QTL analysis for thousand-grain weight under terminal drought stress in bread wheat (<Emphasis Type="Italic">Triticum</Emphasis><Emphasis Type="Italic">aestivum</Emphasis> L.)

Grain yield under post-anthesis drought stress is one of the most complex traits, which is inherited quantitatively. The present study was conducted to identify genes determining post-anthesis drought stress tolerance in bread wheat through Quantitative Trait Loci (QTLs) analysis. Two cultivated bread wheat accessions were selected as parental lines. Population phenotyping was carried out on 133 F_2:3 families. Two field experiments and two experiments in the greenhouse were conducted at IPK-Gatersleben, Germany with control and post-anthesis stress conditions in each experiment. Thousand-grain weight was recorded as the main wheat yield component, which is reduced by post-anthesis drought stress. Chemical desiccation was applied in three experiments as simulator of post-anthesis drought stress whereas water stress was applied in one greenhouse experiment. Analysis of variance showed significant differences among the F_2:3 families. The molecular genetic linkage map including 293 marker loci associated to 19 wheat chromosomes was applied for QTL analysis. The present study revealed four and six QTLs for thousand-grain weight under control and stress conditions, respectively. Only one QTL on chromosome 4BL was common for both conditions. Five QTLs on chromosomes 1AL, 4AL, 7AS, and 7DS were found to be specific to the stress condition. Both parents contributed alleles for drought tolerance. Taking the known reciprocal translocation of chromosomes 4AL/7BS into account, the importance of the short arms of homoeologous group 7 is confirmed for drought stress.     

Identification of common QTL for resistance to <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis> in three doubled haploid populations of <Emphasis Type="Italic">Brassica napus</Emphasis> (L.)

Sclerotinia stem rot (SR) is one of the most devastating diseases of canola/rapeseed. Quantitative trait loci (QTL) analyses were carried out to identify loci responsible for resistance to SR in three doubled haploid DH populations (H1, H2 and H3). Petiole inoculation technique PIT was used to evaluate the all populations for resistance to SR. Genetic maps were developed using sequence related amplified polymorphism SRAP and simple sequence repeat SSR markers. Genetic maps of the H1 and H2 populations were developed using 508 and 478 markers, respectively. Previously published genetic map of the H3 population was also used in this study. The QTL analysis was carried out for each replicate separately as well as on the average of all the replicates. The numbers of identified QTL in each analysis varied from four to six in the H1 population, three to six in the H2 population and two to six in the H3 population. A number of common QTL were identified between the replicates of each population. Two common QTL were identified on linkage group A7 and C6 between the H1 and H3 populations and one QTL on A9 between the H2 and H3 populations. We are the first to report, identification of common QTL between different populations of Brassica napus.     

Genome-wide SNP-based association mapping of resistance to <Emphasis Type="Italic">Phytophthora sojae</Emphasis> in soybean (<Emphasis Type="Italic">Glycine max</Emphasis> (L.) Merr.)

Phytophthora root rot (PRR) is among the most important soybean (Glycine max (L.) Merr.) diseases worldwide, and the host displays complex genetic resistance. A genome-wide association study was performed on 337 accessions from the Yangtze-Huai soybean breeding germplasm to identify resistance regions associated with PRR resistance using 60,862 high-quality single nucleotide polymorphisms markers. Twenty-six significant SNP-trait associations were detected on chromosomes 01 using a mixed linear model with the Q matrix and K matrix as covariates. In addition, twenty-six SNPs belonged to three adjacent haplotype blocks according to a linkage disequilibrium blocks analysis, and no previous studies have reported resistance loci in this 441 kb region. The real-time RT-PCR analysis of the possible candidate genes showed that two genes (Glyma01g32800 and Glyma01g32855) are likely involved in PRR resistance. Markers associated with resistance can contribute to marker-assisted selection in breeding programs. Analyses of candidate genes can lay a foundation for exploring the mechanism of P. sojae resistance.     

