QTL Mapping by Whole Genome Re-sequencing and Analysis of Candidate Genes for Nitrogen Use Efficiency in Rice.
ABSTRACT: Nitrogen is a major nutritional element in rice production. However, excessive application of nitrogen fertilizer has caused severe environmental pollution. Therefore, development of rice varieties with improved nitrogen use efficiency (NUE) is urgent for sustainable agriculture. In this study, bulked segregant analysis (BSA) combined with whole genome re-sequencing (WGS) technology was applied to finely map quantitative trait loci (QTL) for NUE. A key QTL, designated as qNUE6 was identified on chromosome 6 and further validated by Insertion/Deletion (InDel) marker-based substitutional mapping in recombinants from F2 population (NIL-13B4 × GH998). Forty-four genes were identified in this 266.5-kb region. According to detection and annotation analysis of variation sites, 39 genes with large-effect single-nucleotide polymorphisms (SNPs) and large-effect InDels were selected as candidates and their expression levels were analyzed by qRT-PCR. Significant differences in the expression levels of LOC_Os06g15370 (peptide transporter PTR2) and LOC_Os06g15420 (asparagine synthetase) were observed between two parents (Y11 and GH998). Phylogenetic analysis in Arabidopsis thaliana identified two closely related homologs, AT1G68570 (AtNPF3.1) and AT5G65010 (ASN2), which share 72.3 and 87.5% amino acid similarity with LOC_Os06g15370 and LOC_Os06g15420, respectively. Taken together, our results suggested that qNUE6 is a possible candidate gene for NUE in rice. The fine mapping and candidate gene analysis of qNUE6 provide the basis of molecular breeding for genetic improvement of rice varieties with high NUE, and lay the foundation for further cloning and functional analysis.
Project description:To identify the genomic regions for yield and NUE of rice genotypes and lines with promising yield under low N, a recombinant inbred population (RIL) developed between BPT5204 (a mega variety known for its quality) and PTB1 (variety with high NUE) was evaluated for consecutive wet and dry seasons under low nitrogen (LN) and recommended nitrogen (RN) field conditions. A set of 291 RILs were characterized for 24 traits related to leaf, agro-morphological, yield, N content and nitrogen use efficiency indices. More than 50 RILs were found promising with grain yield >10 g under LN. Parental polymorphism survey with 297 SSRs and selective genotyping revealed five genomic regions associated with yield under LN, which were further saturated with polymorphic SSRs. Thirteen promising SSRs were identified out of 144 marker trait associations under LN using single marker analysis. Composite interval mapping showed 37 QTL under LN with five pleiotropic QTL. A major stable pleiotropic (RM13201-RM13209) from PTB1 spanning 825.4 kb region associated with straw N % (SNP) in both treatments across seasons and yield and yield related traits in WS appears to be promising for the MAS. Another major QTL (RM13181-RM13201) was found to be associated with only relative trait parameters of biomass, grain and grain nitrogen. These two major pleiotropic QTL (RM13201-RM13209 and RM13181-RM13201) on chromosome 2 were characterized for their positive allele effect and could be deployed for the development of rice varieties with NUE.
Project description:Over-application of nitrogen fertilizer in fields has had a negative impact on both environment and human health. Domesticated rice varieties with high nitrogen use efficiency (NUE) reduce fertilizer for sustainable agriculture. Here, we perform genome-wide association analysis of a diverse rice population displaying extreme nitrogen-related phenotypes over three successive years in the field, and identify an elite haplotype of nitrate transporter OsNPF6.1HapB that enhances nitrate uptake and confers high NUE by increasing yield under low nitrogen supply. OsNPF6.1HapB differs in both the protein and promoter element with natural variations, which are differentially trans-activated by OsNAC42, a NUE-related transcription factor. The rare natural allele OsNPF6.1HapB, derived from variation in wild rice and selected for enhancing both NUE and yield, has been lost in 90.3% of rice varieties due to the increased application of fertilizer. Our discovery highlights this NAC42-NPF6.1 signaling cascade as a strategy for high NUE and yield breeding in rice.
Project description:In crops, nitrogen directly determines productivity and biomass. However, the improvement of nitrogen utilization efficiency (NUE) is still a major challenge in modern agriculture. Here, we report the characterization of are1, a genetic suppressor of a rice fd-gogat mutant defective in nitrogen assimilation. ARE1 is a highly conserved gene, encoding a chloroplast-localized protein. Loss-of-function mutations in ARE1 cause delayed senescence and result in 10-20% grain yield increases, hence enhance NUE under nitrogen-limiting conditions. Analysis of a panel of 2155 rice varieties reveals that 18% indica and 48% aus accessions carry small insertions in the ARE1 promoter, which result in a reduction in ARE1 expression and an increase in grain yield under nitrogen-limiting conditions. We propose that ARE1 is a key mediator of NUE and represents a promising target for breeding high-yield cultivars under nitrogen-limiting condition.
