Evaluation of Grain-Filling-Related Traits Using Taichung 65 x DV85 Chromosome Segment Substitution Lines (TD-CSSLs) of Rice.

Grain yield of rice consists of sink capacity and grain filling. There are some genes known to contribute to sink capacity, but few genes associated with grain filling are known. We conducted a genetic analysis on yield-related traits by using a chromosome segment substitution line population that have introgression from DV85, an aus variety of rice, in the background of T65, a japonica variety. Refined whole-genome genotypes of the 43 TD-CSSLs were obtained by genotyping-by-sequencing. The effects of previously detected quantitative trait loci (QTLs), qNSC1 and qNSC2, were confirmed by the amount of non-structural carbohydrate (NSC) at 5 days after heading (DAH). The CSSL for qSWTR11, the QTL for decrease in shoot weight during the maturity stage, showed the highest NSC at 5 DAH and lowest at 35 DAH. The brown rice yield of these lines were not stably significant. Most of the sink-related traits correlated between the 2 tested years, but most of the grain-filling traits did not show correlation between the 2 years. Correlation analysis revealed that the sink capacity is stable and primarily determines the yield, and grain filling is more affected by the environment. In addition, biomass production before heading and during the maturity stage contributes to higher yield in TD-CSSLs, and the amount of translocation of stem reserve does not affect much to the yield. We conclude that higher NSC at the heading stage and rapid decrease in shoot biomass during the maturity stage did not directly contribute to the yield formation in the japonica genetic background.

Utilization of Genotyping-by-Sequencing (GBS) for Rice Pre-Breeding and Improvement: A Review.

Molecular markers play a crucial role in the improvement of rice. To benefit from these markers, genotyping is carried out to identify the differences at a specific position in the genome of individuals. The advances in sequencing technologies have led to the development of different genotyping techniques such as genotyping-by-sequencing. Unlike PCR-fragment-based genotyping, genotyping-by-sequencing has enabled the parallel sequencing and genotyping of hundreds of samples in a single run, making it more cost-effective. Currently, GBS is being used in several pre-breeding programs of rice to identify beneficial genes and QTL from different rice genetic resources. In this review, we present the current advances in the utilization of genotyping-by-sequencing for the development of rice pre-breeding materials and the improvement of existing rice cultivars. The challenges and perspectives of using this approach are also highlighted.

A Novel Combination of Genes Causing Temperature-Sensitive Hybrid Weakness in Rice.

Reproductive isolation is an obstacle for plant breeding when a distant cross is demanded. It can be divided into two main types based on different growth stages: prezygotic isolation and postzygotic isolation. The hybrid weakness, which is a type of postzygotic isolation, can become a problem in crop breeding. In order to overcome reproductive isolation, it is necessary to elucidate its mechanism. In this study, genetic analysis for low temperature-dependent hybrid weakness was conducted in a rice F2 population derived from Taichung 65 (T65, Japonica) and Lijiangxintuanheigu (LTH, Japonica). The weak and severe weak plants in F2 showed shorter culm length, late heading, reduced panicle number, decreased grain numbers per panicle, and impaired root development in the field. Our result also showed that hybrid weakness was affected by temperature. It was observed that 24°C enhanced hybrid weakness, whereas 34°C showed recovery from hybrid weakness. In terms of the morphology of embryos, no difference was observed. Therefore, hybrid weakness affects postembryonic development and is independent of embryogenesis. The genotypes of 126 F2 plants were determined through genotyping-by-sequencing and a linkage map consisting of 862 single nucleotide polymorphism markers was obtained. Two major quantitative trait loci (QTLs) were detected on chromosomes 1 [hybrid weakness j 1 (hwj1)] and 11 [hybrid weakness j 2 (hwj2)]. Further genotyping indicated that the hybrid weakness was due to an incompatible interaction between the T65 allele of hwj1 and the LTH allele of hwj2. A large F2 populations consisting of 5,722 plants were used for fine mapping of hwj1 and hwj2. The two loci, hwj1 and hwj2, were mapped in regions of 65-kb on chromosome 1 and 145-kb on chromosome 11, respectively. For hwj1, the 65-kb region contained 11 predicted genes, while in the hwj2 region, 22 predicted genes were identified, two of which are disease resistance-related genes. The identified genes along these regions serve as preliminary information on the molecular networks associated with hybrid weakness in rice.

