Genetic mapping of a major gene affecting onion bulb fructan content

The non-structural dry matter content of onion bulbs consists principally of fructose, glucose, sucrose and fructans. The objective of this study was to understand the genetic basis for the wide variation observed in the relative amounts of these carbohydrates. Bulb carbohydrate composition was evaluated in progeny from crosses between high dry matter storage onion varieties and sweet, low dry matter varieties. When samples were analysed on a dry weight basis, reducing sugar and fructan content exhibited high negative correlations and bimodal segregation suggestive of the action of a major gene. A polymorphic SSR marker, ACM235, was identified which exhibited strong disequilibrium with bulb fructan content in F_2:3 families from the ‘W202A’ × ‘Texas Grano 438’ mapping population evaluated in two environments. This marker was mapped to chromosome 8 in the interspecific population ‘Allium cepa × A. roylei’. Mapping in the ‘Colossal Grano PVP’ × ‘Early Longkeeper P12’ F₂ population showed that a dominant major gene conditioning high-fructan content lay in the same genomic region. QTL analysis of total bulb fructan content in the intraspecific mapping population ‘BYG15-23’ × ‘AC43’ using a complete molecular marker map revealed only one significant QTL in the same chromosomal region. This locus, provisionally named Frc, may account for the major phenotypic differences in bulb carbohydrate content between storage and sweet onion varieties.     

QTL analysis for pseudostem pungency in bunching onion (Allium fistulosum)

Low pungency is one of the most important agronomic traits in bunching onion (Allium fistulosum L.). Although the degree of pungency can be evaluated indirectly using a colorimetric test for pyruvic acid, DNA markers linked to low-pungency quantitative trait loci (QTLs) are still desired. In this study, we evaluated pungency in the bunching onion pseudostem through six trials conducted over 3?years using an F _2:3 population. QTL analysis based on the genetic linkage map revealed that the major pungency QTL was located within a 24.2-cM interval on Chr. 2a. The low-pungency parent-derived allele at AFAT04B03, a simple sequence repeat locus linked to the pungency QTL, was rare among commercial bunching onion cultivars. In addition, individuals homozygous for the low-pungency parent-derived allele at AFAT04B03 were significantly less pungent than those that were homozygous or heterozygous. Thus, these findings suggest that AFAT04B03 is an effective selection marker for low pungency in bunching onion breeding.     

Quantitative trait locus (QTL) mapping reveals a role for unstudied genes in Aspergillus virulence

Infections caused by the fungus Aspergillus are a major cause of morbidity and mortality in immunocompromised populations. To identify genes required for virulence that could be used as targets for novel treatments, we mapped quantitative trait loci (QTL) affecting virulence in the progeny of a cross between two strains of A. nidulans (FGSC strains A4 and A91). We genotyped 61 progeny at 739 single nucleotide polymorphisms (SNP) spread throughout the genome, and constructed a linkage map that was largely consistent with the genomic sequence, with the exception of one potential inversion of ～527 kb on Chromosome V. The estimated genome size was 3705 cM and the average intermarker spacing was 5.0 cM. The average ratio of physical distance to genetic distance was 8.1 kb/cM, which is similar to previous estimates, and variation in recombination rate was significantly positively correlated with GC content, a pattern seen in other taxa. To map QTL affecting virulence, we measured the ability of each progeny strain to kill model hosts, larvae of the wax moth Galleria mellonella. We detected three QTL affecting in vivo virulence that were distinct from QTL affecting in vitro growth, and mapped the virulence QTL to regions containing 7-24 genes, excluding genes with no sequence variation between the parental strains and genes with only synonymous SNPs. None of the genes in our QTL target regions have been previously associated with virulence in Aspergillus, and almost half of these genes are currently annotated as "hypothetical". This study is the first to map QTL affecting the virulence of a fungal pathogen in an animal host, and our results illustrate the power of this approach to identify a short list of unknown genes for further investigation.     

Genetic mapping reveals a single major QTL for bacterial wilt resistance in Italian ryegrass (Lolium multiflorum Lam.)

