Molecular characterization and expression analysis of the Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> exchanger gene family in <Emphasis Type="Italic">Medicago truncatula</Emphasis>

One important mechanism plants use to cope with salinity is keeping the cytosolic Na⁺ concentration low by sequestering Na⁺ in vacuoles, a process facilitated by Na⁺/H⁺ exchangers (NHX). There are eight NHX genes (NHX1 through NHX8) identified and characterized in Arabidopsis thaliana. Bioinformatics analyses of the known Arabidopsis genes enabled us to identify six Medicago truncatula NHX genes (MtNHX1, MtNHX2, MtNHX3, MtNHX4, MtNHX6, and MtNHX7). Twelve transmembrane domains and an amiloride binding site were conserved in five out of six MtNHX proteins. Phylogenetic analysis involving A. thaliana, Glycine max, Phaseolus vulgaris, and M. truncatula revealed that each individual MtNHX class (class I: MtNHX1 through 4; class II: MtNHX6; class III: MtNHX7) falls under a separate clade. In a salinity-stress experiment, M. truncatula exhibited ~?20% reduction in biomass. In the salinity treatment, sodium contents increased by 178 and 75% in leaves and roots, respectively, and Cl^? contents increased by 152 and 162%, respectively. Na⁺ exclusion may be responsible for the relatively smaller increase in Na⁺ concentration in roots under salt stress as compared to Cl^?. Decline in tissue K⁺ concentration under salinity was not surprising as some antiporters play an important role in transporting both Na⁺ and K ⁺ . MtNHX1, MtNHX6, and MtNHX7 display high expression in roots and leaves. MtNHX3, MtNHX6, and MtNHX7 were induced in roots under salinity stress. Expression analysis results indicate that sequestering Na⁺ into vacuoles may not be the principal component trait of the salt tolerance mechanism in M. truncatula and other component traits may be pivotal.     

<Emphasis Type="Italic">OsNHX2</Emphasis>, an Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporter gene,can enhance salt tolerance in rice plants through more effective accumulation of toxic Na<Superscript>+</Superscript> in leaf mesophyll and bundle sheath cells

The Na⁺/H⁺ antiporters play an important role in salt tolerance in plants. However, the functions of OsNHXs in rice except OsNHX1 have not been well studied. Using the gain- and loss-of-function strategies, we studied the potential role of OsNHX2 in salt tolerance in rice. Overexpression of OsNHX2 (OsNHX2-OE) in rice showed the significant tolerance to salt stress than wild-type plants and OsNHX2 knockdown transgenic plants (OsNHX2-KD). Under salt treatments of 300-mM NaCl for 5 days, the plant fresh weights, relative water percentages, shoot heights, Na⁺ contents, K⁺ contents, and K⁺/Na⁺ ratios in leaves of OsNHX2-OE transgenic plants were higher than those in wild-type plants, while no differences were detected in roots. K⁺/Na⁺ ratios in rice leaf mesophyll cells and bundle sheath cells were higher in OsNHX2-OE transgenic plants than in wild-type plants and OsNHX2-KD transgenic plants. Our data indicate that OsNHX2 plays an important role in salt stress based on leaf mesophyll cells and bundle sheath cells and can be served in genetically engineering crop plants with enhanced salt tolerance.     

Development and validation of KASP markers for the greenbug resistance gene <Emphasis Type="Italic">Gb7</Emphasis> and the Hessian fly resistance gene <Emphasis Type="Italic">H32</Emphasis> in wheat

Key message

Greenbug and Hessian fly are important pests that decrease wheat production worldwide. We developed and validated breeder-friendly KASP markers for marker-assisted breeding to increase selection efficiency.

