Use of synteny to identify candidate genes underlying QTL controlling stomatal traits in faba bean (Vicia faba L.)

Key message

We have identified QTLs for stomatal characteristics on chromosome II of faba bean by applying SNPs derived from M. truncatula , and have identified candidate genes within these QTLs using synteny between the two species.

Abstract

Faba bean (Vicia faba L.) is a valuable food and feed crop worldwide, but drought often limits its production, and its genome is large and poorly mapped. No information is available on the effects of genomic regions and genes on drought adaptation characters such as stomatal characteristics in this species, but the synteny between the sequenced model legume, Medicago truncatula, and faba bean can be used to identify candidate genes. A mapping population of 211 F₅ recombinant inbred lines (Mélodie/2 × ILB 938/2) were phenotyped to identify quantitative trait loci (QTL) affecting stomatal morphology and function, along with seed weight, under well-watered conditions in a climate-controlled glasshouse in 2013 and 2014. Canopy temperature (CT) was evaluated in 2013 under water-deficit (CTd). In total, 188 polymorphic single nucleotide polymorphisms (SNPs), developed from M. truncatula genome data, were assigned to nine linkage groups that covered ~928 cM of the faba bean genome with an average inter-marker distance of 5.8 cM. 15 putative QTLs were detected, of which eight (affecting stomatal density, length and conductance and CT) co-located on chromosome II, in the vicinity of a possible candidate gene—a receptor-like protein kinase found in the syntenic interval of M. truncatula chromosome IV. A ribose-phosphate pyrophosphokinase from M. truncatula chromosome V, postulated as a possible candidate gene for the QTL for CTd, was found some distance away in the same chromosome. These results demonstrate that genomic information from M. truncatula can successfully be translated to the faba bean genome.     

Fine genetic mapping of a locus controlling short internode length in melon (Cucumis melo L.)

Compact and dwarfing vining habits in melon (Cucumis melo L.; 2n = 2x = 24) may have commercial importance since they can contribute to the promotion of concentrated fruit set and can be planted in higher plant densities than standard vining types. A study was designed to determine the genetics of dwarfism associated with a diminutive (short internodes) melon mutant line PNU-D1 (C. melo ssp. cantalupensis). PNU-D1 was crossed with inbred wild-type melon line PNU-WT1 (C. melo ssp. agrestis), and resultant F₁ progeny were then self-pollinated to produce an F₂ population that segregated as dwarf and vining plant types. Primary stem length of F₂ progeny assessed under greenhouse conditions indicated that a single recessive gene, designated mdw1, controlled dwarfism in this population. To identify the chromosomal location associated with mdw1, an simple sequence repeat (SSR)-based genetic linkage map was constructed using 94 F₂ progeny. Using 76 SSR markers positioned on 15 linkage groups spanning 462.84 cM, the location of mdw1 was localized to Chromosome 7. Using the putative dwarfing-associated genes, fine genetic mapping of the mdw1 genomic region was facilitated with 1,194 F₂ progeny that defined the genetic distance between mdw1 and cytokinin oxidase gene, a candidate gene for compact growth habit (cp) in cucumber, to be 1.7 cM. The candidate gene ERECTA (serin/threonine kinase) and UBI (ubiquitin) were also mapped to genomic regions flanking mdw1 at distances of 0.6 and 1.2 cM, respectively.     

Effect of the herbicide terbutryn on meiosis,yield and mitotic chromosomes in C2 plants ofVicia faba L.

The herbicide terbutryn induced up to 11.3 % chromosomal abnormalities in cells undergoing meiosis inVicia faba. The mean number of pods/plant and the mean number of seeds/pod were considerably reduced, but the average seed dry mass was not or was only slightly affected by this herbicide. In C₂ plants chromosomal abnormalities were observed in few but significant numbers of cells.     

