The E3 ubiquitin ligase CSIT1 regulates critical sterility-inducing temperature by ribosome-associated quality control to safeguard two-line hybrid breeding in rice

Two-line hybrid breeding can fully utilize heterosis in crops.In thermo-sensitive genic male sterile(TGMS)lines,low critical sterility-inducing temperature(CSIT)is vital to safeguard the production of two-line hybrid seeds in rice(Oryza sativa),but the molecular mechanism determining CSIT is unclear.Here,we report the cloning of CSIT1,which encodes an E3 ubiquitin ligase,and show that CSIT1 modulates the CSIT of thermo-sensitive genic male sterility 5(tms5)-based TGMS lines through ribosome-associated quality control(RQC).Biochemical assays demonstrated that CSIT1 binds to the 80S ribosomes and ubiquitinates abnormal nascent polypeptides for degradation in the RQC process.Loss of CSIT1 function inhibits the possible damage of tms5 to the ubiquitination system and protein translation,resulting in enhanced accumulation of anther-related proteins such as catalase to suppress abnormal accumulation of reactive oxygen species and premature programmed cell death in the tapetum,thereby leading to a much higher CSIT in the tms5-based TGMS lines.Taken together,our findings reveal a regulatory mechanism of CSIT,providing new insights into RQC and potential targets for future two-line hybrid breeding.

Confectionery Hybrid Sunflower Breeding in Russia

Confectionery sunflower hybrid breeding program started at VNIIMK in 1999. Aim of our study was to evaluate sunflower lines and hybrids, developed in this program. Best restorer lines were crossed with two lines with cytoplasmic male sterility （CMS-lines） used as testers. Hybrids were tested for seed yield, husk content and 1,000 seeds weight. Two-year hybrid trial allows us to define the most prominent hybrid combination （VK-905 A ~ K-4） and parental lines （CMS-line VK-905 A and restorer lines K-4, K-3 and K-I） for the future sunflower breeding work. It was further showed that confectionery hybrids with CMS-line VK-905 A do not show decrease in seed size with the increase in plant density.

The Breeding of Hybrid Rape Variety Youyan 924 with Strong Heterosis and High Oil

The rape project of ＂Development and Application of Cabbage Type Rape Hybrids with Strong Heterosis＂ which belongs to National 863 Program aimed to breed the rape variety whose rapeseed yield or oil yield increased more than 10% compared with check （CK） variety, or oil yield increased more than 3 percentage points compared with CK. In the regional trial in the middle react,es of the Yangtze River for two years, the average rapeseed yield and oil yield of Youyan 924 which is a hybrid rape variety respectively reached 2 695.95 and 1 264.35 kg/hm2, which respectively increased by 5.22% and 13.4% compared with those of the other test- ing varieties in the same group, and respectively increased by 10.06% and 23.68% compared with those of Zhongyouza 2 which belongs to CK, thus reaching the standard of hybrid rape variety with strong heterosis; the average oil percentage of the variety in the two years was 46.63%,which increased by 3.44 percentage points compared with that of all the other testing varieties in the same group, and in- creased by 5.14 percentage points compared with that of Zhongyouza 2, and the oil percentage of it in the production testing reached 49.21%. Thus Youyan 924 is a variety with strong heterosis and high oil, moreover, its contents of erucic acid and glucosinolate are 0,25% and 20.27 μmol/g, respectively. From the data aggregation of national regional trial, it has the advantages of high oil yield, high yielding ability, reaching the standards of low erucic acid and low glucosinolate, strong lodging re- sistance, good disease resistance aqd moderate mature period.

Breeding of Hybrid Hebei Cashmere Goat

[Objective] To improve the performance of Hebei cashmere goats and thus promote the development of cashmere industry. [Method] Through grading hybridization, the F2 generation was obtained using Liaoning cashmere goats as male parents and local cashmere goats as female parents. Then fixed cross was performed. After selection, the body size indexes, weight and cashmere yield were determined. The quality of cash mere, lambing rate, and net cashmere rate were also analyzed. [ Result] Compared with the local cashmere goats, the hybrid cashmere goats had robust physique and good body size indexes; the weight and cashmere yield increased significantly; cashmere fiber was longer significantly; the lambing rate and net cashmere rate were improved. [ Conclusion] After breeding, the cashmere goats have good quality and high yield of cashmere, and their performance is significantly improved.

