Screening for scab resistance by RAPD markers in cultivars of apple (Malus spp.)

Genetic markers are a much faster and more practical alternative to classical methods for the identification of genes for scab resistance present in different apple cultivars. In our study, 28 scab-resistant cultivars, four wild sources of the resistance genes and 10 susceptible cultivars were screened for the presence of the RAPD fragments OPM18/900, OPD20/600 and OPA15/900, which are reported to be linked to the Vf gene. All three marker fragments were successfully amplified with different protocols in Vf-resistant cultivars including ‘M. floribunda 821’. No marker fragments were amplified in susceptible cultivars, three out of four Va-resistant cultivars, three out of four Vm-resistant cultivars, two Vr-resistant cultivars, ‘Antonovka PI 172612’ and ‘M. pumila R 12740-7A’. All three markers were found in the cv. ‘Nova Easygro’, reported to possess the Vr gene, and the cv. ‘Reglindis’, reported to be Va-resistant. M. atrosanguinea of unknown origin showed the presence of OPD20/600 and OPA 15/900 marker bands. The cvs. ‘Nova Easygro’, ‘Reglindis’ and M. atrosanguinea are probably carriers of the VF gene.     

The use of a modified bulk segregant analysis to identify a molecular marker linked to a scab resistance gene in apple

Almost two-hundred random sequence decamer-primers were used to screen a pair of bulked samples and the donor parent Malus floribunda clone 821 for markers linked to the V_f gene conferring resistance to apple scab (Venturia inaequalis (Cke.) Wint.). A single primer was identified which generated a PCR fragment, OPK16/1300, from the donor parent M. floribunda clone 821 and the scab-resistant selections/cultivars bulk, but not from the scab-susceptible recurrent parent bulk. Co-segregation analysis using a segregating apple progeny and polymorphism analysis of individual scab-resistant Coop selections/cultivars confirmed that this marker was linked to the scab-resistance gene V_f with a recombination frequency of 4.3%. OPK16/1300 was then cloned and sequenced. Sequence-specific primers of 25 oligonucleotides based on the marker were synthesized, and used in turn to screen M. floribunda clone 821, scab-susceptible apple cultivars, scab-resistant apple cultivars, and scab-resistant Coop selections. A pair of sequence-specific primers of clone OPK16/1300 amplified a distinct single band of the same size as the RAPD clone. Thus, a sequence characterized amplified region (SCAR) marker was developed which can be used to identify polymorphisms of OPK16/1300 based on the presence or absence of a single band. This revised version was published online in July 2006 with corrections to the Cover Date.     

Development of reliable PCR markers for the selection of the Vf gene conferring scab resistance in apple

Early selection of scab-resistant apple seedlings can be enhanced by the use of markers tightly linked to the Vf resistance gene. Two sequence characterized amplified regions (SCAR) markers have been obtained from previously described random amplified polymorphic DNA (RAPD) markers. AM19-SCAR is a codominant marker, while AM19-SCAR is dominant, as is the RAPD from which it was derived. A highly detailed map in the vicinity of the Vf gene was built through the cumulative analysis of about 600 seedlings from six different controlled crosses. The usefulness of these and other SCAR markers will be discussed in relation to combining the traditional phenotypic selection with MAS. The availability of two codominant, tightly linked markers flanking both sides of the resistance gene (AL07-SCAR and M18-CAPS) also makes it easy to identify the seedlings homozygous for the resistance gene.     

Early determination of genotypes for apple scab resistance by forced flowering of test cross progenies

Summary Apple selections with different major genes for resistance to apple scab (Venturia inaequalis) derived from Malus floribunda and M. pumila were crossed with each other. The progenies were screened as young seedlings for their reaction to V. inaequalis race 1. A gene for resistance from M. pumila, causing stellate necrotic (SN) lesions, was epistatic to a second gene for resistance from M. floribunda, causing irregular chlorotic (Chl) lesions. Although in most cases SN, Chl and susceptible phenotypes were clearly distinct, occasionally reactions were difficult to characterize or varied from one inoculation to another. Selected seedlings showing resistant or susceptible reactions were forced to flower in 16–20 months in the greenhouse and test crossed with susceptible cultivars. Test cross seedlings were screened for scab reaction. The presence of both genes for resistance in a resistant plant was indicated by presence of both Chl and SN resistant phenotypes in the test cross progeny. Chi-square analysis of four large progenies produced a good fit to the expected ratio. The use of the forced flowering technique to determine scab resistance genotypes in 28 months demonstrated its value in breeding apples with multiple disease resistance.     

