A prolific and robust whole-genome genotyping method using PCR amplification via primer-template mismatched annealing.

Whole-genome genotyping methods are important for breeding. However, it has been challenging to develop a robust method for simultaneous foreground and background genotyping that can easily be adapted to different genes and species. In our study, we accidently discovered that in adapter ligation-mediated PCR, the amplification by primer-template mismatched annealing (PTMA) along the genome could generate thousands of stable PCR products. Based on this observation, we consequently developed a novel method for simultaneous foreground and background integrated genotyping by sequencing (FBI-seq) using one specific primer, in which foreground genotyping is performed by primer-template perfect annealing (PTPA), while background genotyping employs PTMA. Unlike DNA arrays, multiple PCR, or genome target enrichments, FBI-seq requires little preliminary work for primer design and synthesis, and it is easily adaptable to different foreground genes and species. FBI-seq therefore provides a prolific, robust, and accurate method for simultaneous foreground and background genotyping to facilitate breeding in the post-genomics era.

Pedigree-based analysis of derivation of genome segments of an elite rice reveals key regions during its breeding.

Analyses of genome variations with high-throughput assays have improved our understanding of genetic basis of crop domestication and identified the selected genome regions, but little is known about that of modern breeding, which has limited the usefulness of massive elite cultivars in further breeding. Here we deploy pedigree-based analysis of an elite rice, Huanghuazhan, to exploit key genome regions during its breeding. The cultivars in the pedigree were resequenced with 7.6× depth on average, and 2.1 million high-quality single nucleotide polymorphisms (SNPs) were obtained. Tracing the derivation of genome blocks with pedigree and information on SNPs revealed the chromosomal recombination during breeding, which showed that 26.22% of Huanghuazhan genome are strictly conserved key regions. These major effect regions were further supported by a QTL mapping of 260 recombinant inbred lines derived from the cross of Huanghuazhan and a very dissimilar cultivar, Shuanggui 36, and by the genome profile of eight cultivars and 36 elite lines derived from Huanghuazhan. Hitting these regions with the cloned genes revealed they include numbers of key genes, which were then applied to demonstrate how Huanghuazhan were bred after 30 years of effort and to dissect the deficiency of artificial selection. We concluded the regions are helpful to the further breeding based on this pedigree and performing breeding by design. Our study provides genetic dissection of modern rice breeding and sheds new light on how to perform genomewide breeding by design.

Streamlined whole-genome genotyping through NGS-enhanced thermal asymmetric interlaced (TAIL)-PCR.

Whole-genome genotyping (WGG) stands as a pivotal element in genomic-assisted plant breeding. Nevertheless, sequencing-based approaches for WGG continue to be costly, primarily owing to the high expenses associated with library preparation and the laborious protocol. During prior development of foreground and background integrated genotyping by sequencing (FBI-seq), we discovered that any sequence-specific primer (SP) inherently possesses the capability to amplify a massive array of stable and reproducible non-specific PCR products across the genome. Here, we further improved FBI-seq by replacing the adapter ligated by Tn5 transposase with an arbitrary degenerate (AD) primer. The protocol for the enhanced FBI-seq unexpectedly mirrors a simplified thermal asymmetric interlaced (TAIL)-PCR, a technique that is widely used for isolation of flanking sequences. However, the improved TAIL-PCR maximizes the primer-template mismatched annealing capabilities of both SP and AD primers. In addition, leveraging of next-generation sequencing enhances the ability of this technique to assay tens of thousands of genome-wide loci for any species. This cost-effective, user-friendly, and powerful WGG tool, which we have named TAIL-PCR by sequencing (TAIL-peq), holds great potential for widespread application in breeding programs, thereby facilitating genome-assisted crop improvement.

Linking differential domain functions of the GS3 protein to natural variation of grain size in rice.

