Identification and marker‐assisted introgression of QTL conferring resistance to bacterial leaf blight in cowpea (Vigna unguiculata (L.) Walp.)

Bacterial leaf blight (BLB), caused by Xanthomonas axonopodis pv. vignicola (Xav), is widespread in major cowpea [Vigna unguiculata (L.) Walp.] growing regions of the world. Considering the resource poor nature of cowpea farmers, development and introduction of cultivars resistant to the disease is the best option. Identification of DNA markers and marker‐assisted selection will increase precision of breeding for resistance to diseases like bacterial leaf blight. Hence, an attempt was made to detect QTL for resistance to BLB using 194 F_2 : 3 progeny derived from the cross ‘C‐152’ (susceptible parent) × ‘V‐16’ (resistant parent). These progeny were screened for resistance to bacterial blight by the leaf inoculation method. Platykurtic distribution of per cent disease index scores indicated quantitative inheritance of resistance to bacterial leaf blight. A genetic map with 96 markers (79 SSR and 17 CISP) constructed from the 194 F₂ individuals was used to perform QTL analysis. Out of three major QTL identified, one was on LG 8 (qtlblb‐1) and two on LG 11 (qtlblb‐2 and qtlblb‐3). The PCR product generated by the primer VuMt337 encoded for RIN2‐like mRNA that positively regulate RPM1‐ and RPS2‐dependent hypersensitive response. The QTL qtlblb‐1 explained 30.58% phenotypic variation followed by qtlblb‐2 and qtlblb‐3 with 10.77% and 10.63%, respectively. The major QTL region on LG 8 was introgressed from cultivar V‐16 into the bacterial leaf blight susceptible variety C‐152 through marker‐assisted backcrossing (MABC).     

Development of a kompetitive allele‐specific PCR marker for selection of the mutated Wx‐D1d allele in wheat breeding

Waxy (Wx) protein is a key enzyme for synthesis of amylose in endosperm. Amylose content in wheat grain influences the quality of end‐use products. Seven alleles have been described at the Wx‐D1 locus, but only two of them (Wx‐D1b, Wx‐D1e) were genotyped with codominant markers. The waxy wheat line K107Wx1 developed by treating ‘Kanto 107’ seeds with ethyl methanesulphonate carries the Wx‐D1d allele. However, no molecular basis supports this nomenclature. In the present study, DNA sequence analysis confirmed that a single nucleotide polymorphism in the sixth exon of Wx‐D1 changed tryptophan at position 301 into a termination codon. Based on this sequence variation, a PCR‐based KASP marker was developed to detect this point mutation using 68 BC₈F₁ plants and 297 BC₈F₂ lines derived from the cross ‘Ningmai 14’*9/K107Wx1. Combined with codominant markers for the Wx‐A1 and Wx‐B1 alleles, waxy and non‐waxy near‐isogenic lines were distinguished. The KASP marker was efficient in identifying the mutant allele and can be used to transfer waxiness to elite lines.     

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.     

Amplified fragment length polymorphism- and simple sequence repeat-based molecular tagging and mapping of greenbug resistance gene Gb3 in wheat

The greenbug, Schizaphis graminum (Rondani), is the most economically damaging aphid pest of wheat in the southern Great Plains of the USA. In this study, the single, dominant greenbug resistance gene, Gb3, was molecularly tagged and genetically mapped using amplified fragment length polymorphism (AFLP) and simple sequence repeat(SSR) markers. Three AFLP loci were associated with the Gb3 locus in linkage analysis with 75 F_2:3 families from the cross between two near‐isogenic lines (NILs) for Gb3,‘TXGBE273’ and ‘TXGBE281′. Two of these loci, XMgcc Pagg and Xmagg Patg cosegregate with Gb3 in the population analysed. Further analysis indicated that XMgcc Pagg and Xmagg Patg are specific for the Gb3 locus in diverse genetic backgrounds. Two SSR markers, Xgwm111 and Xgwm428 previously mapped in wheat chromosome 7D, were shown to be linked with Gb3, 22.5 cM and 33.1 cM from Gb3, respectively, in an F₂ population of ‘Largo’בTAM 107’, suggesting that Gb3 is located in the long arm of chromosome 7D. The two AFLP markers cosegregating with Gb3 are valuable tools in developing molecular markers for marker‐assisted selection of greenbug resistance in wheat breeding.     