Bacterial wilt caused by Xanthomonas translucens pv. graminis (Xtg) is a major disease of economically important forage crops such as ryegrasses and fescues. Targeted breeding based on seedling inoculation has resulted in cultivars with considerable levels of resistance. However, the mechanisms of inheritance of resistance are poorly understood and further breeding progress is difficult to obtain. This study aimed to assess the relevance of the seedling screening in the glasshouse for adult plant resistance in the field and to investigate genetic control of resistance to bacterial wilt in Italian ryegrass (Lolium multiflorum Lam.). A mapping population consisting of 306 F₁ individuals was established and resistance to bacterial wilt was assessed in glasshouse and field experiments. Highly correlated data (r = 0.67–0.77, P < 0.01) between trial locations demonstrated the suitability of glasshouse screens for phenotypic selection. Analysis of quantitative trait loci (QTL) based on a high density genetic linkage map consisting of 368 amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers revealed a single major QTL on linkage group (LG) 4 explaining 67% of the total phenotypic variance (Vp). In addition, a minor QTL was observed on LG 5. Field experiments confirmed the major QTL on LG 4 to explain 43% (in 2004) to 84% (in 2005) of Vp and also revealed additional minor QTLs on LG 1, LG 4 and LG 6. The identified QTLs and the closely linked markers represent important targets for marker-assisted selection of Italian ryegrass.     

QTL mapping of temperature sensitivity reveals candidate genes for thermal adaptation and growth morphology in the plant pathogenic fungus Zymoseptoria tritici

Different thermal environments impose strong, differential selection on populations, leading to local adaptation, but the genetic basis of thermal adaptation is poorly understood. We used quantitative trait locus (QTL) mapping in the fungal wheat pathogen Zymoseptoria tritici to study the genetic architecture of thermal adaptation and identify candidate genes. Four wild-type strains originating from the same thermal environment were crossed to generate two mapping populations with 263 (cross 1) and 261 (cross 2) progeny. Restriction site-associated DNA sequencing was used to genotype 9745 (cross 1) and 7333 (cross 2) single-nucleotide polymorphism markers segregating within the mapping population. Temperature sensitivity was assessed using digital image analysis of colonies growing at two different temperatures. We identified four QTLs for temperature sensitivity, with unique QTLs found in each cross. One QTL had a logarithm of odds score >11 and contained only six candidate genes, including PBS2, encoding a mitogen-activated protein kinase kinase associated with low temperature tolerance in Saccharomyces cerevisiae. This and other QTLs showed evidence for pleiotropy among growth rate, melanization and growth morphology, suggesting that many traits can be correlated with thermal adaptation in fungi. Higher temperatures were highly correlated with a shift to filamentous growth among the progeny in both crosses. We show that thermal adaptation has a complex genetic architecture, with natural populations of Z. tritici harboring significant genetic variation for this trait. We conclude that Z. tritici populations have the potential to adapt rapidly to climate change and expand into new climatic zones.     

Tests of candidate genes in breed cross populations for QTL mapping in livestock

In recent years, several F₂ crosses between outbred lines of livestock have been developed to identify quantitative trait loci (QTL). These populations are valuable for further genetic analysis, including positional candidate gene loci (CGL). Analysis of CGL in F₂ populations is, however, hindered by extensive between-breed linkage disequilibrium (LD). The objectives here were to develop and evaluate three tests for CGL in simulated F₂ breed-cross populations. 1) A standard association test, based on the fixed effect of CGL genotype. This test was significant for CGL at considerable distances from the QTL. 2) A marker-assisted association test, based on a test at the CGL of the fixed effect of CGL genotype in a breed-cross QTL interval mapping model. This removed the impact of between-breed LD, but was not powerful in detecting CGL closely linked to the QTL, unless the CGL was the QTL. 3) An F-drop test, comparing F ratios for a QTL at the CGL with and without the CGL included as fixed effect. It had low power to distinguish close from distant CGL. Power to distinguish two CGL within 10 cM from the QTL was limited and little improved by including QTL effects associated with markers to remove between-breed LD, although the power was greater when one of the CGL was the causative mutation. Therefore, while we conclude that candidate gene tests in QTL mapping populations must be interpreted with caution, we now have a clearer picture of the value of candidate gene tests in these populations.     

