A Rapid and Simplified Method to Isolate Specific Regulators Based on Biotin-Avidin Binding Affinities in Crops.

Transcription factors (TFs) are indispensable components of transcriptional regulatory pathways involved in crop growth and development. Herein, we developed a new method for the identification of upstream TFs specific to genes in crops based on the binding affinities of biotin and avidin. First, we constructed and verified the new biotin and avidin system (BAS) by a coprecipitation assay. Subsequently, the feasibility of DNA-based BAS (DBAS) was further proved by in vivo and in vitro assays. Furthermore, we cloned the promoter of rice OsNRT1.1B and the possible regulators were screened and identified. Additionally, partial candidates were validated by the electrophoresis mobility shift assay (EMSA), yeast one-hybrid, and luciferase activity assays. Remarkably, the results showed that the candidates PIP3 and PIP19 both responded to nitrate immediately and overexpression of PIP3 caused retard growth, which indicates that the candidates are functional and the new DBAS method is useful to isolate regulators in crops.

Transcriptomic and Physiological Analyses of Two Rice Restorer Lines under Different Nitrogen Supplies Provide Novel Insights into Hybrid Rice Breeding.

Improving plant nitrogen-use efficiency (NUE) has great significance for various crops, particularly in hybrid breeding. Reducing nitrogen inputs is key to achieving sustainable rice production and mitigating environmental problems. In this study, we analyzed the transcriptomic and physiological changes in two indica restorer lines (Nanhui511 [NH511] and Minghui23 [MH23]) under high nitrogen (HN) and low nitrogen (LN) conditions. Compared to MH23, NH511 was more sensitive to different nitrogen supplies and exhibited higher nitrogen uptake and NUE under HN conditions by increasing lateral root and tiller numbers in the seedling and maturation stages, respectively. NH511 also exhibited a lower survival rate than MH23 when planted in a chlorate-containing hydroponic solution, indicating its HN uptake ability under different nitrogen-supply conditions. Transcriptomic analysis showed that NH511 has 2456 differentially expressed genes, whereas MH23 had only 266. Furthermore, these genes related to nitrogen utilization showed differential expression in NH511 under HN conditions, while the opposite was observed in MH23. Our findings revealed that NH511 could be regarded as elite rice and used for breeding high-NUE restorer lines by regulating and integrating nitrogen-utilization genes, which provides novel insights for the cultivation of high-NUE hybrid rice.

Genome-wide identification of nitrate-responsive microRNAs by small RNA sequencing in the rice restorer cultivar Nanhui 511.

Rice productivity relies heavily on nitrogen fertilization, and improving nitrogen use efficiency (NUE) is important for hybrid rice breeding. Reducing nitrogen inputs is the key to achieving sustainable rice production and reducing environmental problems. Here, we analyzed the genome-wide transcriptomic changes in microRNAs (miRNAs) in the indica rice restorer cultivar Nanhui 511 (NH511) under high (HN) and low nitrogen (LN) conditions. The results showed that NH511 is sensitive to nitrogen supplies and HN conditions promoted the growth its lateral roots at the seedling stage. Furthermore, we identified 483 known miRNAs and 128 novel miRNAs by small RNA sequencing in response to nitrogen in NH511. We also detected 100 differentially expressed genes (DEGs), including 75 upregulated and 25 downregulated DEGs, under HN conditions. Among these DEGs, 43 miRNAs that exhibited a 2-fold change in their expression were identified in response to HN conditions, including 28 upregulated and 15 downregulated genes. Additionally, some differentially expressed miRNAs were further validated by qPCR analysis, which showed that miR443, miR1861b, and miR166k-3p were upregulated, whereas miR395v and miR444b.1 were downregulated under HN conditions. Moreover, the degradomes of possible target genes for miR166k-3p and miR444b.1 and expression variations were analyzed by qPCR at different time points under HN conditions. Our findings revealed comprehensive expression profiles of miRNAs responsive to HN treatments in an indica rice restorer cultivar, which advances our understanding of the regulation of nitrogen signaling mediated by miRNAs and provides novel data for high-NUE hybrid rice cultivation.

Field performance on grain yield and quality and genetic diversity of overwintering cultivated rice (Oryza sativa L.) in southwest China.

