Molecular mapping of greenbug (Schizaphis graminum) resistance gene Rsg1 in barley

The greenbug, Schizaphis graminum (Rondani) is an extremely damaging aphid pest of barley (Hordeum vulgare L.) particularly in the southern Great Plains of the USA. The simply inherited, dominant resistance gene Rsg1 is in all greenbug‐resistant US barley cultivars. In this study, we conducted molecular mapping of Rsg1 using an F_2:3 population derived from a cross between the greenbug‐resistant Post 90*4/R015 and susceptible CI2260 inbred lines. Segregation of host responses to greenbug biotype E infestation confirmed that a single dominant gene is responsible for greenbug resistance in Post 90*4/R015. Simple sequence repeat (SSR) markers evenly distributed along the seven barley chromosomes were employed for the construction of a framework genetic map. Linkage analysis placed the Rsg1 locus in the long arm of chromosome 3H (3HL) flanked by SSR markers Bmag0877 and GBM1420 that were 35 cM apart. Polymorphic single‐nucleotide polymorphism (SNP) markers in 3HL were identified from an Illumina GoldenGate SNP assay and used for targeted mapping to locate Rsg1 to an 8.4‐cM interval. Comparative analysis identified syntenic genomic regions in Brachypodium distachyon chromosome 2, in which 37 putative genes were annotated including a NB‐LRR‐type resistance gene homologue that may be a potential candidate gene for the Rsg1 locus of barley. Results from this study offer a starting point for fine mapping and cloning of this aphid resistance gene in barley.     

Detection and genotyping of SNPs tightly linked to two disease resistance loci, Rsv1 and Rsv3, of soybean

Allele‐specific polymerase chain reaction (AS‐PCR) for assaying single nucleotide polymorphisms (SNPs) would be more widely used with increased availability of AS primers sufficient to distinguish between SNP alleles. AS‐PCR could be a means unambiguously to detect the presence or absence of PCR products. Examples are given here of the detection and genotyping of SNPs in the genomic DNA fragments tightly linked to two soybean mosaic virus resistance genes, Rsv1 and Rsv3, with a modified AS‐PCR procedure in soybean. The modified AS‐PCR that introduces an additional base mismatch closest to the 3′‐end of the AS primers and uses publicly available microsatellite markers as positive controls directly determined SNP alleles from primary PCR of genomic DNAs. It was demonstrated that a set of AS primers designed from two adjacent SNP loci could simultaneously detect the two SNP loci. Using the modified procedure, many SNP loci in eight soybean parental lines and F₂ individuals of three mapping populations could be genotyped. The modified AS‐PCR procedure could greatly facilitate small‐to‐medium scale marker‐assisted selection programmes for agronomically important genes.     

A mutation in Waxy gene affects amylose content,starch granules and kernel characteristics of barley (Hordeum vulgare)

Waxy barley referred to as low‐amylose or amylose‐free has special advantages in nutrition composition and food processing. Waxy gene encoding granule–bound starch synthase I (GBSSI) is responsible for amylose synthesis in barley. The G³⁹³⁵‐to‐T in Waxy gene has been previously found in amylose–free barley. In this study, G³⁹³⁵‐to‐T was proved to co‐segregate with the waxy phenotype of barley, but has no obvious effect on GBSSI catalytic activity and starch chain length distribution. However, recombinant inbred lines with G³⁹³⁵‐to‐T in Waxy gene are of significant modification in starch granules morphology and pasting properties, increase of grain β‐glucan content, and decrease of thousand kernel weight along with lower kernel width. A polymerase chain reaction with confronting two–pair primers marker was developed for economic and efficient screening of G³⁹³⁵‐to‐T. This study provides the basis for cultivar improvement of waxy barley then fully developing its potential value and utility in food processing.     

