis remain unclear. In addition, useful PH-related genes must be urgently identified to facilitate ideal plant architecture breeding in rice (Oryza sativa L.). In the present study, to explore rice quantitative trait loci (QTLs) and heterosis-related loci of PH in rice, we developed a high-generation (>F15 ) population of 272 recombinant inbred lines (RIL) from a cross of two elite varieties, Luohui 9 (indica/xian) × RPY geng (japonica/geng), and two testcross hybrid populations derived from the crosses of RILs and two cytoplasmic male sterile lines (YTA [indica] and Z7A [japonica]). Using deep resequencing data, a high-density genetic map containing 4758 bin markers was constructed, with a total map distance of 2356.41 cM. Finally, 31 PH-related QTLs for different PH component lengths or tiller numbers across five seasons were identified. Two major environment-specific PH QTLs were stably detected in Hainan (qPH-3.1) or Hubei (qPH-5.1), which have undergone significant functional alterations in rice with changes in geographical environment. Based on comparative genomics, gene function annotation, homolog identification, and existing literature (pioneering studies), candidate genes for multiple QTLs were fine-mapped, and the candidate genes qPH-3.1 and qPH-5.1 for PH were further validated using CRISPR-Cas9 gene editing. Specifically, qPH-3.1 was characterized as a pleiotropic gene, and the qPH-3.1 knockout line showed reduced PH, delayed heading, a decreased seed setting rate, and increased tiller numbers. Importantly, 10 PH heterosis-related QTLs were identified in the testcross populations, and a better-parent heterosis locus (qBPH-5.2) completely covered qPH-5.1. Furthermore, the cross results of fixed-genotype RILs verified the dominant effects of qPH-3.1 and qPH-5.1. Together, these findings further our understanding of the genetic mechanisms of PH and offer multiple highly reliable gene targets for breeding rice varieties with ideal architecture and high yield potential in the immediate future.

Bin mapping-based QTL analyses using three genetic populations derived from indica-japonica crosses uncover multiple grain shape heterosis-related loci in rice.

Exploitation of heterosis between indica and japonica has important significance in scientific research and agriculture application. However, the molecular mechanism of grain shape heterosis in indica-japonica hybrid remains unknown in rice (Oryza sativa L.). To reveal the genetic mechanism of grain shape in indica--japonica hybrid, we constructed a high-generation recombinant inbred line (RIL) population and two testcross hybrid populations derived from the cross of RILs and two cytoplasmic male sterile material (YTA and Z7A) and then performed a bin mapping-based quantitative trait locus (QTL) mapping of multiple grain shape traits, such as grain length (GL), grain width (GW), and grain length-to-width ratio (GLWR). A total of sixteen QTLs and 30 heterosis-related QTLs of grain shape traits were detected. We found that GS3, GS5, and OsPPKL2 were also correlated with grain shape both in RILs and two testcross hybrid populations. Homologous gene analysis emphasized two candidate grain shape-associated genes (LOC_Os06g14260 and LOC_Os04g51950). Our findings uncover multiple grain shape heterosis-related loci and provides a new insight into heterosis mechanism of grain shape in rice.

Uncovering the Novel QTLs and Candidate Genes of Salt Tolerance in Rice with Linkage Mapping, RTM-GWAS, and RNA-seq.

Salinity is a major abiotic stress that limits plant growth and crop productivity. Indica rice and japonica rice show significant differences in tolerance to abiotic stress, and it is considered a feasible method to breed progeny with stronger tolerance to abiotic stress by crossing indica and japonica rice. We herein developed a high-generation recombinant inbred lines (RILs) from Luohui 9 (indica) X RPY geng (japonica). Based on the high-density bin map of this RILs population, salt tolerance QTLs controlling final survival rates were analyzed by linkage mapping and RTM-GWAS methods. A total of seven QTLs were identified on chromosome 3, 4, 5, 6, and 8. qST-3.1, qST-5.1, qST-6.1, and qST-6.2 were novel salt tolerance QTLs in this study and their function were functionally verified by comparative analysis of parental genotype RILs. The gene aggregation result of these four new QTLs emphasized that the combination of the four QTL synergistic genotypes can significantly improve the salt stress tolerance of rice. By comparing the transcriptomes of the root tissues of the parents' seedlings, at 3 days and 7 days after salt treatment, we then achieved fine mapping of QTLs based on differentially expressed genes (DEGs) identification and DEGs annotations, namely, LOC_Os06g01250 in qST-6.1, LOC_Os06g37300 in qST-6.2, LOC_Os05g14880 in qST-5.1. The homologous genes of these candidate genes were involved in abiotic stress tolerance in different plants. These results indicated that LOC_Os05g14880, LOC_Os06g01250, and LOC_Os06g37300 were the candidate genes of qST-5.1, qST-6.1, and qST-6.2. Our finding provided novel salt tolerance-related QTLs, candidate genes, and several RILs with better tolerance, which will facilitate breeding for improved salt tolerance of rice varieties and promote the exploration tolerance mechanisms of rice salt stress.