Approaches for breeding apples with durable disease resistance

Summary In most apple breeding programmes resistance to diseases has a high priority. The main emphasis is put on scab and mildew resistance. Recent evidence about the risk of breakdown of the Vf scab resistance demonstrates the need to search for new approaches in disease resistance breeding. The following options might be considered: discover and exploit new sources of resistance to enlarge the genetic basis of resistance; accumulate different functional resistances in one genotype; develop orchard designs in order to increase diversity in the host-pathogen interactions and establish a resistance management system. To implement these strategies the genetic structure of the resistance must be examined by using modern methods of molecular biology. Resistance donors and advanced selections should be examined under high natural inoculum pressure at several locations around the world. We examined the possibility of identifying polygenic or partial scab resistance in apple. Nineteen apple varieties and selections, among them the differential hosts for scab races 1 to 5, were artificially inoculated in the glasshouse with scab conidia. The leaf symptoms were analysed macroscopically and microscopically There were significant differences which are actually exploited in our breeding programme. Conclusions are drawn towards new approaches for breeding and growing apples with durable disease resistance.     

Evidence of gene introgression in apple using RAPD markers

Summary A genomic remnant of Malus floribunda clone 821 introgressed into the cultivated apple M. x domestica Borkh. was identified using randomly amplified polymorphic DNA (RAPD) markers obtained by the polymerase chain reaction (PCR). Using a set of 59 oligonucleotide decamer primers, polymorphic DNA markers were identified among three pooled DNA samples. Based on the presence or absence of bands among bulked apple scab-resistant selections and cultivars, bulked scab-susceptible cultivars, and a M. floribunda clone 821 sample, one primer, A 15, identified amplified fragments in the scab-resistant bulked sample that was also unique to the M. floribunda clone 821. The unique band from M. floribunda clone 821 was amplified in four out of 17 scab-resistant selections/cultivars. This RAPD, designated OA15₉₀₀, identifies an introgressed fragment that has as yet no known function.     

Faba bean breeding for resistance against biotic stresses: Towards application of marker technology

Summary Faba beans are adversely affected by numerous fungal diseases leading to a steady reduction in the cultivated area in many countries. Major diseases such as Ascochyta blight (Ascochyta fabae), rust (Uromyces viciae-fabae), chocolate spot (Botrytis fabae), downy mildew (Peornospora viciae) and foot rots (Fusarium spp.) are considered to be the major constraints to the crop. Importantly, broomrape (Orobanche crenata), a very aggressive parasitic angiosperm, is the most damaging and widespread enemy along the Mediterranean basin and Northern Africa. Recent mapping studies have allowed the identification of genes and QTLs controlling resistance to some of these diseases. In case of broomrape, 3 QTLs explained more than 70% of the phenotypic variance of the trait. Concerning Ascochyta, two QTLs located in chromosomes 2 and 3 explained 45% of variation. A second population sharing the susceptible parental line also revealed two QTLs, one of them likely sharing chromosomal location and jointly contributing with a similar percentage of the total phenotypic variance. Finally, several RAPD markers linked to a gene determining hypersensitive resistance to race 1 of the rust fungus U. viciae-fabae have also been reported. The aim of this paper is to review the state of the art of gene technology for genetic improvement of faba bean against several important biotic stresses. Special emphasis is given on the application of marker technology, and Quantitative Trait Loci (QTL) analysis for Marker-Assisted Selection (MAS) in the species. Finally, the potential use of genomic tools to facilitate breeding in the species is discussed. The combined approach should expedite the future development of lines and cultivars with multiple disease resistance, one of the top priorities in faba bean research programs.     

