Cloning a novel reduced-height (Rht) gene TaOSCA1.4 from a QTL in wheat.

Reducing plant height (PH) is one of the core contents of the "Green Revolution", which began in the 1960s in wheat. A number of 27 reduced-height (Rht) genes have been identified and a great number of quantitative trait loci (QTLs) for PH have been mapped on all 21 chromosomes. Nonetheless, only several genes regulated PH have been cloned. In this study, we found the interval of QTL QPh-1B included an EST-SSR marker swes1079. According to the sequence of swes1079, we cloned the TaOSCA1.4 gene. We developed a CAPS marker to analyze the variation across a natural population. The result showed that the PH was significantly different between the two haplotypes of TaOSCA1.4-1B under most of the 12 environments and the average values of irrigation and rainfed conditions. This result further demonstrated that TaOSCA1.4 was associated with PH. Then, we validated the TaOSCA1.4 via RNAi technology. The average PHs of the wild-type (WT), RNAi lines 1 (Ri-1) and 2 (Ri-2) were 94.6, 83.6 and 79.2 cm, respectively, with significant differences between the WT and Ri-1 and Ri-2. This result indicated that the TaOSCA1.4 gene controls PH. TaOSCA1.4 is a constitutively expressed gene and its protein localizes to the cell membrane. TaOSCA1.4 gene is a member of the OSCA gene family, which regulates intracellular Ca2+ concentration. We hypothesized that knock down mutants of TaOSCA1.4 gene reduced regulatory ability of Ca2+, thus reducing the PH. Furthermore, the cell lengths of the knock down mutants are not significantly different than that of WT. We speculate that TaOSCA1.4 gene is not directly associated with gibberellin (GA), which should be a novel mechanism for a wheat Rht gene.

Identification of candidate genes for Fusarium head blight resistance from QTLs using RIL population in wheat.

Fusarium head blight (FHB) stands out as one of the most devastating wheat diseases and leads to significantly grain yield losses and quality reductions in epidemic years. Exploring quantitative trait loci (QTL) for FHB resistance is a critical step for developing new FHB-resistant varieties. We previously constructed a genetic map of unigenes (UG-Map) according to the physical positions using a set of recombinant-inbred lines (RILs) derived from the cross of 'TN18 × LM6' (TL-RILs). Here, the number of diseased spikelets (NDS) and relative disease index (RDI) for FHB resistance were investigated under four environments using TL-RILs, which were distributed across 13 chromosomes. A number of 36 candidate genes for NDS and RDI from of 19 stable QTLs were identified. The average number of candidate genes per QTL was 1.89, with 14 (73.7%), two (10.5%), and three (15.8%) QTLs including one, two, and 3-10 candidate genes, respectively. Among the 24 candidate genes annotated in the reference genome RefSeq v1.1, the homologous genes of seven candidate genes, including TraesCS4B02G227300 for QNds/Rdi-4BL-4553, TraesCS5B02G303200, TraesCS5B02G303300, TraesCS5B02G303700, TraesCS5B02G303800 and TraesCS5B02G304000 for QNds/Rdi-5BL-9509, and TraesCS7A02G568400 for QNds/Rdi-7AL-14499, were previously reported to be related to FHB resistance in wheat, barely or Brachypodium distachyon. These genes should be closely associated with FHB resistance in wheat. In addition, the homologous genes of five genes, including TraesCS1A02G037600LC for QNds-1AS-2225, TraesCS1D02G017800 and TraesCS1D02G017900 for QNds-1DS-527, TraesCS1D02G018000 for QRdi-1DS-575, and TraesCS4B02G227400 for QNds/Rdi-4BL-4553, were involved in plant defense responses against pathogens. These genes should be likely associated with FHB resistance in wheat.

QTL Mapping and Candidate Gene Identifying for N, P, and K Use Efficiency at the Maturity Stages in Wheat.