Effects of salt stress on nitrogenase activity and growth of four legumes

The effects of salt stress on nitrogenase (N2-ase) activity, growth and nitrogen content ofVicia faba (L.),Medicago sativa (L.) Merrill,Glycine max andVigna sinensis (L.) were investigated. Four levels of salinity were applied and salt treatments were imposed on inoculated and N-fertilized plants.M. sativa tolerated mild levels of salinity but higher salt concentrations depressed N₂-ase activity of this species. The other three legumes were considerably affected by salt treatments, and N₂-ase activity was significantly reduced by salinity. Vicia faba, carrying elongate nodules, could restore a partial N₂-ase activity upon recovery from salt stress whereasG. max andV. sinesis, both with spherical nodules, could not regain significant activity when salinity was removed. Salt stress retarded growth of both inoculated and N-fertilized plants. The nitrogen content of both treatments was also affected by salinity and the effect was more severe for inoculated than N-fertilized plants.     

Development of SCAR markers linked to a gene controlling absence of tannins in faba bean

Faba beans are inexpensive, nutrient-dense sources of plant protein, but anti-nutritional factors such as condensed tannins reduce the biological value of their protein. Two recessive genes, zt-1 and zt-2, control the absence of tannins in faba bean seeds and also determine a white flower character on the plant. However, crosses between them produce coloured F₁ plants with tannins that contaminate the crop. Therefore, it is important to identify the gene present in all tannin-free cultivars and gene bank accessions to enable breeders to choose appropriate genitors for their crosses. The aim of this study was the identification of markers linked to zt-1, one of the genes governing free tannin content in faba bean. A segregating F₂ population derived from the cross between the coloured flower and high tannin content genotype Vf6 and a zt-1 line was developed and characterized phenotypically. Bulked Segregant Analysis (BSA) was used to identify Random Amplified Polymorphic DNA (RAPD) markers linked to the zt-1 gene. Four RAPD loci (OPC5₅₅₁, OPG15₆₀₀, OPG11₁₁₇₁ and OPAF20₇₇₆) showed polymorphism between the contrasting bulks. The markers were sequenced to develop specific Sequence Characterised Amplified Regions (SCARs). Amplification of SCC5₅₅₁ produced a single product which was only observed in the white flowered and zero tannin content genotypes, whereas SCAR SCG11₁₁₇₁only produced a band in F₂ plants with coloured flower and high tannin content. SCARs SCC5₅₅₁ and SCG11₁₁₇₁ were tested for their applicability for routine screening in 37 faba bean genotypes differing in flower colour and tannin content. SCC5₅₅₁, allowed the prediction of the zt-1 genotypes with a 95% of accuracy, underscoring the potential of this SCAR marker as a cost-effective tool for MAS in large faba bean breeding populations.     

Effects of rhizobial inoculation, cropping systems and growth stages on endophytic bacterial community of soybean roots

Interspecific interactions and soil nitrogen supply levels affect intercropping productivity. We hypothesized that interspecific competition can be alleviated by increasing N application rate and yield advantage can be obtained in competitive systems. A field experiment was conducted in Wuwei, Gansu province in 2007 and 2008 to study intercropping of faba bean/maize, wheat/maize, barley/maize and the corresponding monocultures of faba bean (Vicia faba L.), wheat (Triticum aestivum L.), barley (Hordeum vulgare L.) and maize (Zea mays L.) with N application rates of 0, 75, 150, 225 and 300 kg N ha^?1. Total land equivalent ratios (TLER) were 1.22 for faba bean/maize, 1.16 for wheat/maize, and 1.13 for barley/maize intercropping over the 2-year study period. Maize was overyielding when intercropped with faba bean, but underyielding when intercropped with wheat or barley according to partial land equivalent ratios (PLER) based on grain yields of individual crops in intercropping and sole cropping. There was an interspecific facilitation between intercropped faba bean and maize, and interspecific competition between maize and either wheat or barley. The underyielding of maize was higher when intercropped with barley than with wheat. Fertilizer N alleviated competitive interactions in intercrops with adequate fertilizer N at 225 kg ha^?1. Yield advantage of intercropping can be acquired with adequate nitrogen supply, even in an intensive competitive system such as barley/maize intercropping. This is important when using intercropping to develop intensive farming systems with high inputs and high outputs.     