Project description:The indiscriminate use of nitrogenous fertilizers continues unabated for commercial crop production, resulting in air and water pollution. The development of rice varieties with enhanced nitrogen use efficiency (NUE) will require a thorough understanding of the molecular basis of a plant's response to low nitrogen (N) availability. The global expression profiles of root tissues collected from low and high N treatments at different time points in two rice genotypes, Pokkali and Bengal, with contrasting responses to N stress and contrasting root architectures were examined. Overall, the number of differentially expressed genes (DEGs) in Pokkali (indica) was higher than in Bengal (japonica) during low N and early N recovery treatments. Most low N DEGs in both genotypes were downregulated whereas early N recovery DEGs were upregulated. Of these, 148 Pokkali-specific DEGs might contribute to Pokkali's advantage under N stress. These DEGs included transcription factors and transporters and were involved in stress responses, growth and development, regulation, and metabolism. Many DEGs are co-localized with quantitative trait loci (QTL) related to root growth and development, chlorate-resistance, and NUE. Our findings suggest that the superior growth performance of Pokkali under low N conditions could be due to the genetic differences in a diverse set of genes influencing N uptake through the regulation of root architecture.
Project description:Nitrogen (N) fertilizers are a major input cost in rice production, and its excess application leads to major environmental pollution. Development of rice varieties with improved nitrogen use efficiency (NUE) is essential for sustainable agriculture. Here, we report the results of field evaluations of marker-free transgenic NERICA4 (New Rice for Africa 4) rice lines overexpressing barley alanine amino transferase (HvAlaAT) under the control of a rice stress-inducible promoter (pOsAnt1). Field evaluations over three growing seasons and two rice growing ecologies (lowland and upland) revealed that grain yield of pOsAnt1:HvAlaAT transgenic events was significantly higher than sibling nulls and wild-type controls under different N application rates. Our field results clearly demonstrated that this genetic modification can significantly increase the dry biomass and grain yield compared to controls under limited N supply. Increased yield in transgenic events was correlated with increased tiller and panicle number in the field, and evidence of early establishment of a vigorous root system in hydroponic growth. Our results suggest that expression of the HvAlaAT gene can improve NUE in rice without causing undesirable growth phenotypes. The NUE technology described in this article has the potential to significantly reduce the need for N fertilizer and simultaneously improve food security, augment farm economics and mitigate greenhouse gas emissions from the rice ecosystem.
Project description:Improving nitrogen use efficiency (NUE) represents one of the main goals to reduce N input in maximizing crop yield for sustainable agriculture. A NUE key strategy is the exploitation of genetic variation in available germplasm together with the understanding of molecular mechanisms governing this complex trait. Thus, NUE, its components, nitrogen uptake efficiency (NUpE) and nitrogen utilization efficiency (NUtE), and NUE-related traits heritability were evaluated in ancient (Cappelli, Capeiti, Russello, and Mazzancoio) and modern (Messapia, Tiziana, Svevo, and Normanno) wheat genotypes for tackling nitrogen (N) and/or water limitation in both growth chamber and field experiments. Our results exhibited a reduction of NUE, NUpE, and NUtE under water and combined (nitrogen + water) stress in all the genotypes, as expected. The contribution of genetic variability on phenotypic variation was significant for NUtE, harvest index, post-anthesis nitrogen uptake (PANU), and biomass production traits. Moreover, the stress tolerance indexes, calculated and bi-plotted for N and water stresses, exhibited two distinct clusters for many traits as then confirmed by principal component analysis. Although modern varieties showed higher crop yield and NUE under conventional N and water regimes, ancient varieties exhibited best performances to cope with both stresses, mainly under water limitation. Finally, the usage index, which takes into account total biomass increase, underlined that old genotypes were less affected by both stresses during crop cycle. In particular, these genotypes showed the best performances for NUE and its components under both stresses at stem elongation and milk ripening as shown also by PANU. In addition, at these stages, nitrate and ammonium transporter gene expressions in the root were performed, showing the highest activity in ancient varieties. In conclusion, the identification of NUE traits during a specific crop cycle stage, under both N and water limitation, will help in the breeding of more resilient varieties in Mediterranean sustainable agriculture by reducing N supply.