WUSCHEL-related homeobox family genes in rice control lateral root primordium size.

The development of a plastic root system is essential for stable crop production under variable environments. Rice plants have two types of lateral roots (LRs): S-type (short and thin) and L-type (long, thick, and capable of further branching). LR types are determined at the primordium stage, with a larger primordium size in L-types than S-types. Despite the importance of LR types for rice adaptability to variable water conditions, molecular mechanisms underlying the primordium size control of LRs are unknown. Here, we show that two WUSCHEL-related homeobox (WOX) genes have opposing roles in controlling LR primordium (LRP) size in rice. Root tip excision on seminal roots induced L-type LR formation with wider primordia formed from an early developmental stage. QHB/OsWOX5 was isolated as a causative gene of a mutant that is defective in S-type LR formation but produces more L-type LRs than wild-type (WT) plants following root tip excision. A transcriptome analysis revealed that OsWOX10 is highly up-regulated in L-type LRPs. OsWOX10 overexpression in LRPs increased the LR diameter in an expression-dependent manner. Conversely, the mutation in OsWOX10 decreased the L-type LR diameter under mild drought conditions. The qhb mutants had higher OsWOX10 expression than WT after root tip excision. A yeast one-hybrid assay revealed that the transcriptional repressive activity of QHB was lost in qhb mutants. An electrophoresis mobility shift assay revealed that OsWOX10 is a potential target of QHB. These data suggest that QHB represses LR diameter increase, repressing OsWOX10 Our findings could help improve root system plasticity under variable environments.

Development of an Aus-Derived Nested Association Mapping (Aus-NAM) Population in Rice.

A genetic resource for studying genetic architecture of agronomic traits and environmental adaptation is essential for crop improvements. Here, we report the development of a rice nested association mapping population (aus-NAM) using 7 aus varieties as diversity donors and T65 as the common parent. Aus-NAM showed broad phenotypic variations. To test whether aus-NAM was useful for quantitative trait loci (QTL) mapping, known flowering genes (Ehd1, Hd1, and Ghd7) in rice were characterized using single-family QTL mapping, joint QTL mapping, and the methods based on genome-wide association study (GWAS). Ehd1 was detected in all the seven families and all the methods. On the other hand, Hd1 and Ghd7 were detected in some families, and joint QTL mapping and GWAS-based methods resulted in weaker and uncertain peaks. Overall, the high allelic variations in aus-NAM provide a valuable genetic resource for the rice community.

Marker-Assisted Introgression and Stacking of Major QTLs Controlling Grain Number (Gn1a) and Number of Primary Branching (WFP) to NERICA Cultivars.

The era of the green revolution has significantly improved rice yield productivity. However, with the growing population and decreasing arable land, rice scientists must find new ways to improve rice productivity. Although hundreds of rice yield-related QTLs were already mapped and some of them were cloned, only a few were utilized for actual systematic introgression breeding programs. In this study, the major yield QTLs Grain Number 1a (Gn1a) and Wealthy Farmer's Panicle (WFP) were introgressed and stacked in selected NERICA cultivars by marker-assisted backcross breeding (MABB). The DNA markers RM3360, RM3452, and RM5493 were used for foreground selection. At BC3F4 and BC3F5 generation, a combination of marker-assisted selection and phenotypic evaluation were carried out to select lines with target alleles and traits. Further, genotyping-by-sequencing (GBS) was conducted to validate the introgression and determine the recurrent parent genome recovery (RPGR) of the selected lines. The Gn1a and/or WFP introgression lines showed significantly higher numbers of spikelets per panicle and primary branching compared to the recurrent parents. In addition, lines with Gn1a and/or WFP alleles were comparatively similar to the recurrent parents (RP) in most yield-related traits. This study demonstrates the success of utilizing yield QTLs and marker-assisted selection to develop and improve rice cultivars.

Mutation of OUR1/OsbZIP1, which encodes a member of the basic leucine zipper transcription factor family, promotes root development in rice through repressing auxin signaling.