Identification of a novel recessive gene for resistance to powdery mildew (Blumeria graminis f. sp. hordei) in barley (Hordeum vulgare)

One of the most important diseases of barley (Hordeum vulgare) is powdery mildew, caused by Blumeria graminis f. sp. hordei. Spring barley line 173-1-2 was selected from a Moroccan landrace and revealed broad-spectrum resistance to powdery mildew. The objective of this study was to map and characterize the gene for seedling powdery mildew resistance in this line. After crossing with the susceptible cultivar ‘Manchuria’, genetic analysis of F₂ and F₃ families at the seedling stage revealed powdery mildew resistance in line 173-1-2 conditioned by a single recessive gene. Molecular analysis of non-segregating homozygous resistant and homozygous susceptible F₂ plants conducted on the DArTseq platform (Diversity Arrays Technology Pty Ltd) identified significant markers which were converted to allele-specific PCR markers and tested among 94 F₂ individuals. The new resistance gene was mapped on the long arm of chromosome 6H. No other powdery mildew recessive resistance gene has been located on 6H so far. Therefore, we concluded that the 173-1-2 barley line carries a novel recessive resistance gene designated as mlmr.     

Molecular breeding for the BaMMV/BaYMV resistance gene ym4 in winter barley

The aim of this investigation was to develop a procedure for the largescale molecular breeding for ym4, allowing resistance to BaMMV/BaYMV to be fixed in early breeding generations of winter barley. A codominant STS marker derived from the restriction fragment length polymorphism marker MWG838 for the ym4 resistance gene was combined with a new and easy procedure for preparing leaf samples for polymerase chain reaction (PCR), theoretically allowing one person to extract DNA from 5000 samples in a single day. In the procedure for molecular breeding for ym4, all steps, including leaf sampling, DNA extraction, PCR amplification and digestion with restriction enzyme were assembled in microtitre plates allowing multipipetting throughout the procedure, including the loading of gels. The method is amenable to further automation with the aid of a robot arm. Double haploid (DH) lines, as well as F₂ and F₄ breeding lines were analysed and, based on markers, homozygous and heterozygous BaMMV/BaYMV resistant plants were identified for further breeding. The winter barley breeding programmes were modified to include marker-based selection for BaMMV/BaYMV resistance on DH or on F₂ individuals, which had been preselected for mildew and leaf rust resistance.     

Gene dosage effect of the wheat Wx alleles and their interaction on amylose synthesis in the endosperm

To find out gene dose effect of each of the three homoeologous Wx genes and their interaction on the production of granule-bound starch synthase (GBSS I) and amylose biosynthesis in the endosperm, Chinese Spring and its near-isogenic waxy types were crossed reciprocally and, obtained a plant population with varying doses of each Wx gene. The amount of GBSSI was increased linearly with increasing gene dose of either of Wxloci. In each of the three Wx loci, the change in amylose content was linear up to 3 doses, with a more potent capacity ofWx-B1a at any dose. Higher level of amylose production was observed in the reciprocal F₁ grains than the expected effect of dose/s of each gene or additive effect of different allelic combination by artificially blend starches which have amylose produced by equivalent number ofWx alleles to that of relevant F₁ cross. When Wx-B1a and Wx-A1a were combined, increase in amylase content was not in proportion to increase in gene dosage. The enhanced amylase synthesis was shown by 2-gene and 3-geneinteraction, indicating that not only type of the three Wx genes and its dose but the interaction among them have significant roles in determining the amylose content. This revised version was published online in July 2006 with corrections to the Cover Date.     