Inheritance of resistance to the peach root‐knot nematode (Meloidogyne floridensis) in interspecific crosses between peach (Prunus persica) and its wild relative (Prunus kansuensis)

The peach root‐knot nematode, Meloidogyne floridensis (MF), infects majority of available nematode‐resistant peach rootstocks which are mostly derived from peach (Prunus persica) and Chinese wild peach (P. davidiana). Interspecific hybridization of peach with its wild relative, Kansu peach (P. kansuensis), offers potential for broadening the resistance spectrum in standard peach rootstocks. We investigated the inheritance of resistance to MF in segregating populations of peach (‘Okinawa’ or ‘Flordaguard’) × P. kansuensis. A total of 379 individuals from 13 F₂ and BC₁F₁ families were challenged with a pathogenic MF isolate “MFGnv14” and were classified as resistant (R) or susceptible (S) based on root galling intensity. Segregation analyses in F₂ progeny revealed the involvement of a major locus with a dominant or recessive allele determining resistance in progeny segregating 3R:1S and 1R:3S, respectively. Testcrosses with a homozygous‐susceptible peach genotype (‘Flordaguard’ or ‘UFSharp’) confirmed P. kansuensis as a source of new resistance and the heterozygous allelic status of P. kansuensis at the locus conferring resistance to MF. We propose a single‐locus dominant/recessive model for the inheritance of resistance.     

Genetic markers linked to Striga gesnerioides resistance for the improvement of Ghanaian cowpea (Vigna unguiculata) cultivars

The parasitic plant, Striga gesnerioides (Willd.) Vatke, is one of the most important constraints of cowpea production and food security in West Africa. Currently, few Striga resistant cowpea varieties have been developed that are well‐adapted to the dry savannah regions of Ghana. While genes conferring resistance to Striga races SG1, SG3 and SG5 have been mapped, the genetic locus of resistance to the race of Striga found in Ghana (SG‐GH) has not been characterized. Here, we report identification of genetic markers linked to SG‐GH resistance and define the relationship of this locus to SG3 resistance in recombinant inbred line populations generated from crosses between Striga resistant IT97K‐499‐35 and the Striga susceptible varieties Apagbaala and SARC‐LO2. The populations were genotyped with five genetic markers associated with SG3 and SG5 Striga resistance genes and a genetic map was developed. Genes conferring resistance to SG‐GH and SG3 mapped 4.2 cM from each other on chromosome Vu11. The identification of genetic markers linked to SG‐GH resistance will facilitate the marker‐assisted development of high‐quality Striga resistant cowpea varieties in Ghana.     

Breeding cowpea for resistance to Striga gesnerioides in the Nigerian dry savannas using marker‐assisted selection

Historically, conventional breeding has been the primary strategy used to develop a number of Striga‐resistant varieties currently grown in the Sahel of Western Africa. In this study, we have successfully developed and applied a marker‐assisted selection strategy that employs a single backcross programme to introgress Striga resistance into farmer preferred varieties of cowpea for the Nigeria savannas. In this strategy, we have introduced the Striga resistance gene from the donor parent IT97K‐499‐35 into an elite farmer preferred cowpea cultivar ‘Borno Brown’. The selected 47 BC₁F₂ populations confirmed the recombinants with desirable progeny having Striga resistance gene(s). The 28 lines selected in the BC₁F_2:4 generation with large seed size, brown seed coat colour and carrying marker alleles were evaluated in the field for resistance to Striga resistance. This led to the selection of a number of desirable improved lines that were immune to Striga having local genetic background with higher yield than those of their parents and standard varieties.     

