Pairs Of Near-isogenic Lines Research Articles

Phenotypic and genotypic characterization of near-isogenic lines targeting a major 4BL QTL responsible for pre-harvest sprouting in wheat

BackgroundResistance to pre-harvest sprouting (PHS) is one of the major objectives in wheat breeding programs. However, the complex quantitative nature of this trait presents challenges when breeding for PHS resistance. Characterization of PHS using near-isogenic lines (NILs) targeting major quantitative trait locus/loci (QTL/QTLs) can be an effective strategy for the identification of responsible genes and underlying mechanisms.ResultsIn this study, multiple pairs of NILs were developed and confirmed for a major QTL located on the 4BL chromosome arm that contributes to PHS resistance in wheat, using a combined heterogeneous inbred family method and a fast generation cycling system. Phenotypic characterization of these confirmed NILs revealed significant differences in PHS resistance between the isolines, where the presence of the resistant allele increased the resistance to sprouting on spikes by 54.0–81.9% (average 70.8%) and reduced seed germination by 59.4–70.5% (average 66.2%). The 90 K SNP genotyping assay on the confirmed NIL pairs identified eight SNPs on 4BL, associated with five candidate genes; two of the candidate genes were related to seed dormancy. Agronomic traits in the NIL pairs were investigated; both plant height and grain number per spike were positively correlated with PHS susceptibility.ConclusionsThis study confirmed multiple pairs of NILs and identified SNPs between PHS isolines, which are valuable resources for further fine-mapping of this locus to clone the major genes for PHS resistance and investigate the possible functional regulation of these genes on important agronomic traits, such as plant height and grain number per spike.

Temporal expression of candidate genes at the Co-1 locus and their interaction with other defense related genes in common bean

The presence of multiple resistance genes in common bean and the diverse race spectrum of Colletotrichum lindemuthianum suggests that a broad spectrum of resistance mechanisms needs to be deployed in resistance breeding. However, limited studies on the interaction of anthracnose resistance genes and other plant defense genes exist in common bean. Therefore, we analyzed four candidate genes of Co-12 allele at the Co-1 locus and plant defense genes via quantitative real time PCR, to determine the fine-tuned timing and magnitude of expression of the resistance reaction. In the present study, a pair of near isogenic lines (NILs; T9576R and T9576S) that differ for the Co-12 resistance allele, was used that upon infection with race 73 of C. lindemuthianum show differential gene expression between the NILs. Our aim was to identify a particular gene that is effective against race 73 among other candidate genes located at the Co-1 locus. Gene expression analysis revealed higher expression levels of the Phvul.001G243800 gene. Expression was nearly 144 fold higher in the resistant NIL, than in the susceptible NIL and could be a possible candidate gene effective against race 73. In addition, we found that initially the other defense genes are activated which suggests that both defense and candidate gene activation is necessary to effect resistance against the anthracnose pathogen of common bean.

Comparison of Three Alleles at a Major Solid Stem QTL for Wheat Stem Sawfly Resistance and Agronomic Performance in Hexaploid Wheat

ABSTRACTSolid stems provide resistance to the wheat stem sawfly (WSS, Cephus cinctus Nort.) in wheat (Triticum aestivum L.). A major quantitative trait locus, designated Qss.msub‐3BL, controls most of the variation for stem solidness. Two alleles that differ for temporal expression of stem solidness have been identified. An allele derived from the cultivar ‘Rescue’, designated Qss.msub‐3BL.b, results in solid stems throughout stem development. An allele derived from the cultivar ‘Conan’, designated Qss.msub‐3BL.c, causes high stem solidness early in plant development, but solidness declines as the plant matures. The objective of this study was to develop near‐isogenic lines (NILs) derived from marker‐assisted backcrossing to compare the impact of Qss.msub‐3BL alleles in different genetic backgrounds on stem solidness, WSS resistance, and agronomic traits. The Conan‐derived allele Qss.msub‐3BL.c resulted in lower WSS infestation than the Rescue‐derived allele Qss.msub‐3BL.b in two genetic backgrounds. Both alleles showed a high level of early stem solidness, though this declined as the stem matured for NIL with the Conan‐derived allele. The Conan‐derived allele Qss.msub‐3BL.c resulted in lower WSS infestation and stem cutting than the allele for hollow stems in two genetic backgrounds. Based on yield trials grown over 2 yr, there were no significant differences in grain yield between the NIL pairs. Our results indicate the Conan‐derived allele at Qss.msub‐3BL results in lower infestation of WSS and does not significantly affect agronomic performance, thus providing a novel source of resistance for the development of WSS‐resistant cultivars.

