Identification of qGL4.1 and qGL4.2, two closely linked QTL controlling grain length in rice.

Grain size is an important appearance quality trait in rice, which also affects grain yield. In this study, a recombinant inbred line (RIL) population derived from a cross between indica variety 9311 and japonica variety Cypress was constructed. And 181 out of 600 RILs were sequenced, and a high-density genetic map containing 2842 bin markers was constructed, with a total map length of 1500.6 cM. A total of 10 quantitative trait loci (QTL) related to grain length (GL), grain width (GW), grain length-to-width ratio (LWR), and 1000-grain weight (TGW) were detected under two environments. The genetic effect of qGL4, a minor QTL for GL and TGW, was validated using three heterogeneous inbred family (HIF) segregation populations. It was further dissected into two closed linked QTL, qGL4.1 and qGL4.2. By progeny testing, qGL4.1 and qGL4.2 were successfully delimited to intervals of 1304-kb and 423-kb, respectively. Our results lay the foundation for the map-based cloning of qGL4.1 and qGL4.2 and provide new gene resources for the improvement of grain yield and quality in rice. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-024-01447-y.

CRISPR/Cas9-mediated editing of Wx and BADH2 genes created glutinous and aromatic two-line hybrid rice.

Amylose content (AC) is one of the physicochemical indexes of rice quality, which is largely determined by the Waxy (Wx) gene. Fragrance in rice is favored because it adds good flavor and a faint scent. Loss of function of the BADH2 (FGR) gene promotes the biosynthesis of 2-acetyl-1-pyrroline (2AP), which is the main compound responsible for aroma in rice. Here, we used a CRISPR/Cas9 system to simultaneously knock out Wx and FGR genes in 1892S and M858, which are the parents of an indica two-line hybrid rice, Huiliangyou 858 (HLY858). Four T-DNA-free homozygous mutants (1892Swxfgr-1, 1892Swxfgr-2, M858wxfgr-1, and M858wxfgr-2) were obtained. The 1892Swxfgr and M858wxfgr were crossed to generate double mutant hybrid lines HLY858wxfgr-1 and HLY858wxfgr-2. Size-exclusion chromatography (SEC) data indicated that true AC of the wx mutant starches ranged from 0.22 to 1.63%, much lower than those of the wild types (12.93 to 13.76%). However, the gelatinization temperature (GT) of the wx mutants in backgrounds of 1892S, M858, and HLY858 were still high, and showed no significant differences with the wild type controls. The aroma compounds 2AP content in grains of HLY858wxfgr-1 and HLY858wxfgr-2 were 153.0 µg/kg and 151.0 µg/kg, respectively. In contrast, 2AP was not detected in grains of HLY858. There were no significant differences in major agronomic traits between the mutants and HLY858. This study provides guidelines for cultivation of ideal glutinous and aromatic hybrid rice by gene editing.

Albino seedling lethality 4; Chloroplast 30S Ribosomal Protein S1 is Required for Chloroplast Ribosome Biogenesis and Early Chloroplast Development in Rice.

BACKGROUND: Ribosomes responsible for transcription and translation of plastid-encoded proteins in chloroplasts are essential for chloroplast development and plant growth. Although most ribosomal proteins in plastids have been identified, the molecular mechanisms regulating chloroplast biogenesis remain to be investigated. RESULTS: Here, we identified albinic seedling mutant albino seedling lethality 4 (asl4) caused by disruption of 30S ribosomal protein S1 that is targeted to the chloroplast. The mutant was defective in early chloroplast development and chlorophyll (Chl) biosynthesis. A 2855-bp deletion in the ASL4 allele was verified as responsible for the mutant phenotype by complementation tests. Expression analysis revealed that the ASL4 allele was highly expressed in leaf 4 sections and newly expanded leaves during early leaf development. Expression levels were increased by exposure to light following darkness. Some genes involved in chloroplast biogenesis were up-regulated and others down-regulated in asl4 mutant tissues compared to wild type. Plastid-encoded plastid RNA polymerase (PEP)-dependent photosynthesis genes and nuclear-encoded phage-type RNA polymerase (NEP)-dependent housekeeping genes were separately down-regulated and up-regulated, suggesting that plastid transcription was impaired in the mutant. Transcriptome and western blot analyses showed that levels of most plastid-encoded genes and proteins were reduced in the mutant. The decreased contents of chloroplast rRNAs and ribosomal proteins indicated that chloroplast ribosome biogenesis was impaired in the asl4 mutant. CONCLUSIONS: Rice ASL4 encodes 30S ribosomal protein S1, which is targeted to the chloroplast. ASL4 is essential for chloroplast ribosome biogenesis and early chloroplast development. These data will facilitate efforts to further elucidate the molecular mechanism of chloroplast biogenesis.

