Pedigree genome data of an early-matured Geng/japonica glutinous rice mega variety Longgeng 57.

By using PacBio HiFi technology, we produced over 700 Gb of long-read sequencing (LRS) raw data; and by using Illumina paired-end whole-genome shotgun (WGS) sequencing technology, we generated more than 70 Gb of short-read sequencing (SRS) data. With LRS data, we assembled one genome and then generate a set of annotation data for an early-matured Geng/japonica glutinous rice mega variety genome, Longgeng 57 (LG57), which carries multiple elite traits including good grain quality and wide adaptability. Together with the SRS data from three parents of LG57, pedigree genome variations were called for three representative types of genes. These data sets can be used for deep variation mining, aid in the discovery of new insights into genome structure, function, and evolution, and help to provide essential support to biological research in general.

Identification of reliable QTLs and designed QTL breeding for grain shape and milling quality in the reciprocal introgression lines in rice.

Milling quality (MQ) and grain shape (GS) of rice (Oryza sativa L.) are correlated traits, both determine farmers' final profit. More than one population under multiple environments may provide valuable information for breeding selection on these MQ-GS correlations. However, suitable analytical methods for reciprocal introgression lines with linkage map for this kind of correlation remains unclear. In this study, our major tasks were (1) to provide a set of reciprocal introgression lines (composed of two BC2RIL populations) suitable for mapping by linkage mapping using markers/bins with physical positions; (2) to test the mapping effects of different methods by using MQ-GS correlation dissection as sample case; (3) to perform genetic and breeding simulation on pyramiding favorite alleles of QTLs for representative MQ-GS traits. Finally, with four analysis methods and data collected under five environments, we identified about 28.4 loci on average for MQ-GS traits. Notably, 52.3% of these loci were commonly detected by different methods and eight loci were novel. There were also nine regions harboring loci for different MQ-GS traits which may be underlying the MQ-GS correlations. Background independent (BI) loci were also found for each MQ and GS trait. All these information may provide useful resources for rice molecular breeding.

The elite variations in germplasms for soybean breeding.

The genetic base of soybean cultivars (Glycine max (L.) Merr.) has been narrowed through selective domestication and specific breeding improvement, similar to other crops. This presents challenges in breeding new cultivars with improved yield and quality, reduced adaptability to climate change, and increased susceptibility to diseases. On the other hand, the vast collection of soybean germplasms offers a potential source of genetic variations to address those challenges, but it has yet to be fully leveraged. In recent decades, rapidly improved high-throughput genotyping technologies have accelerated the harness of elite variations in soybean germplasm and provided the important information for solving the problem of a narrowed genetic base in breeding. In this review, we will overview the situation of maintenance and utilization of soybean germplasms, various solutions provided for different needs in terms of the number of molecular markers, and the omics-based high-throughput strategies that have been used or can be used to identify elite alleles. We will also provide an overall genetic information generated from soybean germplasms in yield, quality traits, and pest resistance for molecular breeding.

Simultaneous improvement and genetic dissection of drought and submergence tolerances in rice (Oryza sativa L.) by selective introgression.

Introduction: Drought and submergence are contrasting abiotic stresses that often occur in the same rice crop season and cause complete crop failure in many rain-fed lowland areas of Asia. Methods: To develop rice varieties with good tolerances to drought and submergence, 260 introgression lines (ILs) selected for drought tolerance (DT) from nine BC2 populations were screened for submergence tolerance (ST), resulting in 124 ILs with significantly improved ST. Results: Genetic characterization of the 260 ILs with DNA markers identified 59 DT quantitative trait loci (QTLs) and 68 ST QTLs with an average 55% of the identified QTLs associated with both DT and ST. Approximately 50% of the DT QTLs showed 'epigenetic' segregation with very high donor introgression and/or loss of heterozygosity (LOH). Detailed comparison of the ST QTLs identified in ILs selected only for ST with ST QTLs detected in the DT-ST selected ILs of the same populations revealed three groups of QTLs underlying the relationship between DT and ST in rice: a) QTLs with pleiotropic effects on both DT and ST; b) QTLs with opposite effects on DT and ST; and c) QTLs with independent effects on DT and ST. Combined evidence identified most likely candidate genes for eight major QTLs affecting both DT and ST. Moreover, group b QTLs were involved in the Sub1regulated pathway that were negatively associated with most group aQTLs. Discussion: These results were consistent with the current knowledge that DT and ST in rice are controlled by complex cross-talks between or among different phytohormone-mediated signaling pathways. Again, the results demonstrated that the strategy of selective introgression was powerful and efficient for simultaneous improvement and genetic dissection of multiple complex traits, including DT and ST.