Use of near-isogenic lines to precisely map and validate a major QTL for grain weight on chromosome 4AL in bread wheat (Triticum aestivum L.).

This study precisely mapped and validated a major quantitative trait locus (QTL) on chromosome 4AL for thousand-grain weight in wheat using multiple near-isogenic lines. Thousand-grain weight (TGW) is an essential yield component. Following the previous identification of a major QTL for TGW within the interval of 15.7cM (92.7-108.4cM) on chromosome 4AL using the Nongda3338 (ND3338)/Jingdong6 (JD6) doubled haploid population, the aim of this study was to perform more precise mapping and validate the genetic effect of the QTL. Multiple near-isogenic lines (NILs) were developed using ND3338 as the recurrent parent through marker-assisted selection. Based on five independent BC3F3:4 segregating populations derived from BC3F3 plants with different heterozygous segments for the target QTL site and the results of genotyping analysis performed using the Wheat660K SNP array, it was possible to delimit the QTL region to a physical interval of approximately 6.5Mb (677.11-683.61Mb, IWGSC Ref Seq v1.0). Field trials across multiple environments showed that NILsJD6 had a consistent effect on increasing the TGW by 5.16-27.48% and decreasing the grain number per spike (GNS) by 3.98-32.91% compared to the corresponding NILsND3338, which exhibited locus-specific TGW-GNS trade-offs. Moreover, by using RNA sequencing (RNA-Seq) of whole grains at 10days after pollination stage of multiple NILs, we found that differentially expressed genes between the NIL pairs were significantly enriched for cell cycle and the replication of chromosome-related genes, hence affecting cell division and cell proliferation. Overall, our results provide a basis for map-based cloning of the major QTL and determining the mechanisms underlying TGW-GNS trade-offs in wheat, which would help to fine-tune these two components and maximize the grain yield for breeders.

Identification and mapping of expressed genes associated with the 2DL QTL for fusarium head blight resistance in the wheat line Wuhan 1

BackgroundFusarium head blight (FHB) is a problem of great concern in small grain cereals, especially wheat. A quantitative trait locus (QTL) for FHB resistance (FHB_SFI) located on the long arm of chromosome 2D in the spring wheat genotype Wuhan 1 is a resistance locus which has potential to improve the FHB resistance of bread wheat since it confers effective resistance to wheat breeding lines. Recently, differentially expressed genes (DEG) have been identified by comparing near isogenic lines (NIL) carrying the susceptible and resistant alleles for the 2DL QTL, using RNA-Seq. In the present study, we aimed to identify candidate genes located within the genetic interval for the 2DL QTL for FHB resistance, as assessed by single floret inoculation (FHB_SFI), and possibly contributing to it.ResultsCombining previous and additional bioinformatics analyses, 26 DEG that were located on chromosome arm 2DL were selected for further characterization of their expression profile by RT-qPCR. Seven of those DEG showed a consistent differential expression profile between either three pairs of near isogenic lines or other genotypes carrying the R and S alleles for the 2DL QTL for FHB resistance. UN25696, which was identified in previous expression work using microarray was also confirmed to have a differential expression pattern. Those eight candidate genes were further characterized in 85 lines of a double haploid mapping population derived from the cross Wuhan 1/Nyubai, the population where the 2DL QTL was originally identified. The expression QTL for gene Traes_2DL_179570792 overlapped completely with the mapping interval for the 2DL QTL for FHB_SFI while the expression QTL for UN25696 mapped near the QTL, but did not overlap with it. None of the other genes had a significant eQTL on chromosome 2DL. Higher expression of Traes_2DL_179570792 and UN25696 was associated with the resistant allele at that locus.ConclusionsOf the 26 DEG from the 2DL chromosome further characterized in this study, only two had an expression QTL located in or near the interval for the 2DL QTL. Traes_2DL_179570792 is the first expression marker identified as associated with the 2DL QTL.

