A SCAR marker linked to the ToMV resistance gene, Tm22, in tomato

A polymerase chain reaction (PCR)-based co-dominant marker was developed which is tightly linked to Tm2². This dominant locus confers resistance to ToMV in tomato. Random-amplified-polymorphic DNA (RAPD) screening was carried out with DNA from ToMV-susceptible and resistant tomato near-isogenic lines. A polymorphic band linked to ToMV resistance was observed. The polymorphic fragment was cloned and the DNA sequences of both ends determined. Specific PCR primers were designed from these sequences. PCR amplification with the specific primers resulted in an amplified band (SCAR) in both susceptible and resistant tomato lines. The amplified band from the susceptible lines could, however, be discerned from that of the resistant ones after cleavage with the restriction enzyme Hind III. In an F₂ population of 90, the polymorphic markers co-segregated with susceptibility or resistance, as determined by biological assays for ToMV resistance. The reported SCAR marker is linked to ToMV resistance not only in cultivars derived from American lineage, but also from European lineage. This method enables the distinction of homozygous and heterozygous individual plants in segregating populations, and provides a convenient and rapid assay for both selection and quality control during breeding programs and hybrid seed production, respectively. This revised version was published online in July 2006 with corrections to the Cover Date.     

Validation of molecular markers for pathogen resistance in potato

DNA markers have a large potential to improve efficiency and precision of conventional plant breeding programmes based on marker‐assisted selection (MAS). In our study, we have evaluated the predictive abilities of the SCAR marker RYSC3 and the CAPS marker GP122₅₆₄ with regard to the PVY resistance genes Ry_adg and Ry_sto, respectively, and of marker TG689 linked to H1 conferring resistance to Globodera rostochiensis and marker HC associated with high levels of G. pallida resistance. The evaluations were made in 28 cultivars and accessions and in 219 progeny genotypes descending from ten different crosses. We observed in all evaluated cultivars and accessions the expected marker patterns according to their phenotypic classification into resistant and susceptible genotypes. However, in part considerable discrepancies were observed when analysing progeny of controlled crosses involving these resistance sources, particularly with respect to H1. Based on these results, practical aspects for the efficient implementation of marker‐assisted selection are discussed, which consider the genetic origin of the material, costs aspects and methodology applied.     

Evaluation of the Ph‐3 gene‐specific marker developed for marker‐assisted selection of late blight‐resistant tomato

Late blight caused by Phytophthora infestans is one of the most destructive diseases of tomato (Solanum lycopersicum L.) that mainly occurs in cool and wet environments. With the spread of the A2 mating type and new clonal lineages, fewer fungicides provide effective control of the disease, which has increased its worldwide threat. Host resistance could contribute significantly to sustainable disease control. Ph‐3 is a race‐specific late blight resistance gene commonly used in commercial tomato breeding. Availability of precise and easy to use gene‐based markers would facilitate selection. In this study, a Ph‐3 on‐gene cleaved amplified polymorphic sequence (CAPS) marker, Ph3.gsm/HincII, was developed based on the published gene sequence of Ph‐3. The effectiveness of the marker was evaluated along with other published Ph‐3 markers using an F₉ recombinant inbred line (RIL) population derived from NC 23E‐2(93) × L3708. Markers Ph3.gsm/HincII and TG328/BstNI accurately genotyped the RIL population for Ph‐3. In addition, Ph3.gsm/HincII was able to differentiate variable susceptible alleles. This reliable codominant DNA marker would be very useful in marker‐assisted selection, particularly for resistance gene pyramiding.     

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 a SCAR marker linked with a MYMV resistance gene in mungbean (Vigna radiata L. Wilczek)