A well-developed root system is essential for efficient water uptake, particularly in drought-prone environments. However, the molecular mechanisms underlying the promotion of root development are poorly understood. We identified and characterized a rice mutant, outstanding rooting1 (our1), which exhibited a well-developed root system. The our1 mutant displayed typical auxin-related phenotypes, including elongated seminal root and defective gravitropism. Seminal root elongation in the our1 mutant was accelerated via the promotion of cell division and elongation. In addition, compared with the wild type, the density of short and thin lateral roots (S-type LRs) was reduced in the our1 mutant, whereas that of long and thick LRs (L-type LRs) was increased. Expression of OUR1, which encodes OsbZIP1, a member of the basic leucine zipper transcription factor family, was observed in the seminal root tip and sites of LR emergence, wherein attenuation of reporter gene expression levels controlled by the auxin response promoter DR5 was also observed in the our1 mutant. Taken together, our results indicate that the our1 gene promotes root development by suppressing auxin signaling, which may be a key factor contributing to an improvement in root architecture.

QTL analysis for carbon assimilate translocation-related traits during maturity in rice (Oryza sativa L.).

Problems with carbon assimilate translocation from source organs to sink (grains) during ripening cause yield losses in rice (Oryza sativa L.), especially in high-sink-capacity varieties. We conducted a genetic analysis of traits related to such translocation by using recombinant inbred lines. Shoot weight (SW) of T65, a japonica parent, was retained from heading to late maturity, whereas that of DV85, an aus parent, was greater than that of T65 at 5 days after heading (DAH) and then decreased until 20 DAH. This difference was observed clearly under standard-fertilizer but not low-fertilizer conditions. Non-structural carbohydrate (NSC) contents in the parents showed a tendency similar to that for SW. QTL analysis revealed pleiotropic QTLs on chromosomes 5 and 10, probably by associations with heading date QTLs. A QTL associated with harvest index and NSC at 5 DAH was detected on chromosome 1. By considering the temporal changes of the traits, we found a QTL for decrease in SW on chromosome 11; the DV85 allele of this QTL facilitated assimilate translocation and suppressed biomass growth. A suggestive QTL for NSC decrease was located on chromosome 2. These QTLs could represent potential targets for controlling carbon assimilate translocation in breeding programs.

OsPIN2, which encodes a member of the auxin efflux carrier proteins, is involved in root elongation growth and lateral root formation patterns via the regulation of auxin distribution in rice.

Auxin flow is important for different root developmental processes such as root formation, emergence, elongation and gravitropism. However, the detailed information about the mechanisms regulating the auxin flow is less well understood in rice. We characterized the auxin transport-related mutants, Ospin-formed2-1 (Ospin2-1) and Ospin2-2, which exhibited curly root phenotypes and altered lateral root formation patterns in rice. The OsPIN2 gene encodes a member of the auxin efflux carrier proteins that possibly regulates the basipetal auxin flow from the root tip toward the root elongation zone. According to DR5-driven GUS expression, there is an asymmetric auxin distribution in the mutants that corresponded with the asymmetric cell elongation pattern in the mutant root tip. Auxin transport inhibitor, N-1-naphthylphthalamic acid and Ospin2-1 Osiaa13 double mutant rescued the curly root phenotype indicating that this phenotype results from a defect in proper auxin distribution. The typical curly root phenotype was not observed when Ospin2-1 was grown in distilled water as an alternative to tap water, although higher auxin levels were found at the root tip region of the mutant than that of the wild-type. Therefore, the lateral root formation zone in the mutant was shifted basipetally compared with the wild-type. These results reflect that an altered auxin flow in the root tip region is responsible for root elongation growth and lateral root formation patterns in rice.

A major locus involved in the formation of the radial oxygen loss barrier in adventitious roots of teosinte Zea nicaraguensis is located on the short-arm of chromosome 3.