Comprehensive transcriptomics, proteomics, and metabolomics analyses of the mechanisms regulating tiller production in low-tillering wheat.

Tiller development in low-tillering wheat is related to several differentially expressed genes, proteins, and metabolites, as determined by an integrated omics approach combining transcriptome analysis, iTRAQ, and HPLC-MS on multiple NILs. Tillering is an important aspect of plant morphology that affects spike number, thereby contributing to the final crop yield. However, the mechanisms inhibiting tiller production in low-tillering wheat are poorly characterized. To investigate this aspect of wheat biology, two pairs of near-isogenic lines were developed, and an integrated omics approach combining transcriptome analysis, isobaric tags for relative and absolute quantification, and high-performance liquid chromatography-mass spectrometry were used to compare the free-tillering and low-tillering caused by an allele at Qltn.sicau-2D in wheat samples. Overall, 474 genes, 166 proteins, and 28 metabolites were identified as tillering-associated differentially expressed genes, proteins, and metabolites (DEGs, DEPs, and DEMs, respectively). Functional analysis indicated that the abundance of DEGs/DEPs/DEMs was related to lignin and cellulose metabolism, cell division, cell cycle processes, and glycerophospholipid metabolism; three transcription factor families, GRAS, GRF, and REV, might be related to the decrease in tillering in low-tillering wheat. These findings contribute to improve our understanding of the mechanisms responsible for the inhibition of tiller development in low-tillering wheat cultivars.

Multiple Near-Isogenic Lines Targeting a QTL Hotspot of Drought Tolerance Showed Contrasting Performance Under Post-anthesis Water Stress.

The complex quantitative nature of drought-related traits is a major constraint to breed tolerant wheat varieties. Pairs of near-isogenic lines (NILs) with a common genetic background but differing in a particular locus could turn quantitative traits into a Mendelian factor facilitating our understanding of genotype and phenotype interactions. In this study, we report our fast track development and evaluation of NILs from C306 × Dharwar Dry targeting a wheat 4BS QTL hotspot in C306, which confers drought tolerance following the heterogeneous inbreed family (HIF) analysis coupled with immature embryo culture-based fast generation technique. Molecular marker screening and phenotyping for grain yield and related traits under post-anthesis water stress (WS) confirmed four isoline pairs, viz., qDSI.4B.1-2, qDSI.4B.1-3, qDSI.4B.1-6, and qDSI.4B.1-8. There were significant contrasts of responses between the NILs with C306 QTL (+NILs) and the NILs without C306 QTL (−NILs). Among the four confirmed NIL pairs, mean grain yield per plant of the +NILs and −NILs showed significant differences ranging from 9.61 to 10.81 and 6.30 to 7.56 g, respectively, under WS condition, whereas a similar grain yield was recorded between the +NILs and −NILs under well-watered condition. Isolines of +NIL and −NIL pairs showed similar chlorophyll content (SPAD), assimilation rate (A), and transpiration rate (Tr) at the beginning of the stress. However, the +NILs showed significantly higher SPAD (12%), A (66%), stomatal conductance (75%), and Tr (97%) than the −NILs at the seventh day of stress. Quantitative RT-PCR analysis targeting the MYB transcription factor gene Triticum aestivum MYB 82 (TaMYB82), within this genomic region which was retrieved from the wheat reference genome TGACv1, also revealed differential expression in +NILs and –NILs under stress. These results confirmed that the NILs can be invaluable resources for fine mapping of this QTL, and also for cloning and functional characterization of the gene(s) responsible for drought tolerance in wheat.