Nitrogen (N), phosphorus (P), and potassium (K) are the three most important mineral nutrients for crop growth and development. We previously constructed a genetic map of unigenes (UG-Map) based on their physical positions using a RIL population derived from the cross of "TN18 × LM6" (TL-RILs). In this study, a total of 18 traits related to mineral use efficiency (MUE) of N/P/K were investigated under three growing seasons using TL-RILs. A total of 54 stable QTLs were detected, distributed across 19 chromosomes except for 3A and 5B. There were 50 QTLs associated with only one trait, and the other four QTLs were associated with two traits. A total of 73 candidate genes for stable QTLs were identified. Of these, 50 candidate genes were annotated in Chinese Spring (CS) RefSeq v1.1. The average number of candidate genes per QTL was 1.35, with 45 QTLs containing only one candidate gene and nine QTLs containing two or more candidate genes. The candidate gene TraesCS6D02G132100 (TaPTR gene) for QGnc-6D-3306 belongs to the NPF (NRT1/PTR) gene family. We speculate that the TaPTR gene should regulate the GNC trait.

ATP-dependent DNA helicase (TaDHL), a Novel Reduced-Height (Rht) Gene in Wheat.

In wheat, a series of dwarf and semi-dwarf plant varieties have been developed and utilized worldwide since the 1960s and caused the 'Green Revolution'. To date, 25 reduced-height (Rht) genes have been identified, but only several genes for plant height (PH) have been isolated previously. In this study, we identified a candidate gene, ATP-dependent DNA helicase (TaDHL-7B), for PH via QTL mapping and genome-wide association study (GWAS) methods. We knocked out this gene using the CRISPR/Cas9 system in variety 'Fielder'. Two homozygous mutant genotypes, AAbbDD (-5 bp) and AAbbDD (-1 bp), were obtained in the T2 generation. The PH values of AAbbDD (-5 bp) and AAbbDD (-1 bp) were significantly reduced compared with the wild-type (WT, 'Fielder'), indicating that TaDHL-7B is a novel Rht gene that controls the PH. This is the first time that a PH gene of wheat has been isolated with a non-hormone pathway, providing a new insight into the genetic control of PH. The TaDHL gene reduced the PH without a yield penalty. It could be used to improve the lodging resistance and yield in wheat breeding programs.

CRISPR/Cas9-Mediated Disruption of Xylanase inhibitor protein (XIP) Gene Improved the Dough Quality of Common Wheat.

The wheat dough quality is of great significance for the end-use of flour. Some genes have been cloned for controlling the protein fractions, grain protein content, starch synthase, grain hardness, etc. Using a unigene map of the recombinant inbred lines (RILs) for "TN 18 × LM 6," we mapped a quantitative trait locus (QTL) for dough stability time (ST) and SDS-sedimentation values (SV) on chromosome 6A (QSt/Sv-6A-2851). The peak position of the QTL covered two candidate unigenes, and we speculated that TraesCS6A02G077000 (a xylanase inhibitor protein) was the primary candidate gene (named the TaXip gene). The target loci containing the three homologous genes TaXip-6A, TaXip-6B, and TaXip-6D were edited in the variety "Fielder" by clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9). Two mutant types in the T2:3 generation were obtained (aaBBDD and AAbbdd) with about 120 plants per type. The SVs of aaBBDD, AAbbdd, and WT were 31.77, 27.30, and 20.08 ml, respectively. The SVs of the aaBBDD and AAbbdd were all significantly higher than those of the wild type (WT), and the aaBBDD was significantly higher than the AAbbdd. The STs of aaBBDD, AAbbdd, and WT were 2.60, 2.24, and 2.25 min, respectively. The ST for the aaBBDD was significantly higher than that for WT and was not significantly different between WT and AAbbdd. The above results indicated that XIP in vivo can significantly affect wheat dough quality. The selection of TaXip gene should be a new strategy for developing high-quality varieties in wheat breeding programs.

QTL mapping for quality traits using a high-density genetic map of wheat.