Mapping of quantitative trait loci controlling powdery mildew resistance in Mungbean (<Emphasis Type="Italic">Vigna radiata</Emphasis> (L.) Wilczek)

Powdery mildew disease in mungbean is caused by the fungus, Erysiphe polygoni D.C. We identified two quantitative trait loci (QTLs) controlling resistance to the disease in a RIL population of 190 F₇ lines. The population was developed from the cross between a susceptible cultivar, “Kamphaeng Saen 1” and a resistant line, “VC6468-11-1A”. Reaction to the disease was evaluated for resistance in field and greenhouse conditions. Results from analysis of variance revealed that 15 SSR loci on three linkage groups (LG) associated with the resistance. Composite interval mapping consistently identified two QTLs on two LGs, qPMR-1 and qPMR-2, conferring the resistance. qPMR-1 and qPMR-2 accounted for 20.10 and 57.81% of the total variation for plant response to the disease, respectively. Comparison based on common markers used in our and previous studies suggested that qPMR-2 is possibly the same as the major QTL reported earlier using another resistant source. The SSR markers flanking and closely linked to qPMR-1 (CEDG282 and CEDG191) and qPMR-2 (MB-SSR238 and CEDG166) are useful for marker-assisted selection for mungbean resistance to powdery mildew.     

Epistasis and heritability of resistance to <Emphasis Type="Italic">Phytophthora nicotianae</Emphasis> in pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.)

Gene effects of resistance to two isolates of Phytophthora nicotianae in two crosses of pepper were investigated using separate generation means analysis. Additive-dominance models were inadequate in all cases. Digenic parameter models were adequate in three cases and the probability of goodness of fit of models was negatively correlated with the aggressiveness of the pathogen. None of these models explained variation among generation means in the combined cross Beldi × CM334 with P. nicotianae isolate Pn_2. Additive × additive, dominance × dominance and dominance × additive effects were significant in most cases. Additive and dominance effects (of negative sign) contribute more to resistance than to susceptibility. Additive variance was greater than environmental and dominance variance and ranged from 0.038 to 0.224. Narrow-sense heritabilities were dependent upon the cross and inoculate and ranged from 86 to 92%. The results of this study indicate that selection with more aggressive isolates of the pathogen will be useful for enhancing resistance in pepper.     

Identification of quantitative trait loci involved in resistance to <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">syringae</Emphasis> in pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.)

Pseudomonas syringae is the main pathogen responsible for bacterial blight disease in pea and can cause yield losses of 70%. P. syringae pv. pisi is prevalent in most countries but the importance of P. syringae pv. syringae (Psy) is increasing. Several sources of resistance to Psy have been identified but genetics of the resistance is unknown. In this study the inheritance of resistance to Psy was studied in the pea recombinant inbred line population P665 × ‘Messire’. Results suggest a polygenic control of the resistance and two quantitative trait loci (QTL) associated with resistance, Psy1 and Psy2, were identified. The QTL explained individually 22.2 and 8.6% of the phenotypic variation, respectively. In addition 21 SSR markers were included in the P665 × ‘Messire’ map, of which six had not been mapped on the pea genome in previous studies.     

Characterisation of resistance to crenate broomrape (<Emphasis Type="Italic">Orobanche crenata</Emphasis> Forsk.) in <Emphasis Type="Italic">Lathyrus cicera</Emphasis> L.

Field, pots and mini-rhizotrons experiments revealed the existence of useful levels of avoidance and resistance to Orobanche crenata Forsk. within Lathyrus cicera L. accessions, in addition to escape due to precocity or to reduced root biomass. This resistance was characterized by several phenotypic mechanisms of defence which acted alone or in combination, including low stimulation of parasitic seed germination, prevention of the radicle penetration inside the root and prevention of a correct connection by the parasite with the host vascular tissue being visible by reduced development of established parasitic tubercles or abortion of the parasitic tubercles formed.     

