Xihong Shen

Transcriptome analysis of rice root heterosis by RNA-Seq

BackgroundHeterosis is a phenomenon in which hybrids exhibit superior performance relative to parental phenotypes. In addition to the heterosis of above-ground agronomic traits on which most existing studies have focused, root heterosis is also an indispensable component of heterosis in the entire plant and of major importance to plant breeding. Consequently, systematic investigations of root heterosis, particularly in reproductive-stage rice, are needed. The recent advent of RNA sequencing technology (RNA-Seq) provides an opportunity to conduct in-depth transcript profiling for heterosis studies.ResultsUsing the Illumina HiSeq 2000 platform, the root transcriptomes of the super-hybrid rice variety Xieyou 9308 and its parents were analyzed at tillering and heading stages. Approximately 391 million high-quality paired-end reads (100-bp in size) were generated and aligned against the Nipponbare reference genome. We found that 38,872 of 42,081 (92.4%) annotated transcripts were represented by at least one sequence read. A total of 829 and 4186 transcripts that were differentially expressed between the hybrid and its parents (DGHP) were identified at tillering and heading stages, respectively. Out of the DGHP, 66.59% were down-regulated at the tillering stage and 64.41% were up-regulated at the heading stage. At the heading stage, the DGHP were significantly enriched in pathways related to processes such as carbohydrate metabolism and plant hormone signal transduction, with most of the key genes that are involved in the two pathways being up-regulated in the hybrid. Several significant DGHP that could be mapped to quantitative trait loci (QTLs) for yield and root traits are also involved in carbohydrate metabolism and plant hormone signal transduction pathways.ConclusionsAn extensive transcriptome dataset was obtained by RNA-Seq, giving a comprehensive overview of the root transcriptomes at tillering and heading stages in a heterotic rice cross and providing a useful resource for the rice research community. Using comparative transcriptome analysis, we detected DGHP and identified a group of potential candidate transcripts. The changes in the expression of the candidate transcripts may lay a foundation for future studies on molecular mechanisms underlying root heterosis.

Identification of qRL7, a major quantitative trait locus associated with rice root length in hydroponic conditions

Root system development is an important target for improving yield in rice. Active roots that can take up nutrients more efficiently are essential for improving grain yield. In this study, we performed quantitative trait locus (QTL) analyses using 215 recombinant inbred lines derived from a cross between Xieqingzao B (XB), a maintainer line with short roots and R9308, a restorer line with long roots. Only a QTLs associated with root length were mapped on chromosomes 7. The QTL, named qRL7, was located between markers RM3859 and RM214 on chromosome 7 and explained 18.14–18.36% of the total phenotypic variance evaluated across two years. Fine mapping of qRL7 using eight BC3F3 recombinant lines mapped the QTL to between markers InDel11 and InDel17, which delimit a 657.35 kb interval in the reference cultivar Nipponbare. To determine the genotype classes for the target QTL in these BC3F3 recombinants, the root lengths of their BC3F4 progeny were investigated, and the result showed that qRL7 plays a crucial role in root length. The results of this study will increase our understanding of the genetic factors controlling root architecture, which will help rice breeders to breed varieties with deep, strong and vigorous root systems.

Identification of Transcriptome SNPs for Assessing Allele-Specific Gene Expression in a Super-Hybrid Rice Xieyou9308

Hybridization, a common process in nature, can give rise to a vast reservoir of allelic variants. Combination of these allelic variants may result in novel patterns of gene action and is thought to contribute to heterosis. In this study, we analyzed genome-wide allele-specific gene expression (ASGE) in the super-hybrid rice variety Xieyou9308 using RNA sequencing technology (RNA-Seq). We identified 9325 reliable single nucleotide polymorphisms (SNPs) distributed throughout the genome. Nearly 68% of the identified polymorphisms were CT and GA SNPs between R9308 and Xieqingzao B, suggesting the existence of DNA methylation, a heritable epigenetic mark, in the parents and their F1 hybrid. Of 2793 identified transcripts with consistent allelic biases, only 480 (17%) showed significant allelic biases during tillering and/or heading stages, implying that trans effects may mediate most transcriptional differences in hybrid offspring. Approximately 67% and 62% of the 480 transcripts showed R9308 allelic expression biases at tillering and heading stages, respectively. Transcripts with higher levels of gene expression in R9308 also exhibited R9308 allelic biases in the hybrid. In addition, 125 transcripts were identified with significant allelic expression biases at both stages, of which 74% showed R9308 allelic expression biases. R9308 alleles may tend to preserve their characteristic states of activity in the hybrid and may play important roles in hybrid vigor at both stages. The allelic expression of 355 transcripts was highly stage-specific, with divergent allelic expression patterns observed at different developmental stages. Many transcripts associated with stress resistance were differently regulated in the F1 hybrid. The results of this study may provide valuable insights into molecular mechanisms of heterosis.