Isoenzymes of lactate dehydrogenase in the root growth zones ofVicia faba L.

The present studies of the LDH isoenzymes have been made as a part of our studies on the respiration systems of the roots ofVicia faba. All the root zones had 5 LDH isoenzymes and 2 antibands, but there were differences in their relative quantity. Cathodic isoenzymes (LDH₅, LDH₄) prevailed in the meristematic zone, whereas the anodic ones (LDH₁) prevailed in the others. The different enzyme activity in individual zones is probably connected with the transition of the anaerobic metabolism in the meristem to the aerobic one in the differentiating parts of the root.     

Maintenance of plant cell membrane integrity and function by the immobilisation of protoplasts in alginate matrices

Isolated protoplasts (Vicia faba) suspended in a mannitol solution over a time period of about 10 d undergo a process of aging characterised by increased ethane formation, decrease in oxygen evolution and CO₂ uptake, pigment degradation and a change in certain enzyme activities (ribulose-1,5-bisphosphate carboxylase and protease). The technique of immobilisation of protoplasts in a cross-linked alginate matrix offers a way to delay the appearance of the indicators mentioned. Various possible mechanisms for the increased stability of protoplasts are discussed.     

Formation of ascochitine by plant pathogens of the genus Ascochyta

Strains of fungi isolated from pulse crops: pea (Pisum sativum) and faba beans (Vicia faba) plants with symptoms of Ascochyta blight, footrot and stems lesions have been examined under laboratory conditions for their ability to produce ascochitine and metabolites toxic toArtemia salina. BothAscochyta pisi Lib and Ascochyta fabae LK Jones isolates formed ascochitine in yields of 20–480mg/kg. The highest yield of ascochitine was produced on rice and the lowest on maize grain. Ascochyta pinodes andPhoma medicaginis varpinodella (LK Jones) Boerema (formerlyAscochyta pinodella LK Jones) did not produce ascochitine. Crystalline ascochitine was found to be of moderate toxicity toArtemia salina larvae (LC₅₀ = 85μg/cm³BSM*). Extracts ofPhoma medicaginis var,pinodella cultures were found to be highly toxic toArtemia salina.     

Ethylene-enhanced ethylene oxidation inVicia faba

The alkene oxygenase (AO) of fababean (Vicia faba L.) converts ethylene to ethylene oxide. Treatment of fababeans with 10μl/liter ethylene increases the activity of this enzyme within 2 hours of ethylene treatment. Though other alkenes were taken up by fababean seedlings, ethylene was the most active in inducing AO activity. The ability of ethylene to increase AO was blocked 60% by cycloheximide, an inhibitor of protein synthesis, and 35% by AgNO₃, an inhibitor of ethylene action.     

Estimation of linkage in trisomic inheritance

 Based on F₂ families derived from selfed F₁ trisomic plants we have developed a genetic model to estimate linkage relationships between pairs of loci located on the extra chromosome. Genotypic frequencies of each class expected in a trisomic F₂ family have been calculated and the maximum-likelihood equations for recombination-fraction estimation have been derived for a variety of genetic situations. Morton’s test of homogeneity was used to compare recombination fractions estimated between loci exhibiting trisomic segregation to those obtained in families where the same loci showed Mendelian segregation. This method has been applied to an analysis of morphological, isozyme and RAPD data from faba bean (Vicia faba L.). Received: 11 October 1996 / Accepted: 21 March 1997     

Allometric equations for maximum filtration rate in blue mussels Mytilus edulis and importance of condition index