Yellow mosaic disease (YMD) caused by mungbean yellow mosaic virus (MYMV) is the most important disease of mungbean, causing great yield loss. The present investigation was carried out to study the inheritance and identify molecular markers linked with MYMV resistance gene by using F₁, F₂ and 167 F_2 : 8 recombinant inbred lines (RILs) developed from the cross ‘TM‐99‐37’ (resistant) × Mulmarada (susceptible). The F₁ was susceptible, F₂ segregated in 3S:1R phenotypic ratio and RILs segregated in 1S:1R ratio in the field screening indicating that the MYMV resistance gene is governed by a single recessive gene. Of the 140 RAPD primers, 45 primers showing polymorphism in parents were screened using bulked segregant analysis. Three primers amplified specific polymorphic fragments viz. OPB‐07_600, OPC‐06₁₇₅₀ and OPB‐12₈₂₀. The marker OPB‐07₆₀₀ was more closely linked (6.8 cM) with a MYMV resistance gene as compared to OPC‐06₁₇₅₀ (22.8 cM) and OPB‐12₈₂₀ (25.2 cM). The resistance‐specific fragment OPB‐07₆₀₀ was cloned, sequenced and converted into a sequence‐characterized amplified region (SCAR) marker and validated in twenty genotypes with different genetic backgrounds.     

Development of SCAR markers linked to the Ns locus in potato

A random amplified polymorphic DNA marker OPG17₄₅₀ linked to the Ns gene that confers resistance of potato to potato virus S (PVS), was used to develop sequence‐characterized amplified region (SCAR) markers. After cloning and sequencing of OPG17₄₅₀ new polymerase chain reaction (PCR) primers were designed to generate dominant (SCG17₃₂₁) and codominant (SCG17₄₄₈) markers. For SCG17₄₄₈, polymorphism between susceptible and resistant genotypes was recovered after digestion of the marker with the restriction enzyme Muni. In addition to the band corresponding to ‘susceptible’ allele that does not contain the Muni cleavage site, two bands of approximately 251 bp and 197 bp were observed in the resistant genotypes. The usefulness of these SCAR markers was verified in diploid potatoes possessing the Ns locus from clone G‐LKS 678147/60, and in tetraploid potatoes derived from G‐LKS 678147/60 and from clone MPI 65118/3.     

Inheritance and mapping of resistance against Cowpea mild mottle virus strain D1 in soybean

Cowpea mild mottle virus (CPMMV) is an emerging severe disease of soybean. The resistant genotypes, DS 12‐5 and SL958, were crossed with susceptible genotypes F4C7‐32 and JS335, respectively. Resistance reactions of sap‐inoculated F₂ plants and individual F₂ plant‐derived F₃ families indicated that resistance was controlled by a single dominant gene. Molecular mapping with bulked segregant analysis showed that Satt635 and UO8405 are linked to resistance gene which is located on linkage group H.     

Wheat streak mosaic virus resistance in eight wheat germplasm lines

Wheat streak mosaic virus (WSMV) is a destructive pathogen in wheat (Triticum aestivum L.). Host resistance is the most effective way to control this virus. To date, Wsm2 is the only wheat resistance gene that is genetically mapped. The objective of this study was to identify germplasm lines that might carry resistance genes different from Wsm2. Eight newly reported resistant germplasm lines were examined by allelic tests. To validate the allelic test results, five of them were further analysed for the inheritance of WSMV resistance. A Wsm2‐linked marker was also genotyped on populations developed for the inheritance study. Our results suggested that the WSMV resistance in lines CItr9358, PI225288, PI243652, PI245439, PI245526 and PI478095 was controlled by either Wsm2 or a gene very closely linked to Wsm2. The resistance in PI243753 and PI321730, however, is likely controlled by a gene different from, but linked to Wsm2. The resistance in PI321730 might also involve some minor genes. This study provided useful information for breeders to select appropriate resistant lines to improve WSMV resistance in wheat.     

Genetic mapping and inheritance of Russian wheat aphid resistance gene in accession IG 100695

The Russian wheat aphid (RWA), Diuraphis noxia (Kurdjumov), is an important pest of small‐grain cereals, particularly wheat, worldwide. The most efficient strategy against the RWA is to identify sources of resistance and to introduce them into susceptible wheat genotypes. This study was conducted to determine the mode of inheritance of the RWA resistance found in ICARDA accession IG 100695, to identify wheat microsatellite markers closely linked to the gene and to map the chromosomal location of the gene. Simple sequence repeat (SSR) marker scores were identified in a mapping population of 190 F₂ individuals and compared, while phenotypic screening for resistance was performed in F_2 : 3 families derived from a cross between ‘Basribey’ (susceptible) and IG 100695 (resistant). Phenotypic segregation of leaf chlorosis and rolling displayed the effect of a single dominant gene, temporarily denoted Dn100695, in IG 100695. Dn100695 was mapped on the short arm of chromosome 7D with four linked SSR markers, Xgwm44, Xcfd14, Xcfd46 and Xbarc126. Dn100695 and linked SSR markers may be useful for improving resistance for RWA in wheat breeding.     