<Emphasis Type="Italic">Agrobacterium</Emphasis> mediated transformation of indica rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) for insect resistance

The rice leaffolder (RLF), Cnaphalocrocis medinalis is an important pest of rice that causes severe damage in many areas of the world. The plants were transformed with fully modified (plant codon optimized) synthetic Cry1C coding sequences as well as with the hpt and gus genes, coding for hygromycin phosphotransferase and β-glucuronidase, respectively. Cry1C sequences placed under the control of doubled 35S promoter plus the AMV leader sequence, and hpt and gus genes driven by cauliflower mosaic virus 35S promoter, were used in this study. Embryogenic calli after cocultivation with Agrobacterium were selected on the medium containing hygromycin B. A total of 67 hygromycin-resistant plants were regenerated. PCR and Southern blot analyses of primary transformants revealed the stable integration of Cry1C coding sequences into the rice genome with predominant single copy integration. R₁ progeny plants disclosed a monogenic pattern (3:1) of transgene segregation as confirmed by molecular analyses. These transgenic lines were highly resistant to rice leaffolder (RLF), Cnaphalocrocis medinalis as revealed by insect bioassay.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation of pigeon pea [<Emphasis Type="Italic">Cajanus cajan</Emphasis> (L.) Millsp.] for resistance to legume pod borer <Emphasis Type="Italic">Helicoverpa armigera</Emphasis>

Agrobacterium-mediated genetic transformation was performed using embryonic axes explants of pigeon pea. Both legume pod borer resistant gene (cry1Ac) and plant selectable marker neomycine phosphor transferase (nptII) genes under the constitutive expression of the cauliflower mosaic virus 35S promoter (CaMV35S) assembled in pPZP211 binary vector were used for the experiments. An optimum average of 44.61% successfully hardened dot blot Southern hybridization positive plants were obtained on co-cultivation media supplemented with 200 μM acetosyringone without L-cysteine. The increased transformation efficiency from a baseline of 11.53% without acetosyringone to 44.61% with acetosyringone was further declined with the addition of different concentrations of L-cysteine to co-cultivation media. Transgenic shoots were selected on 50 and 75 mg L⁻¹ kanamycin. Rooting efficiency was 100% on half-strength Murashige and Skoog medium with 20 g L⁻¹ sucrose and 0.5 mg L⁻¹ indole butyric acid in the absence of kanamycin. Furthermore, 100% seed setting was found among all the transgenic events. The plants obtained were subjected to multi- and nochoice tests to determine the behavioral responses and mortality through Helicoverpa armigera bioassays on the leaf and relate their relationship with the expression of cry1Ac protein which was found to be less in leaf as compared to the floral buds, anther, pod, and seed.     

Identification of DNA markers linked to an induced mutated gene conferring resistance to powdery mildew in pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.)

We have recently induced two powdery mildew (Erysiphe pisi Syd) resistant mutants in Pisum sativum L. via ethylnitrosourea (ENU) mutagenesis. Both mutations (er1mut1 and er1mut2) affected the same locus er1 that determines most of the identified natural sources of powdery mildew resistance (PMR) in this crop. The mutated gene er1mut2 was mapped to a linkage group of 16 DNA markers combining three main strategies: near isogenic lines (NILs) analysis, bulked segregant analysis and genetic mapping of randomly identified polymorphic markers, together with three DNA-markers techniques: ISSR, RAPDs and AFLPs. Markers located closer to the PMR locus, OPO06_1100y (0.5 cM), OPT06₄₈₀ (3.3 cM) and AGG/CAA₁₂₅ (5.5 cM), were cloned and converted into SCAR markers. Markers AH1R₈₅₀ and AHR_920y were found to be allelic and converted into the co-dominant marker ScAH1 (16.3 cM). Two previously known DNA markers, ScOPE16₁₆₀₀ and A5_420y, were mapped at 9.6 and 23.0 cM from the PMR locus, respectively. The novel markers identified in this study are currently being transferred to a new F2 mapping population derived from a cross between the induced PMR mutant line F(er1mut2) and a more genetically distant susceptible line of Pisum sativum var. arvense.     

Genetic analysis of resistance to flower bud thrips (<Emphasis Type="Italic">Megalurothrips sjostedti</Emphasis>) in cowpea (<Emphasis Type="Italic">Vigna unguiculata</Emphasis> [L.] Walp.)

