Loss of function of OsL1 gene cause early flowering in rice under short-day conditions.

Heading date is one of the important agronomic traits that affects rice yield. In this study, we cloned a new rice B3 family gene, OsL1, which regulates rice heading date. Importantly, osl1-1 and osl1-2, two different types of mutants of OsL1 were created using the gene editing technology CRISPR/Cas9 system and exhibited 4 days earlier heading date than that of the wild type under short-day conditions. Subsequently, the plants overexpressing OsL1, OE-OsL1, showed a 2-day later heading date than the wild type in Changsha and a 5-day later heading date in Lingshui, but there was no significant difference in other yield traits. Moreover, the results of subcellular localization study indicated that OsL1 protein was located in the nucleus and the expression pattern analysis showed that OsL1 gene was expressed in rice roots, stems, leaves, and panicles, and the expression level was higher at the root and weak green panicle. In addition, the OsL1 gene was mainly expressed at night time under short-light conditions. The transcriptomic analysis indicated that OsL1 might be involved in the Hd1-Hd3a pathway function. Together, our results revealed that the cloning and functional analysis of OsL1 can provide new strategy for molecular design breeding of rice with suitable fertility period. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-024-01444-1.

EMBRYO SAC DEVELOPMENT 1 affects seed setting rate in rice by controlling embryo sac development.

Seed setting rate is one of the critical factors that determine rice yield. Grain formation is a complex biological process, whose molecular mechanism is yet to be improved. Here we investigated the function of an OVATE family protein, Embryo Sac Development 1 (ESD1), in the regulation of seed setting rate in rice (Oryza sativa) by examining its loss-of-function mutants generated via clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated9 (Cas9) technology. ESD1 was predominantly expressed at Stage 6 of panicle development, especially in the ovules. esd1 mutants displayed reduced seed setting rates with normal stamen development and pollen tube growth but abnormal pistil group. Investigation of embryo sacs revealed that during the mitosis of functional megaspores, some egg cells degraded during differentiation in esd1 mutants, thereby hindering subsequent fertilization process and reducing seed setting rate. In addition, the transcriptional level of O. sativa anaphase-promoting complex 6, a reported embryo sac developing gene, was significantly reduced in esd1 mutants. These results support that ESD1 is an important modulator of ESD and seed setting rate in rice. Together, this finding demonstrates that ESD1 positively regulates the seed setting rate by controlling ESD in rice and has implications for the improvement of rice yield.

A novel strategy for creating a new system of third-generation hybrid rice technology using a cytoplasmic sterility gene and a genic male-sterile gene.

Heterosis utilization is the most effective way to improve rice yields. The cytoplasmic male-sterility (CMS) and photoperiod/thermosensitive genic male-sterility (PTGMS) systems have been widely used in rice production. However, the rate of resource utilization for the CMS system hybrid rice is low, and the hybrid seed production for the PTGMS system is affected by the environment. The technical limitations of these two breeding methods restrict the rapid development of hybrid rice. The advantages of the genic male-sterility (GMS) rice, such as stable sterility and free combination, can fill the gaps of the first two generations of hybrid rice technology. At present, the third-generation hybrid rice breeding technology is being used to realize the application of GMS materials in hybrid rice. This study aimed to use an artificial CMS gene as a pollen killer to create a smart sterile line for hybrid rice production. The clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) technology was used to successfully obtain a CYP703A3-deficient male-sterile mutant containing no genetically modified component in the genetic background of indica 9311. Through young ear callus transformation, this mutant was transformed with three sets of element-linked expression vectors, including pollen fertility restoration gene CYP703A3, pollen-lethality gene orfH79 and selection marker gene DsRed2. The maintainer 9311-3B with stable inheritance was obtained, which could realize the batch breeding of GMS materials. Further, the sterile line 9311-3A and restorer lines were used for hybridization, and a batch of superior combinations of hybrid rice was obtained.

Structural variations of a new fertility restorer gene, Rf20, underlie the restoration of wild abortive-type cytoplasmic male sterility in rice.

