CLD1/SRL1 modulates leaf rolling by affecting cell wall formation, epidermis integrity and water homeostasis in rice.

Leaf rolling is considered as one of the most important agronomic traits in rice breeding. It has been previously reported that SEMI-ROLLED LEAF 1 (SRL1) modulates leaf rolling by regulating the formation of bulliform cells in rice (Oryza sativa); however, the regulatory mechanism underlying SRL1 has yet to be further elucidated. Here, we report the functional characterization of a novel leaf-rolling mutant, curled leaf and dwarf 1 (cld1), with multiple morphological defects. Map-based cloning revealed that CLD1 is allelic with SRL1, and loses function in cld1 through DNA methylation. CLD1/SRL1 encodes a glycophosphatidylinositol (GPI)-anchored membrane protein that modulates leaf rolling and other aspects of rice growth and development. The cld1 mutant exhibits significant decreases in cellulose and lignin contents in secondary cell walls of leaves, indicating that the loss of function of CLD1/SRL1 affects cell wall formation. Furthermore, the loss of CLD1/SRL1 function leads to defective leaf epidermis such as bulliform-like epidermal cells. The defects in leaf epidermis decrease the water-retaining capacity and lead to water deficits in cld1 leaves, which contribute to the main cause of leaf rolling. As a result of the more rapid water loss and lower water content in leaves, cld1 exhibits reduced drought tolerance. Accordingly, the loss of CLD1/SRL1 function causes abnormal expression of genes and proteins associated with cell wall formation, cuticle development and water stress. Taken together, these findings suggest that the functional roles of CLD1/SRL1 in leaf-rolling regulation are closely related to the maintenance of cell wall formation, epidermal integrity and water homeostasis.

A guanine insert in OsBBS1 leads to early leaf senescence and salt stress sensitivity in rice (Oryza sativa L.).

KEY MESSAGE: A rice receptor-like kinase gene OSBBS1/OsRLCK109 was identified; this gene played vital roles in leaf senescence and the salt stress response. Early leaf senescence can cause negative effects on rice yield, but the underlying molecular regulation is not fully understood. bilateral blade senescence 1 (bbs1), an early leaf senescence mutant with a premature senescence phenotype that occurs mainly performing at the leaf margins, was isolated from a rice mutant population generated by ethylmethane sulfonate (EMS) treatment. The mutant showed premature leaf senescence beginning at the tillering stage and exhibited severe symptoms at the late grain-filling stage. bbs1 showed accelerated dark-induced leaf senescence. The OsBBS1 gene was cloned by a map-based cloning strategy, and a guanine (G) insertion was found in the first exon of LOC_Os03g24930. This gene encodes a receptor-like cytoplasmic kinase and was named OsRLCK109 in a previous study. Transgenic LOC_Os03g24930 knockout plants generated by a CRISPR/Cas9 strategy exhibited similar early leaf senescence phenotypes as did the bbs1 mutant, which confirmed that LOC_Os03g24930 was the OsBBS1 gene. OsBBS1/OsRLCK109 was expressed in all detected tissues and was predominantly expressed in the main vein region of mature leaves. The expression of OsBBS1 could be greatly induced by salt stress, and the bbs1 mutant exhibited hypersensitivity to salt stress. In conclusion, this is the first identification of OsRLCKs participating in leaf senescence and playing critical roles in the salt stress response in rice (Oryza sativa L.).

The ferredoxin-dependent glutamate synthase (OsFd-GOGAT) participates in leaf senescence and the nitrogen remobilization in rice.

Ferredoxin-dependent glutamate synthase (Fd-GOGAT, EC 1.4.7.1) plays major roles in photorespiration and primary nitrogen assimilation. However, due to no mutant or knockdown lines of OsFd-GOGAT have been reported in rice (Oryza sativa L.), the contribution of OsFd-GOGAT to rice foliar nitrogen metabolism remains little up-to-date. Here, we isolated a rice premature leaf senescence mutant named gogat1, which was reduced in 67% of the total GOGAT enzyme activity in leaves. The gogat1 mutant exhibited chlorosis under natural condition, while showed less extent premature leaf senescence under low light treatment. The gogat1 locus was mapped to a 54.1 kb region on chromosome 7, and the sequencing of OsFd-GOGAT showed one substitution (A to T) at the 3017th nucleotide of the open reading frame, leading to the amino-acid substitution of leucine changed to histidine. The gogat1 mutant showed reduced seed setting rate, while the grain protein content in gogat1 mutant was significantly higher than that in wild type. Meanwhile, during the grain-filling stage, total amino acids in the up three leaves and the upmost internode were increased dramatically. The results in this study suggested that OsFd-GOGAT might participate in nitrogen remobilization during leaf senescence, which provides a potential way to improve nitrogen use efficiency in rice.

