Identification of novel alleles from wild barley for the improvement of alpha‐amylase and related malt quality traits

Germplasms consisting of 64 wild barley accessions (Hordeum vulgare ssp. spontaneum) were examined for polymorphisms in α‐amylase using both isoelectric focusing (IEF) and thermostability assays. Wide variation was found for the high pI α‐amylase with 20 IEF band patterns identified. Enzyme activity and thermostability assays showed large differences among α‐amylase isoenzymes. Two wild accessions Tel‐Shoket CPI 77146‐32 and Afiq CPI 77128‐41 showed superior enzyme activity and thermostability compared with commercial varieties such as ‘Baudin’, ‘Flagship’ or ‘Navigator’. The functionality of the Tel‐Shoket allele was validated in backcross lines with ‘Flagship’ as the recurrent parent. The Tel‐Shoket allele at the amy1 locus increased α‐amylase thermostability at 75°C by 8.4% and α‐amylase activity in kilned malt by 18.7%. The introgression of the wild allele also led to significant improvements in fermentability, hot water extract and viscosity. Gene sequencing showed that there are three single nucleotide polymorphisms in the Tel‐Shoket amy1 sequence, which can be used as diagnostic markers for the selection of this allele in breeding programmes.     

An SSR marker in the nitrate reductase gene of common bean is tightly linked to a major gene conferring resistance to common bacterial blight

A simple sequence repeat (SSR) marker composed of a tetra nucleotide repeat is tightly linked to a major gene of common bean (Phaseolus vulgaris L.) conferring resistance to common bacterial blight (CBB) incited by Xanthomonas axonopodis pv. phasoli (Xap). This SSR is located in the third intron region of the common bean nitrate reductase (NR) gene, which is mapped to linkage group (LG) H7, corresponding to LG B7 of the bean Core map. Co-segregation analysis between the SSR marker and CBB resistance in a recombinant inbred line (RIL) population demonstrated a tight linkage between the NR gene-specific marker and the major gene for CBB resistance. In total, the marker explained approximately 70% of the phenotypic variation in the population. Because it is co-dominant, this SSR marker should be more efficient for marker-assisted selection (MAS) than dominant/recessive random amplified polymorphic DNA (RAPD) or sequence characterized amplified region (SCAR) markers that have been developed, especially for early generation selection. This revised version was published online in August 2006 with corrections to the Cover Date.     

Evaluation of cultivated barley (Hordeum vulgare) germplasm for the presence of thermostable alleles of β-amylase

A cleaved amplified polymorphic sequence marker was used to detect the alleles Bmy‐Sd2H and Bmy‐Sd3 identifying highly thermostable isoforms of the enzyme b‐amylase, which improves fermentability during brewing. Among the 889 accessions of barley (Hordeum vulgare) investigated, and two accessions of H. spontaneum a total of 166 accessions were identified carrying the superior b‐amylase alleles. These thermostable alleles of b‐amylase were most frequently observed in six‐rowed varieties originating from Asia, especially Japan, with 61.9% of the accessions from Asia carrying the alleles of interest. Additional six‐rowed barleys carrying the relevant alleles were identified among accessions from America, Africa and the Near East. In the European varieties, the percentage of accessions with the alleles of interest was 5.1% with a strong predominance in two‐rowed spring barleys. A pedigree analysis identified the cross ‘Binder’ x ‘Gull’ as the main source of the thermostable b‐amylase alleles in European varieties. The data suggest that an improvement of malting quality in barley could be achieved by introduction of the Bmy1‐Sd2H and Bmy1‐Sd3 alleles into the European breeding programmes.     