Validation of QTL for grain weight using MAS-derived pairs of NILs in bread wheat (Triticum aestivum L.)

Near isogenic lines (NILs) are ideal material for a variety of genetic studies including validation of specific QTL. In the present study, eight pairs of NILs for grain weight were developed, seven in the background of Raj3765, and one in the background of K9107. For this purpose, marker-assisted selection (MAS) was used for the transfer of three grain weight QTL (QGw.ccsu-1A.2, QGw.ccsu-1A.3 and QGw.ccsu-1B.1) that were earlier identified in our laboratory. Two genotypes of each of the eight pairs of NILs, differed for QTL alleles (QTLHgw derived from the donor parent and the QTLLgw derived from the recipient parent). Each pair of NILs involved a solitary QTL except one NIL, which differed for all the three QTL. The difference in thousand grain weight (TGW) in two NILs of an individual pair ranged from 2.8 to 7.5 g, thus validating the effect of the QTL for TGW, although the quantum of difference did not always match the phenotypic variance of the corresponding QTL. As expected, the NILs which involved all the three QTL had the maximum difference of 7.5 g in TGW, and the NILs which involved QTL, QGw.ccsu-1A.2 had minimum average difference of 2.8 g for TGW. The NILs produced during the present study may be used in future for MAS and for fine mapping of TGW QTL.

H3K4/K9 acetylation and Lr28-mediated expression of six leaf rust responsive genes in wheat (Triticum aestivum).

Development of leaf rust-resistant cultivars is a priority during wheat breeding, since leaf rust causes major losses in yield. Resistance against leaf rust due to Lr genes is partly controlled by epigenetic modifications including histone acetylation that is known to respond to biotic/abiotic stresses. In the present study, enrichment of H3K4ac and H3K9ac in promoters of six defense responsive genes (N-acetyltransferase, WRKY 40, WRKY 70, ASR1, Peroxidase 12 and Sarcosine oxidase) was compared with their expression in a pair of near-isogenic lines (NILs) for the gene Lr28 following inoculation with leaf rust pathotype '77-5'; ChIP-qPCR was used for this purpose. The proximal and distal promoters of these genes contained a number of motifs that are known to respond to biotic stresses. The enrichment of two acetylation marks changed with passage of time; changes in expression of two of the six genes (N-acetyltransferase and peroxidase12), largely matched with changes in H3K4/H3K9 acetylation patterns of the two promoter regions. For example, enrichment of both the marks matched with higher expression of N-acetyltransferase gene in susceptible NIL and the deacetylation (H3K4ac) largely matched with reduced gene expression in resistant NIL. In peroxidase12, enrichment of H3K4ac and H3K9ac largely matched with higher expression in both the NILs. In the remaining four genes, changes in H3 acetylation did not always match with gene expression levels. This indicated complexity in the regulation of the expression of these remaining four genes, which may be controlled by other epigenetic/genetic regulatory mechanisms that need further analysis.

The impact of elevated CO2 on acid-soil tolerance of hexaploid wheat (Triticum aestivum L.) genotypes varying in organic anion efflux