The discovery of a wild abortive-type cytoplasmic male sterile line and the breeding of its restorer line have led to the commercialization of three-line hybrid rice, which has contributed greatly to global food security. However, the molecular mechanisms underlying fertility abortion and the restoration of wild abortive-type cytoplasmic male sterile lines largely remain elusive. In this study, we cloned a restorer gene, Rf20, following a genome-wide association study analysis of the core parent lines of three-line hybrid rice. We found that Rf20 was present in all core parental lines, but different haplotypes and structural variants of its gene resulted in differences in Rf20 expression levels between sterile and restored lines. Rf20 could restore fertility in the wild abortive-type cytoplasmic male sterile line and was found to be responsible for fertility restoration in some cytoplasmic male sterile lines under high temperature. In addition, we found that Rf20 encodes a pentatricopeptide repeat protein that competes with WA352 for binding with COX11. This interaction enhances COX11's function as a scavenger of reactive oxygen species, which in turn restores pollen fertility. In this study, a new model of pentatricopeptide repeat proteins involved in the fertility recovery of cytoplasmic male sterile lines was proposed, which provides an important theoretical basis for the breeding of strong restorer lines and for overcoming high-temperature fertility recovery of some three-line sterile lines.

Mutation at Different Sites of Metal Transporter Gene OsNramp5 Affects Cd Accumulation and Related Agronomic Traits in Rice (Oryza sativa L.).

OsNramp5 is a key gene involved in the control of the uptake of Cd, Mn, and other metal ions by rice root cells. The functional deficiency of this gene can significantly reduce the accumulation of Cd in rice grains, but the effects of its mutation on agronomic traits such as yield and quality have not been investigated comprehensively yet. In the present study, three Huanghuazhan-based OsNramp5 mutants [LCH1 (Low Cadmium Huanghuazhan 1), LCH2 (Low Cadmium Huanghuazhan 2), and LCH3 (Low Cadmium Huanghuazhan 3)] were obtained using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) technology. The mutation-type analysis showed that LCH1, LCH2, and LCH3 encoded defective OsNramp5 protein sequences containing only 76aa, 176aa, and 266aa, respectively. The determination of metal content and the statistics of related agronomic traits revealed that the functionally deficient OsNramp5 not only significantly reduced the accumulation of Cd in the grains of the mutants but also affected rice yield and quality. However, with the decrease of OsNramp5 mutation degree, its effects on chlorenchyma Mn accumulation, yield, and quality were also diminished. Additionally, we also found that the increase in the concentration of Mn in the soil restored the phenotype of the declined yield and quality due to the functional deficiency of OsNramp5. Our findings provide novel insights into and new materials for breeding rice varieties with low Cd accumulation and excellent agronomic traits under severe Cd pollution environment.

Characterization of the global transcriptome for cotton (Gossypium hirsutum L.) anther and development of SSR marker.

Cotton is an important fiber plant, and it's attractive to elucidate the molecular mechanism of anther development due to the close relationship between the anther fertility and boll-setting, and also fiber yield. In the present paper, 47.2 million paired-end reads with average length of 82.87 bp from the anthers of TM-1 (Gossypium hirsutum L.), a genetic standard line, were generated through transcriptome sequencing, and 210,965 unigenes of more than 100 bp were obtained. BLAST, KEGG, COG, and GO analyses showed that the genes were enriched in the processes of transcription, translation, and post-translation as well as hormone signal transduction, the transcription factor families, and cell wall-related genes mainly participating in cell expansion and carbohydrate metabolism. Further analysis identified 11,153 potential SSRs. A suit of 5122 primer pair sequences were designed, and 82 of 300 randomly selected primer pairs produced reproducible amplicons that were polymorphic among 22 cotton accessions from G. hirsutum, Gossypium barbadense and Gossypium arboreum. The UPGMA clustering analysis further confirmed high quality and effectiveness of these novel SSR markers. The present study provided insights into the transcriptome profile of the cotton and established a public information platform for functional genomics and molecular breeding.