QTL mapping based on the embryo and maternal genetic systems for non-essential amino acids in rapeseed (Brassica napus L.) meal.

BACKGROUND: Non-essential amino acids are a good source of nitrogen and also very important contributors to the metabolic process. Analysis of quantitative trait locus (QTL) simultaneously located on the amphidiploid embryo and maternal plant nuclear genomes for non-essential amino acid contents in rapeseed meal across different environments was conducive to further clarify the genetic mechanism of seed quality traits. RESULTS: Twenty-eight QTLs associated with arginine (five QTLs), histidine (four QTLs), glutamic acid (three QTLs), glycine (three QTLs), proline (three QTLs), alanine (four QTLs) and aspartic acid (six QTLs) contents were identified in present study. All of these QTLs had significant additive main effects from embryo and maternal plant nuclear genomes with eight of them showing significant embryo dominance main effects and 12 showing notable QTL × environment interaction effects. Among them, 12 QTLs were major QTLs which could explain 13.27-35.71% of the phenotypic variation. Specially, five QTL clusters associated with several QTLs related to multiple traits were distributed on chromosomes A1, A4, A5, A7 and C2. CONCLUSION: Non-essential amino acids in rapeseed meal could be simultaneously controlled by the genetic effects from the QTLs which were located on the chromosomes both in the embryo and maternal plant genetic systems.

Differential iron-bioavailability with relation to nutrient compositions in polished rice among selected Chinese genotypes using Caco-2 cell culture model.

Genotypic variation of iron bioavailability and the relationship between iron bioavailability and nutrient composition in polished rice among 11 rice genotypes were assessed using an in vitro digestion/Caco-2 cell model. The results indicated that significant differences in iron bioavailability were detected among tested rice genotypes, with a 3-fold range, suggesting a possibility of selecting high bioavailable iron by plant breeding. Although iron bioavailability was not significantly correlated with Fe concentration in polished rice among tested rice genotypes, the results also indicated that most of the iron dense genotypes showed relatively high ferritin formation in Caco-2 cell and transported iron. Additionally, iron bioavailability in polished rice was enhanced by addition of ascorbic acid, with a much wider range of Fe bioavailability variation in polished rice with addition of ascorbic acid than that without addition of ascorbic acid. The positive relationship between iron bioavailability in polished rice and cysteine concentration (R = 0.669) or sulfur (S) concentration (R = 0.744) among tested rice genotypes, suggests that cysteine and sulfur concentration in polished rice could be used as an indicator for high iron bioavailability.

[Promotion of tuition quality in Genetics by developing an excellent instructional system].

This article, which is based on the requirements for developing the course of Genetics as an outstanding course, summarizes the experience of engendering an excellent instructional system, expatiates on the effects of its application to the teaching process of "Genetics" and next step work for continuing this instructional system. The course quality of "Genetics" has been improved under the excellent instructional system by including different teaching methods, renovating the teaching contents, innovating the teaching means, developing a practical courseware, writing extractive textbooks, reforming experimental teaching, and constructing an instructional network, together with teaching methods related to scientific research.

[Morphological characteristics and gene mapping of a novel bent pedicel branch (bpb1) mutant in rice].