Evaluation of the waxy endosperm trait in proso millet (Panicum miliaceum)*

The entire USDA‐ARS maintained collection of 650 accessions of proso millet (Panicum miliaceum L.) was evaluated for the presence of accessions with waxy (amylose‐free) endosperm starch. Six accessions, five of which derived from mainland China, were identified. Segregation ratios for waxy endosperm were evaluated in F₂ and F₃ populations derived from crosses between two waxy accessions, PI 436625 (Lung Shu 16) and PI 436626 (Lung Shu 18), and several wild‐type accessions. The waxy trait was found to be under the control of duplicate recessive alleles at two loci, herein designated wx‐1 and wx‐2. Wild‐type alleles at these loci were designated Wx‐1 and Wx‐2. Iodine‐binding revealed a mean grain‐starch amylose concentration of 3.5% in waxy lines and 25.3% in wild‐type proso. Expression of the granule‐bound starch synthase (waxy protein) in waxy lines was reduced to approximately one‐tenth that of wild‐type accessions. The waxy accessions identified now are available for the introgression of this trait into breeding lines adapted to the Great Plains of North America.     

A novel aphid resistance locus in cowpea identified by combining SSR and SNP markers

The utility of combining simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) marker genotyping was determined for genetically mapping a novel aphid (Aphis craccivora) resistance locus in cowpea breeding line SARC 1‐57‐2 and for introgressing the resistance into elite cultivars by marker‐assisted backcrossing (MABC). The locus was tagged with codominant SSR marker CP 171F/172R with a recombination fraction of 5.91% in an F₂ population from ‘Apagbaala’ x SARC 1‐57‐2. A SNP‐genotyped biparental recombinant inbred line population was genotyped for CP 171F/172R, which was mapped to position 11.5 cM on linkage group (LG) 10 (physical position 30.514 Mb on chromosome Vu10). Using CP 171F/172R for foreground selection and a KASP‐SNP‐based marker panel for background selection in MABC, the resistance from SARC 1‐57‐2 was introduced into elite susceptible cultivar ‘Zaayura’. Five BC₄F₃ lines of improved ‘Zaayura’ that were isogenic except for the resistance locus region had phenotypes similar to SARC 1‐57‐2. This study identified a novel aphid resistance locus and demonstrated the effectiveness of integrating SSR and SNP markers for trait mapping and marker‐assisted breeding.     

Development and application of a new codominant PCR marker for detecting 1BL·1RS wheat–rye chromosome translocations

The 1BL·1RS translocation has been widely used in wheat breeding programmes throughout the world. Unfortunately, this translocation has frequently resulted in unsatisfactory grain processing quality. Two primer combinations derived from the published sequence of a ω‐secalin gene on 1RS gave polymerase chain reaction (PCR) fragments 0.4 and 1.1 kb in size. Both fragments can be used to quickly detect 1BL·1RS translocations. By combining the PCR assay resulting in the 1.1‐kb fragment from 1RS and a PCR assay resulting in a 0.6‐kb fragment from the Glu‐B3 gene on 1BS, plants homozygous for the 1BL 1RS could clearly be distinguished from the heterozygous ones. This codominant marker was successfully applied to genotype a segregating F₂ population and a local cultivar collection.     

AFLP-based STS markers closely linked to a fertility restoration locus (Rfm1) for cytoplasmic male sterility in barley

The Rfm1 gene restores the fertility of the msm1 and msm² male‐sterile cytoplasms in barley. Rfm1 is located on the short arm of chromosome 6H. To develop molecular markers tightly linked to Rfm1 for use in sophisticated marker‐assisted selection and map‐based cloning, an amplified fragment‐length polymorphism (AFLP) marker system with isogenic lines and a segregating BC₁F₁ population was used. Nine hundred primer combinations were screened and a linkage map was constructed around the Rfm1 locus by using 25 recombinant plants selected from 214 BC₁F₁ plants. Three AFLP markers were identified, e34m2, e46m19 and e48m17, linked to the locus. The most closely linked markers were e34m2, at 1.0 cM distally and e46m19, at 1.1 cM proximally. The two AFLP markers were converted to dominant STS markers. These markers should accelerate programmes for breeding restorer lines and will be useful for map‐based cloning.     