Investigation and genetic mapping of a Glomerella leaf spot resistance locus in apple

Apple Glomerella leaf spot (GLS) is a severe fungal disease that damages apple leaves during the summer in China. Breeding new apple varieties that are resistant to the disease is considered the best way of controlling GLS. Fine mapping and tightly linked marker are critically essential for the preselection of resistant seedlings. In this study, a population of 207 F₁ individuals derived from a cross between ‘Golden Delicious’ and ‘Fuji’ was used to construct a fine simple sequence repeat (SSR)‐based genetic linkage map. The position of R_gls, a locus responsible for resistance to GLS, was identified on apple linkage group (LG) 15 using SSR markers CH05g05 and CH01d08, which was adapted from a published set of 300 SSR markers that were developed using the bulked segregant analysis (BSA) method. These two SSR markers flanked the gene, and its recombination rate was 8.7% and 23.2%, respectively. A total of 276 newly developed SSR markers around the target region and designed from the genome apple assembly contig of LG15 were screened. Only nine of these were determined to be linked to the R_gls locus. Thus, a total of 11 SSR markers were in linkage with R_gls, and mapped at distances ranging from 0.5 to 33.8 cM. The closest marker to the R_gls locus was S0405127, which showed a genetic distance of approximately 0.5 cM. The first mapping of the gene R_gls was constructed, and the locations of the 11 effective primers in the ‘Golden Delicious’ apple genome sequence were anchored. This result facilitates better understanding of the molecular mechanisms underlying the trait of resistance to GLS and could be used in improving the breeding efficiency of GLS‐resistant apple varieties.     

Identification of a SCAR marker associated with Bm, the beet mosaic virus resistance gene, on chromosome 1 of sugar beet

Beet mosaic virus (BtMV) is an aphid transmitted, viral disease of beet found worldwide. The Bm gene, a resistance gene effective against BtMV, was identified in the sugar beet line 8500 and backcrossed into a C37 background to produce line C719. Three populations were developed from the cross of line C719 with the susceptible line C37 with the intent of developing markers for use in marker‐assisted selection. The F₂ progeny of three crosses were scored for resistance. Two of the three populations conformed to a 3 : 1 ratio, indicating a single gene trait. Sequence characterized amplified region (SCAR) markers were developed by using bulked segregant analysis combined with random amplified polymorphic DNA type markers. The markers showed close association to the Bm resistance gene and were effective in all three populations. The A₁ allele for genetic male sterility also was found to be associated with Bm and the SCAR marker. Development of a single‐nucleotide polymorphism marker from the SCAR sequence was used to validate linkage to chromosome 1 using separate mapping populations. This marker will be useful for the introgression of the Bm gene into germplasm.     

Application of marker‐assisted selection and genome‐wide association scanning to the development of winter food barley germplasm resources

Barley (Hordeum vulgare) is an important component of heart‐healthy whole grain diets because it contains β‐glucan. All current US barley varieties with high β‐glucan are spring habit and have waxy starch. Winter varieties have agronomic advantages but require low‐temperature tolerance (LTT). Vernalization sensitivity (VS) is associated with higher levels of LTT. To rapidly develop fall‐sown varieties with LTT and higher grain β‐glucan, we therefore used marker‐assisted selection (MAS) at the WX and VRN‐H2 loci. The MAS‐derived lines, together with unrelated non‐waxy germplasm developed via phenotypic selection (PS), were used for a genome‐wide association scan (GWAS). The panel was phenotyped for grain β‐glucan, LTT and VS. It was genotyped with 3072 single‐nucleotide polymorphisms (SNPs) and allele‐specific primers. Marker‐assisted selection fixed target alleles at both loci but only one of the target phenotypes (higher β‐glucan percentage) was achieved. Variation for VS and LTT is attributable to (i) incomplete information about VRN‐H1 at the outset of the project and (ii) unexpected allelic variation at VRN‐H3 with a large effect on VS and LTT.     

