The origin of rhizomania resistance in sugar beet

In the last 35 years, breeding has greatly reduced the damages caused by rhizomania in sugar beet crops. After the first encouraging results using the Alba genotypes, the cultivar Rizor represented a substantial step forward and has given good yield improvement in diseased fields in many parts of the world. The original variety and subsequent improved versions continued to offer good performances for about a decade, after which it was surpassed by other hybrids derived in part from the Rizor itself. Further progress in terms of sugar production became possible in 1986, when the Holly monogerm lines were released in USA and Europe. In spite of the incomplete information about the genealogy of the first resistant materials,many evidences and the molecular analyses on the different genotypes suggest a possible common progenitor and lineage. The resistant cultivars have kept the yield at an adequate level, allowing cultivation to continue in countries where the disease has reached epidemic proportions. The case of rhizomania resistance in sugar beet can therefore be considered as one of the most important achievements in plant breeding. This revised version was published online in August 2006 with corrections to the Cover Date.     

Breeding for resistance to rhizomania in sugar beet: A review

Currently rhizomania is the most important disease in sugar beet worldwide, and attack can lead to serious yield losses. The disease is caused by beet necrotic yellow vein virus (BNYVV) that is transmitted by the soil-borne fungus Polymyxa betae. Breeding sugar beet cultivars with resistance to rhizomania is regarded as the most appropriate way to enable continued production of this crop in BNYVV-infested fields and also to slow the spread of the disease. Breeding for resistance started with selection by scoring disease symptoms in field experiments. The development of non-destructive greenhouse tests, with determination of the virus concentration in rootlets using ELISA, has greatly improved the efficiency of selection. In this paper the impact of scientific research on the progress in breeding cultivars with resistance to rhizomania is reviewed. This includes the distribution, composition, and pathogenicity of the virus, the sources of resistance to virus and vector, the genetics of virus resistance, progress with breeding methods, and the use of molecular markers and pathogen-derived resistance. The yields and quality characteristics of recently introduced resistant cultivars now equal those of the commercial susceptible cultivars. This revised version was published online in July 2006 with corrections to the Cover Date.     

Identification and mapping of random amplified polymorphic DNA markers linked to a rhizomania resistance gene in sugar beet (Beta vulgaris L.) by bulked segregant analysis

Bulked segregant analysis was employed to identify random amplified polymorphic DNA (RAPD) markers linked to a gene that confers rhizomania resistance to a sugar beet line created from a Holly Sugar Company breeding population (USA). Polymorphism revealed with 160 arbitrary 10-mer oligonucleotide primers was screened in two bulks produced by separately pooling the individual DNAs from the six most resistant and the six most susceptible plants of an F₂ population segregating for rhizomania resistance. A study of the F₂ individuals showed that 19 primers generated 44 polymorphic markers which were then grouped into nine linkage groups. By analysis of variance, 12 were shown to have a significant effect upon the level of resistance and were mapped on a segment 22.3 cM long. A quantitative trait locus (QTL) of resistance was identified and located in a 4.6cM interval between two markers. It accounted for 67.4% of the observed variation and almost all the genetic variation. These results suggest that the identified QTL corresponds to a unique major gene conditioning the Holly resistance studied, which we have named Rz-l.     

Molecular markers linked to rhizomania resistance in sugar beet, Beta vulgaris, from two different sources map to the same linkage group