Abstract

Greenbug (Schizaphis graminum Rondani) and Hessian fly [Mayetiola destructor (Say)] are two major destructive insect pests of wheat (Triticum aestivum L.) throughout wheat production regions in the USA and worldwide. Greenbug and Hessian fly infestation can significantly reduce grain yield and quality. Breeding for resistance to these two pests using marker-assisted selection (MAS) is the most economical strategy to minimize losses. In this study, doubled haploid lines from the Synthetic W7984 × Opata M85 wheat reference population were used to construct linkage maps for the greenbug resistance gene Gb7 and the Hessian fly resistance gene H32 with genotyping-by-sequencing (GBS) and 90K array-based single nucleotide polymorphism (SNP) marker data. Flanking markers were closely linked to Gb7 and H32 and were located on chromosome 7DL and 3DL, respectively. Gb7-linked markers (synopGBS773 and synopGBS1141) and H32-linked markers (synopGBS901 and IWB65911) were converted into Kompetitive Allele Specific PCR (KASP) assays for MAS in wheat breeding. In addition, comparative mapping identified syntenic regions in Brachypodium distachyon, rice (Oryza sativa), and sorghum (Sorghum bicolor) for Gb7 and H32 that can be used for fine mapping and map-based cloning of the genes. The KASP markers developed in this study are the first set of SNPs tightly linked to Gb7 and H32 and will be very useful for MAS in wheat breeding programs and future genetic studies of greenbug and Hessian fly resistance.

     

QTL-seq and marker development for resistance to <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">niveum</Emphasis> race 1 in cultivated watermelon

Fusarium wilt, caused by the fungus Fusarium oxysporum f. sp. niveum (Fon), is one of the predominant diseases of watermelon. Resistance to Fon race 1 is conferred by a single major quantitative trait locus (QTL), Fo-1.1, but resolution of this region has been poor due to low marker density. In this study, a combination of whole genome resequencing of bulked segregants (QTL-seq analysis) followed by QTL mapping with kompetitive allele specific PCR (KASP) markers developed across Fo-1.1 successfully increased the resolution from 2.03 to 1.56 Mb and 315 kb, respectively. The linkage of the KASP markers to Fon race 1 resistance across a wide range of watermelon germplasm was validated in a set of elite watermelon cultivars. The linked markers described here provide a breeder-friendly toolkit immediately available for high-throughput genotyping in large-scale breeding programs for fine mapping and incorporation of Fon race 1 resistance in watermelon.     

Molecular markers for adult plant leaf rust resistance gene <Emphasis Type="Italic">Lr48</Emphasis> in wheat

Leaf rust of wheat, caused by Puccinia triticina, is an important disease throughout the world. The adult plant leaf rust resistance gene Lr48 reported in CSP44 was previously mapped in chromosome 2B, but the marker–gene association was weak. In this study, we confirmed the location of Lr48 to be in the short arm of chromosome 2B and identified closely linked markers suitable for use in breeding. The CSP44/WL711 recombinant inbred line (RIL) population (90 lines) showed monogenic segregation for Lr48. Twelve resistant and 12 susceptible RILs were used for selective genotyping using an iSelect 90K Infinium SNP assay. Closely linked SNPs were converted into Kompetitive allele-specific primers (KASP) and tested on the parental lines. KASP markers giving clear clusters for alternate genotypes were assayed on the entire RIL population. SNP markers IWB31002, IWB39832, IWB34324, IWB72894 and IWB36920 co-segregated with Lr48 and the marker IWB70147 was mapped 0.3 cM proximal to this gene. Closely linked KASP markers were tested on a set of Australian and Nordic wheat genotypes. The amplification of SNP alleles alternate to those linked with Lr48 in the majority of the Australian and Nordic wheat genotypes demonstrated the usefulness of these markers for marker-assisted pyramiding of Lr48 with other rust resistance genes.     

Homoeologous recombination-based transfer and molecular cytogenetic mapping of powdery mildew-resistant gene <Emphasis Type="Italic">Pm57</Emphasis> from <Emphasis Type="Italic">Aegilops searsii</Emphasis> into wheat

Key message

Pm57, a novel resistant gene against powdery mildew, was transferred into common wheat from Ae. searsi and further mapped to 2S ^s #1L at an interval of FL0.75 to FL0.87.