The Utilization of HB-red Flower in Hybrid Cotton Breeding

The HB-red flower trait came from the filial generation of the interspecific cross of upland cotton(Gossypium hirsutum L.) and G.bickii.It exhibits pink petals and filaments,with a large

Development and application of marker-assisted reverse breeding using hybrid maize germplasm

Humankind has been through different periods of agricultural improvement aiming at enhancing our food supply and the performance of food crops. In recent years, whole genome sequencing and deep understanding of genetic and epigenetic mechanisms have facilitated new plant breeding approaches to meet the challenge of growing population, dwindling resources, and changing climate. Here we proposed a simple and fast molecular breeding method, marker-assisted reverse breeding（MARB）, which will revert any maize hybrid into inbred lines with any level of required similarity to its original parent lines. Since all the pericarp DNA of a hybrid is from the maternal parent, whereas one half of the embryo DNA is from the maternal parent and the other half from the paternal parent, so we firstly extract DNA from seed embryo and pericarp of a selected elite hybrid separately and then we derived the genotypes of the two parents with high-density single nucleotide polymorphism（SNP） chips. The following marker-assisted selection was performed based on an Illumina low-density SNP chip designed with 192 SNPs polymorphic between the two parental genotypes, which were uniformly distributed on 10 maize chromosomes. This method has the advantages of fast speed, fixed heterotic mode, and quick recovery of beneficial parental genotypes compared to traditional pedigree breeding using elite hybrids. Meanwhile, MARB has the advantage of not requiring sophisticated transformation and double haploid（DH） technologies over RNA interference（RNAi）-mediated reverse breeding. In addition, MARB can also be used with feed corn harvested from big farms, which is often similar to F_2 populations, and the relevant transgenes in the population can be eliminated by marker-assisted selection. As a result, the whole global commercial maize hybrids can be utilized as germplasm for breeding with MARB technology. Starting with an F_2 population derived from an elite hybrid, our experiment indicates that with three cycles of marker-assisted selection, selected lines could recover over 80% of the parental genotypes and associated beneficial genes in a fixed heterotic mode. The success application of MARB in maize suggests that this technology is applicable to any hybrid crop to breed new inbreds with improved hybrid performance but the same heterotic mode. As chip technology becomes cheap, it would be expected that polymorphism screening and following marker-assisted selection could be done with one all-purpose high density chip. Several issues associated with MARB were discussed, including its rationale, efficiency and advantages, along with food/feed and environmental safety issues and applications of MARB in variety protection and marker-assisted plant breeding.

SAR-3——a resource of diploid true breeding hybrid rice

In 1988, we found strain 9003 from compositehybridization of indica and japonica. 9003 pos-sesses characteristics of twin seedlings withfrequency of 15-20% of the investigated popu-lation. The highest frequency was 45.68%.Afterwards, we discovered that some twinseedlings had variabilities in chromosome set.As to a pair of twin plants, it may be 2n-1n,2n-3n, 3n-3n or 1n-1n. The changes in chro-mosome set also occurred in the plants devel-oped from mono-embryo seeds. All of thesevariants nearly account for 1% in our con-trolled experiment. We named the triploidplants SAR-3 and found that SAR-3 could be aresource of diploid true breeding hybrid rice.

Selection of wide compatible restorer lines and their application in hybrid rice breeding

Since the middle of 1980’s, wide compatibility(WC) rice lines have been screened by ricebreeders in China and applied in hybrid ricebreeding program. Several WC lines such asPecos, T984, Lunhui 422, and 02428 withideal agronomic characters were identified. Weincorporated the WC gene into restorer linesby crossing these japonica WC lines with ob-tained indica lines. Some WC restorer lineswith indica-japonica medium type were ob-tained and their application value in intersub-specific hybrid rice breeding were evaluated. 1. Effect of crossing methods on selectionefficiencies of WC restorer lines

A historical review of hybrid rice breeding

The development of germplasm resources and advances in breeding methods have led to steady increases in yield and quality of rice (Oryza sativa L.). Three milestones in the recent history of rice breeding have contributed to these increases: dwarf rice breeding, hybrid rice breeding, and super rice breeding. On the 50th anniversary of the success of three-line hybrid rice,we highlight important scientific discoveries in rice breeding that were made by Chinese scientists and summarize the broader history of the field. We discuss the strategies that could be used in the future to optimize rice breeding further in the hope that China will continue to play a leading role in international rice breeding.