Rhizomania, one of the most important diseases of sugar beet, is caused by beet necrotic yellow vein virus, a Furovirus vectored by the fungus Polymyxa betae Keskin. Reduction of the production losses caused by this disease can only be achieved by using tolerant cultivars. The objective of this study was the identification and mapping of random amplified polymorphic DNA (RAPD) markers linked to a rhizomania resistance gene. The RAPD markers were identified using bulked segregant analysis in a segregating population of 62 individuals derived by intercrossing plants of the resistant commercial hybrid GOLF, and the resistance locus was positioned in a molecular marker linkage map made with a different population of 50 GOLF plants. The resistance locus, Rr1, was mapped to linkage group III of our map of Beta vulgaris L. ssp. vulgaris, which consisted of 76 RAPDs, 20 restriction fragment length polymorphisms (RFLPs), three sequence characterized amplified regions (SCARs) and one sequence tagged site (STS). In total, 101 molecular markers were mapped over 14 linkage groups which spanned 688.4 cM with an average interval length of 8.0 cM. In the combined map, Rr1 proved to be flanked by the RAPD loci RA411₁₈₀₀ and AS7₁₁₀₀ at 9.5 and 18.5cM, respectively. Moreover, in our I₂ population, we found that a set of markers shown by Barzen et al. (1997) to be linked to the ‘Holly’ type resistance gene was also linked to the ‘GOLF’-type resistance gene. These results appeared to indicate that the rhizomania resistance gene present in the GOLF hybrid could be the same gene underlying resistance in ‘Holly’-based resistant genotypes. Two other explanations could be applied: first, that two different alleles at the same locus could have been selected; second, that two different genes at two different but clustered loci underwent the selection process.     

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.     

Development of molecular markers linked to the wheat powdery mildew resistance gene Pm4b and marker validation for molecular breeding

Powdery mildew, caused by Blumeria graminis (DC.) E.O. Speer f. sp. tritici, is an important disease in wheat (Triticum aestivum L.). Bulk segregant analysis (BSA) was employed to identify SRAP (sequence‐related amplified polymorphism), sequence tagged site (STS) and simple sequence repeat (SSR) markers linked to the Pm4b gene, which confers good resistance to powdery mildew in wheat. Out of 240 SRAP primer combinations tested, primer combinations Me8/Em7 and Me12/Em7 yielded 220‐bp and 205‐bp band, respectively, each of them associated with Pm4b. STS‐₂₄₁ also linked to Pm4b with a genetic distance of 4.9 cM. Among the eight SSR markers located on wheat chromosome 2AL, Xgwm382 was found to be polymorphic and linked to Pm4b with a genetic distance of 11.8 cM. Further analysis was carried out using the four markers to investigate marker validation for marker‐assisted selection (MAS). The results showed that a combination of the linked markers STS_?241, Me8/Em7_?220 and Xgwm382 could be used for marker‐assisted selection of the resistance gene Pm4b in wheat breeding programmes.     

The role of molecular markers and marker assisted selection in breeding for organic agriculture

Plant geneticists consider molecular marker assisted selection a useful additional tool in plant breeding programs to make selection more efficient. Standards for organic agriculture do not exclude the use of molecular markers as such, however for the organic sector the appropriateness of molecular markers is not self-evident and is often debated. Organic and low-input farming conditions require breeding for robust and flexible varieties, which may be hampered by too much focus on the molecular level. Pros and contras for application of molecular markers in breeding for organic agriculture was the topic of a recent European plant breeding workshop. The participants evaluated strengths, weaknesses, opportunities, and threats of the use of molecular markers and we formalized their inputs into breeder’s perspectives and perspectives seen from the organic sector’s standpoint. Clear strengths were identified, e.g. better knowledge about gene pool of breeding material, more efficient introgression of new resistance genes from wild relatives and testing pyramided genes. There were also common concerns among breeders aiming at breeding for organic and/or conventional agriculture, such as the increasing competition and cost investments to get access to marker technology, and the need for bridging the gap between phenotyping and genotyping especially with complex and quantitative inherited traits such as nutrient-efficiency. A major conclusion of the authors is that more interaction and mutual understanding between organic and molecular oriented breeders is necessary and can benefit both research communities.     