Evaluation of Czech spring malting barleys with respect to the β-amylase allele incidence

Strong enzymatic activities in the germinating barley grain, together with protein and starch content, are crucial for high extraction values in the resulting malt and, therefore, barley malting quality. The efficient characterization of registered barley cultivars and genetic resources with respect to one of the relevant thermostability enzymes (β‐amylase) is an essential requirement. The template‐directed dye‐terminator incorporation (TDI) assay: based on flourescence resonance energy transfer (TDI‐FRET) ( Chen et al. 1995 ) was used to detect single nucleotide polymorphisms (SNPs) in the β‐amylase coding sequence resulting in low (Sd2L), intermediate (Sd1) and high (Sd2H and Sd3) thermostability enzyme across 84 Czech barley cultivars and genetic resources used over a period of time in the Czech Republic. The incidence of different alleles has changed during the last 100 years. Also the new resources with high thermostabile β‐amylase were identified. They can be used effectively to breed for malting quality improvement.     

RAPD and SCAR markers for powdery mildew resistance gene er in pea

In pea, a single recessive gene (er) on linkage group 6 confers resistance to powdery mildew caused by Erysiphe pisi. The present study aims to identify molecular markers linked to the er gene. Screening of the powdery mildew‐resistant cultivar ‘DMR11’ and its susceptible nearisogenic line for polymorphism revealed linkage of two RAPD primers (OPO‐02 and OPU‐17) to the er gene and a sequence characterized polymorphic region (SCAR) primer, ScOPD‐10₆₅₀ with er in a population of 83 F₂ plants in the order: OPU‐17 ‐ er ‐ ScOPD‐10₆₅₀ ‐ OPO‐02. The markers ScOPD‐10₆₅₀ and OPU‐17 being coupled with the allele causing resistance would substantially increase the efficiency of marker‐assisted selection in peabreeding for powdery mildew.     

Development of simple PCR-based markers linked to the <Emphasis Type="Italic">Ms</Emphasis> locus,a restorer-of-fertility gene in onion (<Emphasis Type="Italic">Allium cepa</Emphasis> L.)

In order to implement reliable marker-assisted selection systems for the restorer-of-fertility locus (Ms) in onions (Allium cepa L.), simple PCR-based codominant markers linked to the Ms locus were developed. Based on the EST probe sequences of previously reported RFLP markers, full-length genomic sequences of the gene encoding putative oligopeptide transporter (OPT) was obtained by RACE. The first intron contained two 108 and 439-bp indel polymorphisms between the two Ms allele-linked OPT alleles. A simple PCR marker for OPT was developed by designing a primer pair on the flanking regions of the 108-bp indel which is created by two tandem repeats. The second simple PCR marker was developed from the EST probe encoding photosystem I subunit O (PsaO). Two 14 and 39-bp tandem repeats were identified from the 5′ upstream sequences of the PsaO-coding gene, which were isolated by genome walking. Three different compositions of these tandem repeats were identified from diverse onion germplasm. A primer set binding to the flanking sequence of these polymorphic repeats was used to amplify three different marker haplotypes. The OPT marker was tightly linked to the Ms locus at a distance of 1.5 cM, but the analysis of the linkage relationship showed little linkage disequilibrium between the marker and the Ms locus. Even so, these simple PCR markers are valuable tools for the marker-assisted selection of segregating individuals in onion F₁ hybrid breeding programs.     

Rph23: A new designated additive adult plant resistance gene to leaf rust in barley on chromosome 7H

We report on a new adult plant resistance (APR) gene Rph23 conferring resistance to leaf rust in barley. The gene was identified and characterized from a doubled haploid population derived from an intercross between the Australian barley varieties Yerong (Y) and Franklin (F). Genetic analysis of adult plant field leaf rust scores of the Y/F population collected over three successive years indicated involvement of two highly additive genes controlling APR, one of which was named Rph23. The gene was mapped to chromosome 7HS positioned at a genetic distance 36.6 cM. Rph23 is closely linked to marker Ebmac0603, which is flanked by markers bPb‐8660 and bPb‐9601 with linkage distances of 0.8 and 5.1 cM, respectively. A PCR‐based marker was optimized for marker‐assisted selection of Rph23, and on the basis of this marker, the gene was postulated as being common in Australian and global barley germplasm. Pedigree and molecular marker analyses indicated that the six‐rowed black Russian landrace ‘LV‐Taganrog’ is the likely origin of Rph23.     