The relationship between body dry weight (W) and shell length (L) of blue mussels, Mytilus edulis, can be expressed by the condition index (CI = W/L ³) which varies from population to population and during the year. Here, we examine the influence of CI on the relationships between maximum filtration rate (F, l h^?1), W (g), and L (mm) as described by the equations: F _W  = aW ^b and F _L  = cL ^d , respectively. This is done by using available and new experimental laboratory data on M. edulis obtained by members of the same research team using different methods and controlled diets of cultivated algal cells. For all data, it was found that F _W  = 6.773W ^0.678 and F _L  = 0.00135L ^2.088 which are very similar to equations for mussels with ‘medium condition’ (CI = 4–6 mg cm^?3): F _W  = 6.567W ^0.681 and F _L  = 0.00150L ^2.051, with b- and d-values within a few percent of the theoretically expected of 2/3 and 2, respectively. Further, based on the present data, we propose a correction factor expressed by the empirical relation F _W /F _L  = 0.3562CI^2/3 which implies that F _W tends to underestimate the actual filtration rate (F _L ) when CI < 4.70 and to overestimate the filtration rate when CI > 4.70.     

Oxidative damage and antioxidant defense system in leaves of Vicia faba major L. cv. Bartom during soil flooding and subsequent drainage

The adaptive reactions of Vicia faba major L. cv. Bartom to 13-27 days soil flooding and to 14 days of drainage following 13-days of soil flooding were studied. Under flooding, oxygen diffusion rate (ODR) in the root zone decreased from 2.28–3.44 to 0.09–0.28?µmol O₂ m^?2 s^?1; the soil redox potential (Eh) decreased from 543 to 70 mV. Upon drainage of flooded soil the ODR and Eh values returned to the control levels. Oxidative damage and defense systems in leaves were assessed by the concentration of thiobarbituric acid reactive substances (TBARs) and by the activities of superoxide dismutase (SOD) and glutathione reductase (GR). Two stages of stress development are described. During the first stage (1–13 days) shoot dry mass did not decrease, the TBARs concentration and SOD activity increased, the GR activity decreased. The second stage (13–27 days) was characterized by a decrease in the TBARs concentration, SOD and GR activities, pigment concentrations and shoot dry mass. Drainage of flooded soil resulted in elevated concentrations of TBARs and also increased the activities of SOD and GR. Increased SOD activity in the first stage of hypoxic stress development and activations of SOD and GR at oxygen re-entry to soil are responsible for tolerance of Vicia faba to hypoxic and post hypoxic stress associated with soil flooding and subsequent drainage.     

Photosynthetic characteristics of female vegetative tissues of Porphyra katadai var. hemiphylla (Bangiales, Rhodophyta) in culture

In this study, chlorophyll fluorescence parameters (?F/F _m′, F _v/F _m) and oxygen evolution of female vegetative tissues of Porphyra katadai var. hemiphylla in unisexual culture (FV) and in mixed culture with male vegetative tissues (FV-M) were followed at 5–20 °C, 10 and 80 μmol photons m^?2 s^?1. The formation of reproductive tissues was closely correlated with decreasing photosynthetic activities. At the same temperature the tissues cultured under 80 μmol photons m^?2 s^?1 showed a greater extent of maturation than those under 10 μmol photons m^?2 s^?1, and their decrease in photosynthesis was also larger. Under the same light intensity the extent of maturation increased with increasing temperature, and both cultures showed higher values of ?F/F _m′ and F _v/F _m at 10 and 15 °C, while their oxygen evolution became negative at 15–20 °C during the later period. Under the same culture condition the maturation of FV-M culture was relatively faster than that of FV culture, while their photosynthetic activity, especially ?F/F _m′, was lower.     