It is unclear how elevated CO2 (eCO2) affects the response of crops to soil acidity. This study examined the effect of eCO2 on acid-soil tolerance of hexaploid wheat genotypes that vary in Al3+ resistance due to differences in root efflux of citrate and malate. Three pairs of near-isogenic lines were grown for 24–25 days under ambient CO2 (400 µmol mol-1) and eCO2 (800 µmol mol-1) in acid soils and hydroponics with various Al3+ concentrations. The lines consisted of pairs that differed in alleles of the TaALMT1 and TaMATE1B genes. Plant growth parameters and rhizosphere soil properties were measured. Elevated CO2 increased the slope of negative correlations between root and shoot biomass and Al3+ concentration in the rhizosphere of a line that has an Al3+-sensitive allele of the TaALMT1 gene conditioning malate efflux (ES8), but did not change that of a near-isogenic sister line with an Al3+-resistant TaALMT1 allele (ET8). Elevated CO2 decreased the relative root length and biomass (% of limed soil) of a line that lacked both malate and citrate efflux (Egret), but did not affect lines that possessed either malate or citrate efflux. Elevated CO2 had no effect on either malate or citrate efflux from root tips of Al3+-resistant lines. Elevated CO2 also increased Al3+ concentration and decreased NH4+ concentration in rhizosphere soil, but decreased concentrations of Al and Zn in shoots. Elevated CO2 decreased acid-soil tolerance of Al3+sensitive genotypes but not of Al3+ resistant genotypes. Malate efflux played a dominant role in conferring acid-soil tolerance to hexaploid wheat.

Characterisation of a novel quantitative trait locus, GN4-1, for grain number and yield in rice (Oryza sativa L.).

A novel QTL for grain number, GN4-1, was identified and fine-mapped to an~190-kb region on the long arm of rice chromosome 4. Rice grain yield is primarily determined by three components: number of panicles per plant, grain number per panicle and grain weight. Among these traits, grain number per panicle is the major contributor to grain yield formation and is a crucial trait for yield improvement. In this study, we identified a major quantitative trait locus (QTL) responsible for rice grain number on chromosome 4, designated GN4-1 (a QTL for Grain Number on chromosome 4), using advanced segregating populations derived from the crosses between an elite indica cultivar 'Zhonghui 8006' (ZH8006) and a japonica rice 'Wuyunjing 8' (WYJ8). GN4-1 was delimited to an~190-kb region on chromosome 4. The genetic effect of GN4-1 was estimated using a pair of near-isogenic lines. The GN4-1 gene from WYJ8 promoted accumulation of cytokinins in the inflorescence and increased grain number per panicle by~17%. More importantly, introduction of the WYJ8 GN4-1 gene into ZH8006 increased grain yield by~14.3 and~11.5% in the experimental plots in 2014 and 2015, respectively. In addition, GN4-1 promoted thickening of the culm and may enhance resistance to lodging. These results demonstrate that GN4-1 is a potentially valuable gene for improvement of yield and lodging resistance in rice breeding.

Development of near-isogenic lines targeting a major QTL on 3AL for pre-harvest sprouting resistance in bread wheat

Resistance to pre-harvest sprouting (PHS) in wheat (Triticum aestivum L.) is one of the most valuable traits in many breeding programs. However, the quantitative nature of inheritance of PHS resistance challenges the study of this trait. Near-isogenic lines (NILs) can turn a complicated quantitative trait into a Mendelian factor (qualitative) and are, therefore, valuable materials for identification of the gene(s) responsible for a specific phenotypic trait and for functional studies of specific loci. Five pairs of NILs were developed and confirmed for a major quantitative trait locus (QTL) located on the long arm of chromosome 3A contributing to PHS resistance in wheat. These NILs were generated by using the heterogeneous inbred family method and a fast generation-cycling system. Significant differences in PHS resistance between the isolines were detected in the NILs. The presence of the PHS-resistance allele from the resistant parent increased resistance to sprouting on spikes by 26.7–96.8%, with an average of 73.8%, and increased seed dormancy by 36.9–87.2%, with an average of 59.9% across the NILs. These NILs are being used for the identification of candidate genes responsible for this major PHS-resistance locus on wheat chromosome arm 3AL.

A study of transcriptome in leaf rust infected bread wheat involving seedling resistance gene Lr28.