Development and validation of molecular markers closely linked to the wheat stripe rust resistance gene YrC591 for marker-assisted selection

Stripe rust resistance gene YrC591, present in wheat cultivar C591, is effective against currently important Puccinia striiformis Westend. f. sp. tritici isolates in China. An F_2:3 population (127 lines) was developed by crossing C591 with susceptible cultivar Taichung 29. Thirty four simple sequence repeat (SSR) and 155 sequence tagged site (STS) markers located on chromosome 7BL were used to perform bulk segregant analysis. Eight SSR markers, cfa2040, wmc273, wmc166, gwm984, barc32 wmc276, barc182 and gwm146, and 6 STS markers, mag1714, mag1757, mag1811, BE425120, BE471173 and BG607810, were polymorphic between the parents and contrasting resistant and susceptible DNA pools. F_2:3 lines were genotyped with these polymorphic markers. Linkage analysis indicated that YrC591 was flanked by Xmag1714 and Xbarc182 with genetic distances of 1.2 and 0.4 cM, respectively. In addition, validation of the SSR markers cfa2040, wmc273 and barc32, and STS markers mag1714 and BE425120 was carried out using wheat lines with C591 as a parent, indicating that these markers should be effective in tracing this gene in marker-assisted selection.     

Inheritance of resistance to angular leaf spot in common bean and validation of the utility of resistance linked markers for marker assisted selection out side the mapping population

Inheritance of resistance to angular leaf spot (ALS) disease caused by Phaeoisariopsis griseola (Sacc.) Ferr was investigated in two common bean cultivars, Mexico 54 and BAT 332. Both Andean and Mesoamerican backgrounds were used to determine the stability of the resistance gene in each of the two cultivars. Resistance to P. griseola was phenotypically evaluated by artificial inoculation with one of the most widely distributed pathotypes, 63–39. Evaluation of the parental genotypes, F₁, F₂ and backcross populations revealed that the resistance to angular leaf spot in the cultivars Mexico 54 and BAT 332 to pathotype 63–39 is controlled by a single dominant gene, when both the Andean and Mesoamerican backgrounds were used. Allelism test showed that ALS resistance in Mexico 54 and BAT 332 to pathotype 63–39 was conditioned by the same resistance locus. Resistant and susceptible segregating populations generated using Mexico 54 resistant parent were selected for DNA extraction and amplification to check for the presence /absence of the SCAR OPN02 and RAPD OPE04 markers linked to the Phg-2 resistance gene. The results indicated that the SCAR OPN02 was not polymorphic in the study populations and therefore of limited application in selecting resistant genotypes in such populations. On the other hand, the RAPD OPE04 marker was observed in all resistant individuals and was absent in those scored susceptible based on virulence data. Use of the RAPD OPE04 marker in marker-assisted selection is underway.     

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.     

Search for molecular markers linked to Liriomyza trifolii resistance in tomato

Resistance to many arthropods, including Liriomyza species, is known to be present in accessions of Lycopersicon hirsutum (f. typicum or f. glabratum). From the cross L. esculentum cv. Moneymaker and L. hirsutum f. glabratum G1561 100 F₂ plants were screened in a no-choice test for resistance to Liriomyza trifolii. The Bulked Segregant Analysis approach was used to find Random Amplified Polymorphic DNA markers linked to resistance. Two markers were located on chromosome 2. Restriction Fragment Length Polymorphisms constructed a more detailed genetic linkage map for part of chromosome 2. Kruskal-Wallis analysis showed that this chromosome harbored a Quantitative Trait Locus (QTL) for number of pupae, number of mines and damage. At least one major QTL is essential for resistance and this QTL is located on chromosome 2 nearby the location of the tomato probe TG451. This revised version was published online in July 2006 with corrections to the Cover Date.     