Genomic Architecture of Yield Performance of an Elite Rice Hybrid Revealed by its Derived Recombinant Inbred Line and Their Backcross Hybrid Populations.

BACKGROUND: Since its development and wide adoption in China, hybrid rice has reached the yield plateau for more than three decades. To understand the genetic basis of heterosis in rice and accelerate hybrid rice breeding, the yield performances of the elite rice hybrid, Quan-you-si-miao (QYSM) were genetically dissected by whole-genome sequencing, large-scale phenotyping of 1061 recombined inbred lines (RILs) and 1061 backcross F1 (BCF1) hybrids derived from QYSM's parents across three environments and gene-based analyses. RESULTS: Genome-wide scanning of 13,847 segregating genes between the parents and linkage mapping based on 855 bins across the rice genome and phenotyping experiments across three environments resulted in identification of large numbers of genes, 639 main-effect QTLs (M-QTLs) and 2736 epistatic QTLs with significant additive or heterotic effects on the trait performances of the combined population consisting of RILs and BCF1 hybrids, most of which were environment-specific. The 324 M-QTLs affecting yield components included 32.7% additive QTLs, 38.0% over-dominant or dominant ones with strong and positive effects and 29.3% under-dominant or incomplete recessive ones with significant negative heterotic effects. 63.6% of 1403 genes with allelic introgression from subspecies japonica/Geng in the parents of QYSM may have contributed significantly to the enhanced yield performance of QYSM. CONCLUSIONS: The parents of QYSM and related rice hybrids in China carry disproportionally more additive and under-dominant genes/QTLs affecting yield traits. Further focus in indica/Xian rice breeding should shift back to improving inbred varieties, while breaking yield ceiling of Xian hybrids can be achieved by one or combinations of the three strategies: (1) by pyramiding favorable alleles of additive genes, (2) by eliminating or minimizing under-dominant loci, and (3) by pyramiding overdominant/dominant genes polymorphic, particularly those underlying inter-subspecific heterosis.

Molecular Dissection of the Gene OsGA2ox8 Conferring Osmotic Stress Tolerance in Rice.

Gibberellin 2-oxidase (GA2ox) plays an important role in the GA catabolic pathway and the molecular function of the OsGA2ox genes in plant abiotic stress tolerance remains largely unknown. In this study, we functionally characterized the rice gibberellin 2-oxidase 8 (OsGA2ox8) gene. The OsGA2ox8 protein was localized in the nucleus, cell membrane, and cytoplasm, and was induced in response to various abiotic stresses and phytohormones. The overexpression of OsGA2ox8 significantly enhanced the osmotic stress tolerance of transgenic rice plants by increasing the number of osmotic regulators and antioxidants. OsGA2ox8 was differentially expressed in the shoots and roots to cope with osmotic stress. The plants overexpressing OsGA2ox8 showed reduced lengths of shoots and roots at the seedling stage, but no difference in plant height at the heading stage was observed, which may be due to the interaction of OsGA2ox8 and OsGA20ox1, implying a complex feedback regulation between GA biosynthesis and metabolism in rice. Importantly, OsGA2ox8 was able to indirectly regulate several genes associated with the anthocyanin and flavonoid biosynthetic pathway and the jasmonic acid (JA) and abscisic acid (ABA) biosynthetic pathway, and overexpression of OsGA2ox8 activated JA signal transduction by inhibiting the expression of jasmonate ZIM domain-containing proteins. These results provide a basis for a future understanding of the networks and respective phenotypic effects associated with OsGA2ox8.