Abstract

Powdery mildew, caused by the fungus Blumeria graminis f. sp. tritici, is one of the most severe foliar diseases of wheat causing reduction in grain yield and quality. Host plant resistance is the most effective and environmentally safe approach to control this disease. Tests of a set of Chinese Spring–Ae. searsii (S^sS^s, 2n?=?2x?=?14) Feldman & Kislev ex K. Hammer disomic addition lines with a mixed isolate of the powdery mildew fungus identified a novel resistance gene(s), designed as Pm57, which was located on chromosome 2S^s#1. Here, we report the development of ten wheat–Ae. searsii recombinants. The wheat chromosomes involved in five of these recombinants were identified by FISH and SSR marker analysis and three of them were resistant to powdery mildew. Pm57 was further mapped to the long arm of chromosome 2S^s#1 at a fraction length interval of FL 0.75 to FL 0.87. The recombinant stocks T2BS.2BL-2S^s#1L 89-346 (TA5108) with distal 2S^s#1L segments of 28% and 89(5)69 (TA5109) with 33% may be useful in wheat improvement. The PCR marker X2L4g9p4/HaeIII was validated to specifically identify the Ae. searsii 2S^s#1L segment harboring Pm57 in T2BS.2BL-2S^s#1L against 16 wheat varieties and advanced breeding lines, and the development of more user-friendly KASP markers is underway.

     

Development of co-dominant KASP markers co-segregating with Ug99 effective stem rust resistance gene <Emphasis Type="Italic">Sr26</Emphasis> in wheat

Stem rust of wheat, caused by Puccinia graminis f. sp. tritici (Pgt), is a threat to global food security due to its ability to cause total crop failures. The Pgt race TTKSK (Ug99) and its derivatives detected in East Africa carry virulence for many resistance genes present in modern cultivars. However, stem rust resistance gene Sr26 remains effective to all races of Pgt worldwide. Sr26 is carried on the Agropyron elongatum (syn. Thinopyrum ponticum) segment 6Ae#1L translocated to chromosome 6AL of wheat. In this study, a recombinant inbred line (RIL) population derived from a cross between the landrace Aus27969 and Avocet S, which carries Sr26, was used to develop co-dominant kompetitive allele-specific polymerase chain reaction (KASP) markers that co-segregate with Sr26. Four KASP markers (sunKASP_216, sunKASP_218, sunKASP_224 and sunKASP_225) were also shown to co-segregate with Sr26 in four additional RIL populations. When tested on Australian cultivars and breeding lines, these markers amplified alleles alternate to that linked with Sr26 in all cultivars known to lack this gene and Sr26-linked alleles in cultivars and genotypes known to carry Sr26. Genotypes WA-1 and WA-1/3*Yitpi carrying the shortest Sr26 translocation segment were positive only for markers sunKASP_224 and sunKASP_225. Our results suggest the four KASP markers are located on the original translocation and sunKASP_224 and sunKASP_225 are located on the shortened version. Therefore, sunKASP_224 and sunKASP_225 can be used for marker-assisted pyramiding of Sr26 with other stem rust resistance genes to achieve durable resistance in wheat.     

Genetic mapping of <Emphasis Type="Italic">Stb19</Emphasis>, a new resistance gene to <Emphasis Type="Italic">Zymoseptoria tritici</Emphasis> in wheat

Key message

A new and dominant R gene Stb19 is identified from a soft wheat cultivar ‘Lorikeet’ and was mapped on the distal region of chromosome 1DS. Two tightly linked KASP markers were also discovered and validated for molecular-assisted breeding programs.

