Shuzhu Tang

Allelic diversities in rice starch biosynthesis lead to a diverse array of rice eating and cooking qualities

More than half of the worlds population uses rice as a source of carbon intake every day. Improving grain quality is thus essential to rice consumers. The three main properties that determine rice eating and cooking quality—amylose content, gel consistency, and gelatinization temperature—correlate with one another, but the underlying mechanism of these properties remains unclear. Through an association analysis approach, we found that genes related to starch synthesis cooperate with each other to form a fine regulating network that controls the eating and cooking quality and defines the correlation among these three properties. Genetic transformation results verified the association findings and also suggested the possibility of developing elite cultivars through modification with selected major and/or minor starch synthesis-related genes.

Deletion in a quantitative trait gene qPE9-1 associated with panicle erectness improves plant architecture during rice domestication.

Rice plant architecture is an important agronomic trait and a major determinant in high productivity. Panicle erectness is the preferred plant architecture in japonica rice, but the molecular mechanism underlying domestication of the erect panicle remains elusive. Here we report the map-based cloning of a major quantitative trait locus, qPE9-1, which plays an integral role in regulation of rice plant architecture including panicle erectness. The R6547 qPE9-1 gene encodes a 426-amino-acid protein, homologous to the keratin-associated protein 5-4 family. The gene is composed of three Von Willebrand factor type C domains, one transmembrane domain, and one 4-disulfide-core domain. Phenotypic comparisons of a set of near-isogenic lines and transgenic lines reveal that the functional allele (qPE9-1) results in drooping panicles, and the loss-of-function mutation (qpe9-1) leads to more erect panicles. In addition, the qPE9-1 locus regulates panicle and grain length, grain weight, and consequently grain yield. We propose that the panicle erectness trait resulted from a natural random loss-of-function mutation for the qPE9-1 gene and has subsequently been the target of artificial selection during japonica rice breeding.

Genomic in situ hybridization (GISH) analyses of Thinopyrum intermedium, its partial amphiploid Zhong 5, and disease-resistant derivatives in wheat

Abstract Genomic in situhybridization (GISH) to root-tip cells at mitotic metaphase, using genomic DNA probes from Thinopyrum intermedium and Pseudoroegneria strigosa, was used to examine the genomic constitution of Th. intermedium, the 56-chromosome partial amphiploid to wheat called Zhong 5 and disease-resistant derivatives of Zhong 5, in a wheat background. Evidence from GISH indicated that Th. intermedium contained seven pairs of St, seven JS and 21 J chromosomes; three pairs of Th. intermedium chromosomes with satellites in their short arms belonging to the St, J, J genomes and homoeologous groups 1, 1, and 5 respectively. GISH results using different materials and different probes showed that seven pairs of added Th. intermedium chromosomes in Zhong 5 included three pairs of St chromosomes, two pairs of JS chromosomes and two pairs of St-JS reciprocal tanslocation chromosomes. A pair of chromosomes, which substituted a pair of wheat chromosomes in Yi 4212 and in HG 295 and was added to 21 pairs of wheat chromosomes in the disomic additions Z1, Z2 and Z6, conferred BYDV-resistance and was identical to a pair of St-JS tanslocation chromosomes (StJS) in Zhong 5. The StJS chromosome had a special GISH signal pattern and could be easily distinguished from other added chromosomes in Zhong 5; it has not yet been possible to locate the BYDV-resistant gene(s) of this translocated chromosome either in the St chromosome portion belonging to homoeologous group 2 or in the JS chromosome portion whose homoeologous group relationship is still uncertain. Among 22 chromosome pairs in disomic addition line Z3, the added chromosome pair had satellites and belonged to the St genome and homoeologous group 1. Disomic addition line Z4 carried a pair of added chromosomes which was composed of a group-7 JS chromosome translocated with a wheat chromosome; this chromosome was different to 7 Ai-1, but was identical to 7 Ai-2. The leaf rust and stem rust resistance genes were located in the distal region of the long arm, whereas the stripe rust resistance gene(s) was located in the short arm or in the proximal region of the long arm of 7 Ai-2. A pair of JS-wheat translocation chromosomes, which originated from the WJS chromosomes in Z4, was added to the disomic addition line Z5; the added chromosomes of Z5 carried leaf and stem rust resistance but not stripe rust resistance; Z5 is a potentially useful source for rust resistance genes in wheat breeding and for cloning these novel rust-resistant genes. GISH analysis using the St genome as a probe has proved advantageous in identifying alien Th. intermedium in wheat.