Development of broad‐spectrum bacterial blight resistance into thermo‐sensitive genic male sterile lines

Two‐line hybrid rice technology is an effective way to increase rice production and improve rice quality. In this study, three bacterial blight (BB) resistance genes, Xa7, Xa21 and Xa23, were introgressed separately into C815S, a popular thermo‐sensitive genic male sterile (TGMS) line to develop five BB‐resistant lines (Hua1005S, Hua1002S, Hua1009S, Hua1006S and Hua1001S) to be resistant against seven races of Xanthomonas oryzae pv. oryzae (Xoo). The two‐line hybrids with heterozygous Xa23 were resistant against seven Xoo strains. But, the hybrids with heterozygous loci for both Xa7 and Xa21 were only resistant against three Xoo strains and were moderately susceptible to the other four strains indicating the role of modifiers influencing the poor expression of dominant BB resistance genes under heterozygous state. Among them, Hua1006S was found to be a promising TGMS line with its higher degree of disease resistance level on account of broad‐spectrum resistance gene Xa23 besides possessing better plant type and rice grain quality features.     

Exploring genetic diversity of rice cultivars for the presence of brown planthopper (BPH) resistance genes and development of SNP marker for Bph18

Brown planthopper (BPH) is the most devastating insect pest in rice‐growing areas. Information on availability of BPH resistance alleles and their sources enhances BPH‐resistant breeding programmes. In this study, 260 highly diversified rice cultivars or breeding lines were screened for the presence of five major BPH resistance genes (Bph10, Bph13, Bph18, Bph20 and Bph21) using gene‐specific markers. The analysis revealed that 137 of the 260 cultivars possess at least one BPH resistance gene. Bph10 was predominant while Bph20 was the least distributed. Moreover, two and three different resistance gene combinations were found in the cultivars. Molecular markers play an important role in molecular breeding programmes. A tightly linked PCR‐based co‐dominant Bph18 marker was developed, which is cost effective and time effective and simpler than available Bph18 CAPS marker (7312.T4A). We strongly believe that the identified BPH‐resistant cultivars can be used as alternative resistance gene sources and also as resource for novel BPH resistance genes. The developed Bph18 marker will be highly useful in molecular breeding applications of BPH‐resistant breeding programmes.     

Identification and characterization of seedling and adult plant resistance to Puccinia hordei in Chinese barley germplasm

Leaf rust of barley, caused by Puccinia hordei, occurs in all barley‐growing regions of Australia causing significant yield losses under epidemic conditions. The development and use of resistant cultivars are the most economical and environmentally sustainable method to control leaf rust which in turn relies on ongoing efforts to identify and characterize new sources of resistance. The aim of this study was to postulate known genes and/or identify new sources of resistance to P. hordei. Fifty‐two genotypes were assessed at the seedling and adult plant growth stages. On the basis of multipathotype tests, 39 genotypes lacked detectable seedling resistance, and nine were postulated to carry the genes Rph2, Rph4, Rph12 and Rph19 singly. Four genotypes carried uncharacterized seedling resistance; however, the gene(s) present in each were ineffective to at least one of the pathotypes used. Field tests at the adult plant growth stage revealed the presence of adult plant resistance (APR) in 12 genotypes. Tests of allelism and marker analysis indicated that resistance genes present in these genotypes were independent of the APR gene Rph20.     

