Molecular markers and plant breeding

The achievements of modern biotechnology allow to modernize significantly the traditional plant breeding. The use of molecular codominant markers reduces considerably the quantity of breeding material and promotes the selection of genotypes, which posses desirable genes in the homozygous state. Molecular marking systems of agronomically important simple and quantitative traits have been developed using mono- and multiloci systems. Markers of the plant type and development rate, alleles of the storage protein genes, Wx-genes, short-stem genes, etc., have been created and tested at the South Biotecnology Center, National Academy of Agricultural Sciences. The technology of the application of DNA-typing for the identification and registration of varieties that has been developed in the South Biotecnology Center is of great importance for the systematization of germplasm sources and the protection of the rights of breeders.     

Development of DNA markers associated with beer foam stability for barley breeding

Traits conferring brewing quality are important objectives in malting barley breeding. Beer foam stability is one of the more difficult traits to evaluate due to the requirement for a relatively large amount of grain to be malted and then the experimental costs for subsequent brewing trials. Consequently, foam stability tends to be evaluated with only advanced lines in the final stages of the breeding process. To simplify the evaluation and selection for this trait, efficient DNA makers were developed in this study. Previous studies have suggested that the level of both of the foam-associated proteins Z4 and Z7 were possible factors that influenced beer foam stability. To confirm the relationship between levels of these proteins in beer and foam stability, 24 beer samples prepared from malt made from 10 barley cultivars, were examined. Regression analyses suggested that beer proteins Z4 and Z7 could be positive and negative markers for beer foam stability, respectively. To develop DNA markers associated with contents of proteins Z4 and Z7 in barley grain, nucleotide sequence polymorphisms in barley cultivars in the upstream region of the translation initiation codon, where the promoter region might be located were compared. As a result, 5 and 23 nucleotide sequence polymorphisms were detected in protein Z4 and protein Z7, respectively. By using these polymorphisms, cleaved amplified polymorphic sequence (CAPS) markers were developed. The CAPS markers for proteins Z4 and Z7 were applied to classify the barley grain content of 23 barley cultivars into two protein Z4 (pZ4-H and pZ4-L) and three protein Z7 (the pZ7-H, pZ7-L and pZ7-L2) haplotypes, respectively. Barley cultivars with pZ4-H showed significantly higher levels of protein Z4 in grain, and those with pZ7-L and pZ7-L2 showed significantly lower levels of protein Z7 in grain. Beer foam stability in the cultivars with pZ4-H and pZ7-L was significantly higher than that with pZ4-L and pZ7-H, respectively. Our results indicate that these CAPS markers provide an efficient selection tool for beer foam stability in barley breeding programs.     

Molecular markers for tracking variation in lipoxygenase activity in wheat breeding

Lipoxygenase (LOX) activity is an important factor determining the color of flour and end-use products of wheat. In the present study, quantitative trait loci (QTL) for LOX activity in common wheat were mapped using 71 doubled haploid (DH) lines derived from a Zhongyou 9507 × CA9632 cross, and SSR markers. Two QTL, QLpx.caas.1AL and QLpx.caas-4B, were identified on chromosomes 1AL and 4B, closely associated with LOX activity. The SSR loci Xwmc312 and Xgwm251 proved to be diagnostic and explained 13.4–25.2% of the phenotypic variance for the 1AL locus and 14.3–27.0% for the 4B locus across four environments. The SSR markers Xgwm251 and Xwmc312 were validated across 198 Chinese wheat cultivars and advanced lines and showed highly significant (P < 0.01) association with LOX activity. We further established a multiplexed PCR with SSR marker combination Xwmc312/Xgwm251 to test these wheat cultivars and advanced lines. The results suggested that the marker combination Xwmc312/Xgwm251 is efficient and reliable for evaluating LOX activity and can be used in marker-assisted selection (MAS) for targeting flour color attributes to noodle and other wheat-based products.     

