Liangming Chen

A gene cluster encoding lectin receptor kinases confers broad-spectrum and durable insect resistance in rice

The brown planthopper (BPH) is the most destructive pest of rice (Oryza sativa) and a substantial threat to rice production, causing losses of billions of dollars annually. Breeding of resistant cultivars is currently hampered by the rapid breakdown of BPH resistance. Thus, there is an urgent need to identify more effective BPH-resistance genes. Here, we report molecular cloning and characterization of Bph3, a locus in rice identified more than 30 years ago that confers resistance to BPH. We show that Bph3 is a cluster of three genes encoding plasma membrane–localized lectin receptor kinases (OsLecRK1-OsLecRK3). Introgression of Bph3 into susceptible rice varieties by transgenic or marker-assisted selection strategies significantly enhanced resistance to both the BPH and the white back planthopper. Our results suggest that these lectin receptor kinase genes function together to confer broad-spectrum and durable insect resistance and provide a resource for molecular breeding of insect-resistant rice cultivars.

The vacuolar processing enzyme OsVPE1 is required for efficient glutelin processing in rice

Rice (Oryza sativa L.) accumulates prolamines and glutelins as its major storage proteins. Glutelins are synthesized on rough endoplasmic reticulum as 57-kDa precursors; they are then sorted into protein storage vacuoles where they are processed into acidic and basic subunits. We report a novel rice glutelin mutant, W379, which accumulates higher levels of the 57-kDa glutelin precursor. Genetic analysis revealed that the W379 phenotype is controlled by a single recessive nuclear gene. Using a map-based cloning strategy, we identified this gene, OsVPE1, which is a homolog of the Arabidopsis betaVPE gene. OsVPE1 encodes a 497-amino-acid polypeptide. Nucleotide sequence analysis revealed a missense mutation in W379 that changes Cys269 to Gly. Like the wild-type protein, the mutant protein is sorted into vacuoles; however, the enzymatic activity of the mutant OsVPE1 is almost completely eliminated. Further, we show that OsVPE1 is incorrectly cleaved, resulting in a mature protein that is smaller than the wild-type mature protein. Taken together, these results demonstrate that OsVPE1 is a cysteine protease that plays a crucial role in the maturation of rice glutelins. Further, OsVPE1 Cys269 is a key residue for maintaining the Asn-specific cleavage activity of OsVPE1.

OsRab5a regulates endomembrane organization and storage protein trafficking in rice endosperm cells.

Rice glutelins are synthesized at the endoplasmic reticulum (ER) as precursors (pro-glutelins), and are transported to protein storage vacuoles, where they are processed into mature proteins. The molecular basis of this process is largely unknown. Here, we report the isolation of a rice mutant, gpa1, that accumulates 57 kDa pro-glutelins in seeds and whose endosperm has a floury appearance. Transmission electron microscopy analysis showed that the gpa1 endosperm cells have an enlarged ER lumen and a smaller protein body II (PBII), and accumulated three types of newly generated subcellular structures. Moreover, a proportion of glutelins in the gpa1 endosperm cells were not delivered to PBII, and instead were mis-targeted to two of the newly generated structures or secreted. The gene corresponding to the gpa1 mutation was found to be OsRab5a, which encodes a small GTPase. In Arabidopsis protoplasts, OsRab5a protein was found to co-localize predominantly with AtVSR2, a molecular marker for the pre-vacuolar compartments (PVC). We conclude that OsRab5a plays an essential role in trafficking of storage protein to PBII, possibly as part of its function in organizing the endomembrane system in developing endosperm cells of rice.

Identification and characterization of a novel Waxy allele from a Yunnan rice landrace