Leaf rust disease causes severe yield losses in wheat throughout the world. During the present study, high-throughput RNA-Seq analysis was used to gain insights into the role of Lr28 gene in imparting seedling leaf rust resistance in wheat. Differential expression analysis was conducted using a pair of near-isogenic lines (NILs) (HD 2329 and HD 2329+Lr28) at early (0h before inoculation (hbi), 24 and 48h after inoculation (hai)) and late stages (72, 96 and 168 hai) after inoculation with a virulent pathotype of pathogen Puccinia triticina. Expression of a large number of genes was found to be affected due to the presence/absence of Lr28. Gene ontology analysis of the differentially expressed transcripts suggested enrichment of transcripts involved in carbohydrate and amino acid metabolism, oxidative stress and hormone metabolism, in resistant and/or susceptible NILs. Genes encoding receptor like kinases (RLKs) (including ATP binding; serine threonine kinases) and other kinases were the most abundant class of genes, whose expression was affected. Genes involved in reactive oxygen species (ROS) homeostasis and several genes encoding transcription factors (TFs) (most abundant being WRKY TFs) were also identified along with some ncRNAs and histone variants. Quantitative real-time PCR was also used for validation of 39 representative selected genes. In the long term, the present study should prove useful in developing leaf rust resistant wheat cultivars through molecular breeding.

A multiple near isogenic line (multi-NIL) RNA-seq approach to identify candidate genes underpinning QTL.

This study demonstrates how identification of genes underpinning disease-resistance QTL based on differential expression and SNPs can be improved by performing transcriptomic analysis on multiple near isogenic lines. Transcriptomic analysis has been widely used to understand the genetic basis of a trait of interest by comparing genotypes with contrasting phenotypes. However, these approaches identify such large sets of differentially expressed genes that it proves difficult to isolate which genes underpin the phenotype of interest. This study tests whether using multiple near isogenic lines (NILs) can improve the resolution of RNA-seq-based approaches to identify genes underpinning disease-resistance QTL. A set of NILs for a major effect Fusarium crown rot-resistance QTL in barley on the 4HL chromosome arm were analysed under Fusarium crown rot using RNA-seq. Differential gene expression and single nucleotide polymorphism detection analyses reduced the number of putative candidates from thousands within individual NIL pairs to only one hundred and two genes, which were differentially expressed or contained SNPs in common across NIL pairs and occurred on 4HL. Our findings support the value of performing RNA-seq analysis using multiple NILs to remove genetic background effects. The enrichment analyses indicated conserved differences in the response to infection between resistant and sensitive isolines suggesting that sensitive isolines are impaired in systemic defence response to Fusarium pseudograminearum.

Characterization of grain protein content gene (GPC-B1) introgression lines and its potential use in breeding for enhanced grain zinc and iron concentration in spring wheat

At least two billion people around the world suffer from micronutrient deficiency, or hidden hunger, which is characterized by iron-deficiency anemia, vitamin A and zinc deficiency. As a key staple food crop, wheat provides 20% of the world’s dietary energy and protein, therefore wheat is an ideal vehicle for biofortification. Developing biofortified wheat varieties with genetically enhanced levels of grain zinc (Zn) and iron (Fe) concentrations, and protein content provides a cost-effective and sustainable solution to the resource-poor wheat consumers. Large genetic variation for Fe and Zn were found in the primitive and wild relatives of wheat, the potential high Zn and Fe containing genetic resources were used as progenitors to breed high-yielding biofortified wheat varieties with 30–40% higher Zn content. Grain protein content (GPC) determines processing and end-use quality of wheat for making diverse food products. The GPC-B1 allele from Triticum turgidum L. var. dicoccoides have been well characterized for the increase in GPC and the associated pleiotropic effect on grain Zn and Fe concentrations in wheat. In this study effect of GPC-B1 allele on grain Zn and Fe concentrations in wheat were measured in different genetic backgrounds and two different agronomic management practices (with- and without foliar Zn fertilization). Six pairs of near-isogenic lines differing for GPC-B1 gene evaluated at CIMMYT, Mexico showed that GPC-B1 influenced marginal increase for grain Zn, Fe concentrations, grain protein content and slight reduction in kernel weight and grain yield. However, the magnitude of GPC and grain Zn and Fe reductions varied depending on the genetic background. Introgression of GPC-B1 functional allele in combination with normal or delayed maturity alleles in the CIMMYT elite wheat germplasm has the potential to improve GPC and grain Zn and Fe concentrations without the negative effect on grain yield due to early senescence and accelerated maturity.