Molecular mapping of greenbug (Schizaphis graminum) resistance gene Rsg1 in barley

The greenbug, Schizaphis graminum (Rondani) is an extremely damaging aphid pest of barley (Hordeum vulgare L.) particularly in the southern Great Plains of the USA. The simply inherited, dominant resistance gene Rsg1 is in all greenbug‐resistant US barley cultivars. In this study, we conducted molecular mapping of Rsg1 using an F_2:3 population derived from a cross between the greenbug‐resistant Post 90*4/R015 and susceptible CI2260 inbred lines. Segregation of host responses to greenbug biotype E infestation confirmed that a single dominant gene is responsible for greenbug resistance in Post 90*4/R015. Simple sequence repeat (SSR) markers evenly distributed along the seven barley chromosomes were employed for the construction of a framework genetic map. Linkage analysis placed the Rsg1 locus in the long arm of chromosome 3H (3HL) flanked by SSR markers Bmag0877 and GBM1420 that were 35 cM apart. Polymorphic single‐nucleotide polymorphism (SNP) markers in 3HL were identified from an Illumina GoldenGate SNP assay and used for targeted mapping to locate Rsg1 to an 8.4‐cM interval. Comparative analysis identified syntenic genomic regions in Brachypodium distachyon chromosome 2, in which 37 putative genes were annotated including a NB‐LRR‐type resistance gene homologue that may be a potential candidate gene for the Rsg1 locus of barley. Results from this study offer a starting point for fine mapping and cloning of this aphid resistance gene in barley.     

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

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

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.     

Effect of 3B, 5A and 3A QTL for Fusarium head blight resistance on agronomic and quality performance of Canadian winter wheat

The objectives of this study were to investigate (i) the correlations between Fusarium head blight (FHB) index, deoxynivalenol (DON) accumulation and percentage of Fusarium‐damaged kernels (FDK) with agronomic and quality traits and (ii) the effect associated with the presence of single QTLs for FHB resistance on agronomic and quality traits in winter wheat. The population was derived from the cross between ‘RCATL33' (FHB resistance derived from ‘Sumai 3’ and ‘Frontana’) and ‘RC Strategy’. Parental lines and recombinant inbred lines (RILs) were genotyped with SSR markers associated with the 3B, 5A and 3A QTLs. The population was planted in FHB‐inoculated nurseries and in agronomy trials. Lines in the 3B QTL class had the lowest FHB index, DON content and FDK level and did not have a significantly lower yield, thousand kernel weight or protein content compared with the lines grouped in other QTL classes (including no QTL class). Marker‐assisted selection of the 3B QTL for FHB resistance into high‐yielding FHB‐susceptible winter wheat is the recommended approach for the development of lines with increased FHB resistance without significant yield and quality penalties.     

Stripe rust resistance gene Yr18 and its suppressor gene in Chinese wheat landraces

The slow‐rusting and mildewing gene Yr18/Lr34/Pm38/Sr57 confers partial, durable resistance to multiple fungal pathogens and has its origins in China. A number of diagnostic markers were developed for this gene based on the gene sequence, but these markers do not always predict the presence of the resistant phenotype as some wheat varieties with the gene are susceptible to stripe rust in China. We hypothesized that these varieties have a suppressor of Yr18. This study was undertaken to determine the presence of Yr18, the suppressor and/or another resistance gene in 144 Chinese wheat landraces using molecular markers and stripe rust field data. Forty‐three landraces were predicted to have Yr18 based on the presence of the markers, but had final disease severities higher than 70%, indicating that this gene may be under the influence of a suppressor. Four of these landraces, ‘Sichuanyonggang 2’, ‘Baikemai’, ‘Youmai’ and ‘Zhangsihuang’, were chosen for genetic studies. Crosses were made between the lines and ‘Avocet S’, with further crosses of Sichuanyonggang 2 ×  ‘Huixianhong’ and Sichuanyonggang 2 ×  ‘Chinese Spring’. The F₁ plants of Sichuanyonggang 2/Chinese Spring was susceptible indicating the presence of a dominant suppressor gene. The results of genetic analyses of F_2:3 and BC₁F₂ families derived from these crosses indicated the presence of Yr18, a Yr18 suppressor and another additive resistance gene. The Yr18 region in Sichuanyonggang 2 was sequenced to ensure that it contained the functional allele. This is the first report of a suppressor of Yr18/Lr34/Pm38/Sr57 gene with respect to stripe rust response.     