Understanding the field performance on grain yield and quality and the genetic diversity of overwintering (OW) cultivated rice (Oryza sativa L.) across main crop (MC) and ratooning crop (RC) is the premise to make strategies for the future OW rice variety improvement in rice production. The present field experiments were conducted in RC of 2016, in MC of both 2017 and 2018, and RC in 2019 to identify genotypes OW rice that perform stable in terms of grain yield and quality across different climate conditions. The grain yield plant-1 (GYP) and its components in six genotypes of OW rice exhibited significant difference across the 4 years (P ≤ 0.05), the maximum GYP in OW6 rice was harvested (60.28 g) in MC of 2017, but the minimum GYP in OW1 rice was harvested (33.01 g) in MC of 2018. Within six genotypes of OW rice, four grain shape traits displayed a relative small significant difference, four grain quality traits exhibited a relative small significant difference except for chalkiness rate (CR), there 226 pairs of significant PCC values between GYP and its components were calculated in all tested rice and varied from six in OW6 to eleven in OW1, there 130 pairs of significant PCC values among the four grain shape traits were calculated and ranged from twenty-one in OW1, 3, 5 to twenty-three in OW2, there 118 pairs of significant PCC values among the four grain quality traits were calculated and ranged from seventeen in OW2 to twenty-three in OW1. The numbers, directions, and size of PCC values for the grain yield and quality characters in all tested rice displayed a series of irregular variations. Six genotypes of OW rice were apparently distinguished by employing 196 pairs of simple-sequence repeats (SSRs) markers and exhibited abundant genetic diversity at the DNA level. Data from this study provide an extensive archive for the future exploration and innovation of overwintering cultivated rice variety.

Construction of three half-sib SSR linkage maps derived from overwintering cultivated rice and segregation distortion loci mapping.

Segregation distortion is a common phenomenon that has been observed in genetics and plant breeding; however, the mechanism of segregation distortion is unknown. In the present study, three half-sib F2 populations derived from three japonica overwinter (perennial) rice varieties (W1, W2, and W3) crossed to the indica rice variety Minghui725 (MH725) were developed to construct three half-sib linkage maps. We established linkage map lengths of 2032.8, 2317.4, and 2108.7 cM with average intervals of 20.1, 20.5, and 19.7 cM using 101, 113, and 107 SSR markers in W1/MH725, W2/MH725, and W3/MH725, respectively. Discrepancies in marker order and genetic linkage distance occurred in the three half-sib linkage maps due to segregation distortion. A total of 88 markers exhibited segregation distortion across the three linkage maps at P < 0.01 level, 42 segregation distortion loci (SDLs) were detected across the three half-sib populations and exhibited variable LOD value that ranged from 3.2 (SDL2f) to 30.1 (SDL5d), and 13 of the 42 SDLs were repeatedly located at the same chromosomal regions of the previously published hybrid sterility quantitative trait loci. Data from this study provide an extensive archive for investigating the genetic characteristic of overwintering cultivated rice and the future exploration and innovation of overwintering rice breeding.

Locating QTLs controlling overwintering trait in Chinese perennial Dongxiang wild rice.

Overwintering (OW) is the process by which rice passes through the winter season and germinates in the following spring. OW is also a typical quantitative inheritance trait. Currently, the molecular genetic basis of OW trait in Chinese perennial Dongxiang wild rice (DXWR) still remains to be known. In this study, a linkage map consisting of 139 simple sequence repeat (SSR) markers was constructed using an F2 population derived from a cross between DXWR and 93-11. This map covered the rice genome by approximately 1778.72 cM with approximately 12.80 cM average interval. The phenotype data of OW trait were investigated for QTL analysis in the following spring of 2017. The gene ontology (GO) annotation of the M-QTL was performed through the rice genome annotation project system. A major QTL-qOW6 was flanked by RM20069 (16,542,428 bp) and RM3498 (20,982,059 bp) on chromosome 6 and detected repeatedly by both inclusive composite interval and single-marker analysis mapping with an LOD score of 9.45 and explained 22.22% of phenotypic variance. In addition, two small QTLs (qOW2 and qOW3) controlling OW trait were detected on the second and third chromosomes, respectively. No epistatic interaction was detected between these QTLs, suggesting their unique genetic model. A total of 183 candidate genes at qOW6 locus were involved in 887 GO terms. Among them, 52 candidate genes were involved in response to stress. The other 28 candidate genes were related to cell membrane, which might affect the OW trait in perennial DXWR. These results may establish the foundation for understanding the genetic mechanism about OW trait and provide a novel gene resource for OW rice variety improvement.

[Research progress of Ypsilandra thibetica, a medicinal plant of Liliaceae].

Ypsilandra thibetica belongs to the family Liliaceae. Its whole plant has the medicinal functions of heat-clearing and detoxifying, relieving congestion and other effects, and is used as the folk medicine to cure scrofula, dysuria embolism and other symptoms. Previous chemical studies revealed that its major and active ingredient is steroidal saponin. Up to now, more than fifty steroidal saponins, mainly composed of spirostan and furostanol types, have been described. Pharmacological and clinical studies have demonstrated that Y. thibetica has anti-tumor, uterine contractions, hemostatic and antibacterial activities, in particular for the treatment of a variety of gynecological hemorrhagic diseases. In an effort to provide references for the advanced research and development of this species, this paper summarized the research progress on its pharmacognosy, including botany and authentication, its isolated secondary metabolites, biological activities and pharmacological applications. In addition, some advantages of this species which could be potentially used as a substitute for Paridis Rhizoma, one of ingredients of the well-known drug "Yunnan Baiyao", together with the future prospect are also briefly included.