Sequence tagged site markers linked to the sbm1 gene for resistance to pea seedborne mosaic virus in pea

Resistance to pea seed‐borne mosaic virus (PSbMV) pathotype P‐1 in peas is conferred by sbm1 with recessive inheritance. PSbMV is an economically important pathogen with world‐wide distribution that causes significant losses in pea yield and reduces seed and produce quality. The sbm1 gene was previously mapped to linkage group VI on molecular linkage maps of the pea genome. To improve plant breeders’ ability to develop varieties resistant to PSbMV, two random amplified polymorphic DNA markers (G05_2537 and L01_910) and one restriction fragment length polymorphism (P446) linked to sbm1 have been identified. The genomic sequences for these markers have been characterized and the information used to develop three simple polymerase chain reaction‐based STS (sequence tagged site) assays. Linkage analysis in two F₂ populations showed that the most tightly linked of these three STS loci (sG05_2537) is approximately 4 cM from sbm1. Characterization of a collection of resistant and susceptible germplasm demonstrated a strong correlation between STS alleles and sbm1 alleles, indicating the utility of these markers for marker‐assisted selection in breeding programmes using a range of germplasm sources.     

User‐friendly markers linked to Fusarium wilt race 1 resistance Fw gene for marker‐assisted selection in pea

Fusarium wilt is one of the most widespread diseases of pea. Resistance to Fusarium wilt race 1 was reported as a single gene, Fw, located on linkage group III. The previously reported AFLP and RAPD markers linked to Fw have limited usage in marker‐assisted selection due to their map distance and linkage phase. Using 80 F₈ recombinant inbred lines (RILs) derived from the cross of Green Arrow × PI 179449, we amplified 72 polymorphic markers between resistant and susceptible lines with the target region amplified polymorphism (TRAP) technique. Marker–trait association analysis revealed a significant association. Five candidate markers were identified and three were converted into user‐friendly dominant SCAR markers. Forty‐eight pea cultivars with known resistant or susceptible phenotypes to Fusarium wilt race 1 verified the marker–trait association. These three markers, Fw_Trap_480, Fw_Trap_340 and Fw_Trap_220, are tightly linked to and only 1.2 cM away from the Fw locus and are therefore ideal for marker‐assisted selection. These newly identified markers are useful to assist in the isolation of the Fusarium wilt race 1 resistance gene in pea.     

Molecular and chemical analysis of β-amylase-less mutant barley in Tibet

The molecular and biochemical aspects of Tibetan β‐amylase‐less mutant barley were investigated. An enzyme assay showed that there were eight β‐amylase‐less mutant lines in the Tibetan landrace barley. Following Southern analysis and polymerase chain reaction, it is suggested that these mutants result from the same insert mutation in the latter part of the β‐amylase structural gene. The α‐glucosidase activity level of the mutant line was high in the initial period of germination, and the glucose ratio in the sugar conformation was remarkably high in the germinated seed of the mutant line. These results suggest that this β‐amylase‐less mutant line could be a new breeding material for beer production.     

Development of cleaved amplified polymorphic sequence (CAPS) markers linked to a green rice leafhopper resistance gene, Grh3(t)

The chromosomal location of the resistance gene for green rice leafhopper (GRLH), an injurious insect for rice, has been determined and RFLP markers closely linked to this gene have been identified. The susceptible japonica rice variety ‘Nipponbare’ was crossed with a resistant japonica rice line ‘Aichi42’, in which green rice leaf hopper resistance had been introduced from an indica variety ‘Rantaj‐emas2’, and the 100 F₂ plants obtained were used for linkage analysis. The green rice leafhopper resistance gene, Grh3(t), was mapped between RFLP markers C288B and C133A on chromosome 6 and co‐segregated with C81. Of the RFLP markers tightly linked to Grh3(t), C81 was converted to a SCAR marker and C133A to a cleaved amplified polymorphic sequence marker that could distinguish the heterozygous genotype to establish an effective marker‐aided selection system for the GRLH resistance gene.     