Abstract

A new R gene, designated as Stb19, provides resistance to Zymoseptoria tritici in wheat. This new dominant gene resides on the short arm of chromosome 1D, exhibiting complete resistance to three Z. tritici isolates, WAI332, WAI251, and WAI161, at the seedling stage. A genetic linkage map, based on an F_2:3 population of ‘Lorikeet’ and ‘Summit,’ found the Stb19 gene at a 9.3 cM region on 1DS, closely linked with two Kompetitive Allele-Specific PCR markers, snp_4909967 and snp_1218021. Further, the two markers were tested and validated in another F_2:3 population and 266 different wheat accessions, which gave over 95% accuracy of resistance/susceptibility prediction. Combined with the physical location of the identified SNPs and the previous evidence of gene order on chromosome 1DS (centromere–Sr45–Sr33–Lr21–telomere), Stb19 is proposed to be located between Sr33 and Lr21. Thus, the newly discovered Stb19 along with the KASP markers represents an increase in genetic resources available for wheat breeding resistance to Z. tritici.

     

An Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporter gene from wheat plays an important role in stress tolerance

A vacuole Na⁺/H⁺ antiporter gene TaNHX2 was obtained by screening the wheat cDNA library and by the 5′-RACE method. The expression of TaNHX2 was induced in roots and leaves by treatment with NaCl, polyethylene glycol (PEG), cold and abscisic acid (ABA). When expressed in a yeast mutant (Δnhx1), TaNHX2 suppressed the salt sensitivity of the mutant, which was deficient in vacuolar Na⁺/H⁺ antiporter, and caused partial recovery of growth of Δnhx1 in NaCl and LiC1 media. The survival rate of yeast cells was improved by overexpressing the TaNHX2 gene under NaCl, KCl, sorbitol and freezing stresses when compared with the control. The results imply that TaNHX2 might play an important role in salt and osmotic stress tolerance in plant cells.     

<Superscript>1</Superscript>H, <Superscript>15</Superscript>N, <Superscript>13</Superscript>C resonance assignments for pyrazinoic acid binding domain of ribosomal protein S1 from <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>

Ribosomal protein S1 of Mycobacterium tuberculosis (MtRpsA) binds to ribosome and mRNA, and plays significant role in the regulation of translation initiation, conventional protein synthesis and transfer-messenger RNA (tmRNA) mediated trans-translation. It has been identified as the target of pyrazinoic acid (POA), a bactericidal moiety from hydrolysis of pyrazinamide, which is a mainstay of combination therapy for tuberculosis. POA prevented the interactions between the C-terminal S1 domain of MtRpsA (residues 280–368, MtRpsA^CTD_S1) and tmRNA; so that POA can inhibit the trans-translation, which is a key component of multiple quality control pathways in bacteria. However, the details of molecular mechanism and dynamic characteristics for MtRpsA^CTD_S1 interactions with POA, tmRNA or mRNA are still unclear. Here we present the ¹H, ¹⁵N, ¹³C resonance assignments of MtRpsA^CTD_S1 as well as the secondary structure information based on backbone chemical shifts, which lay foundation for further solution structure determination, dynamic properties characterization and interactions investigation between MtRpsA^CTD_S1 and tmRNA, RNA or POA.     

Adult plant stripe rust resistance gene <Emphasis Type="Italic">Yr71</Emphasis> maps close to <Emphasis Type="Italic">Lr24</Emphasis> in chromosome 3D of common wheat