Cowpea is an important legume in sub-Saharan Africa where its protein rich grains are consumed. Insect pests constitute a major constraint to cowpea production. Flower bud thrips (FTh) is the first major pest of cowpea at the reproductive stage and if not controlled with insecticides is capable of reducing grain yield significantly. Information on the inheritance of resistance to FTh is required to facilitate breeding of resistant cultivars. The genetics of resistance was studied in crosses of four cowpea lines. Maternal effect was implicated while frequency distributions of the F₂ and backcross generations suggest quantitative inheritance. Additive, dominance and epistatic gene effects made large contributions and since improved inbred lines are the desired product, selection should not be too severe in the early generations to allow for desirable gene recombination. This study suggested that some of the genes involved in the control of resistance to FTh are different in TVu1509 and Sanzi. Broad sense heritability ranged from 56% to 73%. Choice of maternal parent in a cross will be critical to the success of resistance breeding.     

Successful induction of trigenomic hexaploid <Emphasis Type="Italic">Brassica</Emphasis> from a triploid hybrid of <Emphasis Type="Italic">B.</Emphasis><Emphasis Type="Italic">napus</Emphasis> L. and <Emphasis Type="Italic">B. nigra</Emphasis> (L.) Koch

A triploid hybrid with an ABC genome constitution, produced from an interspecific cross between Brassica napus (AACC genome) and B. nigra (BB genome), was used as source material for chromosome doubling. Two approaches were undertaken for the production of hexaploids: firstly, by self-pollination and open-pollination of the triploid hybrid; and secondly, by application of colchicine to axillary meristems of triploid plants. Sixteen seeds were harvested from triploid plants and two seedlings were confirmed to be hexaploids with 54 chromosomes. Pollen viability increased from 13% in triploids to a maximum of 49% in hexaploids. Petal length increased from 1.3 cm (triploid) to 1.9 cm and 1.8 cm in the two hexaploids and longest stamen length increased from 0.9 cm (triploid) to 1.1 cm in the hexaploids. Pollen grains were longer in hexaploids (43.7 and 46.3 μm) compared to the triploid (25.4 μm). A few aneuploid offsprings were also observed, with chromosome number ranging from 34 to 48. This study shows that trigenomic hexaploids can be produced in Brassica through interspecific hybridisation of B. napus and B. nigra followed by colchicine treatment.     

Genetic effects of introgression genomic components from Sea Island cotton (<Emphasis Type="Italic">Gossypium barbadense</Emphasis> L.) on fiber related traits in upland cotton (<Emphasis Type="Italic">G</Emphasis>. <Emphasis Type="Italic">hirsutum</Emphasis> L.)

The germplasm with exotic genomic components especially from Sea Island cotton (Gossypium barbadense L. Gb) is the dominant genetic resources to enhance fiber quality of upland cotton (G. hirsutum L., Gh). Due to low efficiency of phenotypic evaluation and selection on fiber quality, genetic dissection of favorable alleles using molecular markers is essential. Genetic dissection on putative Gb introgressions related to fiber traits were conducted by SSR markers with mapping populations derived from a cross between Luyuan343 (LY343), a superior fiber quality introgression line (IL) with genomic components from Gb, and an elite Upland cotton cv. Lumianyan#22 (LMY22). Among 82 polymorphic loci screened out from 4050 SSRs, 42 were identified as putative introgression alleles. A total of 29 fiber-related QTLs (23 for fiber quality and six for lint percentage) were detected and most of which clustered on the putative Gb introgression chromosomal segments of Chr.2, Chr.16, Chr.23 and Chr.25. As expected, a majority of favorable alleles of fiber quality QTLs (12/17, not considering the QTLs for fiber fineness) came from the IL parent and most of which (11/12) were conferred by the introgression genomic components while three of the six (3/6) favorable alleles for lint percentage came from the Gh parent. Validation of these QTLs using an F₈ breeding population from the same cross made previously indicated that 13 out of 29 QTLs showed considerable stability. The results suggest that fiber quality improvement using the introgression components could be facilitated by marker-assisted selection in cotton breeding program.     