Rice pedicels are tightly associated with the yield of grain. In the present study, a novel and stable pedicel mutant bpb1 (bent pedicel branch 1) was obtained from the wild type "Zhenong 7" after 60Co γ-ray treatment. The mutant had the typical phenotype of bent pedicel branches with multiple abnormal phenotypes, such as longer pedicels, short panicles, and dwarfism. Detail examination using scanning electron microscopy revealed that the pedicel epidermal hairs and stomas in the mutant were smaller than those in the wild type. The epidermal and sclerenchymatous cells were arranged irregularly, and the cells in the bend region of pedicels became smaller and arranged closely. The transverse observation of the mutant pedicel branches showed that the small vascular bundles arranged differently from those of the wild type. Genetic analysis indicated that the abnormal phenotypes were controlled by a single recessive gene. Using the F2 mapping population from the bpb1 mutant crossed with the japonica rice variety "Zhenongda 104", the bpb1 gene was mapped in a 343 kb region between two SSR markers, RM21537 and RM21552, at the long arm of chromosome 7. Because no homologous gene was found in this region until now, bpb1 might be a novel gene related to the pedicel development and growth. This study could be beneficial to future cloning and functional analysis of the bpb1 gene.

[Improving experimental teaching facilities and opening up of laboratories in order to raise experimental teaching quality of genetics].

Advanced teaching facilities and the policy of opening laboratories to students play an important role in raising the quality in the experimental teaching of Genetics. This article introduces the superiority of some advanced instruments and equipment (such as digital microscope mutual laboratory system, flow cytometry, and NIRSystems) in the experimental teaching of genetics, and illustrates with examples the significance of exposing students to experiments in developing their creative consciousness and creative ability. This article also offers some new concepts on the further improvement upon teaching in the laboratory.

[Study on the global and local calibration methods of tryptophan content in rice by near infrared reflectance spectroscopy].

Tryptophan is one of the essential amino acids in human, and also considered as an important limiting amino acid in rice. Alkali hydrolysis method was used to determine the tryptophan content of 272 representative samples selected from the 1 256 samples in four years. Based on the milled rice flour spectroscopy, by comparing the prediction abilities of the equations with global calibrations and local calibration, the global calibration method with MPLS and the local calibration method were better than others. Their calibration models could be introduced to application because of low standard errors of prediction for both methods (0.007%) and high coefficients of determination (87.1% and 87.4%, respectively). Furthermore, the prediction ability of the equation based on the brown rice spectroscopy could be still used for determination application though their effects were worse than that of the milled rice flour spectroscopy. It was concluded that the equation could be directly used to estimate the content of tryptophan in milled rice, and the technology of near infrared reflectance spectroscopy developed in this study could be applied in selecting breeding materials for higher tryptophan content and in quality control during rice processing.

[Compilation and practice for "Genetics" multimedia textbook].

Textbook is an important tool for generating knowledge and is closely related to the teaching effect. Now the multimedia teaching method has become the common norm in teaching process of higher education. This paper describes the compilation of different forms of "Genetics" multimedia textbook, the selection of contents and knowledge, the arrangement of knowledge module and systematic configuration. The characteristics of compiling process of multimedia textbook are also discussed.

[Exploration and practice of inquiring teaching for the experiments of genetics].

Aiming for fostering talents with the sense and ability of creation, the current wave of experimental teaching reform demands significant improvements in genetics. We proposed a train of thoughts about transforming the testing and verifying experiments of genetics into inquiring forms with comprehensive or designing property, and gave some examples to carry out inquiring teaching for the experiments of genetics.

Rice SDSFL1 plays a critical role in the regulation of plant structure through the control of different phytohormones and altered cell structure.

Semi-dwarfism is one of the most important agronomic traits for many cereal crops. In the present study, a mutant with semi-dwarf and short flag leaf 1, sdsfl1, was identified and characterized. The sdsfl1 mutant demonstrated some distinguished structural alterations, including shorter plant height and flag leaf length, increased tiller numbers and flag leaf width, and decreased panicle length compared with those of wild type (WT). Genetic analysis suggested that the mutant traits were completely controlled by a single recessive gene. The SDSFL1 gene was mapped to the long arm of chromosome 3 within a region of 44.6 kb between InDel markers A3P8.3 and A3P8.4. The DNA sequence analysis revealed that there was only a T to C substitution in the coding region of LOC_Os03g63970, resulting in the substitution of Tryptophan (Try) to Arginine (Arg) and encoding a GA 20 oxidase 1 protein of 372 amino acid residues. Photosynthesis analysis showed that the photosynthetic rate (Pn), stomatal conductance (Gs), and intercellular CO2 concentration (Ci) were significantly increased in sdsfl1. Chlorophyll a (Chl a), total Chl, and carotenoid contents were significantly increased in sdsfl1 compared with those in WT. sdsfl1 carried a reduced level of GA3 but reacted to exogenously applied gibberellins (GA). Moreover, the levels of abscisic acid (ABA), indole 3-acetic acid (IAA), and salicylic acid (SA) were notably improved in sdsfl1, whereas there was no noteworthy change in jasmonic acid (JA). The results thus offer a visible foundation for the molecular and physiological analysis of the SDSFL1 gene, which might participate in various functional pathways for controlling plant height and leaf length in rice breeding.