Molecular breeding for grain yield in barley: an evaluation of QTL effects in a spring barley cross

 We report results from a breeding strategy designed to accumulate favorable QTL alleles for grain yield identified in the SteptoeבMorex’ (SM) barley germplasm. Two map lines (SM73 and SM145) from the original mapping population were selected based on their marker genotype and QTL structure. When crossed, these lines would be expected to produce progeny with most favorable QTL alleles. One hundred doubled haploid (DH) lines from the F₁ hybrid of this cross were genotyped with ten RFLP markers and one morphological marker defining grain yield to monitor QTL segregation. A subset of 24 lines representing various combinations of putatively favorable and unfavorable QTL alleles, together with Steptoe, ‘Morex’, SM73, and SM145, were phenotyped for grain yield in five environments. Multiple regression procedures were used to explore phenotype and genotype relationships. Most target QTLs showed significant effects. However, significance and magnitude of QTL effects and favorable QTL allele phase varied across environments. All target QTLs showed significant QTL-by-environment interaction (QTL×E), and the QTL on chromosome 2 expressed alternative favorable QTL alleles in different environments. Digenic epistatic effects were also detected between some QTL loci. For traits such as grain yield, marker-assisted selection efforts may be better targeted at determining optimum combinations of QTL alleles rather than pyramiding alleles detected in a reference mapping population. Received: 2 June 1998 / Accepted: 17 September 1998     

Identification of polypeptide markers of barley yellow dwarf virus resistance and susceptibility genes in non-infected barley (Hordeum vulgare) plants

Summary Barley yellow dwarf (BYDV) is a group a closely related viruses which cause economic losses in a wide range of graminaceous species throughout the world. Barley plants can be protected from the effects of BYDV by the Yd2 resistance gene. Plants which contain the Yd2 gene also contain a constitutively expressed polypeptide which was not found in any plants without Yd2. Conversely, BYDV susceptible plants contain another constitutively expressed polypeptide which was not found in any of the BYDV-resistant lines examined. These two polypeptides appear to have the same molecular weight (as assessed by SDS-PAGE) and only slightly different iso-electric points. They also appear to contain an extensive range of similar antigenic determinants. Both polypeptides were found in F₁ hybrids made from resistant and susceptible plants. We suggest that these two polypeptides are the products of two allelic genes. Analysis of near-isogenic lines showed that the locus which controls the Yd2 resistance gene and the locus controlling the synthesis of the two polypeptides may be within ± 9 cM of each other. We have developed a Western blot technique which allows assessment of barley lines, 4-days after seed imbibition, for the presence of the Yd2 gene.     

Investigating successive Australian barley breeding populations for stable resistance to leaf rust

Key message

Genome-wide association studies of barley breeding populations identified candidate minor genes for pairing with the adult plant resistance gene Rph20 to provide stable leaf rust resistance across environments.

Abstract

Stable resistance to barley leaf rust (BLR, caused by Puccinia hordei) was evaluated across environments in barley breeding populations (BPs). To identify genomic regions that can be combined with Rph20 to improve adult plant resistance (APR), two BPs genotyped with the Diversity Arrays Technology genotyping-by-sequencing platform (DArT-seq) were examined for reaction to BLR at both seedling and adult growth stages in Australian environments. An integrated consensus map comprising both first- and second-generation DArT platforms was used to integrate QTL information across two additional BPs, providing a total of four interrelated BPs and 15 phenotypic data sets. This enabled identification of key loci underpinning BLR resistance. The APR gene Rph20 was the only active resistance region consistently detected across BPs. Of the QTL identified, RphQ27 on chromosome 6HL was considered the best candidate for pairing with Rph20. RphQ27 did not align or share proximity with known genes and was detected in three of the four BPs. The combination of RphQ27 and Rph20 was of low frequency in the breeding material; however, strong resistance responses were observed for the lines carrying this pairing. This suggests that the candidate minor gene RphQ27 can interact additively with Rph20 to provide stable resistance to BLR across diverse environments.