Protein- and starch-related quality traits, which are quantitatively inherited and significantly influenced by the environment, are critical determinants of the end-use quality of wheat. We constructed a high-density genetic map containing 10,739 loci (5,399 unique loci) using a set of 184 recombinant inbred lines (RILs) derived from a cross of 'Tainong 18 × Linmai 6' (TL-RILs). In this study, a quantitative trait loci (QTLs) analysis was used to examine the genetic control of grain protein content, sedimentation value, farinograph parameters, falling number and the performance of the starch pasting properties using TL-RILs grown in a field for three years. A total of 106 QTLs for 13 quality traits were detected, distributed on the 21 chromosomes. Of these, 38 and 68 QTLs for protein- and starch-related traits, respectively, were detected in three environments and their average values (AV). Twenty-six relatively high-frequency QTLs (RHF-QTLs) that were detected in more than two environments. Twelve stable QTL clusters containing at least one RHF-QTL were detected and classified into three types: detected only for protein-related traits (type I), detected only for starch-related traits (type II), and detected for both protein- and starch-related traits (type III). A total of 339 markers flanked with 11 QTL clusters (all except C6), were found to be highly homologous with 282 high confidence (HC) and 57 low confidence (LC) candidate genes based on IWGSC RefSeq v 1.0. These stable QTLs and RHF-QTLs, especially those grouped into clusters, are credible and should be given priority for QTL fine-mapping and identification of candidate genes with which to explain the molecular mechanisms of quality development and inform marker-assisted breeding in the future.

The Hot QTL Locations for Potassium, Calcium, and Magnesium Nutrition and Agronomic Traits at Seedling and Maturity Stages of Wheat under Different Potassium Treatments.

Potassium (K) is one of the most important mineral nutrients for wheat. In this study, the effects of low K (LK) treatments and the quantitative trait loci (QTLs) for K, calcium (Ca), and magnesium (Mg) use efficiency traits, both at the seedling and maturity stages of wheat, were investigated. The set of "Tainong 18 × Linmai 6" recombinant inbred lines (RILs) were used to identify the QTLs under different K treatments using hydroponic culture and field trials. The majority of K concentrations and content-related traits at seedling and maturity stages decreased with reduced K supply, but the K use efficiency-related traits increased. In contrast, with reduced K supply, the contents of Ca and Mg increased, while the Ca and Mg use efficiency decreased. A total of 217 QTLs for seedling traits and 89 QTLs for adult traits were detected. Four relatively high-frequency QTLs (RHF-QTLs) and 18 QTL clusters (colocation of QTLs for more than two traits) were detected. Eight clusters were detected for K-, Ca-, and Mg-related traits simultaneously. This means that these traits might be controlled by the same QTL. In addition, we highlight that 4B might be an important chromosome regulating the nutrition of K, Ca, and Mg in wheat. The 4B chromosome and four hot QTL clusters, which located 45 QTLs, might be important potential targets for further investigation.

QTL Mapping for Phosphorus Efficiency and Morphological Traits at Seedling and Maturity Stages in Wheat.

Phosphorus (P) efficiency (PE), which comprises phosphorus uptake (PupE) and utilization efficiency (PutE), is considered as one of the most important factors for crop yield. In the present study, 11 seedling traits and 13 maturity traits related to wheat PE and morphology were investigated using a set of recombinant inbred lines (RILs) derived from the cross of "TN 18 × LM 6," under hydroponic culture trials and field trials at low P (LP) and normal P (NP) levels in two different years, respectively. The LP input reduced of biomass, yield and PupE traits, but increased PutE traits. A total of 163 QTLs for seedling and maturity traits under different P levels and their AV, and 15 QTLs for relative traits were detected on 21 chromosomes. Of these, 49 and 63 QTLs for were detected specially in LP and NP treatments, respectively. We found 11 relatively high-frequency QTLs (RHF-QTLs) and four important QTL clusters, which may be the potential targets for marker-assisted selection (MAS) in wheat breeding programs for PE. Favorable relationships for breeding programs were found in the four important QTL clusters, which allow the possibility of improving the morphological traits and PutE simultaneously. A total of 29 markers which associated with 51 QTLs were found highly homologous with EST sequences, which suggested that they were potential functional loci. We suggested that the four biomass traits (SDW, RDW, TDW, and RSDW), five yield traits (SN, PH, TGW, GWP, and StWP) and two relative traits (Rstwp and Rgwp) can be considered as the primary indexes for the evaluation of PE for they are easy to identify on a large-scale.