Simultaneous mapping of epistatic QTL in chickens reveals clusters of QTL pairs with similar genetic effects on growth

We used simultaneous mapping of interacting quantitative trait locus (QTL) pairs to study various growth traits in a chicken F2 intercross. The method was shown to increase the number of detected QTLs by 30 % compared with a traditional method detecting QTLs by their marginal genetic effects. Epistasis was shown to be an important contributor to the genetic variance of growth, with the largest impact on early growth (before 6 weeks of age). There is also evidence for a discrete set of interacting loci involved in early growth, supporting the previous findings of different genetic regulation of early and late growth in chicken. The genotype-phenotype relationship was evaluated for all interacting QTL pairs and 17 of the 21 evaluated QTL pairs could be assigned to one of four clusters in which the pairs in a cluster have very similar genetic effects on growth. The genetic effects of the pairs indicate commonly occurring dominance-by-dominance, heterosis and multiplicative interactions. The results from this study clearly illustrate the increase in power obtained by using this novel method for simultaneous detection of epistatic QTL, and also how visualization of genotype-phenotype relationships for epistatic QTL pairs provides new insights to biological mechanisms underlying complex traits.     

Genetic dissection of floral traits in anemone-type chrysanthemum by QTL mapping

Brassica oleracea comprises several important subspecies, including cabbage, broccoli, cauliflower, Chinese kale, and kohlrabi. The petal color of Chinese kale is mostly white and sometimes yellow. To explore the genetic basis of petal color variation in Chinese kale, F₂ and BC₁ (backcross) populations were constructed from the cross of two inbred lines, 2114 (yellow petal) and 2116 (white petal). Genetic analysis of the F₂ and BC₁ populations demonstrated that yellow petal color was controlled by a single recessive nuclear gene, termed cpc-2. Insertion-deletion (InDel) markers, designed based on the parental resequencing data, were used to map cpc-2. The fine mapping results indicated that the cpc-2 gene was located in a 569-kb interval on chromosome C03 flanked by InDel markers ZB636 and ZB692, with genetic distances of 0.3 cM and 0.6 cM, respectively. By analyzing the nucleotide variations and annotations of the genes in this interval, a CCD4 family gene was predicted to be a candidate for cpc-2 and renamed BoCCD4.2. In addition, insertion of the CACTA-like transposable element (TE3) interrupted the function of the BoCCD4 gene, which may have resulted in the loss of function of BoCCD4 and the petal color transition from white to yellow. The TE3 insertion in the BoCCD4 gene was also present in 63 cabbage inbred lines among 159 accessions, which revealed that the TE3-type null allele of BoCCD4 formed before the divergence of the two subspecies cabbage and Chinese kale and that Chinese kale evolved much earlier than cabbage. This study lays the foundation for cloning BoCCD4.2 and revealing the molecular mechanism underlying petal color formation in Chinese kale.

     

Genetic analysis of rice CMS-WA fertility restoration based on QTL mapping

 The inheritance of fertility restoration of rice cytoplasmic male sterility of the wild abortive type was studied by means of QTL mapping. The two segregating populations examined showed high frequencies of highly sterile and highly fertile progenies, but a low frequency of partially sterile and partially fertile progenies. The distributions suggested that fertility restoration was mainly controlled by major genes. Based on a linkage map constructed with 57 RFLP and 61 AFLP markers on a B₁F₁ population, composite interval mapping (CIM) revealed that the fertility was restored by the additive effects of two restorer loci located on chromosome 10. One QTL, tightly linked to RFLP marker C1361 in the middle of the long arm of chromosome 10, explained 71.5% of the phenotypic variance. The second QTL was located between RFLP markers R2309 and RG257 on the short arm and explained 27.3% of the phenotypic variance. Similar results were obtained using the simple interval mapping (SIM) methods. Recived: 8 January 1998/Accepted: 22 April 1998     

QTL mapping in three tropical maize populations reveals a set of constitutive and adaptive genomic regions for drought tolerance

