Yuqing He

Field performance of transgenic elite commercial hybrid rice expressing Bacillus thuringiensis δ-endotoxin

Here we describe development of transgenic elite rice lines expressing a Bt fusion gene derived from cryIA(b) and cryIA(c) under the control of rice actinI promoter. The lines used in the study were indica CMS restorer line of Minghui 63 and its derived hybrid rice Shanyou 63. The level of Bt fusion protein CryIA(b)/CryIA(c) detected in Minghui 63 (T51-1) plants was 20 ng/mg soluble protein. The Bt Shanyou 63 was field-tested in natural and repeated heavy manual infestation of two lepidopteran insects, leaffolder and yellow stem borer. The transgenic hybrid plants showed high protection against both insect pests without reduced yield.

Natural variation in GS5 plays an important role in regulating grain size and yield in rice

Increasing crop yield is one of the most important goals of plant science research. Grain size is a major determinant of grain yield in cereals and is a target trait for both domestication and artificial breeding. We showed that the quantitative trait locus (QTL) GS5 in rice controls grain size by regulating grain width, filling and weight. GS5 encodes a putative serine carboxypeptidase and functions as a positive regulator of grain size, such that higher expression of GS5 is correlated with larger grain size. Sequencing of the promoter region in 51 rice accessions from a wide geographic range identified three haplotypes that seem to be associated with grain width. The results suggest that natural variation in GS5 contributes to grain size diversity in rice and may be useful in improving yield in rice and, potentially, other crops.

Chalk5 encodes a vacuolar H+-translocating pyrophosphatase influencing grain chalkiness in rice

Grain chalkiness is a highly undesirable quality trait in the marketing and consumption of rice grain. However, the molecular basis of this trait is poorly understood. Here we show that a major quantitative trait locus (QTL), Chalk5, influences grain chalkiness, which also affects head rice yield and many other quality traits. Chalk5 encodes a vacuolar H+-translocating pyrophosphatase (V-PPase) with inorganic pyrophosphate (PPi) hydrolysis and H+-translocation activity. Elevated expression of Chalk5 increases the chalkiness of the endosperm, putatively by disturbing the pH homeostasis of the endomembrane trafficking system in developing seeds, which affects the biogenesis of protein bodies and is coupled with a great increase in small vesicle-like structures, thus forming air spaces among endosperm storage substances and resulting in chalky grain. Our results indicate that two consensus nucleotide polymorphisms in the Chalk5 promoter in rice varieties might partly account for the differences in Chalk5 mRNA levels that contribute to natural variation in grain chalkiness.

A High-Density SNP Genotyping Array for Rice Biology and Molecular Breeding

A high-density single nucleotide polymorphism (SNP) array is critically important for geneticists and molecular breeders. With the accumulation of huge amounts of genomic re-sequencing data and available technologies for accurate SNP detection, it is possible to design high-density and high-quality rice SNP arrays. Here we report the development of a high-density rice SNP array and its utility. SNP probes were designed by screening more than 10 000 000 SNP loci extracted from the re-sequencing data of 801 rice varieties and an array named RiceSNP50 was produced on the Illumina Infinium platform. The array contained 51 478 evenly distributed markers, 68% of which were within genic regions. Several hundred rice plants with parent/F1 relationships were used to generate a high-quality cluster file for accurate SNP calling. Application tests showed that this array had high genotyping accuracy, and could be used for different objectives. For example, a core collection of elite rice varieties was clustered with fine resolution. Genome-wide association studies (GWAS) analysis correctly identified a characterized QTL. Further, this array was successfully used for variety verification and trait introgression. As an accurate high-throughput genotyping tool, RiceSNP50 will play an important role in both functional genomics studies and molecular breeding.

Pyramiding and evaluation of the brown planthopper resistance genes Bph14 and Bph15 in hybrid rice

The brown planthopper (BPH) is the most devastating insect pest in rice-producing areas. Shanyou 63 has become a widely cultivated hybrid in China over the last two decades; however, this line has become increasingly susceptible to bacterial blight (BB), blast, and BPH, resulting in a rapid decline in its use in rice production. In this study, a molecular marker-assisted selection (MAS) introgression of Bph14 and Bph15 was performed to improve the BPH resistance of Minghui 63 and its derived hybrids such as Shanyou 63. The effect of pyramiding genes was then comprehensively evaluated using three tests that comprised seedbox screening, feeding rate, and antixenosis for settling in the field. The results showed that the improved hybrids containing a single BPH resistance gene showed enhanced resistance (lower resistance score, honeydew weight and number of BPH settling) compared to conventional hybrids, while pyramiding two genes provided even higher resistance. Moreover, both Bph14 and Bph15 are partial dominance genes, and have a strong dosage effect on the resistance to BPH in the hybrid background, which is useful for breeding BPH-resistant hybrids. Field trial data demonstrated that yields of improved hybrid rice were higher than or similar to the control (Shanyou 63) under natural field conditions. These improved versions could be used in breeding programs for “green super rice.”