Isolation and characterization of the TaSnRK2.10 gene and its association with agronomic traits in wheat (Triticum aestivum L.).

Sucrose non-fermenting 1-related protein kinases (SnRKs) comprise a major family of signaling genes in plants and are associated with metabolic regulation, nutrient utilization and stress responses. This gene family has been proposed to be involved in sucrose signaling. In the present study, we cloned three copies of the TaSnRK2.10 gene from bread wheat on chromosomes 4A, 4B and 4D. The coding sequence (CDS) is 1086 bp in length and encodes a protein of 361 amino acids that exhibits functional domains shared with SnRK2s. Based on the haplotypes of TaSnRK2.10-4A (Hap-4A-H and Hap-4A-L), a cleaved amplified polymorphic sequence (CAPS) marker designated TaSnRK2.10-4A-CAPS was developed and mapped between the markers D-1092101 and D-100014232 using a set of recombinant inbred lines (RILs). The TaSnRK2.10-4B alleles (Hap-4B-G and Hap-4B-A) were transformed into allele-specific PCR (AS-PCR) markers TaSnRK2.10-4B-AS1 and TaSnRK2.10-4B-AS2, which were located between the markers D-1281577 and S-1862758. No diversity was found for TaSnRK2.10-4D. An association analysis using a natural population consisting of 128 winter wheat varieties in multiple environments showed that the thousand grain weight (TGW) and spike length (SL) of Hap-4A-H were significantly higher than those of Hap-4A-L, but pant height (PH) was significantly lower.

Molecular genetic analysis of grain protein content and flour whiteness degree using RILs in common wheat.

Grain protein content (GPC) and flour whiteness degree (FWD) are important qualitative traits in common wheat. Quantitative trait locus (QTL) mapping for GPC and FWD was conducted using a set of 131 recombinant-inbred lines derived from the cross 'Chuan 35050 × Shannong 483' in six environmental conditions. A total of 22 putative QTLs (nine GPC and 13 FWD) were identified on 12 chromosomes with individual QTL explaining 4.5-34.0% phenotypic variation. Nine QTLs (40.9%) were detected in two or more environments. The colocated QTLs were on chromosomes 1B and 4B. Among the QTLs identified for GPC, QGpc.sdau-4A from the parent Shannong 483 represented some important favourable QTL alleles. QGpc.sdau-2A.1 and QFwd.sdau-2A.1 had a significant association with both GPC and FWD. The markers detected on top of QTL regions could be potential targets for marker-assisted selection.

Insights into the origin and evolution of the plant hormone signaling machinery.

Plant hormones modulate plant growth, development, and defense. However, many aspects of the origin and evolution of plant hormone signaling pathways remain obscure. Here, we use a comparative genomic and phylogenetic approach to investigate the origin and evolution of nine major plant hormone (abscisic acid, auxin, brassinosteroid, cytokinin, ethylene, gibberellin, jasmonate, salicylic acid, and strigolactone) signaling pathways. Our multispecies genome-wide analysis reveals that: (1) auxin, cytokinin, and strigolactone signaling pathways originated in charophyte lineages; (2) abscisic acid, jasmonate, and salicylic acid signaling pathways arose in the last common ancestor of land plants; (3) gibberellin signaling evolved after the divergence of bryophytes from land plants; (4) the canonical brassinosteroid signaling originated before the emergence of angiosperms but likely after the split of gymnosperms and angiosperms; and (5) the origin of the canonical ethylene signaling pathway postdates shortly the emergence of angiosperms. Our findings might have important implications in understanding the molecular mechanisms underlying the emergence of land plants.