Despite numerous published reports of quantitative trait loci (QTL) for drought-related traits, practical applications of such QTL in maize improvement are scarce. Identifying QTL of sizeable effects that express more or less uniformly in diverse genetic backgrounds across contrasting water regimes could significantly complement conventional breeding efforts to improve drought tolerance. We evaluated three tropical bi-parental populations under water-stress (WS) and well-watered (WW) regimes in Mexico, Kenya and Zimbabwe to identify genomic regions responsible for grain yield (GY) and anthesis-silking interval (ASI) across multiple environments and diverse genetic backgrounds. Across the three populations, on average, drought stress reduced GY by more than 50 % and increased ASI by 3.2 days. We identified a total of 83 and 62 QTL through individual environment analyses for GY and ASI, respectively. In each population, most QTL consistently showed up in each water regime. Across the three populations, the phenotypic variance explained by various individual QTL ranged from 2.6 to 17.8 % for GY and 1.7 to 17.8 % for ASI under WS environments and from 5 to 19.5 % for GY under WW environments. Meta-QTL (mQTL) analysis across the three populations and multiple environments identified seven genomic regions for GY and one for ASI, of which six mQTL on chr.1, 4, 5 and 10 for GY were constitutively expressed across WS and WW environments. One mQTL on chr.7 for GY and one on chr.3 for ASI were found to be ‘adaptive’ to WS conditions. High throughput assays were developed for SNPs that delimit the physical intervals of these mQTL. At most of the QTL, almost equal number of favorable alleles was donated by either of the parents within each cross, thereby demonstrating the potential of drought tolerant × drought tolerant crosses to identify QTL under contrasting water regimes.     

Genetic mapping of Vibrio cholerae enterotoxin structural genes

The structural genes which constitute the cholera toxin operon, ctxAB, were genetically mapped in the Vibrio cholerae El Tor strain RV79. This strain of V. cholerae contains two copies of the ctx operon located on a 7-kilobase-pair tandemly duplicated region. We began by isolating a vibriophage VcA1 insertion mutation in one of the two ctxA genes located in this region. The mutant carrying this ctxA::VcA1 insertion, DC24, was converted to a VcA1-facilitated donor by introduction of the conjugal plasmid pSJ15, which carries an inserted copy of a defective VcA1-like prophage. The donor characteristics of DC24(pSJ15) indicated that the ctxA::VcA1 insertion mutation was near the trp region of the V. cholerae chromosome. Subsequent RV79 three-factor crosses were performed between VcA1-facilitated donors and recipient strains carrying one of two structural gene mutations in ctx, either delta ctxA23P Kmr or delta ctx-7922. The former was constructed by an in vivo marker exchange procedure and could be scored either by its kanamycin resistance phenotype or by its lack of DNA sequences homologous to the ctxA region. The delta ctx-7922 mutation is a total deletion of both ctx copies of strain RV79. The three-factor cross data strongly suggest that the two ctx loci of RV79 map between the nal and his genes of V. cholerae in the trp nal his linkage group. Physical analysis and heterologous crosses between an RV79 El Tor donor and a 569B classical recipient indicates that one of the two 569B ctx operon copies maps in the same region as the RV79 ctx loci (i.e., linked to nal). Together with previously published observations, these data show that the ctx structural genes are not closely linked to other genes known to affect toxin production in V. cholerae.     

Bacillus subtilis cysteine synthetase is a global regulator of the expression of genes involved in sulfur assimilation

The synthesis of L-cysteine, the major mechanism by which sulfur is incorporated into organic compounds in microorganisms, occupies a significant fraction of bacterial metabolism. In Bacillus subtilis the cysH operon, encoding several proteins involved in cysteine biosynthesis, is induced by sulfur starvation and tightly repressed by cysteine. We show that a null mutation in the cysK gene encoding an O-acetylserine-(thiol)lyase, the enzyme that catalyzes the final step in cysteine biosynthesis, results in constitutive expression of the cysH operon. Using DNA microarrays we found that, in addition to cysH, almost all of the genes required for sulfate assimilation are constitutively expressed in cysK mutants. These results indicate that CysK, besides its enzymatic role in cysteine biosynthesis, is a global negative regulator of genes involved in sulfur metabolism.     