     

Molecular markers linked to papaya ring spot virus resistance and Fusarium race 2 resistance in melon

In melon, the Fom-1 gene confers monogenic resistance against the soil-borne fungus Fusarium oxysporum f. sp. melonis, races 0 and 2, while the closely linked Prv gene specifies resistance against the papaya ring spot virus. Markers linked to these resistance (R) genes were identified using two recombinant inbred line populations, derived from crosses between Cucumis melo Védrantais and C. melo PI 161375, and between C. melo Védrantais and C. melo PI 414723, respectively. Using bulked segregant analysis, as well as systematic scoring of the mapping populations, we developed two amplified fragment length polymorphism markers, two random amplified polymorphic DNA markers and five restriction fragment length polymorphism (RFLP) markers linked to this locus. Four of the RFLP sequences bear homology to nucleotide-binding site–leucine-rich repeat R genes, indicating the presence of a significant R-gene cluster in this locus. Our study provides the most closely linked markers published so far for these important traits. It also improves the resolution of the whole linkage group IX, which was difficult to order in our previous studies. Two of the markers were converted to cleaved amplified polymorphic sequence markers to facilitate their application in marker-assisted selection. Testing these two markers in several melon lines revealed different marker haplotypes in the melon germplasm and supported multiple, independent origin of the Fusarium races 0 and 2 resistance trait.     

Molecular markers for leaf rust resistance genes in wheat

Over 100 genes of resistance to rust fungi: Puccinia recondita f. sp. tritici, (47 Lr - leaf rust genes), P. striiformis (18 Yr - yellow rust genes) and P. graminis f. sp. tritici (41 Sr - stripe rust genes) have been identified in wheat (Triticum aestivum L.) and its wild relatives according to recent papers. Sixteen Lr resistance genes have been mapped using restriction fragments length polymorphism (RFLP) markers on wheat chromosomes. More than ten Lr genes can be identified in breeding materials by sequence tagged site (STS) specific markers. Gene Lrk 10, closely linked to gene Lr 10, has been cloned and its function recognized. Available markers are presented in this review. The STS, cleaved amplified polymorphic sequence (CAPS) and sequence characterized amplified regions (SCAR) markers found in the literature should be verified using Triticum spp. with different genetic background. Simple sequence repeats (SSR) markers for Lr resistance genes are now also available.     

Molecular breeding of polymerases for resistance to environmental inhibitors

Potent inhibitors limit the use of PCR assays in a wide spectrum of specimens. Here, we describe the engineering of polymerases with a broad resistance to complex environmental inhibitors using molecular breeding of eight different polymerase orthologues from the genus Thermus and directed evolution by CSR in the presence of inhibitors. Selecting for resistance to the inhibitory effects of Neomylodon bone powder, we isolated 2D9, a chimeric polymerase comprising sequence elements derived from DNA polymerases from Thermus aquaticus, Thermus oshimai, Thermus thermophilus and Thermus brockianus. 2D9 displayed a striking resistance to a broad spectrum of complex inhibitors of highly divergent composition including humic acid, bone dust, coprolite, peat extract, clay-rich soil, cave sediment and tar. The selected polymerase promises to have utility in PCR-based applications in a wide range of fields including palaeobiology, archaeology, conservation biology, forensic and historic medicine.     

Molecular marker analyses of powdery mildew resistance in barley

Powdery mildew, caused byEryisphe graminis f. sp.hordei, is one of the most important diseases of barley (Hordeum vulgare). A number of loci conditioning resistance to this disease have been reported previously. The objective of this study was to use molecular markers to identify chromosomal regions containing genes for powdery mildew resistance and to estimate the resistance effect of each locus. A set of 28 F₁ hybrids and eight parental lines from a barley diallel study was inoculated with each of five isolates ofE. graminis. The parents were surveyed for restriction fragment length polymorphisms (RFLPs) at 84 marker loci that cover about 1100 cM of the barley genome. The RFLP genotypes of the F₁s were deduced from those of the parents. A total of 27 loci, distributed on six of the seven barley chromosomes, detected significant resistance effects to at least one of the five isolates. Almost all the chromosomal regions previously reported to carry genes for powdery mildew resistance were detected, plus the possible existence of 1 additional locus on chromosome 7. The analysis indicated that additive genetic effects are the most important component in conditioning powdery mildew resistance. However, there is also a considerable amount of dominance effects at most loci, and even overdominance is likely to be present at a number of loci. These results suggest that quantitative differences are likely to exist among alleles even at loci which are considered to carry major genes for resistance, and minor effects may be prevalent in cultivars that are not known to carry major genes for resistance.     