Characterization of broad‐spectrum resistance to Soybean mosaic virus in soybean [Glycine max (L.) Merr.] cultivar ‘RN‐9’

Soybean mosaic virus (SMV) can cause serious yield losses in soybean. Soybean cultivar ‘RN‐9’ is resistant to 15 of 21 SMV strains. To well‐characterize this invaluable broad‐spectrum SMV‐resistance, populations (F₁, F₂ and F_2:3) derived from resistant (R) × susceptible (S) and R × R crosses were tested for SMV‐SC18 resistance. Genetic analysis revealed that SC18 resistance in ‘RN‐9’ plus two elite SMV‐resistant genotypes (‘Qihuang No.1’ and ‘Kefeng No.1’) are controlled by independently single dominant genes. Linkage analysis showed that the resistance of ‘RN‐9’ to SMV strains SC10, SC14, SC15 and SC18 is controlled by more than one gene(s). Moreover, Rsc10‐r and Rsc18‐r were both positioned between the two simple sequence repeats markers Satt286 and Satt277, while Rsc14‐r was fine‐mapped in 136.8‐kb genomic region containing sixteen genes, flanked by BARCSOYSSR_06_0786 and BARCSOYSSR_06_0790 at genetic distances of 3.79 and 4.14 cM, respectively. Allelic sequence comparison showed that Cytochrome P450‐encoding genes (Glyma.06g176000 and Glyma.06g176100) likely confer the resistance to SC14 in ‘RN‐9’. Our results would facilitate the breeding of broad‐spectrum and durable SMV resistance in soybeans.     

Identification of new sources of resistance to Striga gesnerioides in cowpea germplasm

There have been reports of breakdown of striga resistance in previously resistant cowpea cultivars in Burkina Faso. This could be attributed to new striga races emerging or to an increase in the aggressiveness of current striga races. Therefore, cowpea genotypes were evaluated in fields infested with S. gesnerioides at three striga hot spots in Burkina Faso and in pots under artificial infestation with striga races SR 1, SR 5 and SR Kp to identify new, adapted and striga‐resistant sources. Cowpea genotypes showed differential reactions for striga resistance over sites and for striga races in pot experiments, indicating differences in the races involved, and SR Kp was reported as a new race. Resistant sources conferring site‐specific or multiple striga‐race resistance were identified. Genotypes 58‐57, Sanga 2, IT84S‐2049, IT98K‐205‐8, IT93K‐693‐2, KVx771‐10, KVx775‐33‐2, KVx61‐1, Gorom local, Mouride and Melakh conferred resistance to all three striga races. These genotypes are potential donor parents for breeding new, adapted and striga‐resistant genotypes. Cowpea landraces including Moussa local and Niaogo local with farmers' preferred traits were susceptible and need improvement for striga resistance.     

Marker‐assisted selection for rice blast resistance genes Pi2 and Pi9 through high‐resolution melting of a gene‐targeted amplicon

The use of host resistance (R) genes is considered the most cost‐effective option to control the rice blast disease. The two allelic R genes Pi2 and Pi9 confer very broad‐spectrum resistance against blast isolates collected worldwide. However, the two genes have not yet been widely deployed in rice breeding programmes. Availability of specific markers for them would facilitate incorporating the two R genes into new rice lines through marker‐assisted selection. Herein, we report the development and utilization of a robust and specific marker for the Pi2 and Pi9. This marker was derived from polymorphisms within the target gene, and achieved simultaneously distinguish Pi2 and Pi9 from other alleles through high‐resolution melting of a small amplicon. With the marker, we were able to transfer the Pi2 into an elite restorer line through marker‐assisted backcrossing, successfully obtained effective resistance to blast disease, and we were also able to, respectively, incorporate the Pi2 and Pi9 with two other R genes. As the additive effect, blast resistance in these stacking lines harbouring three R genes were significantly improved.     

ISSR and SCAR markers linked to the mungbean yellow mosaic virus (MYMV) resistance gene in blackgram [Vigna mungo (L.) Hepper]

A recombinant inbred line (RIL) mapping population (F₈) was generated by crossing Vigna mungo (cv. TU 94‐2) with Vigna mungo var. silvestris and screened for mungbean yellow mosaic virus (MYMV) resistance. The inter simple sequence repeat (ISSR) marker technique was employed to identify markers linked to the MYMV resistance gene. Of the 100 primers screened, 54 showed amplification of which 36 exhibited polymorphism between the parents TU 94‐2 (resistant) and V. mungo var. silvestris (susceptible). Individual plants from 53 RIL populations were analysed and one marker (ISSR811₁₃₅₇) was identified as tightly linked to the MYMV resistant gene at 6.8 cM. Both the phenotype as well as the ISSR811₁₃₅₇ marker segregated in a 1 : 1 ratio. The ISSR811₁₃₅₇ marker was sequenced and sequence characterized amplified region (SCAR) primers were designed (YMV1‐F and YMV1‐R) to amplify the marker. Screening for the SCAR marker in the RIL population distinguished the MYMV resistant and susceptible plants, agreeing well with the phenotypic data. The ISSR811₁₃₅₇ marker was validated using diverse blackgram genotypes differing in their MYMV reaction. The marker will be useful for the development of MYMV‐resistant genotypes in blackgram.     