Reciprocal adaptation of rice and Xanthomonas oryzae pv. oryzae: cross-species 2D GWAS reveals the underlying genetics.

A 1D/2D genome-wide association study strategy was adopted to investigate the genetic systems underlying the reciprocal adaptation of rice (Oryza sativa) and its bacterial pathogen, Xanthomonas oryzae pv. oryzae (Xoo) using the whole-genome sequencing and large-scale phenotyping data of 701 rice accessions and 23 diverse Xoo strains. Forty-seven Xoo virulence-related genes and 318 rice quantitative resistance genes (QR-genes) mainly located in 41 genomic regions, and genome-wide interactions between the detected virulence-related genes and QR genes were identified, including well-known resistance genes/virulence genes plus many previously uncharacterized ones. The relationship between rice and Xoo was characterized by strong differentiation among Xoo races corresponding to the subspecific differentiation of rice, by strong shifts toward increased resistance/virulence of rice/Xoo populations and by rich genetic diversity at the detected rice QR-genes and Xoo virulence genes, and by genome-wide interactions between many rice QR-genes and Xoo virulence genes in a multiple-to-multiple manner, presumably resulting either from direct protein-protein interactions or from genetic epistasis. The observed complex genetic interaction system between rice and Xoo likely exists in other crop-pathogen systems that would maintain high levels of diversity at their QR-loci/virulence-loci, resulting in dynamic coevolutionary consequences during their reciprocal adaptation.

Identification and allele mining of new candidate genes underlying rice grain weight and grain shape by genome-wide association study.

BACKGROUND: Grain weight and grain shape are important agronomic traits that affect the grain yield potential and grain quality of rice. Both grain weight and grain shape are controlled by multiple genes. The 3,000 Rice Genomes Project (3 K RGP) greatly facilitates the discovery of agriculturally important genetic variants and germplasm resources for grain weight and grain shape. RESULTS: Abundant natural variations and distinct phenotic differentiation among the subgroups in grain weight and grain shape were observed in a large population of 2,453 accessions from the 3 K RGP. A total of 21 stable quantitative trait nucleotides (QTNs) for the four traits were consistently identified in at least two of 3-year trials by genome-wide association study (GWAS), including six new QTNs (qTGW3.1, qTGW9, qTGW11, qGL4/qRLW4, qGL10, and qRLW1) for grain weight and grain shape. We further predicted seven candidate genes (Os03g0186600, Os09g0544400, Os11g0163600, Os04g0580700, Os10g0399700, Os10g0400100 and Os01g0171000) for the six new QTNs by high-density association and gene-based haplotype analyses. The favorable haplotypes of the seven candidate genes and five previously cloned genes in elite accessions with high TGW and RLW are also provided. CONCLUSIONS: Our results deepen the understanding of the genetic basis of grain weight and grain shape in rice and provide valuable information for improving rice grain yield and grain quality through molecular breeding.

A new immunochemical strategy for triple-negative breast cancer therapy.