Validation of molecular markers linked to fertility restorer gene(s) for WA-CMS lines of rice

The present study was carried out with the objective to validate the molecular markers, which have been previously reported to be linked to fertility restorer (Rf) gene(s) for WA-CMS lines of rice. Two mapping populations involving fertility restorer lines for WA-cytoplasm, viz., (i) an F₂ population derived from the cross IR58025A/KMR3R consisting of 347 plants and (ii) a BC₁F₁ population derived from the cross IR62829A/IR10198R//IR62829A consisting of 130 plants were analyzed. Nine SSR and three CAPS markers reported to be linked to Rf genes along with two previously unreported SSR markers were analyzed in the mapping populations. In both the populations studied, the trait of fertility restoration was observed to be under digenic control. Eight SSR markers (RM6100, RM228, RM171, RM216, RM474, RM311, MRG4456 and pRf1&2) showed polymorphism between the parents of the F₂ population, while the SSR markers RM6100 and RM474 showed polymorphism between the parents of both the F₂ and BC₁F₁ populations. Only one CAPS marker, RG146FL/RL was polymorphic between the parents of the BC₁F₁ population. RM6100 was observed to be closely segregating with fertility restoration in both the mapping populations and was located at a distance of ~1.2 cM. The largest phenotypic variation was accounted for the region located between RM311 and RM6100. Using the marker-trait segregation data derived from analysis of both the mapping populations, a local linkage map of the genomic region around Rf-4, a major fertility restoration locus on Chromosome 10 was constructed, and RM6100 was observed to be very close to the gene at a distance of 1.2 cM. The accuracy of the marker RM6100 in predicting fertility restoration was validated in 21 restorers and 18 maintainers. RM6100 amplified the Rf-4 linked allele in a majority of the restorers with a selection accuracy of 94.87%. Through the present study, we have established the usefulness of the marker RM6100 in marker-assisted selection for fertility restoration in segregating populations and identification of restorers while screening rice germplasm for their fertility restoration ability.     

Development of genic molecular markers linked to a rust resistance gene in cultivated groundnut (Arachis hypogaea L.)

Development of molecular markers for different economically important traits in cultivated groundnut has progressed at slow pace. Although many genomic SSR markers were developed in both the wild and cultivated groundnut, the genetic linkage map in the species is still not saturated. Availability of a large number of ESTs in GenBank opened up the possibility of integrating new markers and to identify markers closely linked to agronomic traits. EST-SSR markers are also considered as genic molecular markers. In this study, 259 EST-SSR markers were developed by mining 5,184 Arachis hypogaea ESTs from NCBI database. These EST-SSRs and 34 resistance gene candidate markers were used for association and genetic mapping of rust resistance in cultivated groundnut. From these, Cer2, SSR_GO340445, SSR_HO115759, SSR_GO341324 and RGC 2 had a significant association with rust resistance based on locus-by-locus AMOVA and/or Kruskal?CWallis ANOVA. Some of these associated markers also had protein activity related to biotic stress responses. Through genetic mapping, EST-SSR markers SSR_GO340445 and SSR_HO115759 were found closely linked to a rust resistance gene at 1.9 and 3.8?cM distances, respectively. These markers are thus suitable candidates for marker assisted selection in groundnut. The tight linkage of SSR_GO340445 would be helpful to screen BAC clones and to isolate rust resistance gene in groundnut.     

Identification of a molecular marker linked to an Agropyron elongatum-derived gene Lr19 for leaf rust resistance in wheat

The leaf rust resistance gene Lr19, transferred from Agropyron elongatum into wheat (Triticum aestivum L.) imparts resistance to all pathotypes of leaf rust (Puccinia recondita f.sp. tritici) in South‐east Asia. A segregating F₂ population from a cross between the leaf rust resistant parent ‘HW 2046’ carrying Lr19 and a susceptible parent ‘Agra Local’ was screened in the phytotron against a virulent pathotype 77‐5 of leaf rust with the objective of identifying the molecular markers linked to Lr19. The gene was first tagged with a randomly amplified polymorphic DNA (RAPD) marker S73₇₂₈. The RAPD marker linked to the gene Lr19 which mapped at 6.4 ± 0.035 cM distance, was converted to a sequence characterized amplified region (SCAR) marker. The SCAR marker (SCS73₇₁₉) was specific to Lr19 and was not amplified in the near‐isogenic lines (NILs) carrying other equally effective alien genes Lr9, Lr28 and Lr32 enabling breeders to pyramid Lr19 with these genes.     