The combined effects of temperature and CO2 lead to altered gene expression in Acropora aspera

This study explored the interactive effects of near-term CO₂ increases (40–90 ppm above current ambient) during a simulated bleaching event (34 °C for 5 d) of Acropora aspera by linking physiology to expression patterns of genes involved in carbon metabolism. Symbiodinium photosynthetic efficiency (F _v /F _m ) was significantly depressed by the bleaching event, while elevated pressure of CO₂ (pCO₂) slightly mitigated the effects of increased temperature on F _v /F _m during the final 4 d of the recovery period, however, did not affect the loss of symbionts. Elevated pCO₂ alone had no effect on F _v /F _m or symbiont density. Expression of targeted Symbiodinium genes involved in carbon metabolism and heat stress response was not significantly altered by either increased temperature and/or CO₂. Of the selected host genes, two carbonic anhydrase isoforms (coCA2 and coCA3) exhibited the largest changes, most notably in crossed bleaching and elevated pCO₂ treatments. CA2 was significantly down-regulated on day 14 in all treatments, with the greatest decrease in the crossed treatment (relative expression compared to control = 0.16; p < 0.05); CA3 showed a similar trend, with expression levels 0.20-fold of controls on day 14 (p < 0.05) in the elevated temperature/pCO₂ treatment. The synergistic effects of ocean acidification and bleaching were evident during this study and demonstrate that increased pCO₂ in surface waters will impact corals much sooner than many studies utilising end-of-century pCO₂ concentrations would indicate.     

QTLs for Orobanche spp. resistance in faba bean: identification and validation across different environments

Broomrapes are holoparasitic plants which infect faba bean (Vicia faba L.), among other legumes. Here, we aimed to identify and validate quantitative trait loci (QTLs) for broomrape resistance in the cross 29H × Vf136 and to investigate the existence of common and specific genomic regions against Orobanche crenata and O. foetida. A genetic map including 171 markers was constructed for QTL analyses. Field trials for O. crenata were conducted during three consecutive seasons at Córdoba (Spain) and in a single season at Kafr El-Sheikh (Egypt). QTL analysis for O. foetida was performed using data from a single season at Beja (Tunisia). Seven QTLs for O. crenata were identified. Oc7 on chromosome VI was detected over 3 years at Córdoba, explaining between 22 and 33 % of the phenotypic variation, which make it the most promising candidate for future marker-assisted breeding for broomrape resistance in faba bean. O. crenata QTLs identified at Kafr El-Sheikh did not co-localize with those identified in Córdoba. Environmental differences together with the diversity of parasitic populations between locations may account for the discrepancy. Three QTLs for O. foetida were detected. Co-localization of Oc8 and Of3 in chromosome V confirms a common resistance against both O. crenata and O. foetida, as previously reported.     

Histogenesis and localization of non-specific esterase in root tip

A procedure was developed for satisfactory freeze-sectioning of root tips. The use of Ca formol-fixed material kept and frozen in Holt's syrup is recommended. The existence and different localization of 2 fractions of non—specific esterase was verified in root tips ofVicia faba. The same results were revealed in fixed and unfixed material. The dynamics ofin situ reaction was followed with respect to optimal incubation time. The results with substrates of different chain length support the existence of 2 fraction of the studied enzyme, none of which, concerning substrate specificity, is a lipase. It follows from the present studies inVicia faba and other species (Cucurbita pepo, Lupinus albus, Pisum sativum Zea mays), that non-specific esterase localization is not directly given by histogenesis.     

Characterization of the quantitative trait locus OilA1 for oil content in Brassica napus