Triple-negative breast cancer (TNBC) is a highly diverse group of malignant neoplasms which tend to have poor outcomes, and the development of new targets and strategies to treat these cancers is sorely needed. Antibody-drug conjugate (ADC) therapy has been shown to be a promising targeted therapy for treating many cancers, but has only rarely been tried in patients with TNBC. A major reason the efficacy of ADC therapy in the setting of TNBC has not been more fully investigated is the lack of appropriate target molecules. In this work we were able to identify an effective TNBC target for use in immunotherapy. We were guided by our previous observation that in some breast cancer patients the protein tropomyosin receptor kinase B cell surface protein (TrkB) had become immunogenic, suggesting that it was somehow sufficiently chemically different enough (presumably by mutation) to escaped immune tolerance. We postulated that this difference might well offer a means for selective targeting by antibodies. We engineered site-specific ADCs using a dual variable domain (DVD) format which combines anti-TrkB antibody with the h38C2 catalytic antibody. This format enables rapid, one-step, and homogeneous conjugation of ß-lactam-derivatized drugs. Following conjugation to ß-lactam-derivatized monomethyl auristatin F, the TrkB-targeting DVD-ADCs showed potency against multiple breast cancer cell lines, including TNBC cell lines. In addition, our isolation of antibody that specifically recognized the breast cancer-associated mutant form of TrkB, but not the wild type TrkB, indicates the possibility of further refining the selectivity of anti-TrkB DVD-ADCs, which should enhance their therapeutic index. These results confirmed our supposition that TrkB is a potential target for immunotherapy for TNBC, as well as for other cancers with mutated cell surface proteins.

A cell-cell interaction format for selection of high-affinity antibodies to membrane proteins.

Generating and improving antibodies and peptides that bind specifically to membrane protein targets such as ion channels and G protein-coupled receptors (GPCRs) can be challenging using established selection methods. Current strategies are often limited by difficulties in the presentation of the antigen or the efficiency of the selection process. Here, we report a method for obtaining antibodies specific for whole cell membrane-associated antigens which combines a cell-cell interaction format based on yeast display technology with fluorescence-activated cell sorting of dual fluorescent complexes. Using this method, we were able to direct the affinity maturation of an antagonist antibody specific for the proton-gated ion channel ASIC1a and showed that both the affinity and potency were improved. We were also able to use this method to do kinetic selections to generate clones with better dissociation profiles. In addition, this method was employed successfully to handle the difficult problem of selecting antibodies specific to a GPCR target, the mu-opioid receptor.

Genetic dissection of seedling vigour in a diverse panel from the 3,000 Rice (Oryza sativa L.) Genome Project.

Seedling vigour (SV) is important for direct seeding rice (Oryza sativa L.), especially in a paddy-direct seeding system, but the genetic mechanisms behind the related traits remain largely unknown. Here, we used 744 germplasms, having at least two subsets, for the detection of quantitative trait loci (QTLs) affecting the SV-related traits tiller number, plant height, and aboveground dry weight at three sampling stages, 27, 34, and 41 d after sowing. A joint map based on GAPIT and mrMLM produced a satisfying balance between type I and II errors. In total, 42 QTL regions, containing 18 (42.9%) previously reported overlapping QTL regions and 24 new ones, responsible for SV were detected throughout the genome. Four QTL regions, qSV1a, qSV3e, qSV4c, and qSV7c, were delimited and harboured quantitative trait nucleotides that are responsible for SV-related traits. Favourable haplotype mining for the candidate genes within these four regions, as well as the early SV gene OsGA20ox1, was performed, and the favourable haplotypes were presented with donors from the 3,000 Rice Genome Project. This work provides new information and materials for the future molecular breeding of direct seeding rice, especially in paddy-direct seeding cultivation systems.

Antibody selection using clonal cocultivation of Escherichia coli and eukaryotic cells in miniecosystems.

We describe a method for the rapid selection of functional antibodies. The method depends on the cocultivation of Escherichia coli that produce phage with target eukaryotic cells in very small volumes. The antibodies on phage induce selectable phenotypes in the target cells, and the nature of the antibody is determined by gene sequencing of the phage genome. To select functional antibodies from the diverse antibody repertoire, we devised a selection platform that contains millions of picoliter-sized droplet ecosystems. In each miniecosystem, the bacteria produce phage displaying unique members of the antibody repertoire. These phage interact only with eukaryotic cells in the same miniecosystem, making phage available directly for activity-based antibody selection in biological systems.

Joint Exploration of Favorable Haplotypes for Mineral Concentrations in Milled Grains of Rice (Oryza sativa L.).