Fine mapping of a <Emphasis Type="Italic">Xantha</Emphasis> mutation in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

The recessive mutation of the XANTHA gene (XNT) transforms seedlings and plants into a yellow color, visually distinguishable from normal (green) rice. Thus, it has been introduced into male sterile lines as a distinct marker for rapidly testing and efficiently increasing varietal purity in seed and paddy production of hybrid rice. To identify closely linked markers and eventually isolate the XNT gene, two mapping populations were developed by crossing the xantha mutant line Huangyu B (indica) with two wild type japonica varieties; a total of 1,720 mutant type F₂ individuals were analyzed for fine mapping using polymorphic InDel markers and high dense microsatellite markers. The XNT gene was mapped on chromosome 11, within in a fragment of ~100 kb, where 13 genes are annotated. The NP_001067671.1 gene within the delimited region is likely to be a candidate XNT gene, since it encodes ATP-dependent chloroplast protease ATP-binding subunit clp A. However, no sequence differences were observed between the mutant and its parent. Bioinformatics analysis demonstrated that four chlorophyll deficient mutations that were previously mapped on the same chromosome are located outside the XNT region, indicating XNT is a new gene. The results provide useful DNA markers not only for marker assisted selection of the xantha trait but also its eventual cloning.     

Increased resistance to <Emphasis Type="Italic">Ustilago zeae</Emphasis> and <Emphasis Type="Italic">Fusarium verticilliodes</Emphasis> in maize inbred lines bred for <Emphasis Type="Italic">Fusarium graminearum</Emphasis> resistance

Preliminary field observations in our maize breeding nurseries indicated that breeding for improved resistance to gibberella ear rot (Fusarium graminearum) in maize may indirectly select for resistance to another ear disease, common smut (Ustilago zeae). To investigate this, we compared the disease severity ratings obtained on 189 maize inbreds, eight of which included our inbreds developed with selection for gibberella ear rot resistance after field inoculation and breeding for 8–10 years. No correlation was found between disease severities for the 189 inbreds but the eight gibberella-resistant lines were consistently more resistant to smut. To further examine this relationship and to determine if these eight inbreds would be useful for developing inbreds with either common smut or fusarium ear rot (F. verticilliodes) resistance, we conducted a Griffing’s diallel analysis on six inbreds of maize, four with high levels of gibberella ear rot resistance representing all of the pedigree groups in our eight gibberella lines, and two with very low levels. Our most gibberella ear rot resistant inbreds, CO433 and CO441, had the lowest disease ratings for all three diseases, the consistently largest general combining ability effects and several significant specific combining ability effects. It was concluded that some inbreds bred specifically for gibberella ear rot would also be useful in breeding for resistance to common smut and fusarium ear rot.     

Search in <Emphasis Type="Italic">Solanum</Emphasis> (section <Emphasis Type="Italic">Lycopersicon</Emphasis>) germplasm for sources of broad-spectrum resistance to four <Emphasis Type="Italic">Tospovirus</Emphasis> species

The genus Tospovirus was considered as monotypic with Tomato spotted wilt virus (TSWV) being the only assigned species. However, extensive studies with worldwide isolates revealed that this genus comprises a number of species with distinct virulence profiles. The Neotropical South America is one center of Tospovirus diversity with many endemic species. Groundnut ringspot virus (GRSV), TSWV, Tomato chlorotic spot virus (TCSV), and Chrysanthemum stem necrosis virus (CSNV) are the predominant tomato-infecting species in Brazil. Sources of resistance were found in Solanum (section Lycopersicon) mainly against TSWV isolates from distinct continents, but there is an overall lack of information about resistance to other viral species. One-hundred and five Solanum (section Lycopersicon: Solanaceae) accessions were initially evaluated for their reaction against a GRSV isolate by analysis of symptom expression and systemic virus accumulation using DAS-ELISA. A subgroup comprising the most resistant accessions was re-evaluated in a second assay with TSWV, TCSV, and GRSV isolates and in a third assay with a CSNV isolate. Seven S. peruvianum accessions displayed a broad-spectrum resistance to all viral species with all plants being free of symptoms and systemic infection. Sources of resistance were also found in tomato cultivars with the Sw-5 gene and also in accessions of S. pimpinellifolium, S. chilense, S. arcanum, S. habrochaites, S. corneliomuelleri, and S. lycopersicum. The introgression/incorporation of these genetic factors into cultivated tomato varieties might allow the development of genetic materials with broad-spectrum resistance, as well as with improved levels of phenotypic expression.     