[The screening of mutants induced by physical and chemical factors and construction of mutant population for Brassica napus L].

The mature seeds of rapeseed (Brassica napus L.) from variety Gaoyou 605 were treated with g-rays and Ethyl Methan Sulfonate (EMS). 152 mutants (12.67% of M2 population) with mutative traits, including the mutation of leaf color, leaf shape, plant height, number and angle of branches, diameter of main stalk, color of stalk and flower, number and size of petals, pistil shape, male sterility, bud death and date of bloom were found in screened M2 progenies, which have been identified in M3. The mutants of cotyledon and root traits were also screened by hydroponics culture and their total mutant frequency were estimated at 12.78% and 7.07% in M3, respectively. Identification of M4 showed that these mutations could be inherited stably. The mutant library including the mutants of leaf, plant-type, flower, cotyledon, root and physiological traits had been built in present experiment. These mutants might be used as important germplasm for rapeseed breeding and functional genomics study.

[Creating a software package and improving the multimedia teaching of genetics course].

Multimedia as a pedagogical tool has been widely adopted for improving the quality of teaching. This paper describes the construction of a teaching software package based on the characteristics of the "Genetics" course. It also discusses some particulars of multimedia teaching, the collection of teaching materials, the improvement of teaching effects, and the compilation of a multimedia textbook. How to enhance the quality of the teachers as well as the motivation of the students are also discussed.

[Study on developing calibration models of fatty acid composition in intact rapeseed by near infrared reflectance spectroscopy].

Four hundred ninety seven rapeseed samples, which feature multi-year, multi-loci and highly variant characteristics, were collected as a raw set. The NIR spectra were pretreated by scatter correction and mathematics treatments, and calibration models of fat acid composition of intact rapeseed were developed by using the algorithm method of modified partial least square (MPLS). Meanwhile, three types of sample cups with different capacity were used to screen the suitable calibration model for rapeseed quality breeding. The results showed that the calibration model of 8-gram-sample was the best, and the calibration determination coefficient was in the range of 0.74-0.98. The calibration effects of 3-gram-sample, which were similar to those of 0.6-gram-sample, were good with high determination coefficient (RSQ1, 1-VR) and low error (SEC, SECV). Therefore, the calibration set with multi-year and multi-loci samples can improve the accuracy and repeatability of NIRS models. The fat acids NIRS models of intact rapeseed developed could be introduced into breeding lines' selection, mutant screening and germplasm evaluation.

REL2, A Gene Encoding An Unknown Function Protein which Contains DUF630 and DUF632 Domains Controls Leaf Rolling in Rice.

BACKGROUND: Rice leaves are important energy source for the whole plant. An optimal structure will be beneficial for rice leaves to capture light energy and exchange gas, thus increasing the yield of rice. Moderate leaf rolling and relatively erect plant architecture may contribute to high yield of rice, but the relevant molecular mechanism remains unclear. RESULTS: In this study, we identified and characterized a rolling and erect leaf mutant in rice and named it as rel2. Histological analysis showed that the rel2 mutant has increased number of bulliform cells and reduced size of middle bulliform cells. We firstly mapped REL2 to a 35-kb physical region of chromosome 10 by map-based cloning strategy. Further analysis revealed that REL2 encodes a protein containing DUF630 and DUF632 domains. In rel2 mutant, the mutation of two nucleotide substitutions in DUF630 domain led to the loss-of-function of REL2 locus and the function of REL2 could be confirmed by complementary expression of REL2 in rel2 mutant. Further studies showed that REL2 protein is mainly distributed along the plasma membrane of cells and the REL2 gene is relatively higher expressed in younger leaves of rice. The results from quantitative RT-PCR analysis indicated that REL2 functioning in the leaf shape formation might have functional linkage with many genes associated with the bulliform cells development, auxin synthesis and transport, etc. CONCLUSIONS: REL2 is the DUF domains contained protein which involves in the control of leaf rolling in rice. It is the plasma membrane localization and its functions in the control of leaf morphology might involve in multiple biological processes such as bulliform cell development and auxin synthesis and transport.