Breeding of Zhongyouza 8, a Canola Variety with Large Seeds and High Oil Yield

High oil yield resulted from a combination of high grain yield and high oil content is a prerequisite for the high efficient oilseed rape production. By using irradiation induced mutation and sexual hybridization combined with paired test cross, the fertility, yield and oil content of the three lines of cytoplasmic male sterility have been improved and a new hybrid variety Zhongyouza 8 with high oil yield was developed. It has been testified that the yield of Zhongyouza 8 was significantly higher than that of the control variety Zhongyou 821 with 9.82 and 10.64% increase in the regional trials of Hubei Province and nationwide, respectively. The oil content and oil yield of Zhongyouza 8 were the highest among all the lines involved in Hubei provincial trials, being 42.77% and 1 051.05 kg ha^-1 which was raised by 3% and 161.25 kg ha^-1 compared to the control Zhongyou 821, respectively. The genetic basis for the strong heterosis, and the factors contributing to the yield and oil content increase of Zhongyouza 8 as well as the strategy for high oil yielding variety improvement through increasing seed size were also discussed in this paper.

Breeding of New Large Cocoon and Hypersilkgeneous Tussah Varieties with Eurytopicity

The new tussah varieties have been bred by selecting large, high quality and midterm based on the indexes of high cocoon shell rate and high stress re- sistance. In the meantime, whole cocoon weight, hatchabillty and other economic parameters were considered in new variety breeding. Through 14 generations of hybrid breeding for over 7 years, two new tussah varieties had been successfully obtained. The breeding results of DW were as follows ： the 1 000-cocoon weight was 11.57 kg; the whole cocoon weight was 12.06 g; the cocoon shell rate was 12.22% ; and the larval-pupal integrated survival rate was 95.87%. The breeding re- sults of GH were as follows： the 1 000-cocoon weight was 11.52 kg; the whole cocoon weight was 11.95 g; the cocoon shell rate was 12.66% ; and the larval-pupal integrated survival rate was 90.37%.

Hybridization between salt resistant and salt susceptible genotypes of mungbean(Vigna radiata L.Wilczek)and purity testing of the hybrids using SSRs markers

Six cultivated and two wild genotypes of mungbean（Vigna radiata L. Wilczek） possessing variation for phenotypic and genotypic response for salt tolerance were hybridized. Hybridization results showed successful pod set and significant variations in cross-compatibility of investigated genotypes. Genotypes PLM 380 and PLM 562 showed promising combining ability with all genotypes. Results revealed significant crossing compatibility between V. radiata and V. sublobata. The cross ability ranged from 1.99 to 5.12%（average 3.08%）. Molecular analysis confirmed the hybrids purity. All F_1 seeds were bold, green/shiny green and germinated between 3 to 5 days. Hybrid plants were normal, fertile and healthier over their respective progenitors. Uniform flowering and maturity of the hybrids showed absence of any defect or alteration in plant habit and life cycle of the hybrids. The hybrid plants showed increase in yield characteristics as no. of pods, pod length, 100 seeds weight, and yield per plant. Number of pods containing F_2 seeds ranged from 5–8 per cluster. The F_2 seeds were collected and stored for further research. Present study suggests that salt resistant wild relatives or cultivars of mungbean can be explored by breeding as a source of useful traits/genes providing salt tolerance. This may help in development of best mungbean variety for saline prone areas. Micro-satellite markers linked to the trait/genes may assist breeding by early selection of the genotypes compared to the phenotypic screening.