Grain minerals in rice, especially those in milled grains, are important sources of micro-nutrition elements, such as iron (Fe), zinc (Zn), manganese (Mn), copper (Cu), and selenium (Se), and of toxic heavy metal elements, especially cadmium (Cd), for populations consuming a rice diet. To date, the genetic mechanism underlying grain mineral concentrations (GMCs) in milled grain remains largely unknown. In this report, we adopted a set of 698 germplasms consisting of two subsets [indica/Xian (X-set) and japonica/Geng (G-set)], to detect quantitative trait loci (QTL) affecting GMC traits of Fe, Zn, Cd, Mn, Cu, and Se in milled grains. A total of 47 QTL regions, including 18 loci and 29 clusters (covering 62 Cd loci), responsible for the GMCs in milled grains were detected throughout the genome. A joint exploration of favorable haplotypes of candidate genes was carried out as follows: (1) By comparative mapping, 10 chromosome regions were found to be consistent with our previously detected QTL from linkage mapping. (2) Within eight of these regions on chromosomes 1, 4, 6, 7, and 8, candidate genes were identified in the genome annotation database. (3) A total of 192 candidate genes were then submitted to further haplotype analysis using million-scale single nucleotide polymorphisms (SNPs) from the X-set and the G-set. (4) Finally, 37 genes (19.3%) were found to be significant in the association between the QTL targeting traits and the haplotype variations by pair-wise comparison. (5) The phenotypic values for the haplotypes of each candidate were plotted. Three zinc finger (like) genes within two candidate QTL regions (qFe6-2 and qZn7), and three major GMC traits (Fe, Zn, and Cd) were picked as sample cases, in addition to non-exhausted cross validations, to elucidate this kind of association by trait value plotting. Taken together, our results, especially the 37 genes with favorable haplotype variations, will be useful for rice biofortification molecular breeding.

Identification of QTN and candidate genes for Salinity Tolerance at the Germination and Seedling Stages in Rice by Genome-Wide Association Analyses.

To facilitate developing rice varieties tolerant to salt stress, a panel of 208 rice mini-core accessions collected from 25 countries were evaluated for 13 traits associated with salt tolerance (ST) at the germination and seedling stages. The rice panel showed tremendous variation for all measured ST traits and eight accessions showing high levels of ST at either and/or both the germination and seedling stages. Using 395,553 SNP markers covering ~372 Mb of the rice genome and multi-locus mixed linear models, 20 QTN associated with 11 ST traits were identified by GWAS, including 6 QTN affecting ST at the germination stage and 14 QTN for ST at the seedling stage. The integration of bioinformatic with haplotype analyses for the ST QTN lets us identify 22 candidate genes for nine important ST QTN (qGR3, qSNK1, qSNK12, qSNC1, qSNC6, qRNK2, qSDW9a, qSST5 and qSST9). These candidate genes included three known ST genes (SKC1, OsTZF1 and OsEATB) for QTN qSNK1 qSST5 and qSST9. Candidate genes showed significant phenotypic differences in ST traits were detected between or among 2-4 major haplotypes. Thus, our results provided useful materials and genetic information for improving rice ST in future breeding and for molecular dissection of ST in rice.

Genomic variation in 3,010 diverse accessions of Asian cultivated rice.

Here we analyse genetic variation, population structure and diversity among 3,010 diverse Asian cultivated rice (Oryza sativa L.) genomes from the 3,000 Rice Genomes Project. Our results are consistent with the five major groups previously recognized, but also suggest several unreported subpopulations that correlate with geographic location. We identified 29 million single nucleotide polymorphisms, 2.4 million small indels and over 90,000 structural variations that contribute to within- and between-population variation. Using pan-genome analyses, we identified more than 10,000 novel full-length protein-coding genes and a high number of presence-absence variations. The complex patterns of introgression observed in domestication genes are consistent with multiple independent rice domestication events. The public availability of data from the 3,000 Rice Genomes Project provides a resource for rice genomics research and breeding.