The V f gene for scab resistance in apple is linked to sub-lethal genes

Summary V _f is the most widely used resistance gene in the breeding for scab resistant apple cultivars. Distorted segregation ratios for V _f -resistance have frequently been reported. Here we revealed that sub-lethal genes caused the distorted segregation. The inheritance of V _f was examined in six progenies by testing linked molecular markers. Three progenies showed distorted segregations that could be explained by three sub-lethal genes (sl1, sl2 and sl3), of which sl1, sl2 were closely linked to V _f . The s11 gene was located at about 14 cM from V _f and expressed itself only in the presence of another independently segregating sub-lethal gene sl3. Only the double homozygous recessive genotypes (sl1sl1 sl3sl3) were lethal, which occurred at first as dwarf and poor vigour plants during the first three months after germination. The sl2 gene was also linked to V _f and its lethality was expressed prior to seed germination and also required the homozygous recessive presence of sl3. The map position of sl3 has not yet been identified. The linkage of V _f to sub-lethal genes usually results in a shortage of V _f -resistant progenies. But in some exceptional crosses, it will lead to abundance of resistant seedling.An erratum to this article can be found at     

Pyramiding genes affecting sprouting resistance in Rye by means of marker assisted selection

The 541 × Ot1-3 intercross population and bulked segregant analysis (BSA) were used to search for molecular markers linked to genes underlying sprouting and alpha-amylase activity. Six RAPD markers showing association with studied traits were tested for their potential effectiveness in selecting sprouting resistant genotypes. It was shown that although individual effects of markers were not high, their accumulation in one genotype gives substantial increase in sprouting resistance.     

The perspectives of polygenic resistance in breeding for durable disease resistance

Polygenic resistance is generally quantitative without clear race specific effects. With the onset of molecular markers technologies, the identification of chromosome regions that are involved in quantitative resistance has become feasible. These regions are designated quantitative trait loci (QTLs). The mapping of `major' QTLs can be independent of environment, season, year or race of the challenging pathogen. However, the detection of minor QTLs may be dependent on the `environment'. As QTLs are defined by the position on the genome and the quantitative effect on resistance, they are not informative about the mechanism of resistance. By comparing QTL with the loci that are involved in race specific resistance the coincidence of these loci may suggest a common mechanism. However, the histological characterisation of the resistance is more informative about the resistance mechanism. Estimations about the durability of polygenic, quantitative resistance are still academic as there is hardly any experience with large-scale usage of quantitative resistance over a longer period. The clearest example of non-durable resistance is race specific monogenic resistance that is associated with a hypersensitive response (HR). Hence, there is a great chance that polygenic resistance that is not associated with HR is more durable. In some pathosystems with a long experience with non-durable race specific HR genes, quantitative resistance offers a good alternative and marker-assisted breeding will facilitate the exploitation of these resistance for commercial purposes. This revised version was published online in August 2006 with corrections to the Cover Date.     

Lathyrus improvement for resistance against biotic and abiotic stresses: From classical breeding to marker assisted selection

Summary Several Lathyrus species and in particular Lathyrus sativus (grass pea) have great agronomic potential as grain and forage legume, especially in drought conditions. Grass pea is rightly considered as one of the most promising sources of calories and protein for the vast and expanding populations of drought-prone and marginal areas of Asia and Africa. It is virtually the only species that can yield high protein food and feed under these conditions. It is superior in yield, protein value, nitrogen fixation, and drought, flood and salinity tolerance than other legume crops. Lathyrus species have a considerable potential in crop rotation, improving soil physical conditions; reducing the amount of disease and weed populations, with the overall reduction of production costs. Grass pea was already in use in Neolithic times, and presently is considered as a model crop for sustainable agriculture. As a result of the little breeding effort invested in it compared to other legumes, grass pea cultivation has shown a regressive pattern in many areas in recent decades. This is due to variable yield caused by sensitivity to diseases and stress factors and above all, to the presence of the neurotoxin β-N-oxalyl-L-α,β-diaminopropionic acid (β-ODAP), increasing the danger of genetic erosion. However, both L. sativus and L. cicera are gaining interest as grain legume crops in Mediterranean-type environments and production is increasing in Ethiopia, China, Australia and several European countries. This paper reviews research work on Lathyrus breeding focusing mainly on biotic and abiotic resistance improvement, and lists current developments in biotechnologies to identify challenges for Lathyrus improvement in the future.     