Utilization of Aromatic Rice in Improving Grain Quality of Hybrid Rice(Ⅰ)

To improve grain quality of the high-yielding hybrid rice in China, we introduced the aromatic rice MR365, an improved Indian cultivar, from IRRI in 1984 and began to transfer its aroma and good quality characters into the existing maintainer lines. In the meantime, the research on the inheritance of aroma for increasing the breeding efficiency was also conducted.It was found that the inheritance of aroma in MR 365 and its derivatives was controlled by one pair of recessive major genes based on the KOH-soaking method. There existed disparity in aroma degree among different grains of F2 generation, and different aromatic CMS lines derived from the same aromatic donor had also a little difference in the degree of aroma, which implies that, besides the major genes, aroma may also be affected by the genetic backgrounds or minor genes.Xiangxiang 2A, developed from the cross of V20A//V20B/MR365, is the first aromatic CMS line bred in China. It is not only aromatic but also has good grain quality and combining ability. Using it as female parent, Xiangyou 63 (Xiangxiang 2A / Minghui 63), the first quasi-aromatic hybrid rice combination in China, was developed, and released to farmers in 1995. Xiangyou 63 is characteristic of quasi-aromatic or partially aromatic (because only a portion of or not all grains are aromatic), good grain quality, high-yielding ability, good blast resistance and wide adaptability.

Utilization of aromatic rice in improving grain quality of hybrid rice(Ⅱ)

Xiangxiang 2A has an evident drawback, i.e., instability in male sterility under higher temperature conditions resulting from the existence of minor restoring genes in it, which greatly hampered the extension of its elite hybrid Xiangyou 63 with both high yield and fine quality in commercial production. To improve Xiangxiang 2A, the hybridization of Xiangxiang 2B with V20 B was made again in 1990. A new aromatic CMS line Xinxiang A was successfully developed in 1994. It not only retains the favorable characteristics of Xiangxiang 2A in grain quality and combining ability, but also expresses complete and stable male sterility and high seed production yield potential. Up to now, by using it as female parent, a series of quasi aromatic hybrids have been developed. Some of them have been released to farmers. Because such hybrids can not only yield higher or as high as but also possess a better grain quality than the current common high yielding hybrid rice varieties,so that they are preferred and well welcome by the farmers in China. The planting area under these hybrids is increasing rapidly in China.

The essential roles of sugar metabolism for pollen development and male fertility in plants

Sugar metabolism plays an essential role in plant male reproduction. Defects in sugar metabolism during anther and pollen development often result in genic male sterility(GMS). In this review, we summarize the recent progresses of the sugar metabolism-related GMS genes and their roles during plant anther and pollen development, including callose wall and primexine formation, intine development, pollen maturation and starch accumulation, anther dehiscence, and pollen germination and tube growth. We predict 112 putative sugar metabolic GMS genes in maize based on bioinformatics and RNA-seq analyses, and most of them have peak expression patterns during middle or late anther developmental stages.Finally, we outline the potential applications of sugar metabolic GMS genes in crop hybrid breeding and seed production. This review will deepen our understanding on sugar metabolic pathways in controlling pollen development and male fertility in plants.

Identification of no pollen 1 provides a candidate gene for heterosis utilization in foxtail millet(Setaria italica L.)

Male sterility is a common biological phenomenon in plant kingdom and has been used to generate male-sterile lines, which are important genetic resources for commercial hybrid seed production. Although increasing numbers of male-sterility genes have been identified in rice(Oryza sativa) and Arabidopsis(Arabidopsis thaliana), few male-sterility-related genes have been characterized in foxtail millet(Setaria italica). In this study, we isolated a male-sterile ethyl methanesulfonate-generated mutant in foxtail millet, no pollen 1(sinp1), which displayed abnormal Ubisch bodies, defective pollen exine and complete male sterility. Using bulk segregation analysis, we cloned SiNP1 and confirmed its function with CRISPR/Cas9 genome editing. SiNP1 encoded a putative glucose-methanol-choline oxidoreductase.Subcellular localization showed that the SiNP1 protein was preferentially localized to the endoplasmic reticulum and was predominantly expressed in panicle. Transcriptome analysis revealed that many genes were differentially expressed in the sinp1 mutant, some of which encoded proteins putatively involved in carbohydrate metabolism, fatty acid biosynthesis, and lipid transport and metabolism, which were closely associated with pollen wall development. Metabolome analysis revealed the disturbance of flavonoids metabolism and fatty acid biosynthesis in the mutant. In conclusion, identification of SiNP1 provides a candidate male-sterility gene for heterosis utilization in foxtail millet and gives further insight into the mechanism of pollen reproduction in plants.