Marker‐assisted introgression of lpa2 locus responsible for low‐phytic acid trait into an elite tropical maize inbred (Zea mays L.)

Maize is an important food and feed crop worldwide. Phytic acid (PA), in maize kernel, is an antinutritional factor. PA chelates mineral cations and causes mineral deficiency in humans and phosphorous deficiency in animals. The undigested PA excreted by monogastric animals causes phosphorous eutrophication. Therefore, development of low‐phytate maize is indispensable. The low‐phytate locus (lpa2 allele) has been transferred from low‐phytate mutant line ‘EC 659418’ into an elite inbred UMI 395 through marker‐assisted backcross breeding (MABB). The MABB involved three backcrosses followed by two selfing steps, including ‘foreground selection’, that is, selecting lines with lpa2 allele with the help of a codominant SSR marker ‘umc2230’ and ‘background selection’, that is, selecting plants having genetic background similar to that of the recurrent parent using 50 codominant SSR markers. Two low‐phytate lpa2 lines with genome similar (>90% similarity) to that of recurrent parent have been identified. These lines can be used as parent in future hybridization programmes for obtaining low‐phytate high‐yielding maize hybrids.     

Genome-wide detection of genetic loci associated with soybean aphid resistance in soybean germplasm PI 603712

Soybean aphid (Aphis glycines Matsumura) has become one of the major pests of soybean [Glycine max (L.) Merr.] in North America since 2000. At least four biotypes of soybean aphid have been confirmed in the United States. Genetic characterization of new sources of soybean aphid resistance will facilitate the expansion of soybean gene pool for soybean aphid resistance and thus will help to develop soybean aphid resistant cultivars. To characterize the genetic basis of soybean aphid resistance in PI 603712, a newly identified resistant germplasm line, 142 F₂ plants derived from the cross ‘Roberts’ × PI 603712 and their parents were evaluated for soybean aphid resistance in the greenhouse, and were genotyped with BARCSoySNP6K Illumina Infinium II BeadChip. A genome-wide molecular linkage map was constructed with 1495 polymorphic SNP markers. QTL analysis revealed that PI 603712 possessed two major loci associated with soybean aphid resistance, located on chromosome 7 and 16, respectively. The locus on chromosome 7 was dominantly expressed and positioned about one Mega-base-pair distant from the previously identified resistance locus Rag1. The locus on chromosome 16 was positioned near the previously identified resistance locus Rag3 and expressed partially dominance or additive effect. Interestingly, two minor loci were also detected on chromosomes 13 and 17 but the alleles from PI 603712 decreased the resistance. In developing soybean aphid resistant cultivars through marker-assisted selection, an appropriate combination of resistance loci should be selected when PI 603712 is used as a donor parent of resistance.     

Molecular mapping of black rot resistance locus Xca1bo on chromosome 3 in Indian cauliflower (Brassica oleracea var. botrytis L.)

Black rot is the most devastating disease of cauliflower worldwide causing severe damage to crop. The identification of markers linked to loci that control resistance can facilitate selection of plants for breeding programmes. In the present investigation, F₂ population derived from a cross between ‘Pusa Himjyoti’, a susceptible genotype, and ‘BR‐161’, a resistant genotype, was phenotyped by artificial inoculation using Xcc race 1. Segregation analysis of F₂ progeny indicated that a single dominant locus governed resistance to Xcc race 1 in ‘BR‐161’. Bulk segregant analysis in resistant and susceptible bulks of F₂ progeny revealed seven differentiating polymorphic markers (three RAPD, two ISSR and two SSR) of 102 markers screened. Subsequently, these markers were used to genotype the entire F₂ population, and a genetic linkage map covering 74.7 cM distance was developed. The major locus Xca1bo was mapped in 1.6‐cM interval flanked by the markers RAPD 04₈₃₃ and ISSR 11₆₃₅. The Xca1bo locus was located on chromosome 3. The linked markers will be useful for marker‐assisted resistance breeding in cauliflower.     