Marker-assisted backcross introgression of the Yd2 gene conferring resistance to barley yellow dwarf virus in barley

YLM, a codaominant polymerase chain reaction (PCR) marker linked to Yd2, could substantially improve the precision and efficiency of barley yellow dwarf virus (BYDV) resistance breeding. The aim of this study was to assess the effectiveness of YLM in a marker‐assisted introgression programme and to quantify associations between the presence of Yd2 and other agronomic and quality traits. The Yd2 gene was introgressed into a BYDV‐susceptible background through two cycles of marker‐assisted backcrossing. BC₂ F₂‐derived lines, either carrying or not carrying the YLM allele associated with resistance, were compared in the presence and absence of BYDV. The YLM marker was shown to be effective in the introgression of Yd2. Lines carrying the YLM allele associated with resistance produced significantly fewer leaf symptoms and showed a reduction in yield loss when infected with BYDV. There were no deleterious effects associated with the introgression of Yd2 on grain yield, grain size or malting quality. The implications of marker‐assisted selection for Yd2 on barley improvement are discussed.     

Development of a kompetitive allele‐specific PCR marker for selection of the mutated Wx‐D1d allele in wheat breeding

Waxy (Wx) protein is a key enzyme for synthesis of amylose in endosperm. Amylose content in wheat grain influences the quality of end‐use products. Seven alleles have been described at the Wx‐D1 locus, but only two of them (Wx‐D1b, Wx‐D1e) were genotyped with codominant markers. The waxy wheat line K107Wx1 developed by treating ‘Kanto 107’ seeds with ethyl methanesulphonate carries the Wx‐D1d allele. However, no molecular basis supports this nomenclature. In the present study, DNA sequence analysis confirmed that a single nucleotide polymorphism in the sixth exon of Wx‐D1 changed tryptophan at position 301 into a termination codon. Based on this sequence variation, a PCR‐based KASP marker was developed to detect this point mutation using 68 BC₈F₁ plants and 297 BC₈F₂ lines derived from the cross ‘Ningmai 14’*9/K107Wx1. Combined with codominant markers for the Wx‐A1 and Wx‐B1 alleles, waxy and non‐waxy near‐isogenic lines were distinguished. The KASP marker was efficient in identifying the mutant allele and can be used to transfer waxiness to elite lines.     

Mapping of the Vrn-B1 gene in Triticum aestivum using microsatellite markers

An F₂ population segregating for the dominant gene Vrn‐B1 was developed from the cross of the substitution line ‘Diamant/'Miro‐novskaya 808 5A’ and the winter wheat cultivar ‘Bezostaya 1′. Microsatellite markers (Xgwm and Xbarc) with known map locations on chromosome 5B of common wheat were used for mapping the gene Vrn‐B1. Polymorphism between parental varieties was observed for 28 out of 34 microsatellite markers (82%). Applying the quantitative trait loci mapping approach, the target gene was mapped on the long arm of chromosome 5B, closely linked to Xgwm408. The map position of Vrn‐B1 suggests that the gene is homoeologous to other vernalization response genes located on the homoeologous group 5 chromosomes of wheat, rye and barley.     

Development of SCAR markers linked to a scald resistance gene derived from wild barley

The F₂ progeny of a third backcross(BC₃) line, BC line 240, derived from a Turkish accession of wild barley (Hordeum vulgare ssp. spontaneum),segregated for resistance to scald (Rhynchosporium secalis) in a manner indicating the presence of a single dominant resistance gene. Two SCAR marker slinked to this resistance were developed from AFLP markers. Screens of disomic and ditelosomic wheat-barley addition lines with the SCAR markers demonstrated that the scald resistance gene is located in the centromeric region of barley chromosome 3H,a region previously reported to contain a major scald resistance locus, Rrs1. Markers that flank the Rrs1 locus were used to screen the wild barley-derivedBC₃F₂ population. These markers also flank the wild barley-derived scald resistance, indicating that it maps to the same locus as Rrs1; it may beallelic, or a separate gene within a complex locus. However, BC line 240 does not respond to treatment with the Rhynchosporium secalis avirulence factorNIP1 in the same way as the Rrs1-carrying cultivar Atlas46. This suggests that the scald resistance gene derived from wild barley confers a different specificity of response to theRrs1 allele in Atlas46.In order to increase the durability of scald resistance in the field, we suggest that at least two scald resistances should be combined into barley cultivars before release. The scald resistance gene described here will be of value in the Australian environment, and the several markers linked to it will facilitate pyramiding. This revised version was published online in July 2006 with corrections to the Cover Date.     