Stable Inheritance of the Antisense Waxy Gene in Transgenic Rice with Reduced Amylose Level and Improved Quality

Amylose content in rice endosperm is a key determinant of eating and cooking quality. In the present study, a chimeric antisense construct, which contained a 756-bp antisense Waxy (Wx) gene DNA fragment from rice and the gusA coding sequence, both fused to the 3.1-kb rice Wx promoter, was efficiently introduced into several elite rice cultivars, both of japonica and indica type, via Agrobacterium. More than 200 independent transgenic lines were produced and integration transgene was confirmed by PCR and Southern blotting. Northern blot analysis suggested that the antisense Wx transcript interacted with both the endogenous Wx mature mRNA and unspliced transcripts, but only interaction with the mature mRNA resulted in reduced amylose synthesis. Analysis of GUS activity showed that the gusA fusion gene driven by the rice Wx promoter expressed highly in the endosperm of the transgenic rice plants. Varying degrees of reduction in amylose content, up to 96%, were found in seeds derived from these transformants. Consistently, opaque white seeds, similar to glutinous rice, were observed in several transgenic lines of japonica rice. In transgenic lines derived from indica rice, which usually has a high amylose level, significant reduction of amylose content was also found in the endosperm, but the levels of reduction were lower than those of japonica rice. Genetic analysis demonstrated that transgenes and improved amylose content were stably inherited (up to ninth generation) in these transgenic lines. Several elite transgenic lines with improved amylose level and quality have been selected for field evaluation.

Genetic analysis of starch paste viscosity parameters in glutinous rice (Oryza sativa L.).

Starch paste viscosity plays an important role in estimating the cooking, eating, and processing quality of rice. The inheritance of starch paste viscosity in glutinous rice remains undefined. In the present study, 118 glutinous rice accessions were collected, and the genotypes of 17 starch synthesis-related genes (SSRG) were analyzed by using 43 gene-specific molecular markers. Association analysis indicated that 10 of 17 SSRGs were involved in controlling the rapid visco analyzer (RVA) profile parameters. Among these, the PUL gene was identified to play an important role in control of peak viscosity (PKV), hot paste viscosity (HPV), cool paste viscosity (CPV), breakdown viscosity (BDV), peak time (PeT), and paste temperature (PaT) in glutinous rice. Other SSRGs involved only a few RVA profile parameters. Furthermore, interactions between SSRGs were found being responsible for PeT, PaT, and BDV. Some of the RVA parameters, including PKV, HPV, CPV, CSV, and PaT, were mainly governed by single SSRG, whereas other parameters, such as BDV, SBV, and PeT, were controlled by a few SSRGs, functioning cooperatively. Further, three near-isogenic lines (NIL) of a japonica glutinous cv. Suyunuo as genetic background, with PUL, SSIII-1, and SSIII-2 alleles replaced with those of indica cv. Guichao 2, were employed to verify the genetic effects of the various genes, and the results were consistent with those obtained from the association analysis. These findings indicated that starch paste viscosity in glutinous rice had a complex genetic system, and the PUL gene played an important role in determining the RVA profile parameters in glutinous rice. These results provide important information for potentially improving the quality of glutinous rice.

Identification and characterization of a major QTL responsible for erect panicle trait in japonica rice (Oryza sativa L.)

Panicle erectness (PE) is one of the most important traits for high-yielding japonica cultivars. Although several cultivars with PE trait have been developed and released for commercial production in China, there is little information on the inheritance of PE traits in rice. In the present study, 69 widely cultivated japonica cultivars and a double haploid (DH) population derived from a cross between a PE cultivar (Wuyunjing 8) and a drooping panicle cultivar (Nongken 57) were utilized to elucidate the mechanisms of PE formation and to map PE associated genes. Our data suggested that panicle length (PL) and plant height (PH) significantly affected panicle curvature (PC), with shorter PL and PH resulting in smaller PC and consequently more erect. A putative major gene was identified on chromosome 9 by molecular markers and bulk segregant analysis in DH population. In order to finely map the major gene, all simple sequence repeats (SSR) markers on chromosome 9 as well as 100 newly developed sequence-tagged site (STS) markers were used to construct a linkage group for quantitative trait locus (QTL) mapping. A major QTL, qPE9-1, between STS marker H90 and SSR marker RM5652, was detected, and accounted for 41.72% of PC variation with pleiotropic effect on PH and PL. another QTL, qPE9-2, was also found to be adjacent to qPE9-1. In addition, we found that H90, the nearest marker to qPE9-1, used for genotyping 38 cultivars with extremely erect and drooping panicles, segregated in agreement with PC, suggesting the H90 product was possibly part of the qPE9-1 gene or closely related to it. These data demonstrated that H90 could be used for marker-aided selection for the PE trait in breeding and in the cloning of qPE9-1.

CENTRAL REGION COMPONENT1, a Novel Synaptonemal Complex Component, Is Essential for Meiotic Recombination Initiation in Rice