Low amylose content (AC) is a desirable trait for rice (Oryza sativa L.) cooking quality and is selected in soft rice breeding. To gain a better understanding of the molecular mechanism controlling AC formation, we screened 83 Yunnan rice landraces in China and identified a rice variety, Haopi, with low AC. Genetic analyses and transgenic experiments revealed that low AC in Haopi was controlled by a novel allele of the Wx locus, Wxhp, encoding a granule-bound starch synthase (GBSSI). Sequence comparisons of Wxhp and Wxb alleles (from Nipponbare) showed several nucleotide changes in the upstream regulatory regions (including the promoter, 5′-untranslated region, and first intron 5′ splicing junction site). Interestingly, these changes had no obvious effect on the expression level and splicing efficiency of Wx transcripts. In addition, an examination of the coding region revealed that the Wxhp allele carries an A-to-G change at nucleotide position +497 from the start codon, resulting in an Asp165/Gly165 substitution. The amino acid substitution had no detectable effects on GBSSI activity in vitro; however, it notably reduced the binding of GBSSI to starch granules, resulting in a reduction of AC in rice seeds. Moreover, three other Yunnan landraces with low AC also carry a nucleotide substitution identical to Haopi at the +497 position of the Wx gene, suggesting common ancestry. Based on the single-nucleotide polymorphism, we have developed a new derived cleaved amplified polymorphic sequence marker for use in breeding practice to manipulate AC in rice endosperm.

Fine mapping of stable QTLs related to eating quality in rice (Oryza sativa L.) by CSSLs harboring small target chromosomal segments

Amylose content (AC) and viscosity profile are primary indices for evaluating eating and cooking qualities of rice grain. Using chromosome segment substitution lines (CSSLs), previous studies identified a QTL cluster of genes for rice eating and cooking quality in the interval R727–G1149 on chromosome 8. In this study we report two QTLs for viscosity parameters, respectively controlling setback viscosity (SBV) and consistency viscosity (CSV), located in the same interval using rapid viscosity analyzer (RVA) profile as an indicator of eating quality. Previously reported QTL for AC was dissected into two components with opposite genetic effects. Of four QTLs, qCSV-8 and qAC-8-2 had stable genetic effects across three and four environments, respectively. qSBV-8, qCSV-8 and qAC-8-1 partly overlapped, but were separated from qAC-8-2. Based on data from an Affymetrix rice GeneChip, two genes related to starch biosynthesis at the qAC-8-2 locus were chosen for further quantitative expression analysis. Both genes showed enhanced expression in sub-CSSLs carrying the target qAC-8-2 allele, but not in sub-CSSLs without the target qAC-8-2 allele, indicating their possible role in rice quality determination. Molecular markers closely linked to the two stable QTL provide the opportunity for marker-assisted selection (MAS) in breeding high quality rice.

Marker assisted pyramiding of two brown planthopper resistance genes, Bph3 and Bph27 (t), into elite rice Cultivars

BackgroundBrown planthopper (BPH) is the most destructive insect in rice production. Breeding of resistant cultivars is the most cost-effective and environment-friendly strategy for BPH management; however, resistant cultivars are currently hampered by the rapid breakdown of BPH resistance. Thus, there is an urgent need to use more effective BPH resistance genes or pyramiding different resistance genes to develop more durable resistant rice cultivars.ResultsHere a dominant BPH resistance gene Bph27(t) were introgressed into a susceptible commercial japonica variety Ningjing3 (NJ3) and indica variety 93-11 using marker-assisted selection (MAS), respectively. Further, Bph27(t) and a durable BPH resistance gene Bph3 was pyramided by intercrossing single-gene introgressed lines through MAS. The introgression of BPH resistance genes significantly improved the BPH resistance and reduced the yield loss caused by BPH.ConclusionThe development of single and two genes pyramided lines in this study provides innovative resources for molecular breeding of durable BPH-resistant rice cultivars and BPH management through resistant cultivars.

Mapping and Interaction of QTLs for Thousand-Grain Weight and Percentage of Grains with Chalkiness in Rice

Using a backcross inbred line (BIL) population derived from the cross between Koshihikari (Oryza sativa subsp. japonica) and Kasalath (O. sativa subsp. indica), the main-effect QTLs for thousand-grain weight (TGW) and percentage of grains with chalkiness (PGWC) in Nanjing in 2005 and 2006, and the epistatic effect and the QTL × environment interaction (QE) were analyzed. Correlation analysis showed that there was a significantly positive correlation (r = 0.42 in 2005, and r = 0.35 in 2006, P<0.001) between TGW and PGWC in the BIL population. A total of 11 main-effect QTLs for TGW were identified, including 5 common QTLs in both years and 5 QTLs with significant QE effect. There were 8 pairs of epistatic effect for TGW, and 7 pairs of them with significant QE effect. Six main-effect QTLs and 9 epistatic effects for PGWC were identified, and 3 main-effect QTLs and 4 epistatic effects had QE effect. Three main-effect QTLs simultaneously controlled TGW and PGWC, and their alleles from the same parent increased TGW and PGWC. One epistatic effect had similar effects on both traits. Some main-effect QTLs, such as qTGW-3c, qTGW-4a, and qTGW-6b, only controlled TGW and could be used in breeding programs.