A radial oxygen loss (ROL) barrier in roots of waterlogging-tolerant plants promotes oxygen movement via aerenchyma to the root tip, and impedes soil phytotoxin entry. The molecular mechanism and genetic regulation of ROL barrier formation are largely unknown. Zea nicaraguensis, a waterlogging-tolerant wild relative of maize (Zea mays ssp. mays), forms a tight ROL barrier in its roots when waterlogged. We used Z. nicaraguensis chromosome segment introgression lines (ILs) in maize (inbred line Mi29) to elucidate the chromosomal region involved in regulating root ROL barrier formation. A segment of the short-arm of chromosome 3 of Z. nicaraguensis conferred ROL barrier formation in the genetic background of maize. This chromosome segment also decreased apoplastic solute permeability across the hypodermis/exodermis. However, the IL and maize were similar for suberin staining in the hypodermis/exodermis at 40 mm and further behind the root tip. Z. nicaraguensis contained suberin in the hypodermis/exodermis at 20 mm and lignin at the epidermis. The IL with ROL barrier, however, did not contain lignin in the epidermis. Discovery of the Z. nicaraguensis chromosomal region responsible for root ROL barrier formation has improved knowledge of this trait and is an important step towards improvement of waterlogging tolerance in maize.

RCN1/OsABCG5, an ATP-binding cassette (ABC) transporter, is required for hypodermal suberization of roots in rice (Oryza sativa).

Suberin is a complex polymer composed of aliphatic and phenolic compounds. It is a constituent of apoplastic plant interfaces. In many plant species, including rice (Oryza sativa), the hypodermis in the outer part of roots forms a suberized cell wall (the Casparian strip and/or suberin lamellae), which inhibits the flow of water and ions and protects against pathogens. To date, there is no genetic evidence that suberin forms an apoplastic transport barrier in the hypodermis. We discovered that a rice reduced culm number1 (rcn1) mutant could not develop roots longer than 100 mm in waterlogged soil. The mutated gene encoded an ATP-binding cassette (ABC) transporter named RCN1/OsABCG5. RCN1/OsABCG5 gene expression in the wild type was increased in most hypodermal and some endodermal roots cells under stagnant deoxygenated conditions. A GFP-RCN1/OsABCG5 fusion protein localized at the plasma membrane of the wild type. Under stagnant deoxygenated conditions, well suberized hypodermis developed in wild types but not in rcn1 mutants. Under stagnant deoxygenated conditions, apoplastic tracers (periodic acid and berberine) were blocked at the hypodermis in the wild type but not in rcn1, indicating that the apoplastic barrier in the mutant was impaired. The amount of the major aliphatic suberin monomers originating from C(28) and C(30) fatty acids or ω-OH fatty acids was much lower in rcn1 than in the wild type. These findings suggest that RCN1/OsABCG5 has a role in the suberization of the hypodermis of rice roots, which contributes to formation of the apoplastic barrier.

Does suberin accumulation in plant roots contribute to waterlogging tolerance?

Plants that are adapted to waterlogged conditions develop aerenchyma in roots for ventilation. Some wetland plant species also form an apoplastic barrier at the outer cell layers of roots that reduces radial oxygen loss (ROL) from the aerenchyma and prevents toxic compounds from entering the root. The composition of the apoplastic barrier is not well understood. One potential component is suberin, which accumulates at the hypodermal/exodermal cell layers of the roots under waterlogged soil conditions or in response to other environmental stimuli. However, differences in suberin content and composition between plant species make it difficult to evaluate whether suberin has a role in preventing ROL. In this article, we summarize recent advances in understanding apoplastic barrier formation in roots and, between various plant species, compare the chemical compositions of the apoplastic barriers in relation to their permeability to oxygen. Moreover, the relationship between suberin accumulation and the barrier to ROL is discussed.

SLL1, which encodes a member of the stearoyl-acyl carrier protein fatty acid desaturase family, is involved in cell elongation in lateral roots via regulation of fatty acid content in rice.

We have identified a gene, SHORT LATERAL ROOT LENGTH1 (SLL1), which is important for the elongation of lateral roots in rice. An sll1 mutant has decreased lateral root growth due to a defect in the cell elongation. The SLL1 gene encodes a member of the stearoyl-acyl carrier protein fatty acid desaturase family that is the key regulator of overall fatty acid desaturation in plants. We measured the fatty acid content and found that the 18:0 content in the sll1 mutant root was approximately 4 times that in the wild-type root. When the sll1 mutant was grown at 33 °C, it complemented the mutant phenotype to a moderate level, which reflects the importance of the low 18:0 content in maintaining the cell membrane structure. The SLL1 gene was expressed at the lateral root tip, whereas SLL1 expression was not detected in the elongation zone of the crown roots. These results indicate that the lateral root specific defect in sll1 mutant is caused by the different expression patterns of SLL1 in lateral and crown roots. In addition, SLL1 over-expressers produced significantly longer lateral roots compared to the wild-type, and thus SLL1 gene would be very useful for improving rice root architecture.