Low validation rate of quantitative trait loci for Gibberella ear rot resistance in European maize.

Six quantitative trait loci (QTL) for Gibberella ear rot resistance in maize were tested in two different genetic backgrounds; three QTL displayed an effect in few near isogenic line pairs. Few quantitative trait loci (QTL) mapping studies for Gibberella ear rot (GER) have been conducted, but no QTL have been verified so far. QTL validation is prudent before their implementation into marker-assisted selection (MAS) programs. Our objectives were to (1) validate six QTL for GER resistance, (2) evaluate the QTL across two genetic backgrounds, (3) investigate the genetic background outside the targeted introgressions. Pairs of near isogenic lines (NILs) segregating for a single QTL (Qger1, Qger2, Qger10, Qger13, Qger16, or Qger21) were developed by recurrent backcross until generation BC3S2. Donor parents (DP) carrying QTL were backcrossed to a susceptible (UH009) and a moderately resistant (UH007) recurrent parent. MAS was performed using five SNP markers covering a region of 40cM around each QTL. All NILs were genotyped with the MaizeSNP50 assay and phenotyped for GER severity and deoxynivalenol and zearalenone content. Traits were significantly (P<0.001) intercorrelated. Out of 34 NIL pairs with the UH009 genetic background, three pairs showed significant differences in at least one trait for three QTL (Qger1, Qger2, Qger13). Out of 25 NIL pairs with the UH007 genetic background, five pairs showed significant differences in at least one trait for two QTL (Qger2, Qger21). However, Qger16, Qger10 and Qger13 were most likely false positives. The genetic background possibly affected NIL pairs comparisons due to linkage drag and/or epistasis with residual loci from the DP in non-target regions. In conclusion, validation rates were disappointingly low, which further indicates that GER resistance is controlled by many low-effect QTL.

Differential accumulation of cadmium in near-isogenic lines of durum wheat: no role for phytochelatins.

Certain cultivars of some crops, including durum wheat (Triticum durum Desf.), have a propensity to accumulate cadmium in the grain. In the 1980s, a Canadian wheat breeding program generated five pairs of near-isogenic lines of durum wheat that vary in cadmium-accumulation. Within each pair, one member accumulates twofold to threefold higher concentrations of cadmium in the shoot and grain. However, the physiological explanation for the high-low phenotype is unknown. We studied correlations between concentrations of cadmium and non-protein thiols, including phytochelatins, in these five pairs of near-isogenic lines to test the hypothesis that differential retention of cadmium-binding complexes in the root would explain the phenotype. The expected high-low pattern of cadmium accumulation was found in three of the pairs. In one pair, cadmium was positively correlated with cysteine and glutathione in the roots and with phytochelatins 2 and 4 in the shoots but in another pair cadmium was strongly negatively correlated with phytochelatins 2 and 4 in the shoots and unrelated to cysteine or glutathione. No correlations between concentrations of cadmium and the non-protein thiols were found in the third pair or in the remaining two pairs. The production of phytochelatins is a well-described response to cadmium but the lack of consistent correlation between cadmium and non-protein thiols in these five near-isogenic lines indicates that complexation with non-protein thiols does not explain differential translocation of cadmium in durum wheat.