Increasing seed oil content has become one of the most important breeding criteria in rapeseed (Brassica napus). However, oil content is a complex quantitative trait. QTL mapping in a double haploid population (SG population) emerging from a cross between a German (Sollux) and Chinese (Gaoyou) cultivars revealed one QTL for oil content on linkage group A1 (OilA1), which was mapped to a 17 cM genetic interval. To further validate and characterize the OilA1, we constructed a high-resolution map using B. rapa sequence resources and developed a set of near-isogenic lines (NILs) by employing a DH line SG-DH267 as donor and Chinese parent Gaoyou as recurrent background. The results showed highly conserved synteny order between B. rapa and B. napus within the linkage group A1 and revealed a possible centromere region between two markers ZAASA1-38 and NTP3 (2.5 cM). OilA1 was firstly validated by 250 BC₅F₂ plants and was confirmed in a 10.6 cM interval between the markers ZAASA1-47 and ZAASA1-77. Further substitution mapping was conducted by using two generations of QTL-NILs, 283 lines from eight BC₅F_3:4 families and 428 plants from six BC₅F₄ sub-NILs and thus narrowed the OilA1 interval to 6.9 cM and 4.3 cM (1.4 Mb), respectively. Field investigations with two replications using homozygous BC₅F_3:4 sister sub-NILs indicated that NILs, which carry a Sollux chromosome segment across the target region showed significant higher oil content (1.26 %, p < 0.001) than their sister NILs containing Gaoyou chromosome. The OilA1 locus is of particular interest for breeding purpose in China because 80 % of Chinese cultivars do not carry this desirable allele.     

Identification of large-effect QTL for kernel row number has potential for maize yield improvement

The kernel row number (KRN) per ear is an important component of maize (Zea mays L.) yield. In this study, a line with six kernel rows, MT-6, was used to investigate the genetic basis of KRN by quantitative trait locus (QTL) mapping. MT-6 was derived from a maize inbred line Mo17 and a teosinte entry X26-4 (Zea mays ssp. mexicana), with 23 % of its genome being homologous to X26-4. An MT-6/B73 F₂ segregating population consisting of 266 individuals was genotyped using 192 molecular markers spread evenly across the genome. The same F₂ population, together with its F_2:3 population, was phenotyped for KRN in three environments. Five individual QTL for KRN, including three substantially consistent major QTL detected in all environments, were identified on chromosomes 1, 2, 3, 4, and 5, respectively. These QTL accounted for 39.5–65.0 % of the KRN variation in these populations. Additionally, one pair of epistatic interaction between two loci with additive effects was detected and accounted for about 3 % of KRN variation. These results demonstrate that a few major QTL could substantially affect the evolution of maize KRNs and therefore provide valuable information for our understanding of the mechanism of KRN and the improvement in maize grain yield by molecular breeding.     

Genetic analysis and molecular mapping of low amylose gene du12(t) in rice (Oryza sativa L.)

Key message

We obtained interesting results for genetic analysis and molecular mapping of the du12(t) gene.

Abstract

Control of the amylose content in rice is the major strategy for breeding rice with improved quality. In this study, we conducted genetic analysis and molecular mapping to identify the dull gene in the dull rice, Milyang262. A single recessive gene, tentatively designated as du12(t), was identified as the dull gene that leads to the low amylose character of Milyang262. To investigate the inheritance of du12(t), genetic analysis on an F₂ population derived from a cross between the gene carrier, Milyang262, and a moderate amylose content variety, Junam, was conducted. A segregation ratio of 3:1 (χ ² = 1.71, p = 0.19) was observed, suggesting that du12(t) is a single recessive factor that controls the dull character in Milyang262. Allelism tests confirmed that du12(t) is not allelic to other low amylose controlling genes, wx or du1. Recessive class analysis was performed to localize the du12(t) locus. Mapping of du12(t) was conducted on F₂ and F₃ populations of Baegokchal/Milyang262 cross. Linkage analysis of 120 F₂ plants revealed that RM6926 and RM3509 flank du12(t) at a 2.38-Mb region. To refine the du12(t) locus position, 986 F₂ and 289 F₃ additional normal plants were screened by the flanking markers. Twenty-six recombinant plants were identified and later genotyped with four additional adjacent markers located between RM6926 and RM3509. Finally, du12(t) was mapped to an 840-kb region on the distal region of the long arm of chromosome 6, delimited by SSR markers RM20662 and RM412, and co-segregated by RM3765 and RM176.