Genetic analysis and fine mapping of a semi-dwarf gene in a centromeric region in rice (Oryza sativa L.).

Superior plant architecture is a key means of enhancing yield potential in high yielding varieties. A newly identified recessive gene, named sd-c, controls plant height and tiller number. Genetic analysis of an F2 population from a cross between the semi-dwarf mutant and japonica cv. Houshengheng showed that the sd-c locus was flanked by SSR markers RM27877 and RM277 on chromosome 12. Thirty nine InDel markers were developed in the region and the sd-c gene was further mapped to a 1 cM centromeric region between InDel markers C11 and C12. These sequenced markers can be used to distinguish wild type and mutants and thus can be used in marker-assisted selection. The sd-c mutant decreases culm length by about 26% and doubles the tiller number without changing seed weight. Until now only sd-1 has been used in indica rice breeding programs. The sd-c mutant seems to have no undesirable pleiotropic effects and is therefore a potential genetic resource for breeding semi-dwarf indica rice cultivars.

FLOURY ENDOSPERM8 , encoding the UDP-glucose pyrophosphorylase 1, affects the synthesis and structure of starch in rice endosperm

Cereal opaque-kernel mutants are ideal genetic materials for studying the mechanism of starch biosynthesis and amyloplast development. Here we isolated and identified two allelic floury endosperm 8 (flo8) mutants of rice, named flo8-1 and flo8-2. In the flo8 mutant, the starch content was decreased and the normal physicochemical features of starch were altered. Map-based cloning and subsequent DNA sequencing analysis revealed a single nucleotide substitution and an 8-bp insertion occurred in UDP-glucose pyrophosphorylase 1 (Ugp1) gene in flo8-1 and flo8-2, respectively. Complementation of the flo8-1 mutant restored normal seed appearance by expressing full length coding sequence of Ugp1. RT-qPCR analysis revealed that Ugp1 was ubiquitously expressed. Mutation caused the decreased UGPase activity and affected the expression of most of genes associated with starch biosynthesis. Meanwhile, western blot and enzyme activity analyses showed the comparability of protein levels and enzyme activity of most tested starch biosynthesis related genes. Our results demonstrate that Ugp1 plays an important role for starch biosynthesis in rice endosperm.

Genetic dissection of leaf-related traits using 156 chromosomal segment substitution lines

A two-line super-hybrid rice (Oryza sativa L.) variety [Liangyoupei9 (LYP9)] demonstrated superiority over its both parents, viz. elite inbred lines 93-11 and Pei-ai64S (PA64S), as well as other conventional hybrids, and had long been exploited in China. However, the genetic basis of its leaf-related traits, supposed to be an important component for yield potential, remains elusive. Here, initially a set of chromosome segment substitution lines (CSSLs) was constructed, in which the genome of Pei-ai64S has been introgressed into the background of 93-11. This set was developed by marker aided selection, based on 123 polymorphic SSR markers. The introgressed chromosomal segments presented in the 156 CSSLs covered 96.46% of Pei-ai64S genome. Afterwards, the CSSLs were deployed to assess the genetic basis of leaf size (length and width) and chlorophyll content of top three leaves across five different environments. The CSSLs showed transgressive segregation for all of the traits, and significant correlations were detected among most of the traits. A total of 27 quantitative trait loci (QTL) were identified on ten chromosomes, and three QTL cluster affecting related traits were found on chromosome 3, 6, and 8, respectively. Remarkably, two key QTLs, qALW3-1 and qALW3-2, both controlling the antepenultimate leaf width, were identified in all five environments, and their effect were further validated by CSSLs harboring the two QTL alleles. Our results indicate that developing CSSLs is a powerful tool for genetic dissection of quantitative traits. Meanwhile, the QTLs controlling leaf-related traits uncovered here provide useful information for marker-assisted selection in improving the performance of leaf morphology and photosynthetic ability.