Grain yield in many cereal crops is largely determined by grain size. Here we report the genetic and molecular characterization of GS3, a major quantitative trait locus for grain size. It functions as a negative regulator of grain size and organ size. The wild-type isoform is composed of four putative domains: a plant-specific organ size regulation (OSR) domain in the N terminus, a transmembrane domain, a tumor necrosis factor receptor/nerve growth factor receptor (TNFR/NGFR) family cysteine-rich domain, and a von Willebrand factor type C (VWFC) in the C terminus. These domains function differentially in grain size regulation. The OSR domain is both necessary and sufficient for functioning as a negative regulator. The wild-type allele corresponds to medium grain. Loss of function of OSR results in long grain. The C-terminal TNFR/NGFR and VWFC domains show an inhibitory effect on the OSR function; loss-of-function mutations of these domains produced very short grain. This study linked the functional domains of the GS3 protein to natural variation of grain size in rice.

Genetic Dissection of Rice Ratooning Ability Using an Introgression Line Population and Substitution Mapping of a Pleiotropic Quantitative Trait Locus qRA5.

Ratooning ability is a key factor that influences ratoon rice yield, in the area where light and temperature are not enough for second season rice. In the present study, an introgression line population derived from Minghui 63 as the recipient parent and 02428 as the donor parent was developed, and a high-density bin map containing 4568 bins was constructed. Nine ratooning-ability-related traits were measured, including maximum tiller number, panicle number, and grain yield per plant in the first season and ratoon season, as well as three secondary traits, maximum tiller number ratio, panicle number ratio, and grain yield ratio. A total of 22 main-effect QTLs were identified and explained for 3.26-18.63% of the phenotypic variations in the introgression line population. Three genomic regions, including 14.12-14.65 Mb on chromosome 5, 4.64-5.76 Mb on chromosome 8, and 10.64-15.52 Mb on chromosome 11, were identified to simultaneously control different ratooning-ability-related traits. Among them, qRA5 in the region of 14.12-14.65 Mb on chromosome 5 was validated for its pleiotropic effects on maximum tiller number and panicle number in the first season, as well as its maximum tiller number ratio, panicle number ratio, and grain yield ratio. Moreover, qRA5 was independent of genetic background and delimited into a 311.16 kb region by a substitution mapping approach. These results will help us better understand the genetic basis of rice ratooning ability and provide a valuable gene resource for breeding high-yield ratoon rice varieties.

Diversity and selective sweep in the OsAMT1;1 genomic region of rice.

BACKGROUND: Ammonium is one of the major forms in which nitrogen is available for plant growth. OsAMT1;1 is a high-affinity ammonium transporter in rice (Oryza sativa L.), responsible for ammonium uptake at low nitrogen concentration. The expression pattern of the gene has been reported. However, variations in its nucleotides and the evolutionary pathway of its descent from wild progenitors are yet to be elucidated. In this study, nucleotide diversity of the gene OsAMT1;1 and the diversity pattern of seven gene fragments spanning a genomic region approximately 150 kb long surrounding the gene were surveyed by sequencing a panel of 216 rice accessions including both cultivated rice and wild relatives. RESULTS: Nucleotide polymorphism (Pi) of OsAMT1;1 was as low as 0.00004 in cultivated rice (Oryza sativa), only 2.3% of that in the common wild rice (O. rufipogon). A single dominant haplotype was fixed at the locus in O. sativa. The test values for neutrality were significantly negative in the entire region stretching 5' upstream and 3' downstream of the gene in all accessions. The value of linkage disequilibrium remained high across a 100 kb genomic region around OsAMT1;1 in O. sativa, but fell rapidly in O. rufipogon on either side of the promoter of OsAMT1;1, demonstrating a strong natural selection within or nearby the ammonium transporter. CONCLUSIONS: The severe reduction in nucleotide variation at OsAMT1;1 in rice was caused by a selective sweep around OsAMT1;1, which may reflect the nitrogen uptake system under strong selection by the paddy soil during the domestication of rice. Purifying selection also occurred before the wild rice diverged into its two subspecies, namely indica and japonica. These findings would provide useful insights into the processes of evolution and domestication of nitrogen uptake genes in rice.

Functional markers developed from multiple loci in GS3 for fine marker-assisted selection of grain length in rice.