The role of RAPD markers in breeding for disease resistance in common bean

Diseases are regarded as the leading constraint to increased common bean (Phaseolus vulgaris L.) production worldwide. The range in variability and complexity among bean pathogens can be controlled with different single gene and quantitative resistance sources. Combining these resistance sources into commercial cultivars is a major challenge for bean breeders. To assist breeders, a major effort to identify RAPD markers tightly linked to different genes was undertaken. To date, 23 RAPD and five SCAR markers linked to 15 different resistance genes have been identified, in addition to QTL conditioning resistance to seven major pathogens of common bean. We review the feasibility of using marker-assisted selection (MAS) to incorporate disease resistance into common bean. Indirect selection of single resistance genes in the absence of the pathogen and the opportunity afforded breeders to pyramid these genes to improve their longevity and retain valuable hypostatic genes is discussed. The role of markers linked to the QTL controlling complex resistance and the potential to combine resistance sources using marker based selection is reviewed. Improving levels of selection efficiency using flanking markers, repulsion-phase linkages, co-dominant marker pairs, recombination-facilitated MAS and SCAR markers is demonstrated. Marker-assisted selection for disease resistance in common bean provides opportunities to breeders that were not feasible with traditional breeding methods.     

Association mapping of resistance to Puccinia hordei in Australian barley breeding germplasm

Key message

“To find stable resistance using association mapping tools, QTL with major and minor effects on leaf rust reactions were identified in barley breeding lines by assessing seedlings and adult plants.”

Abstract

Three hundred and sixty (360) elite barley (Hordeum vulgare L.) breeding lines from the Northern Region Barley Breeding Program in Australia were genotyped with 3,244 polymorphic diversity arrays technology markers and the results used to map quantitative trait loci (QTL) conferring a reaction to leaf rust (Puccinia hordei Otth). The F_3:5 (Stage 2) lines were derived or sourced from different geographic origins or hubs of international barley breeding ventures representing two breeding cycles (2009 and 2011 trials) and were evaluated across eight environments for infection type at both seedling and adult plant stages. Association mapping was performed using mean scores for disease reaction, accounting for family effects using the eigenvalues from a matrix of genotype correlations. In this study, 15 QTL were detected; 5 QTL co-located with catalogued leaf rust resistance genes (Rph1, Rph3/19, Rph8/14/15, Rph20, Rph21), 6 QTL aligned with previously reported genomic regions and 4 QTL (3 on chromosome 1H and 1 on 7H) were novel. The adult plant resistance gene Rph20 was identified across the majority of environments and pathotypes. The QTL detected in this study offer opportunities for breeding for more durable resistance to leaf rust through pyramiding multiple genomic regions via marker-assisted selection.     

Prospects for molecular breeding of barley

Data from UK Recommended List Trials showed that the introduction of new cultivars of spring and winter barley has maintained a significant increase in yield over time, whereas there has been no significant improvement in hot water extract, the major determinant of good malting quality, in either crop. Commercial barley breeding is based upon phenotypic selection, and the introduction of molecular breeding methods must either increase the rate of advance, or offer an improvement in the cost‐effectiveness of breeding programmes. Molecular breeding can be applied to either single gene or polygenic characters but is not widely used in commercial barley breeding, other than as a marker for resistance to the Barley Yellow Mosaic Virus complex. There are many reports of potential targets for use in molecular breeding but the few validation studies that have been carried out to date are disappointing. Results from genomics studies are likely to lead to the identification of key candidate genes, which can be associated with economically important characters through co‐location on certain chromosomal regions. Associations between candidate gene sequence haplotypes and phenotypic characteristics is expected to identify allelic combinations, which are most frequently observed in successful cultivars, that can be used in molecular breeding of barley on a commercial scale.     