Australian cultivar Sunco carries three adult plant stripe rust resistance genes. One of these genes corresponded to Yr18 in chromosome 7DS; the second, YrCK, was mapped on chromosome 2D. Here, we describe the characterization of the third adult plant resistance (APR) gene from Sunco. Sunco/2*Avocet S-derived lines SA65 (resistant) and SA67 (susceptible) were crossed and a recombinant inbred line F₆ population was generated. Monogenic segregation among SA65/SA67-derived RIL population was demonstrated and the resistance locus was designated YrSA3. Selective genotyping using an iSelect 90 K Infinium SNP array and SSR markers located YrSA3 on chromosome 3D. Development of KASP markers for SNP loci showing association with YrSA3 allowed construction of a genetic map harboring the resistance gene. Ten KASP markers (KASP_8306, KASP_9142, KASP_10438, KASP_16434, KASP_17207, KASP_20836, KASP_23518, KASP_23615, KASP_57983 and KASP_63653), one SSR marker (gwm114b) and Lr24/Sr24 were mapped 1.8 cM distal to YrSA3. Comparison of marker data indicated that the previously named seedling stripe rust resistance gene Yr45 was located proximal to YrSA3, and therefore the latter was formally designated Yr71. Two recombinants carrying Lr24/Sr24 and Yr71 in combination were identified for use as donor sources in wheat breeding programs. The robustness of gwm114b, KASP_16434, KASP_17207 and KASP_20836 for marker-assisted selection of these genes was demonstrated through tests on 74 Australian wheat cultivars.     

Analysis of compound heterozygotes reveals that the mouse floxed <Emphasis Type="Italic">Pax6</Emphasis><Superscript><Emphasis Type="Italic">tm1Ued</Emphasis></Superscript> allele produces abnormal eye phenotypes

Analysis of abnormal phenotypes produced by different types of mutations has been crucial for our understanding of gene function. Some floxed alleles that retain a neomycin-resistance selection cassette (neo cassette) are not equivalent to wild-type alleles and provide useful experimental resources. Pax6 is an important developmental gene and the aim of this study was to determine whether the floxed Pax6 ^tm1Ued (Pax6 ^fl ) allele, which has a retained neo cassette, produced any abnormal eye phenotypes that would imply that it differs from the wild-type allele. Homozygous Pax6 ^fl/fl and heterozygous Pax6 ^fl/+ mice had no overt qualitative eye abnormalities but morphometric analysis showed that Pax6 ^fl/fl corneas tended be thicker and smaller in diameter. To aid identification of weak effects, we produced compound heterozygotes with the Pax6 ^Sey-Neu (Pax6 ^?) null allele. Pax6 ^fl/? compound heterozygotes had more severe eye abnormalities than Pax6 ^+/? heterozygotes, implying that Pax6 ^fl differs from the wild-type Pax6 ⁺ allele. Immunohistochemistry showed that the Pax6 ^fl/? corneal epithelium was positive for keratin 19 and negative for keratin 12, indicating that it was abnormally differentiated. This Pax6 ^fl allele provides a useful addition to the existing Pax6 allelic series and this study demonstrates the utility of using compound heterozygotes with null alleles to unmask cryptic effects of floxed alleles.     

<Superscript>15</Superscript>N photo-CIDNP MAS NMR analysis of reaction centers of <Emphasis Type="Italic">Chloracidobacterium thermophilum</Emphasis>

Photochemically induced dynamic nuclear polarization (photo-CIDNP) has been observed in the homodimeric, type-1 photochemical reaction centers (RCs) of the acidobacterium, Chloracidobacterium (Cab.) thermophilum, by ¹⁵N magic-angle spinning (MAS) solid-state NMR under continuous white-light illumination. Three light-induced emissive (negative) signals are detected. In the RCs of Cab. thermophilum, three types of (bacterio)chlorophylls have previously been identified: bacteriochlorophyll a (BChl a), chlorophyll a (Chl a), and Zn-bacteriochlorophyll a′ (Zn-BChl a′) (Tsukatani et al. in J Biol Chem 287:5720–5732, 2012). Based upon experimental and quantum chemical ¹⁵N NMR data, we assign the observed signals to a Chl a cofactor. We exclude Zn-BChl because of its measured spectroscopic properties. We conclude that Chl a is the primary electron acceptor, which implies that the primary donor is most likely Zn-BChl a′. Chl a and 8¹-OH Chl a have been shown to be the primary electron acceptors in green sulfur bacteria and heliobacteria, respectively, and thus a Chl a molecule serves this role in all known homodimeric type-1 RCs.