Development of the “Third-Generation” Hybrid Rice in China

Rice is a major cereal crop for China. The development of the ‘‘three-line" hybrid rice system based on cytoplasmic male sterility in the 1970 s(first-generation) and the ‘‘two-line" hybrid rice system based on photoperiod-and thermo-sensitive genic male-sterile lines(second-generation)in the 1980 s has contributed significantly to rice yield increase and food security in China. Here we describe the development and implementation of the ‘‘third-generation" hybrid rice breeding system that is based on a transgenic approach to propagate and utilize stable recessive nuclear male sterile lines, and as such, the male sterile line and hybrid rice produced using such a system is nontransgenic. Such a system should overcome the intrinsic problems of the ‘‘first-generation" and‘‘second-generation" hybrid rice systems and hold great promise to further boost production of hybrid rice and other crops.

Understanding the genetic and molecular constitutions of heterosis for developing hybrid rice

The wide adoption of hybrid rice has greatly increased rice yield in the last several decades.The utilization of heterosis facilitated by male sterility has been a common strategy for hybrid rice development.Here,we summarize our efforts in the genetic and molecular understanding of heterosis and male sterility together with the related progress from other research groups.Analyses of F1 diallel crosses show that strong heterosis widely exists in hybrids of diverse germplasms,and inter-subspecific hybrids often display higher heterosis.Using the elite hybrid Shanyou 63 as a model,an immortalized F2 population design is conducted for systematic characterization of the biological mechanism of heterosis,with identification of loci controlling heterosis of yield and yield component traits.Dominance,overdominance,and epistasis all play important roles in the genetic basis of heterosis;quantitative assessment of these components well addressed the three classical genetic hypotheses for heterosis.Environment-sensitive genic male sterility(EGMS)enables the development of two-line hybrids,and long noncoding RNAs often function as regulators of EGMS.Inter-subspecific hybrids show greatly reduced fertility;the identification and molecular characterization of hybrid sterility genes offer strategies for overcoming inter-subspecific hybrid sterility.These developments have significant implications for future hybrid rice improvement using genomic breeding.

A straight-forward seed production technology system for foxtail millet(Setaria italica)

For autogamous crops, a precondition for using heterosis is to produce sufficient pure male-sterile female parents that can be used to produce hybrid seeds. To date, cytoplasmic male sterility(CMS)and environment-sensitive genic male sterility(EGMS) have been used commercially to exploit heterosis for autogamous species. However, neither CMS nor EGMS has been established for foxtail millet(Setaria italica). Here, we report on the establishment and application of a seed production technology(SPT) system for this crop.First, we established a Ds Red-based SPT system,but found that it was unsuitable because it required the use of a fluorescent device for seed sorting. Instead, we constructed an SPT system with de novo betalain biosynthesis as the selection marker. This allowed us to distinguish transgenic seeds with the naked eye, thereby facilitating the identification of SPT maintainer line seeds. In this system, a seed sorter was not required to obtain sufficient seeds. The key point of the strategy is that the seed pool of the SPT maintainer line is propagated by artificial identification and harvesting of male-fertile individuals in the field, and the male-sterile line seed pool for hybrid production is produced and propagated by free pollination of male-sterile plants with the SPT maintainer line. In a field experiment, we obtained 423.96 kg male-sterile line seeds per acre,which is sufficient to plant 700.18 acres of farmland for hybrid seed production or male-sterile line reproduction. Our study therefore describes a powerful tool for hybrid seed production in foxtail millet, and demonstrates how the SPT system can be used for a small-grained crop with high reproduction efficiency.

MS1 is essential for male fertility by regulating the microsporocyte cell plate expansion in soybean

Male sterility is an essential trait in hybrid seed production,especially for monoclinous and autogamous food crops.Soybean male-sterile ms1 mutant has been known for more than 50 years and could be instrumental in making hybrid seeds.However,the gene responsible for the male-sterile phenotype has remained unknown.Here,we report the map-based cloning and characterization of the MS1 gene in soybean.MS1 encodes a kinesin protein and localizes to the nucleus,where it is required for the male meiotic cytokinesis after telophase Ⅱ.We further substantiated that MS1 colocalizes with microtubules and is essential for cell plate formation in soybean male gametogenesis through immunostaining.Both ms1 and CRISPR/Cas9 knockout mutants show complete male sterility but are otherwise phenotypically normal,making them perfect tools for producing hybrid seeds.The identification of MS1 has the practical potential for assembling the sterility system and speeding up hybrid soybean breeding.