Phosphoproteomic analysis of the posterior silk gland of Bombyx mori provides novel insight into phosphorylation regulating the silk production

To understand phosphorylation event regulating silk synthesis in the posterior silk gland of Bombyx mori, phosphoproteome was profiled in a pair of near-isogenic lines, a normally cocooning strain (IC) and a nakedly pupated strain (IN) that the silk production is much lower than IC. In the posterior silk gland of the IC and IN, 714 and 658 phosphosites resided on 554 and 507 phosphopeptides from 431 and 383 phosphoproteins, were identified, respectively. Of all the phosphosites, the single phosphosite was the dominate phosphorylation form, comprising>60% of all the phosphosites in two phenotypic of silk production. All these phosphosites were classified as acidophilic and proline-directed kinase classes, and three motifs were uniquely identified in the IC. The motif S-P-P might be important for regulating phosphorylation network of silk protein synthesis. The dynamically phosphorylated proteins participated in ribosome, protein transport and energy metabolism suggest that phosphorylation may play key roles in regulating silk protein synthesis and secretion. Furthermore, fibroin heavy chain, an important component of silk protein, was specifically phosphorylated in the IC strain, suggesting its role to ensure the normal formation of silk structure and silk secretion. The data gain new understanding of the regulatory processes of silk protein synthesis and offer as starting point for further research on the silk production at phosphoproteome level. Significance of this studyDespite the knowledge on regulation of silk protein synthesis in the posterior silk gland has gained at the gene or protein levels, how phosphorylation event influences the silk yield is largely unknown. To this end, we constructed a pair of silkworm near-isogenic lines that showed different cocooning phenotypes, and the phosphoproteome of the posterior silk gland of two isolines was compared. Here, we reported the first phosphoproteome data on the silkworm and found several key pathways related protein synthesis are regulated by phosphorylation, thereby influencing the silk production. The data provide valuable resources for further functional assay of targeted protein phosphorylation that regulates the silk synthesis in silkworm.

Identification of the target region including the Foc0 1 /foc0 1 gene and development of near isogenic lines for resistance to Fusarium Wilt race 0 in chickpea

Three recombinant inbred populations (RIP-1, RIP-7 and RIP-10) were used to validate and saturate the target area involved in the resistance to Fusarium oxysporum f.sp. ciceris race 0 (Foc0) in chickpeas. With the aim of physically localizing the genetic area controlling Foc0 resistance, a new genetic map of the linkage group 5 (LG5) and a physical map of the Ca5 pseudomolecule were drawn up and compared. The genetic and physical between CaGM20820 and CaGM20889 (16759541–17501349 bp) within an interval of 2 cM comprising 27 annotated genes. Only two of the annotated genes in this region could mapping results obtained were useful to refine the position of the target area be involved in resistance mechanisms. The three RIPs were used to develop three pairs of near isogenic lines (NILs) for resistance/susceptibility to Foc0. NILs are a valuable tool for fine mapping, refining the target region and selecting candidate genes for resistance to Foc0. The information reported in this paper will help develop diagnostic markers, considered useful tools for MAS in chickpea breeding programs.

Fine mapping and candidate gene analysis of the dominant glandless gene Gl 2 (e) in cotton (Gossypium spp.).

Dominant glandless gene Gl 2 (e) was fine-mapped to a 15kb region containing one candidate gene encoding an MYC transcription factor, sequence and expression level of the gene were analyzed. Cottonseed product is an excellent source of oil and protein. However, this nutrition source is greatly limited in utilization by the toxic gossypol in pigment glands. It is reported that the Gl 2 (e) gene could effectively inhibit the formation of the pigment glands. Here, three F2 populations were constructed using two pairs of near isogenic lines (NILs), which differ nearly only by the gland trait, for fine mapping of Gl 2 (e) . DNA markers were identified from recently developed cotton genome sequence. The Gl 2 (e) gene was located within a 15-kb genomic interval between two markers CS2 and CS4 on chromosome 12. Only one gene was identified in the genomic interval as the candidate for Gl 2 (e) which encodes a family member of MYC transcription factor with 475-amino acids. Unexpectedly, the results of expression analysis indicated that the MYC gene expresses in glanded lines while almost does not express in glandless lines. These results suggest that the MYC gene probably serves as a vital positive regulator in the organogenesis pathway of pigment gland, and low expression of this gene will not launch the downstream pathway of pigment gland formation. This is the first pigment gland-related gene identification in cotton and will facilitate the research on glandless trait, cotton MYC proteins and low-gossypol cotton breeding.