Molecular mapping of a recessive gene for resistance to stripe rust in barley

Barley stripe rust, caused by Puccinia striiformis f. sp. hordei, is one of the most important barley (Hordeum vulgare) diseases in the United States. The disease is best controlled using resistant cultivars. Barley genotype Grannenlose Zweizeilige (GZ) has a recessive gene (rpsGZ) that is effective against all races of P. striiformis f. sp. hordei identified so far in the USA. To develop a molecular map for mapping the gene, F₈ recombinant inbred lines (RILs) were developed from the Steptoe X GZ cross through single-seed descent. Seedlings of the parents and RILs were evaluated for resistance to races PSH-14 and PSH-54 of P. striiformis f. sp. hordei under controlled greenhouse conditions. Genomic DNA was extracted from the parents and 182 F₈ RILs and used for linkage analysis. The resistance gene analog polymorphism (RGAP) technique was used to identify molecular markers for rpsGZ. A linkage group for the gene was constructed with 12 RGAP markers, of which two markers co-segregated with the resistance locus, and two markers were closely linked to the locus with a genetic distance of 0.9 and 2.0 cM, respectively. These four markers were present only in the susceptible parent. The closest marker to the resistance allele was 11.7 cM away. Analyses of two sets of barley chromosome addition lines of wheat with the two RGAP markers that were cosegregating with the susceptibility allele showed that rpsGZ and the markers were located on the long arm of barley chromosome 4H. Further, tests with four simple sequence repeat (SSR) markers confirmed the chromosomal location of the rpsGZ gene and also integrated the RGAP markers into the known SSR-based linkage map of barley. The closest SSR marker EBmac0679 had a genetic distance of 7.5 cM with the gene in the integrated linkage map constructed with the 12 RGAP markers and 4 SSR markers. The information on chromosomal location and molecular markers for rpsGZ should be useful for incorporating this gene into commercial cultivars and combining it with other resistance genes for durable resistance.     

Association mapping of stem rust race TTKSK resistance in US barley breeding germplasm

Key message

Loci conferring resistance to the highly virulent African stem rust race TTKSK were identified in advanced barley breeding germplasm and positioned to chromosomes 5H and 7H using an association mapping approach.

Abstract

African races of the stem rust pathogen (Puccinia graminis f. sp. tritici) are a serious threat to barley production worldwide because of their wide virulence. To discover and characterize resistance to African stem rust race TTKSK in US barley breeding germplasm, over 3,000 lines/cultivars were assessed for resistance at the seedling stage in the greenhouse and also the adult plant stage in the field in Kenya. Only 12 (0.3 %) and 64 (2.1 %) lines exhibited a resistance level comparable to the resistant control at the seedling and adult plant stage, respectively. To map quantitative trait loci (QTL) for resistance to race TTKSK, an association mapping approach was conducted, utilizing 3,072 single nucleotide polymorphism (SNP) markers. At the seedling stage, two neighboring SNP markers (0.8 cM apart) on chromosome 7H (11_21491 and 12_30528) were found significantly associated with resistance. The most significant one found was 12_30528; thus, the resistance QTL was named Rpg-qtl-7H-12_30528. At the adult plant stage, two SNP markers on chromosome 5H (11_11355 and 12_31427) were found significantly associated with resistance. This resistance QTL was named Rpg-qtl-5H-11_11355 for the most significant marker identified. Adult plant resistance is of paramount importance for stem rust. The marker associated with Rpg-qtl-5H-11_11355 for adult plant resistance explained only a small portion of the phenotypic variation (0.02); however, this QTL reduced disease severity up to 55.0 % under low disease pressure and up to 21.1 % under heavy disease pressure. SNP marker 11_11355 will be valuable for marker-assisted selection of adult plant stem rust resistance in barley breeding.     