‘CPAN 1842’: a new source of adult plant leaf rust resistance in bread wheat

There is worldwide interest in adult plant resistance (APR) because of greater durability of APR to the cereal rusts. Peruvian bread wheat genotype ‘CPAN (Coordinated Project Accession Number) 1842’ (LM 50–53) has shown leaf rust resistance in disease screening nurseries since its introduction in 1977. However, it is susceptible at the seedling stage to several Puccinia triticina (Pt) pathotypes including the widely prevalent 77‐5 (121R63‐1) that infects bread wheat. Inheritance studies showed that CPAN 1842 carried a dominant gene for APR to pathotype 77‐5, which was different from Lr12, Lr13, Lr22a, Lr34, Lr35, Lr37, Lr46, Lr48, Lr49 and Lr68, based on the tests of allelism; and from Lr67, based on genotyping with the closely linked SSR marker cfd71. This gene should also be different from Lr22b as the latter is totally ineffective against pathotype 77‐5. CPAN 1842 therefore appears to be a new promising source of leaf rust resistance. Also having resistance to stem rust and stripe rust, this line can contribute to breeding for multiple rust resistances in wheat.     

Evaluation of tomato entries with different combinations of resistance genes to tomato yellow leaf curl disease in Tunisia

Tomato yellow leaf curl virus (TYLCV) is one of the most widespread begomoviruses transmitted by the whitefly Bemisia tabaci that cause tomato yellow leaf curl virus diseases (TYLCDs). TYLCD losses can be especially severe in open‐field tomato (Solanum lycopersicum L.) crops. TYLCV is particularly well known and widespread in the Mediterranean (Med) countries where TYLCD control is mostly based on insecticidal control of B. tabaci populations. Unfortunately, Tunisian B. tabaci populations include Middle East‐Asia Minor I and the Med species that have developed resistance to many classes of insecticides. Therefore, TYLCD‐resistant cultivars are essential for sustainable disease management. Six TYLCD resistance genes (Ty) have been introgressed from wild species into cultivated tomato and are available for breeding. Information on the Ty genes or gene combinations is useful for breeding resistant cultivars. To this end, 14 tomato lines carrying different Ty gene combinations and two susceptible tomato entries were evaluated for TYLCD incidence and severity in two field trials during late season in Tunisia. Entries with Ty‐1/Ty‐3 + Ty‐2 offered the highest levels of resistance in Tunisia.     

Variable resistance of bread wheat (Triticum aestivum) lines carrying 2NS/2AS translocation to wheat blast

Wheat blast disease, caused by Magnaporthe oryzae (anamorph Pyricularia oryzae), produces severe damage to wheat production in South America. It was observed that many resistant cultivars contain the 2NS/2AS translocation from Triticum ventricosum. In this study, we evaluate the presence of the 2NS/2AS translocation in 57 advanced breeding lines and one variety ‘Caninde 1’ from Paraguayan wheat germplasm, using VENTRIUP‐LN2 primers. The germplasm ‘Caninde 1 and 22’ of the breeding lines, found positive for the presence of 2NS/2AS translocation, were inoculated with a single aggressive Magnaporthe pathotype P14‐039, to assess their response to wheat blast infection under controlled conditions. Based on the disease infection score, ten of the breeding lines, ‘Caninde 1’ and ‘Milan’ (positive control), were classified as resistant. Three of the remaining breeding lines were classified as moderately resistant, five as moderately susceptible and other four as susceptible. Our results show that the expression of 2NS/2AS‐based blast resistance is more dependent on genetic background of the inserted germplasm than previously envisioned.