Mechanisms for coping with submergence and waterlogging in rice.

Rice (Oryza sativa L.), unlike other cereals, can grow well in paddy fields and is highly tolerant of excess water stress, from either submergence (in which part or all of the plant is under water) or waterlogging (in which excess water in soil limits gas diffusion). Rice handles submergence stress by internal aeration and growth controls. A quiescence strategy based on Submergence-1A (SUB1A) or an escape strategy based on SNORKEL1 (SK1) and SNORKEL2 (SK2) is used for the growth controls. On the other hand, rice handles waterlogging stress by forming lysigenous aerenchyma and a barrier to radial O2 loss (ROL) in roots in order to supply O2 to the root tip. In this article, we summarize recent advances in understanding the mechanisms of responding to excess water stresses (i.e., submergence and waterlogging) in rice and other gramineous plants.

Ectopic expression of the K+ channel ß subunits from Puccinellia tenuiflora (KPutB1) and rice (KOB1) alters K+ homeostasis of yeast and Arabidopsis.

In this study, we cloned a cDNA for the K+ channel ß subunit from the halophyte Puccinellia tenuiflora and named it KPutB1. KPutB1 was preferentially expressed in the roots and was transiently induced by K+-starvation, salt stress, or the combination of both stresses. By yeast two-hybrid assay, we demonstrated that KPutB1 interacts with PutAKT1, α subunit of an AKT1-type K+ channel of P. tenuiflora. The functional relevance of this interaction on K+-nutrition was investigated by co-expression experiments in yeast under various ionic conditions, and K+ channel α and ß subunit homologues from rice (OsAKT1 and KOB1, respectively) were included for comparison. Yeast co-expressing PutAKT1 and the ß subunits (KPutB1 and KOB1) had better growth and higher K+-uptake ability than yeast expressing PutAKT1 alone. In contrast, yeast co-expressing the ß subunits (KPutB1 and KOB1) with OsAKT1 had slower growth and lower K+ uptake than yeast expressing OsAKT1 alone. Arabidopsis plants over-expressing the K+ channel ß subunit of P. tenuiflora or rice showed increased shoot K+ content and decreased root Na+ content under control, 75 mM NaCl, and K+-starvation stress conditions. These results suggest that ectopic expression of the K+ channel ß subunit could alter K+ and Na+ homeostasis in plants.

Identification of genes expressed in maize root cortical cells during lysigenous aerenchyma formation using laser microdissection and microarray analyses.

• To adapt to waterlogging in soil, some gramineous plants, such as maize (Zea mays), form lysigenous aerenchyma in the root cortex. Ethylene, which is accumulated during waterlogging, promotes aerenchyma formation. However, the molecular mechanism of aerenchyma formation is not understood. • The aim of this study was to identify aerenchyma formation-associated genes expressed in maize roots as a basis for understanding the molecular mechanism of aerenchyma formation. Maize plants were grown under waterlogged conditions, with or without pretreatment with an ethylene perception inhibitor 1-methylcyclopropene (1-MCP), or under aerobic conditions. Cortical cells were isolated by laser microdissection and their mRNA levels were examined with a microarray. • The microarray analysis revealed 575 genes in the cortical cells, whose expression was either up-regulated or down-regulated under waterlogged conditions and whose induction or repression was suppressed by pretreatment with 1-MCP. • The differentially expressed genes included genes related to the generation or scavenging of reactive oxygen species, Ca(2+) signaling, and cell wall loosening and degradation. The results of this study should lead to a better understanding of the mechanism of root lysigenous aerenchyma formation.

Analysis of expressed sequence tags from a NaHCO(3)-treated alkali-tolerant plant, Chloris virgata.