Breeding methodology and practice of super rice in China.

Abstract To meet the growing demand for rice production, the Ministry of Agriculture, P. R. China initiated a nationwide project on the development of super rice in 1996. Through efforts in research, super rice breeding in China has gained significant advances in the aspects of breeding theories, creation of breeding materials and selection and promotion of elite rice varieties. A creative super rice breeding path of the construction of harmonious plant type combined with the utilization of heterosis was proposed. Super rice plays more and more important roles in rice production. Totally, 71 super rice varieties in China have been successfully developed and released to production. These varieties have 12 t/hm2 yield potential in on-farm demonstration fields, 8% to 15% higher than the check varieties with high resistance to pest and medium to high level of grain quality, the total growing area of which has reached 23.7 million hectares, and rice grain production increased by 17.7 million tons from 1996 to 2009. This paper reviewed the development and achievements of super rice breeding in aspects of the breeding methodology and breeding practices, summarized the successful experience of super rice breeding in China and proposed the developmental strategies of super rice for the future.

Mapping of QTLs associated with important agronomic traits using three populations derived from a super hybrid rice Xieyou9308

Xieyou9308 is the first commercial super hybrid rice released in 1996 in China. To clarify its genetic mechanism underlying high yield potential, a recombinant inbred line (RIL) population derived from the cross between the maintainer line XieqingzaoB (XQZB) and the restorer line Zhonghui9308 (ZH9308) and two derived backcross F1 (BCF1) populations were developed for the identification of quantitative trait loci (QTLs) related to ten important agronomic traits (tiller number (TN), heading date (HD), and grain yield per plant (GYPP), etc.). The BCF1 performance was closely correlated with the performance of their parental RILs according to both the analysis of broad-sense heritability (hB2) and phenotypic correlation coefficient (PCC) in the two BCF1 populations, but not proved by QTL analysis. A total of 21 additive-effect main QTLs (M-QTLs), 22 dominant-effect M-QTLs, and 19 dominant-effect M-QTLs were detected with the WinQTLCart 2.50 software for the ten traits in the RIL and two BCF1 populations, respectively. Of theses, three QTLs (qHD7a, qPPP3a, and qPL10) of 21 were detected repeatedly in the RIL and one BCF1 populations, ten QTLs underlying four traits were only detected repeatedly in two BCF1 populations, and nine QTLs controlling more than two traits were detected repeatedly, the additive-effect QTLs and dominant-effect QTLs play an important role in the performance of agronomic traits and no epistatic QTL of additive by additive effect and dominant by dominant-effect was detected for all traits in three populations. This research is valuable for M-QTL related to important agronomic trait in future fine mapping and positional cloning.

Mapping and Comparative Analysis of QTL for Rice Plant Height Based on Different Sample Sizes within a Single Line in RIL Population

To clarify the most appropriate sample size for obtaining phenotypic data for a single line, we investigated the main-effect QTL (M-QTL) of a quantitative trait plant height (ph) in a recombinant inbred line (RIL) population of rice (derived from the cross between Xieqingzao B and Zhonghui 9308) using five individual plants in 2006 and 2009. Twenty-six ph phenotypic datasets from the completely random combinations of 2, 3, 4, and 5 plants in a single line, and five ph phenotypic datasets from five individual plants were used to detect the QTLs. Fifteen M-QTLs were detected by 1 to 31 datasets. Of these, qph7a was detected repeatedly by all the 31 ph datasets in 2006 and explained 11.67% to 23.93% of phenotypic variation; qph3 was detected repeatedly by all the 31 datasets and explained 5.21% to 7.93% and 11.51% to 24.46% of phenotypic variance in 2006 and 2009, respectively. The results indicate that the M-QTL for a quantitative trait could be detected repeatedly by the phenotypic values from 5 individual plants and 26 sets of completely random combinations of phenotypic data within a single line in an RIL population under different environments. The sample size for a single line of the RIL population did not affect the efficiency for identification of stably expressed M-QTLs.