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.     

Molecular markers linked to the Aegilops variabilis-derived root-knot nematode resistance gene Rkn-mn1 in wheat

Aegilops variabilis no. 1 is the only known source of resistance to the root‐knot nematode Meloidogyne naasi in wheat. Previous studies showed that a dominant gene, Rkn‐mn1, was transferred to a wheat translocation line from the donor Ae. variabilis. Random amplified polymorphic DNA (RAPD) analysis was performed on the wheat cultivar ‘Lutin’, on Ae. variabilis, on a resistant disomic addition line and on a resistant translocation line. For genetic and molecular studies, 114‐117 BC₃F₂ plants and F₃‐derived families were tested. Five DNA and one isozyme marker were linked to Rkn‐mn1. Three RAPD markers flanking the Rkn‐mn1 locus were mapped at 0 cM (OpY16_‐1065), 0.8 cM (OpB12_‐1320) and 1.7 cM (OpN20_‐1235), respectively. Since the Rkn‐mn1 gene remained effective, its introduction into different wheat cultivars by marker‐assisted selection is suggested.     

A RAPD-derived STS marker is linked to a bacterial wilt (Burkholderia caryophylli) resistance gene in carnation

Bacterial wilt caused by Burkholderia caryophylli is one of the most important and damaging diseases of carnations (Dianthus caryophyllus) in Japan. We aimed to identify random amplified polymorphic DNA (RAPD) markers associated with the genes controlling bacterial wilt resistance in a resistance-segregating population of 134 progeny plants derived from a cross between Carnation Nou No. 1 (a carnation breeding line resistant to bacterial wilt) and Pretty Favvare (a susceptible cultivar). We screened a total of 505 primers to obtain RAPD markers useful for selecting resistant carnation lines: 8 RAPD markers identified by bulked segregant analysis were linked to a major resistance gene; of these, WG44-1050 had the greatest effect on resistance to bacterial wilt. A locus with large effect on bacterial resistance was mapped around WG44-1050 through QTL analysis. The RAPD marker WG44-1050 was successfully converted to a sequence-tagged site (STS) marker suitable for marker-assisted selection (MAS). Five combinations of primers were designed for specific amplification of WG44-1050. In addition, the STS marker we developed was useful and reliable as a selection marker for breeding for resistance to bacterial wilt, using a highly resistant wild species, D. capitatus ssp. andrzejowskianus and a resistant line, Carnation Nou No. 1, as breeding materials.     

Mapping of rhizoctonia root rot resistance genes in sugar beet using pathogen response‐related sequences as molecular markers

Rhizoctonia root and crown rot caused by the fungus Rhizoctonia solani is a serious disease of sugar beet. An F_2:3 population from a cross between a resistant and a susceptible parent has been tested for R. solani resistance and a genetic map has been constructed from the corresponding F₂ parents. The map encompasses 38 expressed sequence tags (ESTs) with high similarity to genes which are involved in resistance reactions of plants (R‐ESTs) and 25 bacterial artificial chromosomes (BACs) containing nucleotide binding site (NBS)‐motifs typical for disease resistance genes. Three quantitative trait loci (QTL) for R. solani resistance were found on chromosomes 4, 5 and 7 collectively explaining 71% of the total phenotypic variation. A number of R‐ESTs were mapped in close distance to the R. solani resistance QTL. In contrast, the NBS‐BACs mapped to chromosomes 1, 3, 7 and 9 with two major clusters of NBS‐BACs on chromosome 3. No linkage between NBS‐BACs and R. solani resistance QTL was found. The data are discussed with regard to using R‐ESTs and NBS markers for mapping quantitative disease resistances.