Chloris virgata Swartz (C. virgata) is a gramineous wild plant that can survive in saline-alkali areas in northeast China. To examine the tolerance mechanisms of C. virgata, we constructed a cDNA library from whole plants of C. virgata that had been treated with 100 mM NaHCO(3) for 24 h and sequenced 3168 randomly selected clones. Most (2590) of the expressed sequence tags (ESTs) showed significant similarity to sequences in the NCBI database. Of the 2590 genes, 1893 were unique. Gene Ontology (GO) Slim annotations were obtained for 1081 ESTs by BLAST2GO and it was found that 75 genes of them were annotated with GO terms "response to stress", "response to abiotic stimulus", and "response to biotic stimulus", indicating these genes were likely to function in tolerance mechanism of C. virgata. In a separate experiment, 24 genes that are known from previous studies to be associated with abiotic stress tolerance were further examined by real-time RT-PCR to see how their expressions were affected by NaHCO(3) stress. NaHCO(3) treatment up-regulated the expressions of pathogenesis-related gene (DC998527), Win1 precursor gene (DC998617), catalase gene (DC999385), ribosome inactivating protein 1 (DC999555), Na(+)/H(+) antiporter gene (DC998043), and two-component regulator gene (DC998236).

Isolation and characterization of a metallothionein-1 protein in Chloris virgata Swartz that enhances stress tolerances to oxidative, salinity and carbonate stress in Saccharomyces cerevisiae.

Chloris virgata Swartz (C. virgata) is a gramineous wild plant that is found in alkaline soil areas in northeast China and is highly tolerant to carbonate stress. We constructed a cDNA library from C. virgata seedlings treated with NaHCO(3), and isolated a type 1 metallothionein (MT1) gene (ChlMT1: AB294238) from the library. The amino acid sequence of ChlMT1 contained 12 cysteine residues that constituted the Cys-X-Cys (X = amino acid except Cys) motifs in the N- and C-terminal regions. Northern hybridization showed that expression of ChlMT1 was induced by several abiotic stresses, from salts (NaCl and NaHCO(3)), a ROS inducer (paraquat), and metals (CuSO(4), ZnSO(4), and CoCl(2)). ChlMT1 expression in leaf was induced by 200 mM NaCl and 100 mM NaHCO(3). About 5 microM Paraquat, 500 microM Zn(2+), and 500 microM Co(2+) also induced expression of ChlMT1 in leaf after 6 h, and 100 microM Cu(2+) induced it after 24 h. Saccharomyces cerevisiae when transformed with the ChlMT1 gene had dramatically increased tolerances to salts (NaCl and NaHCO(3)) and ROS.

Expression of an NADP-malic enzyme gene in rice (Oryza sativa. L) is induced by environmental stresses; over-expression of the gene in Arabidopsis confers salt and osmotic stress tolerance.

NADP-malic enzyme (NADP-ME, EC 1.1.1.40) functions in many different pathways in plants, and has recently been implicated in plant defense such as in responses to wounding and UV-B radiation. In this study, we isolated a complementary DNA (cDNA) clone by using the differential display method and screening of a root cDNA library of rice (Oryza sativa. L) under carbonate (NaHCO3) stress, and identified it as one of the rice NADP-ME genes (we named it NADP-ME2, GenBank accession no. AB053295). The 5' end of NADP-ME2 was obtained by the 5'-RACE method, and the full-length cDNA had a length of 2217 bp encoding 593 amino acids. Expression of NADP-ME2 mRNA in roots was induced by stress from carbonates (NaHCO3 and Na2CO3, NaCl, and environmental pH changes. NADP-ME2 transcripts increased during 72-h exposures to NaHCO3, NaCl, and PEG stresses. Furthermore, NADP-ME activities in leaves and roots of rice seedlings increased by more than 50% in the presence of carbonates (NaHCO3 and Na2CO3), NaCl, and PEG. These results indicate that rice NADP-ME2 responds to salts and osmotic stresses. Transgenic Arabidopsis plants over-expressing NADP-ME2 were obtained through transformation, screening, Northern analysis and in situ NADP-ME activity assay. Transgenic Arabidopsis plants over-expressing NADP-ME2 grew well in 1/2 x MS medium with 100 mM NaCl or 4% mannitol, whereas growth of wild-type (WT) Arabidopsis seedlings was strongly inhibited. In addition, under 125 mM NaCl stress, the root lengths of transgenic lines were about twice as long as those of the WT. These results suggest that NADP-ME2 has a role in enhancing tolerance of plants to salt and osmotic stress.