OsCOL16, encoding a CONSTANS-like protein, represses flowering by up-regulating Ghd7 expression in rice

Flowering time is an important agronomic trait that coordinates the plant life cycle with regional adaptability and thereby impacts yield potentials for cereal crops. The CONSTANS (CO)-like gene family plays vital roles in the regulation of flowering time. CO-like proteins are typically divided into four phylogenetic groups in rice. Several genes from groups I, III, and IV have been functionally characterized, though little is known about the genes of group II in rice. We report the functional characterization in rice of a constitutive floral inhibitor, OsCOL16, encoding a group-II CO-like protein that delays flowering time and increases plant height and grain yield. Overexpression of OsCOL16 resulted in late heading under both long-day and short-day conditions. OsCOL16 expression exhibits a diurnal oscillation and serves as a transcription factor with transcriptional activation activity. We determined that OsCOL16 up-regulates the expression of the floral repressor Ghd7, leading to down-regulation of the expression of Ehd1, Hd3a, and RFT1. Moreover, genetic diversity and evolutionary analyses suggest that remarkable differences in flowering times correlate with two major alleles of OsCOL16. Our combined molecular biology and phylogeographic analyses revealed that OsCOL16 plays an important role in regulating rice photoperiodic flowering, allowing for environmental adaptation of rice.

Genetic mapping of a QTL controlling source–sink size and heading date in rice

Source size, sink size and heading date (HD) are three important classes of traits that determine the productivity of rice. In this study, a set of recombinant inbred lines (RILs) derived from the cross between an elite indica line Big Grain1 (BG1) and a japonica line Xiaolijing (XLJ) were used to map quantitative trait loci (QTLs) for source-sink size and heading date. Totally, thirty-one QTLs for source size, twenty-two for sink size, four for heading date and seven QTL clusters which included QTLs for multiple traits were identified in three environmental trials. Thirty QTLs could be consistently detected in at least two trials and generally located in the clusters. Using a set of BC4F2 lines, the QTL cluster in C5-1-C5-2 on chromosome 5 was validated to be a major QTL pleiotropically affecting heading date, source size (flag leaf area) and panicle type (neck length of panicle, primary branching number and the ratio of secondary branching number to primary branching number), and was narrowed down to a 309.52Kb region. QTL clusters described above have a large effect on source-sink size and/or heading date, therefore they should be good resources to improve the adaptability and high yield potential of cultivars genetically.

ES7, encoding a ferredoxin-dependent glutamate synthase, functions in nitrogen metabolism and impacts leaf senescence in rice

Glutamate synthase (GOGAT) is a key enzyme for nitrogen metabolism and ammonium assimilation in plants. In this study, an early senescence 7 (es7) mutant was identified and characterized. The leaves of the es7 mutant begin to senesce at the tillering stage about 60day after sowing, and become increasingly senescent as the plants develop at the heading stage. When es7 plants are grown under photorespiration-suppressed conditions (high CO2), the senescence phenotype and chlorophyll content are rescued. qRT-PCR analysis showed that senescence- associated genes were up-regulated significantly in es7. A map-based cloning strategy was used to identify ES7, which encodes a ferredoxin-dependent glutamate synthase (Fd-GOGAT). ES7 was expressed constitutively, and the ES7 protein was localized in chloroplast. qRT-PCR analysis indicated that several genes related to nitrogen metabolism were differentially expressed in es7. Further, we also demonstrated that chlorophyll synthesis-associated genes were significantly down-regulated in es7. In addition, when seedlings are grown under increasing nitrogen concentrations (NH4NO3) for 15days, the contents of chlorophyll a, chlorophyll b and total chlorophyll were significantly lower in es7. Our results demonstrated that ES7 is involved in nitrogen metabolism, effects chlorophyll synthesis, and may also associated with photorespiration, impacting leaf senescence in rice.

QTL Mapping for Hull Thickness and Related Traits in Hybrid Rice Xieyou 9308

We conducted a quantitative trait locus (QTL) analysis of 165 rice recombinant inbred lines derived from a cross between Zhonghui 9308 (Z9308) and Xieqingzao B (XB) in Hainan and Hangzhou, China. Grain thickness (GT), brown rice thickness (BRT), hull thickness (HT) and milling quality were used for QTL mapping. HT was significantly and positively correlated with GT and BRT. Twenty-nine QTLs were detected with phenotypic effects ranging from 2.80% to 21.27%. Six QTLs, qGT3, qBRT3, qBRT4, qHT6.1, qHT8 and qHT11, were detected repeatedly across two environments. Inherited from XB, qHT6.1, qHT8 and qHT11 showed stable expression, explaining 9.92%, 21.27% and 10.83% of the phenotypic variances in Hainan and 9.61%, 6.40% and 6.71% in Hangzhou, respectively. Additionally, the QTL cluster between RM5944 and RM5626 on chromosome 3 was probably responsible for GT and milling quality. The cluster between RM6992 and RM6473 on chromosome 4 played an important role in grain filling. Three near isogenic lines (NILs), X345, X338 and X389, were selected because they contained homozygous fragments from Zhonghui 9308, corresponding to qHT6.1, qHT8 and qHT11, respectively. The hull of XB was thicker than those of X345, X338 and X389. In all the lines, qHT6.1, qHT8 and qHT11 that regulated rice HT were stably inherited with obvious genetic effects.