This work identifies CRC1 (for CENTRAL REGION COMPONENT1), a novel synaptonemal complex component that is essential for the initiation of homologous recombination in meiosis. CRC1 is the rice ortholog of budding yeast Pch2 and mouse TRIP13, but the roles of CRC1 identified here have not been reported for Pch2 or TRIP13. In meiosis, homologous recombination entails programmed DNA double-strand break (DSB) formation and synaptonemal complex (SC) assembly coupled with the DSB repair. Although SCs display extensive structural conservation among species, their components identified are poorly conserved at the sequence level. Here, we identified a novel SC component, designated CENTRAL REGION COMPONENT1 (CRC1), in rice (Oryza sativa). CRC1 colocalizes with ZEP1, the rice SC transverse filament protein, to the central region of SCs in a mutually dependent fashion. Consistent with this colocalization, CRC1 interacts with ZEP1 in yeast two-hybrid assays. CRC1 is orthologous to Saccharomyces cerevisiae pachytene checkpoint2 (Pch2) and Mus musculus THYROID RECEPTOR-INTERACTING PROTEIN13 (TRIP13) and may be a conserved SC component. Additionally, we provide evidence that CRC1 is essential for meiotic DSB formation. CRC1 interacts with HOMOLOGOUS PAIRING ABERRATION IN RICE MEIOSIS1 (PAIR1) in vitro, suggesting that these proteins act as a complex to promote DSB formation. PAIR2, the rice ortholog of budding yeast homolog pairing1, is required for homologous chromosome pairing. We found that CRC1 is also essential for the recruitment of PAIR2 onto meiotic chromosomes. The roles of CRC1 identified here have not been reported for Pch2 or TRIP13.

Development and high-throughput genotyping of substitution lines carring the chromosome segments of indica 9311 in the background of japonica Nipponbare.

Chromosome segment substitution lines (CSSLs) are useful for the precise mapping of quantitative trait loci (QTLs) and dissection of the genetic basis of complex traits. In this study, two whole-genome sequenced rice cultivars, the japonica Nipponbare and indica 9311 were used as recipient and donor, respectively. A population with 57 CSSLs was developed after crossing and back-crossing assisted by molecular markers, and genotypes were identified using a high-throughput resequencing strategy. Detailed graphical genotypes of 38 lines were constructed based on resequencing data. These CSSLs had a total of 95 substituted segments derived from indica 9311, with an average of about 2.5 segments per CSSL and eight segments per chromosome, and covered about 87.4% of the rice whole genome. A multiple linear regression QTL analysis mapped four QTLs for 1000-grain weight. The largest-effect QTL was located in a region on chromosome 5 that contained a cloned major QTL GW5/qSW5 for grain size in rice. These CSSLs with a background of Nipponbare may provide powerful tools for future whole-genome discovery and functional study of essential genes/QTLs in rice, and offer ideal materials and foundations for japonica breeding.

Fine mapping of a major QTL controlling panicle number in rice

Rice panicle number per plant is a grain yield component that directly influences rice yield. The identification of the genes controlling panicle number will play a vital role in high-yield rice breeding. C3074, a chromosome segment substitution line containing an introgression segment of Nipponbare in the genetic background of Guangluai 4, had significantly fewer panicles. Quantitative trait locus (QTL) analysis in F2 and F3 generations derived from a cross between C3074 and Guangluai 4 revealed that qPN1, a major QTL locating on the long arm of chromosome 1, was responsible for this phenotypic variation. qPN1 also affected plant height, panicle length, grain number per panicle and grain yield per plant. Genetic analysis indicated that qPN1 acted as a single Mendelian factor, and that the allele from Nipponbare decreased the panicle number per plant. Fine mapping was carried out with a total of 1,429 recessive individuals from F2 and F3 generations, localizing the gene to a 34.4-kb region containing six annotated genes according to available sequence annotation databases. This information will be helpful for future identification and isolation of the candidate gene. The tightly linked markers that we have developed for qPN1 will be a useful tool in the marker-assisted selection of this gene in rice improvement programs.

Development of gene-tagged markers for quantitative trait loci underlying rice yield components

Higher yields of rice have always been a predominant goal in rice breeding techniques. However, the inheritances of rice yield and its components are still unknown, and no information regarding suitable alleles can be directly provided for improving the rice yield level until three major quantitative trait loci (QTLs) have been cloned and functionally characterized. These QTLs contain Gn1a for grain number per panicle and GS3 and GW2 for grain weight. It has been proven that these three genes show a potential in improving the rice yield level. However, the distribution of suitable alleles on these three loci in rice cultivars and germ plasm are yet to be elucidated, this retards the progress of the utilization of suitable alleles in rice breeding techniques to produce higher yields. In the present study, we developed a set of gene-tagged markers based on the gene mutation sites Gn1a-M1 and Gn1a-M2 for Gn1a, GW2-HapI for GW2, and GS3-PstI for GS3. The results demonstrated that these STS markers could clearly differentiate between the different alleles at each gene locus. A survey of the allele distributions of the abovementioned three genes was performed with 156 cultivars. It was observed that the 5150-Gn1a allele was absent on the Gn1a locus and only two type alleles (Ha-Gn1a and Ko-Gn1a) were present, of which 54.3% indica and 21.5% japonica cultivars contained the Ha-Gn1a allele. Two alleles (MH-GS3 and ZS-GS3) were detected on the GS3 locus, and 48.6% indica and 9.9% japonica cultivars harbored the suitable allele MH-GS3. Further, all the cultivars contained the FA-GW2 allele on GW2, whereas the WY-GW2 allele was not found. These results further suggested that some of the alleles residing in the indica subspecies have introgressed into the japonica group with a very low frequency. The gene-tagged markers developed in the present study can be directly used as a tool for marker-aided selection (MAS) in rice breeding techniques to produce higher yields.