The gene GS3 has major effect on grain size and plays an important role in rice breeding. The C to A mutation in the second exon of GS3 was reported to be functionally associated with enhanced grain length in rice. In the present study, besides the C-A mutation at locus SF28, three novel polymorphic loci, SR17, RGS1, and RGS2, were discovered in the second intron, the last intron and the final exon of GS3, respectively. A number of alleles at these four polymorphic loci were observed in a total of 287 accessions including Chinese rice varieties (Oryza sativa), African cultivated rice (O. glaberrima) and AA-genome wild relatives. The haplotype analysis revealed that the simple sequence repeats (AT)(n) at RGS1 and (TCC)(n) at RGS2 had differentiated in the wild rice whilst the C-A mutation occurred in the cultivated rice recently during domestication. It also indicated that A allele at SF28 was highly associated with long rice grain whilst various motifs of (AT)(n) at RGS1 and (TCC)(n) at RGS2 were mainly associated with medium to short grain in Chinese rice. The C-A mutation at SF28 explained 33.4% of the grain length variation in the whole rice population tested in this study, whereas (AT)(n) at RGS1 and (TCC)(n) at RGS2 explained 26.4 and 26.2% of the variation, respectively. These results would be helpful for better understanding domestication of GS3 and its manipulation for grain size in rice. The genic marker RGS1 based on the motifs (AT)(n) was further validated as a functional marker using two sets of backcross recombinant inbred lines. These results suggested that the functional markers developed from four different loci within GS3 could be used for fine marker-assisted selection of grain length in rice breeding.

Morphological and physiological factors contributing to early vigor in the elite rice cultivar 9,311.

Huanghuazhan (HHZ) and 9,311 are two elite rice cultivars in China. They have achieved high yield through quite different mechanisms. One of the major features that gives high yield capacity to 9,311 is its strong early vigor, i.e., faster establishment of its seedling as well as its better growth in its early stages. To understand the mechanistic basis of early vigor in 9,311, as compared to HHZ the cultivar, we have examined, under controlled environmental conditions, different morphological and physiological traits that may contribute to its early vigor. Our results show that the fresh weight of the seeds, at germination, not only determined the seedling biomass at 10 days after germination (DAG), but was also responsible for ~ 80% of variations in plant biomass between the two cultivars even up to 30 DAG. Furthermore, the 9,311 cultivar had a larger root system, which led to its higher nitrogen uptake capacity. Other noteworthy observations about 9,311 being a better cultivar than HHZ are: (i) Ten out of 15 genes involved in nitrogen metabolism were much more highly expressed in its roots; (ii) it had a higher water uptake rate, promoting better root-to-shoot nitrogen transfer; and (iii) consistent with the above, it had higher leaf photosynthetic rate and stomatal conductance. All of the above identified features explain, to a large extent, why the 9,311, as compared to HHZ, exhibits much more vigorous early growth.

Hairy Leaf 6, an AP2/ERF Transcription Factor, Interacts with OsWOX3B and Regulates Trichome Formation in Rice.

Trichome formation has been extensively studied as a mechanistic model for epidermal cell differentiation and cell morphogenesis in plants. However, the genetic and molecular mechanisms underlying trichome formation (i.e., initiation and elongation) in rice remain largely unclear. Here, we report an AP2/ERF transcription factor, Hairy Leaf 6 (HL6), which controls trichome formation in rice. Functional analyses revealed that HL6 transcriptionally regulates trichome elongation in rice, which is dependent on functional OsWOX3B, a homeodomain-containing protein that acts as a key regulator in trichome initiation. Biochemical and molecular genetic analyses demonstrated that HL6 physically interacts with OsWOX3B, and both of them regulate the expression of some auxin-related genes during trichome formation, in which OsWOX3B likely enhances the binding ability of HL6 with one of its direct target gene, OsYUCCA5. Population genetic analysis indicated that HL6 was under negative selection during rice domestication. Taken together, our findings provide new insights into the molecular regulatory network of trichome formation in rice.

Genome-wide study of an elite rice pedigree reveals a complex history of genetic architecture for breeding improvement.