[Establishment of Genic Male Sterile Lines of Brassica napus by Wide Cross and Their Cytology and Morphology.].

One and three male sterile plants were found in the progenies of the intergeneric hybrids by crossing Brassica napus and Brassica juncea to Orychophragmus violaceus, respectively. After interspecific cross and full-sib crosses, four genic male sterile lines were obtained. The results of cytological observations indicated that somatic chromosome number was 38 in all types of sterile lines, which were considered as the type of B. napus. Most of pollen mother cells (PMC) in all sterile plants were observed normally for chromosome behaviour. But abnormal phenomena of chromosome, which present for chromosome lagging and bridge, were still found at each stage of meiosis in these sterile lines. These lines belonged to complete sterile type. There were distinct differences between sterile plants and normal plants in the flower morphology. Bud death at different degrees was found on anthotaxies in sterile plants. Studies on the flower growth showed that the growths of the pistils in sterile plants were gradually accelerated along with pollen sterility. At the same time there were two slowly growth stages of the pistils growth in normal plants. The prospects of these sterile lines were discussed in this paper.

[Improvement and optimization for "genetics" course teaching].

"Genetics" is one of the main courses for the study scheme of undergraduate students in university. This paper pointed out some problems found in the teaching process according to the characteristics of "Genetics" course. At the same time, the thought on the construction of "Genetics" course were discussed concerning the reform of course contents, the improvement of teaching method, the creation of teaching CAI (computer assisted instruction), the compile of electronic edition for teaching materials, the evaluation of teaching level, the innovation of experimental contents and the methods for improving the teaching effect.

[Genetic diversity of flue-cured tobacco varieties based on ISSR markers].

Genetic diversity was assessed among 24 flue-cured tobacco varieties by ISSR (inter simple sequence repeats). A total of 100 ISSR primers were used to amplify the DNA from these varieties, of which 10 primers produced reproducible amplified products. Using polyacrylamide gel electrophoresis 208 bands were identified, of which 141 bands were polymorphic among the flue-cured tobacco varieties analyzed. Each primer produced 7-37 bands, the length of which ranged 200-2,400 bp. The ratio of polymorphic bands (PPB) was 67.79%. By cluster analysis based on ISSR markers using UPGMA, 24 varieties were divided into 5 major groups, in which the biggest group consisted of 12 varieties derived from Coker319. The genetic similarity index was 0.66-0.85 among 24 flue-cured tobacco varieties. Low genetic diversity among flue-cured tobacco varieties suggested that it is necessary to expand the genetic base of the flue-cured tobacco. 24 varieties could be distinguished by using 2 ISSR markers. The result also indicated that ISSR analysis was suitable for varietal identification and the study on genetic diversity of tobacco germplasm.

[Developmental genetic analysis for transparency of rice (Oryza sativa L.) at different environments].

The developmental behavior of gene expression for transparency of rice (TR) was analyzed from two-year experimental data at different filling stages by using both conditional and unconditional analysis methods for quantitative traits of seeds in cereal crops. It was revealed that the gene expression was most active for the performance of TR at the middle and late filling stages (8-21 d after flowering). The differences of expression at different environments for triploid endosperm nuclear genes, cytoplasmic genes and diploid maternal plant nuclear genes were found for the performance of TR. The phenomena of which some genetic effects were spasmodically expressible were detected among filling stages of rice. Because of the higher additive and cytoplasmic effects along with their interaction effects for TR at the filling stages, the better improving effects for this trait could be expected by selection in rice breeding. Some parents such as Zhenan 3 and 1391 were better than others for improving TR.