Identification of SSR markers linked to the Phytophthora resistance gene Rps1-d in soybean

To identify markers for the Phytophthora resistance gene, Rps1‐d, 123 F_{2 : 3} families were produced from a cross between Glycine max (L.) Merr. ‘Tanbakuro’ (a Japanese traditional black soybean) and PI103091 (Rps1‐d) as an experimental population. The results of virulence tests produced 33 homozygous resistant, 61 segregating and 29 homozygous susceptible F_{2 : 3} families. The chi‐squared test gave a goodness‐of‐fit for the expected ratio of 1 : 2 : 1 for resistant, segregating and susceptible traits, suggesting that the inheritance of Rps1‐d is controlled by a monogenic dominant gene. Simple sequence repeat (SSR) analyses of this trait were carried out using the cultivars ‘Tanbakuro’ and PI103091. Sixteen SSR primers, which produced 19 polymorphic fragments between the two parents, were identified from 41 SSR primers in MLG N. Eight SSR markers were related to Rps1‐d, based on 32 of the 123 F_{2 : 3} families, consisting of 16 homozygous resistant and 16 homozygous susceptible lines. The remaining 91 families were analysed for these eight markers, and a linkage map was constructed using all 123 F_{2 : 3} families. The length of this linkage group is 44.0 cM. The closest markers, Sat_186 and Satt152, are mapped at 5.7 cM and 11.5 cM, respectively, on either side of the Rps1‐d gene. Three‐way contingency table analysis indicates that dual‐marker‐assisted selection using these two flanking markers would be efficient.     

Development of SCAR markers for identification of stem rust resistance gene Sr31 in the homozygous or heterozygous condition in bread wheat

The stem rust resistance gene Sr31, transferred from rye (Secale cereale) into wheat (Triticum aestivum L.) imparts resistance to all the virulent pathotypes of stem rust (Puccinia graminis f. sp. tritici) found in India. Wheat genotypes including carriers and non‐carriers of the Sr31 gene were analysed using arbitrary primed polymerase chain reaction (AP‐PCR). AP‐PCR markers viz. SS30.2_580(H) associated with the Sr31 gene and SS26.1₁₁₀₀ associated with the allele for susceptibility were identified. Linkage between the markers and phenotypes was confirmed by analysing an F₂ population obtained from a cross between a resistant and a susceptible genotype. The markers were tightly linked to the respective alleles. Both the AP‐PCR markers were converted into sequence characterized amplified region (SCAR) markers, viz. SCSS30.2₅₇₆ and SCSS26.1₁₁₀₀ respectively. The markers were validated in two more segregating populations and 49 wheat genotypes. Using both markers it was possible to distinguish the homozygous from the heterozygous carriers of the Sr31 gene in the F₂ generation. The markers developed in this study can be used for pyramiding of the Sr31 gene with other rust resistance genes and in marker‐assisted selection.     

The Yd2 gene and enhanced resistance to barley yellow dwarf virus (BYDV) in winter barley

A breeding programme was developed to obtain barley yellow dwarf virus (BYDV)-resistant winter genotypes using the Yd2 gene. The aim was to incorporate the Yd2 allele into the new high-yielding genotypes to release cultivars that allow barley cultivation in areas where BYDV is endemic. The resistant lines were developed using pedigree selection. An ICARDA resistant line (83RCBB130) carrying the Yd2 gene was crossed with three susceptible, high-yielding winter varieties and their F₁ lines were either selfed or backcrossed to the matching susceptible parent. The best lines selected from subsequent selfing generations were evaluated in replicated trials in the presence or absence of BYDV, starting from F₆ and BC₁F₅ to F₈ and BC₁F₇ generations. Four genotypes with superior agronomic traits and BYDV resistance were selected.     