QTL mapping for seedling traits in wheat grown under varying concentrations of N, P and K nutrients.

Nutrient use efficiency (NuUE), comprising nutrient uptake and utilization efficiency, is regarded as one of the most important factors for wheat yield. In the present study, six morphological, nine nutrient content and nine nutrient utilization efficiency traits were investigated at the seedling stage using a set of recombinant inbred lines (RILs), under hydroponic culture of 12 treatments including single nutrient levels and two- and three-nutrient combinations treatments of N, P and K. For the 12 designed treatments, a total of 380 quantitative trait loci (QTLs) on 20 chromosomes for the 24 traits were detected. Of these, 87, 149 and 144 QTLs for morphological, nutrient content and nutrient utilization efficiency traits were found, respectively. Using the data of the average value (AV) across 12 treatments, 70 QTLs were detected for 23 traits. Most QTLs were located in new marker regions. Twenty-six important QTL clusters were mapped on 13 chromosomes, 1A, 1B, 1D, 2B, 3A, 3B, 4A, 4B, 5D, 6A, 6B, 7A and 7B. Of these, ten clusters involved 147 QTLs (38.7%) for investigated traits, indicating that these 10 loci were more important for the NuUE of N, P and K. We found evidence for cooperative uptake and utilization (CUU) of N, P and K in the early growth period at both the phenotype and QTL level. The correlation coefficients (r) between nutrient content and nutrient utilization efficiency traits for N, P and K were almost all significantly positive correlations. A total of 32 cooperative CUU loci (L1-L32) were found, which included 190 out of the 293 QTLs (64.8%) for the nutrient uptake and utilization efficiency traits, indicating that the CUU-QTLs were common for N, P and K. The CUU-QTLs in L3, L7, L16 and L28 were relatively stable. The CUU-QTLs may explain the CUU phenotype at the QTL level.

Enrichment of a common wheat genetic map and QTL mapping for fatty acid content in grain.

DArT and SSR markers were used to saturate and improve a previous genetic map of RILs derived from the cross Chuan35050 × Shannong483. The new map comprised 719 loci, 561 of which were located on specific chromosomes, giving a total map length of 4008.4 cM; the rest 158 loci were mapped to the most likely intervals. The average chromosome length was 190.9 cM and the marker density was 7.15 cM per marker interval. Among the 719 loci, the majority of marker loci were DArTs (361); the rest included 170 SSRs, 100 EST-SSRs, and 88 other molecular and biochemical loci. QTL mapping for fatty acid content in wheat grain was conducted in this study. Forty QTLs were detected in different environments, with single QTL explaining 3.6-58.1% of the phenotypic variations. These QTLs were distributed on 16 chromosomes. Twenty-two QTLs showed positive additive effects, with Chuan35050 increasing the QTL effects, whereas 18 QTLs were negative with increasing effects from Shannong483. Six sets of co-located QTLs for different traits occurred on chromosomes 1B, 1D, 2D, 5D, and 6B.

Conditional QTL mapping for plant height with respect to the length of the spike and internode in two mapping populations of wheat.