Genome-wide association mapping of Fusarium head blight resistance in contemporary barley breeding germplasm

Utilization of quantitative trait loci (QTL) identified in bi-parental mapping populations has had limited success for improving complex quantitative traits with low to moderate heritability. Association mapping in contemporary breeding germplasm may lead to more effective marker strategies for crop improvement. To test this approach, we conducted association mapping of two complex traits with moderate heritability; Fusarium head blight (FHB) severity and the grain concentration of mycotoxin associated with disease, deoxynivalenol (DON). To map FHB resistance in barley, 768 breeding lines were evaluated in 2006 and 2007 in four locations. All lines were genotyped with 1,536 SNP markers and QTL were mapped using a mixed model that accounts for relatedness among lines. Average linkage disequilibrium within the breeding germplasm extended beyond 4 cM. Four QTL were identified for FHB severity and eight QTL were identified for the DON concentration in two independent sets of breeding lines. The QTL effects were small, explaining 1–3% of the phenotypic variation, as might be expected for complex polygenic traits. We show that using breeding germplasm to map QTL can complement bi-parental mapping studies by providing independent validation, mapping QTL with more precision, resolving questions of linkage and pleiotropy, and identifying genetic markers that can be applied immediately in crop improvement.     

Molecular markers for resistance to Heterodera glycines in advanced soybean germplasm

Germplasm line J87-233 is resistant to soybean cyst nematode (SCN) races 1, 2, 3, 5 and moderately resistant to race 14 with resistance derived from 3 primitive sources, Peking, PI 88788 and PI 90763. F_2:3 progeny of J87-233 and SCN-susceptible Hutcheson cross were evaluated for response to SCN races 1, 2, 3, 5 and 14. Linkage groups (LG) A, B, F, G, J, M, N, S were tested with 215 genomic clones and 45 decamers for parental genotypes. QTL for race 1 and QTL for race 3 were detected on LG A2, the region of BLT65V and SCAR 548/563_{1100/1025,975.} The cluster analysis of 12 soybean cultivars and 38 plant introductions confirmed association of SCAR_{1100/1025,975} with resistance to races 1 and 3, and suggested possible DNA rearrangements that might give rise to new resistance specificities in the region. The highly significant association of K69T marker with SCN race 1 resistance in conjunction with its location, 18.5 cM from the reported QTL, exemplifies the importance of the QTL locus on LG G and suggests expansion of the linkage map in the LG G-terminal region. Detected interaction between loci on LG A2 and LG G, and also with loci on LG F and LG M, may play a significant role in the genotype-specific response to SCN. Identification of two major regions on LG A2 and LG G for SCN resistance shows their applicability to advanced germplasm, however, transmission of molecular marker alleles indicates that applied markers are not yet reliable in revealing all possible recombination events in breeding for SCN resistance.     

Protein markers for anther culturability in barley

Two-dimensional electrophoresis of proteins from a recombinant population of anther culture-derived doubled haploid lines identified 4 loci or linkage groups showing a deviation from an expected 11 segregation. It was hypothesized that these markers are linked to genes involved in the process of haploid plant production and that the deviation was due to a selection for alleles conferring higher anther culture response. To check this hypothesis, the anther culturability of 50 of the doubled haploid lines and their two inbred parents was assessed. It was found that 2 of the loci which had a distortion of segregation showed a significant effect on anther culture response, the most efficient allele being the most frequent in both loci. In addition, 2 more markers associated with anther culturability were found. One of the first mentioned 2 loci and one of the latter 2 were found to be linked to genes involved in both embryoid production and subsequent green plant regeneration. The remaining two were linked to genes involved only in green plant regeneration. Of the 4 favorable alleles 3 were inherited from one parent.