Mapping Co, a gene controlling the columnar phenotype of apple, with molecular markers

The columnar phenotype is a very valuable genetic resource for apple breeding because of its compact growth form determined by the dominant gene Co. Using bulked segregant analysis combined with several DNA molecular marker techniques to screen the F₁ progeny of Spur Fuji × Telamon (heterozygous for Co), 9 new DNA markers (6 RAPD, 1 AFLP and 2 SSRs) linked to the Co gene were identified. A total of 500 10-mer random primers, 56 pairs of selective AFLP primers and 8 SSR primer pairs were screened. One RAPD marker S1142₆₈₂, and the AFLP marker, E-ACT/M-CTA₃₄₆, were converted into SCAR markers designated SCAR₆₈₂ and SCAR₂₁₆, respectively. These markers will enable early selection in progenies where Co is difficult to identify. The Co gene was located between the SSR markers CH03d11 and COL on linkage group 10 of the apple genetic linkage map. Finally, a local genetic map of the region around the Co gene was constructed by linkage analysis of the nine new markers and three markers developed earlier.     

Identification and characterization of a RAPD/SCAR marker linked to a resistance gene for soybean mosaic virus in soybean

Soybean line `ICGR95-5383' [Glycinemax (L.) Merr.] is a newly releasedgermplasm from China and is resistant (R)to soybean mosaic virus (SMV). ICGR95-5383was crossed to the susceptible (S)cultivars `HB1', `Tiefeng21', `Amsoy', and`Williams' to investigate the inheritanceof SMV resistance. The F<sub>1</sub> and F<sub>2</sub>plants were inoculated with SMV-3 (the mostvirulent) strain from Northeast China. Theresults showed that F<sub>1</sub> plants from thefour R × S crosses were necrotic (N) andall F<sub>2</sub> populations segregated in a3(R+N):1S ratio, indicating thatICGR95-5383 carries a single gene withincomplete dominance for resistance to SMV. In a bulked segregant analysis (BSA) of theF<sub>2</sub>population from ICGR95-5383 × HB1, a codominant RAPD marker,OPN11<sub>980/1070</sub>, was found to be linkedto the resistance gene in ICGR95-5383. The980-base pair (bp) fragment OPN11<sub>980</sub>was amplified in the R parent ICGR95-5383,R bulk, and resistant F<sub>2</sub> plants. Theother 1070-bp fragment OPN11<sub>1070</sub> wasamplified in the S parent HB1, S bulk, andsusceptible F<sub>2</sub>plants.OPN11<sub>980/1070</sub> was amplified in theF<sub>1</sub> plants and the necroticF<sub>2</sub> plants from the R×S cross.Segregation analysis of the RAPD marker inthe F<sub>2</sub> population revealed that themarker OPN11<sub>980/1070</sub> is closely linkedto the resistance gene with a map distanceof 3.03 cM. OPN11<sub>980/1070</sub> was clonedand sequenced, and specific PCR primerswere designed to convertOPN11<sub>980/1070</sub> into sequencecharacterized amplified region (SCAR) makerSCN11<sub>980/1070</sub>. SCAR analysis of theF<sub>2</sub> population confirmed thatOPN11<sub>980/1070</sub> and SCN11<sub>980/1070</sub> areat the same locus linked to the SMVresistance gene. The RAPD markerOPN11<sub>980</sub> was used as RFLP probefor southern hybridization to soybeangenomic DNA. Southern analysis showed thatsoybean genome contains low-copy sequenceof OPN11<sub>980</sub>. Using a recombinant inbredmapping population of `Kefeng No.1' (R) ×Nannong1138-2'(S), OPN11_980/1070 was mapped to thesoybean molecular linkage group (MLG) Fbetween the restriction fragment lengthpolymorphism (RFLP) markers B212 (0.7 cM) and K07 (6.7 cM) and 3.03 cM apart from theSMV resistance gene.     