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 and validation of an over‐dominant QTL controlling soybean seed weight using populations derived from Glycine max × Glycine soja

Heterosis, or hybrid vigour, has been used to improve seed yield in several important crops for decades and it has potential applications in soybean. The discovery of over‐dominant quantitative trait loci (QTL) underlying yield‐related traits, such as seed weight, will facilitate hybrid soybean breeding via marker‐assisted selection. In this study, F₂ and F_2 : 3 populations derived from the crosses of ‘Jidou 12’ (Glycine max) × ‘ZYD2738’ (Glycine soja) and ‘Jidou 9’ (G. max) × ‘ZYD2738’ were used to identify over‐dominant QTL associated with seed weight. A total of seven QTL were identified. Among them, qSWT_13_1, mapped on chromosome 13 and linked with Satt114, showed an over‐dominant effect in two populations for two successive generations. This over‐dominant effect was further examined by six subpopulations derived from ‘Jidou12’ × ‘ZYD2738’. The seed weight for heterozygous individuals was 1.1‐ to 1.6‐fold higher than that of homozygous individuals among the six validation populations examined in different locations and years. Therefore, qSWT_13_1 may be a useful locus to improve the yield of hybrid soybean and to understand the molecular mechanism of heterosis in soybean.     

Molecular mapping of a new gene for resistance to frogeye leaf spot of soya bean in 'Peking'

Amplified fragment length polymorphism (AFLP) and microsatellite (simple sequence repeat, SSR) techniques were used to map the _RGSp_eking gene, which is resistant to most isolates of Cercospora sojina in the soya bean cultivar ‘Peking’. The mapping was conducted using a defined F₂ population derived from the cross of ‘Peking’(resistant) בLee’(susceptible). Of 64 EcoRI and MseI primer combinations, 30 produced polymorphisms between the two parents. The F₂ population, consisting of 116 individuals, was screened with the 30 AFLP primer pairs and three mapped SSR markers to detect markers possibly linked to Rcs_Peking. One AFLP marker amplified by primer pair E‐AAC/M‐CTA and one SSR marker Satt244 were identified to be linked to Res_Peking. The gene was located within a 2.1‐cM interval between markers AACCTA178 and Satt244, 1.1 cM from Satt244 and 1.0 cM from AACCTA178. Since the SSR markers Satt244 and Satt431 have been mapped to molecular linkage group (LG) J of soya bean, the Res_Peking resistance gene was putatively located on the LG J. This will provide soya bean breeders an opportunity to use these markers for marker‐assisted selection for frogeye leaf spot resistance in soya bean.     

Genetic linkage map of Chinese native variety faba bean (Vicia faba L.) based on simple sequence repeat markers

Simple sequence repeat (SSR) marker is a powerful tool for construction of genetic linkage map which can be applied for quantitative trait loci (QTL) and marker‐assisted selection (MAS). In this study, a genetic map of faba bean was constructed with SSR markers using a 129 F₂ individuals population derived from the cross of Chinese native variety 91825 (large seed) and K1563 (small seed). By screening 11 551 SSR primers between two parents, 149 primer pairs were detected polymorphic and used for F₂ population analysis. This SSR‐based genetic linkage map consisted of 15 linkage groups with 128 SSR. The map encompassed 1587 cM with an average genetic distance of 12.4 cM. The genetic map generated in this study will be beneficial for genetic studies of faba bean for identification of marker‐locus‐trait associations as well as comparative mapping among faba bean, pea and grasspea.