Breeding signatures of rice improvement revealed by a genomic variation map from a large germplasm collection

Significance Intensive rice breeding over the past 50 y has produced many high-performing cultivars, but our knowledge of the genomic changes associated with such improvement remains limited. By analyzing sequences of 1,479 rice accessions, this study identified genomic changes associated with breeding efforts, referred to as breeding signatures, involving 7.8% of the rice genome. Accumulation of selected regions is positively correlated with yield improvement. The number of selected regions in a line may be used for predicting agronomic potential, and the selected loci may provide useful targets for rice improvement. Intensive rice breeding over the past 50 y has dramatically increased productivity especially in the indica subspecies, but our knowledge of the genomic changes associated with such improvement has been limited. In this study, we analyzed low-coverage sequencing data of 1,479 rice accessions from 73 countries, including landraces and modern cultivars. We identified two major subpopulations, indica I (IndI) and indica II (IndII), in the indica subspecies, which corresponded to the two putative heterotic groups resulting from independent breeding efforts. We detected 200 regions spanning 7.8% of the rice genome that had been differentially selected between IndI and IndII, and thus referred to as breeding signatures. These regions included large numbers of known functional genes and loci associated with important agronomic traits revealed by genome-wide association studies. Grain yield was positively correlated with the number of breeding signatures in a variety, suggesting that the number of breeding signatures in a line may be useful for predicting agronomic potential and the selected loci may provide targets for rice improvement.

Natural variation and artificial selection in four genes determine grain shape in rice

The size of cultivated rice (Oryza sativa) grains has been altered by both domestication and artificial selection over the course of evolutionary history. Several quantitative trait loci (QTLs) for grain size have been cloned in the past 10 yr. To explore the natural variation in these QTLs, resequencing of grain width and weight 2 (GW2), grain size 5 (GS5) and QTL for seed width 5 (qSW5) and genotyping of grain size 3 (GS3) were performed in the germplasms of 127 varieties of rice (O. sativa) and 10-15 samples of wild rice (Oryza rufipogon). Ten, 10 and 15 haplotypes were observed for GW2, GS5 and qSW5. qSW5 and GS3 had the strongest effects on grain size, which have been widely utilized in rice production, whereas GW2 and GS5 showed more modest effects. GS5 showed small sequence variations in O. sativa germplasm and that of its progenitor O. rufipogon. qSW5 exhibited the highest level of nucleotide diversity. GW2 showed signs of purifying selection. The four grain size genes experienced different selection intensities depending on their genetic effects. In the indica population, linkage disequilibrium (LD) was detected among GS3, qSW5 and GS5. The substantial genetic variation in these four genes provides the flexibility needed to design various rice grain shapes. These findings provide insight into the evolutionary features of grain size genes in rice.

Pyramiding and evaluation of three dominant brown planthopper resistance genes in the elite indica rice 9311 and its hybrids

BACKGROUND Brown planthopper (BPH), Nilaparvata lugens Stål, is the most devastating insect pest in rice-producing areas. Three dominant BPH resistance genes (Bph14, Bph15, Bph18) were pyramided into elite indica rice 9311 and its hybrids using marker-assisted selection. Gene effectiveness was evaluated on the basis of seedling and adult rice resistance, honeydew weight and survival rate of BPH. RESULTS All three genes affected BPH growth and development and antibiotic factors, resulting in both seedling and adult resistance. Bph15 had the greatest effect on conferring resistance to BPH. The results showed an additive effect of pyramiding genes, the order of the gene effect being 14/15/18 ≥ 14/15 > 15/18 ≥ 15 > 14/18 ≥ 14 ≥ 18 > none. The pyramided or single-gene introgression hybrids showed greater resistance than conventional hybrids, although the heterozygous genotypes had weaker effects than the corresponding homozygous genotypes. Furthermore, field trial data demonstrated that yields of improved 9311 lines were higher than or similar to that of the control under natural field conditions. These improved versions can be immediately used in hybrid improvement and production. CONCLUSION Compared with controls, pyramided lines and hybrids with three genes showed the strongest resistance to BPH, without a yield decrease.

The QTL controlling amino acid content in grains of rice ( Oryza sativa ) are co-localized with the regions involved in the amino acid metabolism pathway

The improvement of grain quality, such as protein content (PC) and amino acid composition, has been a major concern of rice breeders. We constructed a population of 190 recombinant inbred lines (RILs) from a cross between Zhenshan 97 and Nanyangzhan to map the quantitative trait locus or loci (QTL) for amino acid content (AAC) as characterized by each of the AACs, total essential AAC, and all AAC. Using the data collected from milled rice in 2002 and 2004, we identified 18 chromosomal regions for 19 components of AAC. For 13 of all the loci, the Zhenshan 97 allele increased the trait values. Most QTL were co-localized, forming ten QTL clusters in 2002 and six in 2004. The QTL clusters varied in both effects and locations, and the mean values of variation explained by individual QTL in the clusters ranged from 4.3% to 28.82%. A relatively strong QTL cluster, consisting of up to 19 individual QTL, was found at the bottom of chromosome 1. The major QTL clusters identified for two different years were coincident. A wide coincidence was found between the QTL we detected and the loci involved in amino acid metabolism pathways, including N assimilation and transfer, and amino acid or protein biosynthesis. The results will be useful for candidate gene identification and marker-assisted favorable allele transfer in rice breeding programs.

OsAAP6 functions as an important regulator of grain protein content and nutritional quality in rice

Grains from cereals contribute an important source of protein to human food, and grain protein content (GPC) is an important determinant of nutritional quality in cereals. Here we show that the quantitative trait locus (QTL) qPC1 in rice controls GPC by regulating the synthesis and accumulation of glutelins, prolamins, globulins, albumins and starch. qPC1 encodes a putative amino acid transporter OsAAP6, which functions as a positive regulator of GPC in rice, such that higher expression of OsAAP6 is correlated with higher GPC. OsAAP6 greatly enhances root absorption of a range of amino acids and has effects on the distribution of various amino acids. Two common variations in the potential cis-regulatory elements of the OsAAP6 5′-untranslated region seem to be associated with GPC diversity mainly in indica cultivars. Our results represent the first step toward unravelling the mechanism of regulation underlying natural variation of GPC in rice.