甘蔗抗黄锈病G1标记的分子检测及候选抗病基因WAK的分析

甘蔗黄锈病是屈恩柄锈菌(Puccinia kuehnii Butler)引起的一种世界性真菌病害,导致产量减少和糖分降低,给甘蔗产业造成严重损失。本研究采用抗黄锈病分子标记G1,检测我国和世界上重要的甘蔗栽培品种、野生种和近缘属的抗黄锈病基因,并对扩增的代表性特异条带进行克隆测序、功能注释和聚类分析,推测其抗性基因的起源和进化。G1检测结果表明,国内124份甘蔗栽培品种检测到G1标记的有83份,占66.9%;国外46份甘蔗栽培品种检测到G1标记的有31份,占67.4%。34份甘蔗野生种和近缘属中检测到G1标记的有17份,占50%,其中割手密种含有该基因比例最高,为100%。功能注释揭示,G1标记的候选基因编码一种细胞壁连接的类受体激酶,并在甘蔗栽培品种的单倍体蛋白组数据库中鉴定到3个相似度较高的蛋白,这些蛋白都有细胞壁受体激酶结构的胞外域、跨膜域和激酶活性的胞内域。聚类结果则清晰展示了抗病候选基因的起源及进化关系,具体可分为3组,第1组来源于割手密种和大茎野生种;第2组来源于大茎野生种、热带种和河八王属;第3组来源于割手密种、大茎野生种、中国种和栽培品种。研究结果为抗黄锈病甘蔗品种的选育提供重要的抗源支撑,并为抗性分子机制的解析奠定基础。     

Screening for resistance to Nectria galligena Bres. in cut shoots of apple

Summary To find a fast and reliable test to assess resistance to Nectria galligena in apple, different methods of inoculation were compared using macroconidia of N. galligena and one-year-old cut shoots from mature trees of Cox's Orange Pippin, IVT 69078-19, James Grieve and Jonathan.With the best inoculation method 11 genotypes were screened for resistance. Elstar, Golden Delicious, Jonathan and Lombart's Calville were highly resistant and the level of resistance of Ingrid marie, Gloster, Melrose, IVT 69078-19, Cox's Orange Pippin, James Grieve and Idared decreased in this order.The best inoculation method proved to be simple, giving results within four to nine weeks after inoculation.     

A gene for susceptibility to the fungicide azoxystrobin in apple and a tightly linked microsatellite marker

Various apple cultivars, including 'McIntosh' and 'Cox', are reported to be susceptible to the strobilurin fungicide azoxystrobin whereas others, including 'Delicious' and 'Golden Delicious', are resistant. To investigate the genetic control, progenies from various crosses between these four cultivars were raised and the seedlings tested for response by painting three expanded leaves with 200 mg/l of azoxystrobin solution and noting symptoms about a week later. From the segregations, it was concluded that susceptibility to azoxystrobin is due to a dominant gene, Azs , for which 'McIntosh' and 'Cox' are heterozygous. By scoring the segregation in the mapping progeny 'Fiesta' × 'Totem' and comparing it with the segregation of microsatellite [simple sequence repeat (SSR)] markers, Azs was found to co-segregate with SSR-GD127 on linkage group 12 and this tight linkage was confirmed in the progeny from the cross of 'Golden Delicious' × 'Cox' and 'McIntosh'   ×   'Golden Delicious'. The susceptibility of 'McIntosh', attributed to allele Azs-m , was greater than that of 'Cox', Azs-c , and in the 'McIntosh'   ×   'Cox' progeny the GD127 marker showed that Azs-c is dominant to Azs-m .     

Validation of a major QTL for scab resistance with SSR markers and use of marker-assisted selection in wheat

The objectives of this study were to validate the major quantitative trait locus (QTL) for scab resistance on the short arm of chromosome 3B in bread wheat and to isolate near‐isogenic lines for this QTL using marker‐assisted selection (MAS). Two resistant by susceptible populations, both using ‘Ning7840’ as the source of resistance, were developed to examine the effect of the 3BS QTL in different genetic backgrounds. Data for scab resistance and simple sequence repeat (SSR) markers linked to the resistance QTL were analyzed in the F_2:3 lines of one population and in the F_3:4 lines of the other. Markers linked to the major QTL on chromosome 3BS in the original mapping population (‘Ning7840’/‘Clark’) were closely associated with scab resistance in both validation populations. Marker‐assisted selection for the QTL with the SSR markers combined with phenotypic selection was more effective than selection based solely on phenotypic evaluation in early generations. Marker‐assisted selection of the major QTL during the seedling stage plus phenotypic selection after flowering effectively identified scab resistant lines in this experiment. Near‐isogenic lines for this 3BS QTL were isolated from the F₆ generation of the cross ‘Ning7840’/‘IL89‐7978’ based on two flanking SSR markers, Xgwm389 and Xbarc147. Based on these results, MAS for the major scab resistance QTL can improve selection efficiency and may facilitate stacking of scab resistance genes from different sources.