Genetic parameters for body weight, carcass chemical composition and yield in a broiler-layer cross developed for QTL mapping

The objective of this study was to estimate genetic and phenotypic correlations of body weight at 6 weeks of age (BW6), as well as final carcass yield, and moisture, protein, fat and ash contents, using data from 3,422 F2 chickens originated from reciprocal cross between a broiler and a layer line. Variance components were estimated by the REML method, using animal models for evaluating random additive genetic and fixed contemporary group (sex, hatch and genetic group) effects. The heritability estimates (h(2)) for BW6, carcass yield and percentage of carcass moisture were 0.31 ± 0.07, 0.20 ± 0.05 and 0.33 ± 0.07, respectively. The h(2) for the percentages of protein, fat and ash on a dry matter basis were 0.48 ± 0.09, 0.55 ± 0.10 and 0.36 ± 0.08, respectively. BW6 had a positive genetic correlation with fat percentage in the carcass, but a negative one with protein and ash contents. Carcass yield, thus, appears to have only low genetic association with carcass composition traits. The genetic correlations observed between traits, measured on a dry matter basis, indicated that selection for carcass protein content may favor higher ash content and a lower percentage of carcass fat.     

Genetic analyses of anthocyanin concentrations and intensity of red bulb color among segregating haploid progenies of onion

Higher concentrations of anthocyanins in vegetables are important for attractive appearance and may offer health benefits for consumers. The red color of onion (Allium cepa) bulbs is due primarily to the accumulation of anthocyanins. The goal of this study was to identify chromosome regions that significantly affect concentrations of anthocyanins and soluble solids in onion bulbs. Segregating haploid plants from the cross of yellow (OH1) and red (5225) inbreds were asexually propagated and bulbs were produced in replicated trials across three environments. Concentrations of soluble solids were measured at 30 days after harvest and quantitative analyses revealed a significant region on chromosome 5. Analyses using a binary model for segregation of red versus yellow bulbs revealed a significant region on chromosome 7 and two regions linked in repulsion phase on chromosome 4. These results are consistent with the complementary two-locus model previously proposed to control red versus yellow bulb colors in onion. The region on chromosome 7 mapped to the same location as the R locus, and the regions on chromosome 4 may correspond to the L and L2 loci. The intensity of red bulb color was assessed visually by a panel of evaluators and by amounts of anthocyanins [peonidin 3-glucoside and cyanidin 3-(6″-malonoyl-laminaribioside)] measured by high-performance liquid chromatography. Quantitative analyses using a normal model revealed significant quantitative trait loci on chromosomes 1, 4 and 8 affecting anthocyanin concentrations, and yellow onion contributed beneficial genetic variation to enhance red bulb color. Significant correlations were observed between these anthocyanin concentrations and panel scores, indicating that visual selection should be effective for increasing anthocyanin levels in onion bulbs. These selected populations may be more attractive to consumers, potentially provide health benefits from increased anthocyanin consumption, and be a source of natural colorants.     

Fine mapping a QTL for carbon isotope composition in tomato

Carbon isotope composition (delta(13)C) and leaf water-use efficiency vary in concert in C3 plants, making delta(13)C useful as a proxy for plant water-use efficiency. A QTL for delta(13)C was detected in the Solanum pennellii chromosome fragment of IL5-4, an introgression line with S. lycopersicum cv. M82 background. M82 and IL 5-4 were crossed, and RFLP markers in the target region converted to PCR-based markers. Forty-one recombinants with an introgression fragment ranging in length from 1.1 to 11.4 cM were identified by marker assisted selection (MAS) among approximately 2000 F2 plants. A total of 29 markers were mapped within the introgression fragment unique to IL5-4. These markers divided the about 9 cM target region into nine intervals. A dominant QTL for delta(13)C, designated QWUE5.1 that explained 25.6% of the total phenotypic variance was mapped to an interval about 2.2 cM long. Twenty-one plants with a S. pennellii chromosome fragment shortened to a length of 2.0-9.1 cM by a second recombination event were generated by MAS of 1,125 F4 plants. Two near isogenic lines with high delta(13)C (small negative value) and carrying QWUE5.1 on the shortest introgression fragments (about 7.0 cM) were identified. The markers and genetic stocks developed are valuable for cloning the gene underlying QWUE5.1, MAS of QWUE5.1, and fine-mapping genes/QTL located in this region.