Genetic Dissection and Simultaneous Improvement of Drought and Low Nitrogen Tolerances by Designed QTL Pyramiding in Rice.

Drought and low nitrogen are the most common abiotic stresses limiting rice productivity in the rainfed rice areas of Asia and Africa. Development and adoption of green super rice (GSR) varieties with greatly improved drought tolerance (DT) and low nitrogen tolerance (LNT) are the most efficient way to resolve this problem. In this study, using three sets of trait-specific introgression lines (ILs) in a Xian (indica) variety Huanghuazhan (HHZ) background, we identified nine DT-QTL and seven LNT-QTL by a segregation distortion approach and a genome-wide association study, respectively. Based on performances of DT and LNT and genotypes at the detected QTL, two ILs M79 and M387 with DT and LNT were selected for cross-making to validate the identified QTL and to develop DT and LNT rice lines by pyramiding two DT-QTL (qDT3.9 and qDT6.3) and two LNT-QTL (qGY1 and qSF8). Using four pairs of kompetitive allele specific PCR (KASP) SNP markers, we selected 66 F2 individuals with different combinations of the target DT- and LNT-QTL favorable alleles and they showed expected improvement in DT and/or LNT, which were further validated by the significant improvement in DT and/or LNT of their F3 progeny testing. Based on evaluation of pyramiding lines in F3 lines under drought, low nitrogen (LN) and normal conditions, four promising pyramiding lines having different QTL favorable alleles were selected, which showed significantly improved tolerances to drought and/or LN than HHZ and their IL parents. Our results demonstrated that trait-specific ILs could effectively connect QTL mapping and QTL pyramiding breeding, and designed QTL pyramiding (DQP) using ILs could be more effective in molecular rice breeding for complex quantitative traits.

QTL mapping and candidate gene analysis of peduncle vascular bundle related traits in rice by genome-wide association study.

BACKGROUND: The vascular bundle especially in the peduncle is one of crucial limiting factors of rice yield, and it determines how plants efficiently transport photosynthetic products, mineral nutrients and water from leaf and root to panicle. However, the genetic base of rice vascular bundle related traits in the peduncle still remains unknown. RESULTS: The 423 panel showed substantial natural variations of peduncle vascular bundle. In total, 48 quantitative trait loci/locus (QTL) affecting the eight traits were identified throughout the genome by applying a significance threshold of P < 1.0 × 10- 4. Combined determining linkage disequilibrium (LD) blocks associated with significant SNPs and haplotype analyses allowed us to shortlist six candidate genes for four important QTL regions affecting the peduncle vascular bundle traits, including one cloned gene (NAL1) and three newly identified QTL (qLVN6, qSVN7, and qSVA8.1). Further the most likely candidate genes for each important QTL were also discussed based on functional annotation. CONCLUSIONS: Genetic base on peduncle vascular bundle related traits in rice was systematically dissected, and most likely candidate genes of the known gene NAL1 and the three newly identified QTL (qLVN6, qSVN7, and qSVA8.1) were analyzed. The results provided valuable information for future functional characterization and rice breeding for high yield through optimizing transportation efficiency of photosynthetic products by marker-assisted selection.

Assembly of an early-matured japonica (Geng) rice genome, Suijing18, based on PacBio and Illumina sequencing.

The early-matured japonica (Geng) rice variety, Suijing18 (SJ18), carries multiple elite traits including durable blast resistance, good grain quality, and high yield. Using PacBio SMRT technology, we produced over 25 Gb of long-read sequencing raw data from SJ18 with a coverage of 62×. Using Illumina paired-end whole-genome shotgun sequencing technology, we generated 59 Gb of short-read sequencing data from SJ18 (23.6 Gb from a 200 bp library with a coverage of 59× and 35.4 Gb from an 800 bp library with a coverage of 88×). With these data, we assembled a single SJ18 genome and then generated a set of annotation data. These data sets can be used to test new programs for variation deep mining, and will provide new insights into the genome structure, function, and evolution of SJ18, and will provide essential support for biological research in general.