QTL mapping of agronomically important traits in sorghum (<Emphasis Type="Italic">Sorghum bicolor</Emphasis> L.)

Sorghum is one of the most important cereal crops; it is used to produce feed, sugar, and biofuel. To investigate genetic tradeoffs between grain and stem sugar production, we evaluated plant height, Brix (the percentage of soluble solids in stalk juice), 100-grain weight and flowering time over 3 years in a recombinant inbred line (RIL) population consisting of 189 individuals derived from a cross between the sweet sorghum cultivar ‘Rio’ and grain sorghum ‘BTx623’. We constructed a genetic linkage map (total length, 1418.71 cM; average distance between markers, 11.26 cM), which consisted of 118 simple sequence repeat (SSR) and 8 insertion-deletion (INDEL) markers. A total of 14 QTLs were detected on chromosomes 1, 3, 6, 7, and 9, which included 6 QTLs for plant height; 4 for Brix; and 2 QTLs for each 100-grain weight and flowering time. Eight QTLs were detected at least in 2 years. These results will be useful for future QTL fine mapping and gene mining for these traits, and useful for the improvement of sorghum through molecular marker-assisted selection.     

RDA derived <Emphasis Type="Italic">Oryza minuta</Emphasis>-specific clones to probe genomic conservation across <Emphasis Type="Italic">Oryza</Emphasis> and introgression into rice (<Emphasis Type="Italic">O. sativa</Emphasis> L.)

Molecular markers have been successfully used in rice breeding however available markers based on Oryza sativa sequences are not efficient to monitor alien introgression from distant genomes of Oryza. We developed O. minuta (2n = 48, BBCC)-specific clones comprising of 105 clones (266–715 bp) from the initial library composed of 1,920 clones against O. sativa by representational difference analysis (RDA), a subtractive cloning method and validated through Southern blot hybridization. Chromosomal location of O. minuta-specific clones was identified by hybridization with the genomic DNA of eight monosomic alien additional lines (MAALs). The 37 clones were located either on chromosomes 6, 7, or 12. Different hybridization patterns between O. minuta-specific clones and wild species such as O. punctata, O. officinalis, O. rhizomatis, O. australiensis, and O. ridleyi were observed indicating conservation of the O. minuta fragments across Oryza spp. A highly repetitive clone, OmSC45 hybridized with O. minuta and O. australiensis (EE), and was found in 6,500 and 9,000 copies, respectively, suggesting an independent and exponential amplification of the fragment in both species during the evolution of Oryza. Hybridization of 105 O. minuta specific clones with BB- and CC-genome wild Oryza species resulted in the identification of 4 BB-genome-specific and 14 CC-genome-specific clones. OmSC45 was identified as a fragment of RIRE1, an LTR-retrotransposon. Furthermore this clone was introgressed from O. minuta into the advanced breeding lines of O. sativa.     

Molecular mapping of <Emphasis Type="Italic">Pc68</Emphasis>, a crown rust resistance gene in <Emphasis Type="Italic">Avena</Emphasis><Emphasis Type="Italic">sativa</Emphasis>

Crown rust, which is caused by Puccinia coronata f. sp. avenae, P. Syd. & Syd., is the most destructive disease of cultivated oats (Avena sativa L.) throughout the world. Resistance to the disease that is based on a single gene is often short-lived because of the extremely great genetic diversity of P. coronata, which suggests that there is a need to develop oat cultivars with several resistance genes. This study aimed to identify amplified fragment length polymorphism AFLP markers that are linked to the major resistance gene, Pc68, and to amplify the F₆ genetic map from Pc68/5*Starter × UFRGS8. Seventy-eight markers with normal segregation were discovered and distributed in 12 linkage groups. The map covered 409.4 cM of the Avena sativa genome. Two AFLP markers were linked in repulsion to Pc68: U8PM22 and U8PM25, which flank the gene at 18.60 and 18.83 centiMorgans (cM), respectively. The marker U8PM25 is located in the linkage group 4_12 in the Kanota × Ogle reference oat population. These markers should be useful for transferring Pc68 to genotypes with good agronomic characteristics and for pyramiding crown rust resistance genes.