A Young Seedling Stripe2 phenotype in rice is caused by mutation of a chloroplast-localized nucleoside diphosphate kinase 2 required for chloroplast biogenesis.

Chloroplast development and chlorophyll (Chl) biosynthesis in plants are regulated by many genes, but the underlying molecular mechanisms remain largely elusive. We isolated a rice mutant named yss2 (young seedling stripe2) with a striated seedling phenotype beginning from leaf 2 of delayed plant growth. The mutant developed normal green leaves from leaf 5, but reduced tillering and chlorotic leaves and panicles appeared later. Chlorotic yss2 seedlings have decreased pigment contents and impaired chloroplast development. Genetic analysis showed that the mutant phenotype was due to a single recessive gene. Positional cloning and sequence analysis identified a single nucleotide substitution in YSS2 gene causing an amino acid change from Gly to Asp. The YSS2 allele encodes a NDPK2 (nucleoside diphosphate kinase 2) protein showing high similarity to other types of NDPKs. Real-time RT-PCR analysis demonstrated that YSS2 transcripts accumulated highly in L4 sections at the early leaf development stage. Expression levels of genes associated with Chl biosynthesis and photosynthesis in yss2 were mostly decreased, but genes involved in chloroplast biogenesis were up-regulated compared to the wild type. The YSS2 protein was associated with punctate structures in the chloroplasts of rice protoplasts. Our overall data suggest that YSS2 has important roles in chloroplast biogenesis.

A Young Seedling Stripe2 phenotype in rice is caused by mutation of a chloroplast-localized nucleoside diphosphate kinase 2 required for chloroplast biogenesis

Abstract Chloroplast development and chlorophyll (Chl) biosynthesis in plants are regulated by many genes, but the underlying molecular mechanisms remain largely elusive. We isolated a rice mutant named yss2 (young seedling stripe2) with a striated seedling phenotype beginning from leaf 2 of delayed plant growth. The mutant developed normal green leaves from leaf 5, but reduced tillering and chlorotic leaves and panicles appeared later. Chlorotic yss2 seedlings have decreased pigment contents and impaired chloroplast development. Genetic analysis showed that the mutant phenotype was due to a single recessive gene. Positional cloning and sequence analysis identified a single nucleotide substitution in YSS2 gene causing an amino acid change from Gly to Asp. The YSS2 allele encodes a NDPK2 (nucleoside diphosphate kinase 2) protein showing high similarity to other types of NDPKs. Real-time RT-PCR analysis demonstrated that YSS2 transcripts accumulated highly in L4 sections at the early leaf development stage. Expression levels of genes associated with Chl biosynthesis and photosynthesis in yss2 were mostly decreased, but genes involved in chloroplast biogenesis were up-regulated compared to the wild type. The YSS2 protein was associated with punctate structures in the chloroplasts of rice protoplasts. Our overall data suggest that YSS2 has important roles in chloroplast biogenesis.

Adjuvanticity of compound polysaccharides on chickens against Newcastle disease and avian influenza vaccine.