High-resolution mapping of the nud locus controlling the naked caryopsis in barley

The hulled or naked caryopsis character of barley is an important agronomic trait because of the direct link to its use. A single recessive gene, nud, located on the long arm of chromosome 7H, controls the naked caryopsis character. Previously, linked amplified fragment length polymorphism (AFLP) bands from bulked segregant analysis were screened, and the nud gene was mapped in a population of 151 F₂ plants. In the present study, the aim was to construct a high‐resolution map of the nud gene towards its positional cloning. Two AFLP bands were converted into sequence‐characterized amplified region (SCAR) markers (sKT5 and sKT9), and a previously reported SCAR marker sKT3 was improved for more reliable detection of polymorphism. A total of 2380 segregants derived from five cross‐combinations were analysed, and the nud gene was flanked by sKT3 and sKT9, at the 0.6‐cM proximal and the 0.06‐cM distal side, respectively. The SCAR markers developed in this study should be useful for marker‐assisted selection in naked barley breeding employing crosses between naked and hulled accessions.     

Cold tolerance during juvenile development in sorghum: a comparative analysis by genomewide association and linkage mapping

Improved cold tolerance during the juvenile phase is a major breeding goal to develop new sorghum cultivars suitable as an alternative energy crop in temperate regions. The objectives of this study were to identify marker‐trait associations for cold tolerance in a sorghum diversity panel fingerprinted with 2620 single nucleotide polymorphism (SNP) markers and to detect quantitative trait loci (QTL) in two F_2:3 populations. Traits of interest were dry matter growth rate (DMGR), leaf appearance rate (LAR), chlorophyll content (SPAD) and chlorophyll fluorescence (F_v′/F_m′ and Ф_PSII) in relation to temperature. The association panel comprised 194 genotypes, while the F_2:3 populations consisted of 80 and 92 genotypes. All populations were tested under controlled conditions in a minimum of four temperature regimes ranging from 9.4°C to 20.8°C. QTL were identified for means across environments and regression parameters describing temperature effects. Several marker‐trait associations for mean (m) DMGR, base temperature (T_b) of SPAD and Ф_PSII and temperature effect on LAR were validated by QTL detected in population 1 or 2. Promising QTL regions were found on chromosomes SBI‐01, SBI‐02, SBI‐03, SBI‐04, SBI‐06 and SBI‐09, among them genomic regions where candidate genes involved in the C‐repeat binding pathway or encoding cold‐shock proteins are located.     

Development of AS‐PCR marker based on a key mutation confirmed by resequencing of Wx‐mp in Milky Princess and its application in japonica soft rice (Oryza sativa L.) breeding

Soft rice with low amylose content (AC) ranging by 5–15% is a unique type with special eating and appearance quality and has become popular in the rice market. We resequenced the Wx‐mp, a key allele from Milky Princess, a Japanese low AC variety, and found that the +473 mutation in exon 4 is the key mutation in both Wx‐mp and its ancestor allele, Wx‐mq from Milky Queen. Based on this functional mutation, an allele‐specific PCR (AS‐PCR) marker was developed and proven in a breeding population derived from a cross between a Chinese late variety Nan Keng 46 (Wx‐mp/Wx‐mp) and an early line Ning 63121(Wx‐b/Wx‐b). Based on the marker‐aided selection by our newly developed AS‐PCR marker for Wx‐mp and the known ST10 marker for Stvb‐i, a total of 12 Wx‐mp homozygotes were selected from 198 F₂ progenies, and four of them were immune to rice stripe virus (RSV) with averagely 11.3 days earlier heading than Nan Keng 46 without significant change in grain yield.