[Progress on genetics of rice grain shape trait and its related gene mapping and cloning].

Increase of crop production is the primary goal of crop breeding. Rice grain shape is a quantitative trait that is directly related to yield traits and has a close relationship with quality traits. The evaluation of grain shape is mainly grain length, grain width, grain thickness, length/width, and length/thickness. In recent years, the quantitative genetic research on rice grain shape has made a significant progress and a number of important genes associated with rice grain shape have been cloned. This paper reviews the classic genetic analysis on rice grain traits, QTL mapping, grain shape gene cloning and functional analysis, and their application in rice breeding for super high yield.

Microsatellite analysis of homozygosity progression of heterozygous genotypes segregating in the rice subspecies cross Pei'ai64s/Nipponbare.

Progression to homozygosity of heterozygous genotypes was studied in a cross of the rice subspecies Pei'ai64s and Nipponbare, using a set of 157 polymorphic microsatellite (SSR) markers. The segregation of heterozygous genotypes ranged from 49.13% in the F(2) population to 4.52% in the F(6) population (progression value 11.15%). The heterozygous genotypes were widely distributed in 180 F(2) plants, 330 F(6) lines, and 157 SSR markers. Homozygosity progression showed a wide distribution in plants and SSR markers but not in chromosomes. The segregation of heterozygous genotypes was not significant between populations but varied greatly in F(2) plants, F(6) lines, and SSR markers. The correlation between the progression to homozygosity and the heterozygosity of SSR markers was significant at the chromosome level. The segregation of heterozygous genotypes in plants, SSR markers, and chromosomes was not completely in accordance with Mendel's law. This information will help rice geneticists and breeders to understand heterozygous genotype segregation at the DNA level and to screen special markers for breeding.

[Comparison of genetic linkage maps based on F2F6 populations derived from rice subspecies cross].

A F2 population containing 180 lines, which was derived from the cross between the partially sequenced indica variety "Pei'ai 64S" and the completely sequenced japonica variety "Nipponbare" , was used to construct a genetic linkage framework map (referred to as F2 map), which included 138 microsatellite sites and covered 1737.81 cM of total genomic length, an average distance of 11.90 cM. Single seed descent F2:6 population with 330 lines was used to construct a genetic linkage map (known as F6 map) using 92 markers. The total genomic length and average distance were 2563.5 cM and 27.86 cM, respectively. The F2 and F6 maps differed in linkage groups, mapped markers, sequenced order of markers, ge-netic distance and average distance on the maps. Preliminary analysis about these difference was carried out.

Analysis of segregation distortion of molecular markers in F2 population of rice.

A genetic linkage map comprising 148 SSR markers loci was constructed using an F2 population consisting of 90 lines derived from a sub-specific cross between a japonica variety Nipponbare and an indica variety Guangluai-4. The F2 population showed high significantly distorted segregations. Among these SSR markers, 49 markers(33.11%) showed the genetics distortion(P<0.05). Of them, 36 markers deviated toward male parent indica GuangLuAi-4 and 13 markers toward heterozygote, but none toward the female parent Nipponbare. It was found that the segregation distortion might be caused by gametophyte and zygote. Since most gametophyte loci and sterility loci were mapped in segregation distortion regions, it indicated that the segregation distortion may be caused by these gametophyte loci and sterility loci. Finally, this research also analyzed the skewed segregation of some markers, which had not been mapped on chromosome.

Construction of a microsatellite linkage map with two sequenced rice varieties.

Based on the successful development of new microsatellite markers from the data of two whole-sequenced rice varieties, japonica variety Nipponbare and indica variety 9311, an F2 population of 90 lines, which was derived from a single cross between Nipponbare and 9311, was applied to construct a genetic linkage framework map. The map covered 2 455.7 cM of total genomic length, and consisted of 152 simple sequence repeats (SSRs) loci including 46 pairs of new SSR primers developed by our research institute. The average genetic distance between two markers was 16.16 cM. In addition, markers RM345 and RM494, which have not been mapped on the Temnykh's map et al. (2001) were anchored on the sixth chromosome of this map. We compared this research with maps of Temnykh et al.(2001) and LAN et al. (2003) regarding the aspects of type and size of population, type and quantity of markers, and the marker arrangement order on chromosome, etc. Results indicated that the similarity of marker linear alignment was 93.81% between this map and T-map. Finally, the important significance of using sequenced rice varieties to construct linkage map was also discussed.