Plant height (PH) in wheat is a complex trait; its components include spike length (SL) and internode lengths. To precisely analyze the factors affecting PH, two F(8:9) recombinant inbred line (RIL) populations comprising 485 and 229 lines were generated. Crosses were performed between Weimai 8 and Jimai 20 (WJ) and between Weimai 8 and Yannong 19 (WY). Possible genetic relationships between PH and PH components (PHC) were evaluated at the quantitative trait locus (QTL) level. PH and PHC (including SL and internode lengths from the first to the fourth counted from the top, abbreviated as FIITL, SITL, TITL, and FOITL, respectively) were measured in four environments. Individual and the pooled values from four trials were used in the present analysis. A QTL for PH was mapped using data on PH and on PH conditioned by PHC using IciMapping V2.2. All 21 chromosomes in wheat were shown to harbor factors affecting PH in two populations, by both conditional and unconditional QTL mapping methods. At least 11 pairwise congruent QTL were identified in the two populations. In total, ten unconditional QTL and five conditional QTL that could be detected in the conditional analysis only have been verified in no less than three trials in WJ and WY. In addition, three QTL on the short arms of chromosomes 4B, 4D, and 7B were mapped to positions similar to those of the semi-dwarfing genes Rht-B1, Rht-D1 and Rht13, respectively. Conditional QTL mapping analysis in WJ and WY proved that, at the QTL level, SL contributed the least to PH, followed by FIITL; TITL had the strongest influence on PH, followed by SITL and FOITL. The results above indicated that the conditional QTL mapping method can be used to evaluate possible genetic relationships between PH and PHC, and it can efficiently and precisely reveal counteracting QTL, which will enhance the understanding of the genetic basis of PH in wheat. The combination of two related populations with a large/moderate population size made the results authentic and accurate.

No observed effect of homologous recombination on influenza C virus evolution.

The occurrence of homologous recombination in influenza viruses has been under some debate recently. To determine the extent of homologous recombination in influenza C virus, recombination analyses of all available gene sequences of influenza C virus were carried out. No recombination signal was found. With the previous evidence in influenza A and B viruses, it seems that homologous recombination has minimal or no effect on influenza virus evolution.

Transgenic microalgae as a non-antibiotic bactericide producer to defend against bacterial pathogen infection in the fish digestive tract.

Antibiotics are commonly employed in most fish aquacultures to prevent disease. One major risk in this practice is that antibiotic-resistant pathogens may be selected. Therefore, we wanted to examine the feasibility of producing an economical, non-antibiotic alternative. The microalga Nannochloropsis oculata is an essential phytoplankton used as live feed for fish larvae. We attempted to culture N. oculata in a way that would provide an organism against bacterial pathogenic infection. To test this idea, we constructed an algae-codon-optimized bovine lactoferricin (LFB) fused with a red fluorescent protein (DsRed) driven by a heat-inducible promoter, which is a heat shock protein 70A promoter combined with a ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit 2' promoter from Chlamydomonas reinhardtii. After electroporation, we examined 491 microalgal clones and generated two stable transgenic lines, each expressing a stable transgene inheritance for at least 26 months. This was confirmed by the positive detection of the mRNA transcript and the protein of LFB-DsRed produced by the transgenic microalgae. To test the efficacy of the antimicrobial peptide LFB, medaka fish (Oryzias latipes) were adapted from freshwater to seawater and were fed with the transgenic algae by oral-in-tube delivery method. Bacterial infection with 1 x 10(5)Vibrio parahaemolyticus per fish was induced 6h thereafter by oral-in-tube delivery as well. For medaka fish fed with 1 x 10(8) transgenic algae per fish, the average survival rate after a 24-h period of infection was much higher than that of medaka fed with wild-type algae (85+/-7.1% versus 5+/-7.1%). This result suggests that medaka fish fed with the LFB-containing transgenic microalgae will have bactericidal defense against V. parahaemolyticus infection in its digestive tract.

Cross-species recombination in the haemagglutinin gene of canine distemper virus.

Canine distemper virus (CDV) has high prevalence in the world dog population and poses an important conservation threat to many carnivore species. In this study, extensive phylogenetic and recombination analyses were performed on all available complete haemagglutinin gene sequences and a strain (AF178038) isolated from giant panda was identified as putative recombinant. Interestingly, the mosaic was produced by recombination between genotypes European wildlife and Asia-1 and the recombination event involves viruses infecting different host species. This finding may have important implications for the evolution of CDV.