Improving breeding has been widely utilized in crop breeding and contributed to yield and quality improvement, yet few researches have been done to analyze genetic architecture underlying breeding improvement comprehensively. Here, we collected genotype and phenotype data of 99 cultivars from the complete pedigree including Huanghuazhan, an elite, high-quality, conventional indica rice that has been grown over 4.5 million hectares in southern China and from which more than 20 excellent cultivars have been derived. We identified 1,313 selective sweeps (SSWs) revealing four stage-specific selection patterns corresponding to improvement preference during 65 years, and 1113 conserved Huanghuazhan traceable blocks (cHTBs) introduced from different donors and conserved in >3 breeding generations were the core genomic regions for superior performance of Huanghuazhan. Based on 151 quantitative trait loci (QTLs) identified for 13 improved traits in the pedigree, we reproduced their improvement process in silico, highlighting improving breeding works well for traits controlled by major/major + minor effect QTLs, but was inefficient for traits controlled by QTLs with complex interactions or explaining low levels of phenotypic variation. These results indicate long-term breeding improvement is efficient to construct superior genetic architecture for elite performance, yet molecular breeding with designed genotype of QTLs can facilitate complex traits improvement.

A major QTL, Ghd8, plays pleiotropic roles in regulating grain productivity, plant height, and heading date in rice.

Rice yield and heading date are two distinct traits controlled by quantitative trait loci (QTLs). The dissection of molecular mechanisms underlying rice yield traits is important for developing high-yielding rice varieties. Here, we report the cloning and characterization of Ghd8, a major QTL with pleiotropic effects on grain yield, heading date, and plant height. Two sets of near isogenic line populations were developed for the cloning of Ghd8. Ghd8 was narrowed down to a 20-kb region containing two putative genes, of which one encodes the OsHAP3 subunit of a CCAAT-box binding protein (HAP complex); this gene was regarded as the Ghd8 candidate. A complementary test confirmed the identity and pleiotropic effects of the gene; interestingly, the genetic effect of Ghd8 was dependent on its genetic background. By regulating Ehd1, RFT1, and Hd3a, Ghd8 delayed flowering under long-day conditions, but promoted flowering under short-day conditions. Ghd8 up-regulated MOC1, a key gene controlling tillering and branching; this increased the number of tillers, primary and secondary branches, thus producing 50% more grains per plant. The ectopic expression of Ghd8 in Arabidopsis caused early flowering by 10 d-a situation similar to the one observed by its homolog AtHAP3b, when compared to wild-type under long-day conditions; these findings indicate the conserved function of Ghd8 and AtHAP3b in flowering in Arabidopsis. Our results demonstrated the important roles of Ghd8 in rice yield formation and flowering, as well as its opposite functions in flowering between rice and Arabidopsis under long-day conditions.

A causal C-A mutation in the second exon of GS3 highly associated with rice grain length and validated as a functional marker.

Comparative sequencing of GS3, the most important grain length (GL) QTL, has shown that differentiation of rice GL might be principally due to a single nucleotide polymorphism (SNP) between C and A in the second exon. A total of 180 varieties representing a wide range of rice germplasm were used for association analysis between C-A mutation and GL in order to confirm the potential causal mutation. A cleaved amplified polymorphic sequence (CAPS) marker, SF28, was developed based on the C-A polymorphism in the GS3 gene. A total of 142 varieties carried allele C with GL from 6.4 to 8.8 mm, while the remaining 38 varieties carried allele A with GL from 8.8 to 10.7 mm. Twenty-four unlinked SSR markers were selected to genotype 180 varieties for population structure analysis. Population structure was observed when the population was classified to three subpopulations. Average GL of either genotype A or genotype C within japonica among the three subpopulations had no significant difference from that in indica, respectively, although indica rice had longer grains on average than japonica in the 180 varieties. However, genotype C always had longer grain length on average than genotype A among three subpopulations. The mutation could explain 79.1, 66.4 and 34.7% of GL variation in the three subpopulations, respectively. These results clearly confirmed the mutation between C and A was highly associated with GL. The SF28 could be a functional marker for improvement of rice grain length.