Development of PCR-based markers linked to a restorer gene for cytoplasmic male sterility in radish (Raphanus sativus L.)

A random amplified polymorphic DNA (RAPD) marker named OPC06-1900 was previously found linked to a fertility restorer gene (Rfw) for cytoplasmic male sterility (CMS) in radish (Raphanus sativus L.). The RAPD marker was converted to a dominant sequence characterized amplified region (SCAR) marker SCC06-1894 by molecular cloning and nucleotide sequencing. A BLAST search revealed that the SCAR marker SCC06-1894 showed significant homology to the corresponding regions of Arabidopsis and Brassica sulfate transporter genes. The presence of the intron and exon of the DNA fragment SCC06-1894 was demonstrated by comparing RT-PCR and PCR products. Thus, allele-specific oligonucleotide primers were designed to amplify the SCAR marker SCC06-415. PCR test with F₂ plants and sequence analysis showed that SCC06-1894 and SCC06-415 were allelic, linked to Rfw/rfw gene at 8.0 cM. Nine oligonucleotide primers were designed based on a single radish nuclear restorer gene mRNA. A survey of these primer combinations by bulked segregant analysis (BSA) identified three polymorphisms. The three PCR-based markers were co-segregant in the coupling phase and distant from the Rfw gene by 1.4 cM. These specific markers distributed on both sides of the Rfw gene and will be helpful for breeding new rapseed (Brassica napus L.) restorer lines.     

Development of an STS marker and SSRs suitable for marker-assisted selection for the BaMMV resistance gene rym9 in barley

Based on the RAPD marker OP‐C04H910 which is closely linked to the barley mild mosaic virus (BaMMV) resistance gene rym9 derived from the variety ‘Bulgarian 347’ the marker STS‐C04H910 cosegregating with OP‐C04H910 and generating a single additional band on plants carrying the recessive resistance encoding allele has been developed. Furthermore, the simple sequence repeats (SSRs) WMS6 and HVM67 have been integrated into the genetic map of the rym9 region on chromosome 4HL. Because of their close linkage to rym9 and distinct banding pattern STS‐C04H910 and HVM67 are well‐suited for marker‐ assisted selection, enhanced backcrossing procedures and pyramiding of resistance genes.     

Development of gene‐based markers for the Turnip mosaic virus resistance gene retr02 in Brassica rapa

Turnip mosaic virus (TuMV) is responsible for a serious disease that affects the production of Chinese cabbage. Previous studies have cloned a series of TuMV resistance genes and developed molecular markers. In this study, a derived cleaved amplified polymorphism sequence (dCAPS) marker and a Kompetitive Allele Specific PCR (KASP) marker were developed based on a single recessive gene, retr02, which confers broad‐spectrum TuMV resistance in Chinese cabbage by means of an additional G at the junction of exon 1 and intron 1. The two markers were able to detect the retr02 allele in Chinese cabbage accessions used in breeding programmes. Compared with the dCAPS marker, the KASP marker was flexible, cost‐effective and quick to process, which is likely to be beneficial in establishing high‐throughput assays for marker‐assisted selection.     