Project description:Breeding high-yielding and nitrogen-efficient maize (Zea mays L.) hybrid varieties is a strategy that could simultaneously solve the problems of resource shortages and environmental pollution. We conducted a 2-year field study using four nitrogen application rates (0, 150, 225, and 300 kg N hm-2) and two maize hybrid varieties (ZD958 and QS101) to understand the plant traits related to high grain yields and high nitrogen use efficiency (NUE). We found that ZD958 had a higher grain yield and nitrogen accumulation in the shoots at harvest as well as a higher NUE at lower nitrogen application rates (0 and 150 kg hm-2) than QS101. The grain yields and NUE were almost identical for the two hybrid varieties at nitrogen application rates of 225 and 300 kg N hm-2. Compared with QS101, ZD958 had higher above-ground and below-ground biomass amounts, a deeper root distribution, longer root length, root active absorption area, greater grain filling rate, and higher photosynthetic NUE than QS101 at lower nitrogen application rates. Our results showed that ZD958 can maintain a higher grain yield at lower nitrogen rates in a similar manner to N-efficient maize hybrid varieties. The selection of hybrids such as ZD958 with a deeper root distribution and higher photosynthetic NUE can increase the grain yield and NUE under low nitrogen conditions.
Project description:The yield potential of rice (Oryza sativa L.) has experienced two significant growth periods that coincide with the introduction of semi-dwarfism and the utilization of heterosis. In present study, we determined the annual increase in the grain yield of rice varieties grown from 1936 to 2005 in Middle Reaches of Yangtze River and examined the contributions of RUE (radiation-use efficiency, the conversion efficiency of pre-anthesis intercepted global radiation to biomass) and NUE (nitrogen-use efficiency, the ratio of grain yield to aboveground N accumulation) to these improvements. An examination of the 70-year period showed that the annual gains of 61.9 and 75.3 kg ha(-1) in 2013 and 2014, respectively, corresponded to an annual increase of 1.18 and 1.16% in grain yields, respectively. The improvements in grain yield resulted from increases in the harvest index and biomass, and the sink size (spikelets per panicle) was significantly enlarged because of breeding for larger panicles. Improvements were observed in RUE and NUE through advancements in breeding. Moreover, both RUE and NUE were significantly correlated with the grain yield. Thus, our study suggests that genetic improvements in rice grain yield are associated with increased RUE and NUE.
Project description:To understand variation in the root development traits (total root length (TRL), maximum root length (MRL) and root number) of 18 New Rice for Africa (NERICA) varieties, seedlings were hydroponically grown under deficient and sufficient concentrations of two forms of nitrogen, NH4 + and NO3 -. The donor African rice variety, 'CG14' (Oryza glaberrima Steud.), showed greater TRL and MRL than three background Asian rice varieties (Oryza sativa L.). Wide distribution was observed in all traits of the 18 NERICAs. The 18 NERICAs and parental varieties were classified into three cluster groups by cluster analysis. Cluster Ia included only 'CG14'. Comparative analysis characterized cluster Ib (including 'NERICA7') as an active root elongation group, and cluster II (including 'WAB56-104') as an active primordia development group. QTL analysis of F2 plants developed from a cross between 'WAB56-104' and 'NERICA7' detected two putative quantitative trait loci (QTLs) for root elongation on chromosome 1. Of these, a major QTL, designated as qRL1.4-NERICA7, was an NH4 +-responsive QTL, which was narrowed down to a 0.7-Mbp region through progeny testing using F7 lines. qRL1.4-NERICA7 should help us understand genetic control in NERICAs, and improve root elongation in rice breeding programs.
Project description:The NIN-LIKE PROTEIN (NLP) family of transcription factors were identified as nitrate-responsive cis-element (NRE)-binding proteins, which function as transcriptional activators in the nitrate-regulated expression of downstream genes. This study was aimed at genome-wide analysis of NLP gene family in rice and the expression profiling of NLPs in response to nitrogen (N) supply and deficiency in rice genotypes with contrasting N use efficiency (NUE). Based on in silico analysis, 6 NLP genes (including alternative splice forms 11 NLPs) were identified from rice. Expression of NLPs was promoted by nitrate supply as well as N deficiency (NLP1, NLP3, NLP4 and NLP5). Four rice genotypes APO (high NUE under sufficient N), IR83929-B-B-291-3-1-1 (IR-3-1-1), Nerica-L-42 (NL-42) (High NUE at low N), and Pusa Basmati 1 (PB1, low NUE) to correlate traits governing NUE and expression of NLPs. Analysis of rate of nitrate uptake and expression of N assimilatory and uptake genes established that IR-3-1-1 has high uptake and assimilation efficiency, translating into high NUE, whereas PB1 is efficient in uptake only when N availability is high. Along with the transcriptional upregulation of NLPs, genotype IR-3-1-1, displayed highest expression of OsNRT1.1B gene, the closest rice homologue of nitrate transceptor AtNRT1.1 and plays major role in nitrate uptake, translocation and signaling in rice. The results showed that high NUE rice genotypes has both high Nitrogen uptake efficiency (NUpE) and Nitrogen utilization efficiency (NUtE), resulting from the effective and coordinated signal transduction network involving the rice homologue of nitrate transceptor OsNRT1.1B, the probable primary nitrate response (PNR) regulator OsNLP1 and the master response regulator OsNLP3, a homologue of AtNLP6/7.