This study was conducted to evaluate the effects of compound polysaccharides (cPS) on the immune responses via chicken models. First, in screening experiment, a comprehensive analysis for immunomodulatory activity of four cPSs, including Astragalus polysaccharides (APS), Epimedium polysaccharides (EPS), sulfated APS (sAPS) and sulfated EPS (sEPS), was performed in vitro and in vivo. APS-sEPS was picked out having the best effect on lymphocyte proliferation and raising the antibody titers. Therefore, the adjuvanticities of APS-sEPS on Newcastle disease (ND) and avian influenza (AI) vaccine were further validated. Chickens were administrated with ND or AI vaccines containing APS-sEPS of 150, 100 and 50 mg/kg, respectively, taking oil adjuvant vaccine as control. It was observed ND or AI antibody titers and lymphocyte proliferation were enhanced at 100 mg/kg of APS-sEPS. In conclusion, appropriate dose of APS-sEPS may be a safe and efficacious immune stimulator candidate suitable for vaccines.

Optimization of sulfated modification conditions of tremella polysaccharide and effects of modifiers on cellular infectivity of NDV.

Based on our previous research, sulfated modification conditions of Tremella polysaccharide (TPS), the chlorosulfonic acid to pyridine (CSA-Pry) ratio, reaction temperature and time, were optimized by L(9) (3(4)) orthogonal design taking the yield and degree of sulfation (DS) of modifiers as indexes. Two TPSs, TPS(tp) and TPS(70c), were modified under optimized conditions. The effects of two modifiers, sTPS(tp) and sTPS(70c), on cellular infectivity of NDV were determined by MTT method taking the non-modified TPS(tp), TPS(tc) and TPS(70c) as controls. The results showed that the optimized modification conditions were reaction temperature of 80°C, CSA-Pry ratio of 1:6 and reaction time of 1.5h. Five polysaccharides at proper concentrations could significantly inhibit the infectivity of NDV to CEF. The virus inhibitory rates of sTPS(tp) at 1.563 µg mL(-1) group were the highest and significantly higher than those of other three non-modified polysaccharide groups in three sample-adding modes. This indicated that sulfated modification could significantly improve the antiviral activity of TPS. sTPS(tp) possessed the best efficacy and would be as a component of antiviral polysaccharide drug.

Epimedium polysaccharide and propolis flavone can synergistically stimulate lymphocyte proliferation in vitro and enhance the immune responses to ND vaccine in chickens.

Four prescriptions, epimedium flavone plus propolis flavone (EF-PF), epimedium flavone plus propolis extracts (EF-PE), epimedium polysaccharide plus propolis flavone (EP-PF) and epimedium polysaccharide plus propolis extracts (EP-PE), were prepared and their immune-enhancing effects were compared. In test in vitro, the effects of them on chicken peripheral lymphocyte proliferation were determined by MTT method. The results showed that EP-PF group presented the highest stimulating index at most concentrations. In immune test, 300 14-day-old chickens were randomly divided into six groups and vaccinated with ND vaccine except for blank control (BC) group, re-challenged at 28 days of age. At the same time of the first vaccination, the chickens in four experimental groups were injected, respectively, with four prescriptions. The changes of the lymphocyte proliferation and antibody titer were determined. On day 28 after the first vaccination, the chickens except for BC group were challenged with NDV, the immune protective effect was observed. The results displayed that in EP-PF group, the antibody titers, lymphocyte proliferation and protective rate were the highest, the morbidity and mortality were the lowest. In dose test, 14-day-old chickens were randomly divided into five groups. The treatment and determinations were the same as the immune test except that the chickens in experimental groups were injected, respectively, with high, medium and low doses of EP-PF. The results revealed that in medium dose group, the antibody titers, lymphocyte proliferation and protective rate were the highest, the morbidity and mortality were the lowest. These results indicated that EP and PF possessed synergistically immune enhancement, EP-PF had the best efficacy, especially at medium dose, and would be expected to exploit into a new-type immunopotentiator.