Genetic diversity of Phaseolus vulgaris L. and P. coccineus L. landraces in central Italy

The genetic diversity of 66 Phaseolus genotypes was investigated, which included 14 local varieties of Phaseolus vulgaris and nine local varieties of P. coccineus, collected in Marche, central Italy. Their genetic diversity was assessed using three types of molecular marker: inter simple sequence repeats (ISSRs), nuclear gene‐tagged simple sequence repeats (SSRs) and chloroplast simple sequence repeats (CpSSRs). Phaseolus vulgaris shows a higher genetic diversity than P. coccineus for the SSRs and CpSSRs, but not for the putative neutral ISSR markers. These data suggest that selection by farmers and adaptation to heterogeneous environments has maintained the diversity in landraces of the common bean. Comparing genetic diversity in Marche with that of the American controls reveals that 71% of the local P. vulgaris varieties in Marche are of Andean origin. The two gene pools of the common bean can be found on the same farm, and there is some evidence of past hybridization events between these two gene pools.     

Marker‐assisted backcrossing of a null allele of the α‐subunit of Soybean (Glycine max) β‐conglycinin with a Chinese soybean cultivar (a). The development of improved lines

The development of soybean varieties that lack the β‐conglycinin α‐subunit is an attractive goal because the β‐conglycinin α‐subunit negatively influences the nutrition and gelation of tofu and is a major allergen. To remove this undesirable allergen and simultaneously improve the seed nutritional value and food‐processing quality, marker‐assisted background selection (MABS) was used in backcross breeding to incorporate cgy‐2, a null phenotype version of the gene encoding the β‐conglycinin α‐subunit, from the donor line ‘RiB’ into the genetic background of the Chinese cultivar ‘Dongnong47’ (DN47), a popular high‐oil superfine seed soybean cultivar from Heilongjiang Province, China. In each F₂ (F₂, BC_nF₂) generation of the breeding programme, the offspring that carried the introgressed cgy‐2 were identified by sodium dodecyl sulphate–polyacrylamide gel electrophoresis and rescreened by MABS using simple sequence repeat markers to accelerate recurrent parent genome recovery. Of the 49 advanced backcrossing breeding lines (ABLs), the three best lines, ABL1, ABL2 and ABL3, were selected from the BC₁, BC₂ and BC₃ populations, respectively. The ABLs were evaluated for desirable agronomic characteristics, yield‐related traits, amino acid composition, free amino acid composition and tofu‐processing quality in the mature seeds. All of the ABLs lacked the α‐subunit but grew and reproduced normally without deleterious effects on physiological processes such as seed development and germination. The free amino acid content of ABL1 was significantly higher than that of ‘DN47’, with arginine (Arg) being particularly enriched. Compared to the recurrent parent ‘DN47’, the total protein content of the three ABLs was higher, the amino acid composition of the seed proteins was markedly modified and the yield and hardness of the tofu that was made from the ABLs were significantly increased. MABS combined with stringent phenotypic selection in a backcross breeding programme is a feasible strategy for the genetic engineering of seed protein components to produce allergenic subunit‐deficient variant alleles.     

Development of a CAPS marker for the badh2.7 allele in Sri Lankan fragrant rice (Oryza sativa)

Fragrance in rice is caused by mutations in the badh2 (betaine aldehyde dehydrogenase) gene. It was previously reported that exons 1, 2, 7, 10, 13 and 14 of badh2 are hot spots for various mutations leading to fragrance in most aromatic rice. This study was carried out to sequence the 14th exon of badh2 gene of Sri Lankan aromatic rice varieties that lack the badh2.1 allele. The aims of the study were to predict the aberrant protein structure and to develop a functional DNA marker. In view of this, we sequenced the 14th exon of four traditional aromatic accessions and compared with a published sequence. Four accessions contained a nucleotide ‘G’ insertion in the 14th exon. This novel mutation can be classified as the badh2.7 allele. The predicted three‐dimensional protein structure of the mutant shows loss of part of the oligomerization and coenzyme binding domains, a change that is predicted to result in fragrance. A CAPS‐based novel marker, Bad2.7CAPS, was developed to identify varieties possessing this badh2.7 allele, and it can be utilized in rice breeding programmes.