QTL mapping and candidate gene mining of seed size and seed weight in castor plant (Ricinus communis L.).

BACKGROUND: Castor (Ricinus communis L., 2n = 2x = 20) is an important industrial crop, due to its oil is very important to the global special chemical industry. Seed size and seed weight are fundamentally important in determining castor yield, while little is known about it. In this study, QTL analysis and candidate gene mining of castor seed size and seed weight were conducted with composite interval mapping (CIM), inclusive composite interval mapping (ICIM) and marker enrichment strategy in 4 populations, i.e., populations F2, BC1, S1-1 and S1-2, derived from 2 accessions with significant phenotypic differences. RESULTS: In the QTL primary mapping, 2 novel QTL clusters were detected in marker intervals RCM520-RCM76 and RCM915-RCM950. In order to verify their accuracy and to narrow their intervals, QTL remapping was carried out in populations F2 and BC1. Among them, 44 and 30 QTLs underlying seed size and seed weight were detected in F2 population using methods CIM and ICIM-ADD respectively, including 4-9 and 3-5 ones conferring each trait were identified with a phenotypic variation explained ranged from 37.92 to 115.81% and 32.86-45.98% respectively. The remapping results in BC1 population were consistent with those in F2 population. Importantly, 3 QTL clusters (i.e. QTL-cluster1, QTL-cluster2 and QTL-cluster3) were found in marker intervals RCM74-RCM76 (37.1 kb), RCM930-RCM950 (259.8 kb) and RCM918-RCM920 (172.9 kb) respectively; in addition, all of them were detected again, the former one was found in the S1-2 population, and the latter two were found simultaneously in the populations S1-1 and S1-2. Finally, 6 candidate genes (i.e. LOC8266555, LOC8281168, LOC8281151, LOC8259066, LOC8258591 and LOC8270077) were screened in the above QTL clusters, they were differentially expressed in multiple seed tissues of both parents, signifying the potential role in regulating seed size and seed weight. CONCLUSION: The above results not only provide new insights into the genetic structure of seed size and seed weight in castor, but also lay the foundation for the functional identification of these candidate genes.

Enhancing discoveries of molecular QTL studies with small sample size using summary statistic imputation.

Quantitative trait locus (QTL) analyses of multiomic molecular traits, such as gene transcription (eQTL), DNA methylation (mQTL) and histone modification (haQTL), have been widely used to infer the functional effects of genome variants. However, the QTL discovery is largely restricted by the limited study sample size, which demands higher threshold of minor allele frequency and then causes heavy missing molecular trait-variant associations. This happens prominently in single-cell level molecular QTL studies because of sample availability and cost. It is urgent to propose a method to solve this problem in order to enhance discoveries of current molecular QTL studies with small sample size. In this study, we presented an efficient computational framework called xQTLImp to impute missing molecular QTL associations. In the local-region imputation, xQTLImp uses multivariate Gaussian model to impute the missing associations by leveraging known association statistics of variants and the linkage disequilibrium (LD) around. In the genome-wide imputation, novel procedures are implemented to improve efficiency, including dynamically constructing a reused LD buffer, adopting multiple heuristic strategies and parallel computing. Experiments on various multiomic bulk and single-cell sequencing-based QTL datasets have demonstrated high imputation accuracy and novel QTL discovery ability of xQTLImp. Finally, a C++ software package is freely available at https://github.com/stormlovetao/QTLIMP.

Genome-wide association study of leaf photosynthesis using a high-throughput gas exchange system in rice.

Enhancing leaf photosynthetic capacity is essential for improving the yield of rice (Oryza sativa L.). Although the exploitation of natural genetic resources is considered a promising approach to enhance photosynthetic capacity, genomic factors related to the genetic diversity of leaf photosynthetic capacity have yet to be fully elucidated due to the limitation of measurement efficiency. In this study, we aimed to identify novel genomic regions for the net CO2 assimilation rate (A) by combining genome-wide association study (GWAS) and the newly developed rapid closed gas exchange system MIC-100. Using three MIC-100 systems in the field at the vegetative stage, we measured A of 168 temperate japonica rice varieties with six replicates for three years. We found that the modern varieties exhibited higher A than the landraces, while there was no significant relationship between the release year and A among the modern varieties. Our GWAS scan revealed two major peaks located on chromosomes 4 and 8, which were repeatedly detected in the different experiments and in the generalized linear modelling approach. We suggest that high-throughput gas exchange measurements combined with GWAS is a reliable approach for understanding the genetic mechanisms underlying photosynthetic diversities in crop species.

Identification of genes associated with the high-temperature fermentation trait in the Saccharomyces cerevisiae natural isolate BCC39850.

The fermentative model yeast Saccharomyces cerevisiae has been extensively used to study the genetic basis of stress response and homeostasis. In this study, we performed quantitative trait loci (QTL) analysis of the high-temperature fermentation trait of the progeny from the mating of the S. cerevisiae natural isolate BCC39850 (haploid#17) and the laboratory strain CEN.PK2-1C. A single QTL on chromosome X was identified, encompassing six candidate genes (GEA1, PTK2, NTA1, NPA3, IRT1, and IML1). The functions of these candidates were tested by reverse genetic experiments. Deletion mutants of PTK2, NTA1, and IML1 showed growth defects at 42 °C. The PTK2 knock-out mutant also showed significantly reduced ethanol production and plasma membrane H+ ATPase activity and increased sensitivity to acetic acid, ethanol, amphotericin B (AMB), and ß-1,3-glucanase treatment. The CRISPR-Cas9 system was used to construct knock-in mutants by replacement of PTK2, NTA1, IML1, and NPA3 genes with BCC39850 alleles. The PTK2 and NTA1 knock-in mutants showed increased growth and ethanol production titers at 42 °C. These findings suggest an important role for the PTK2 serine/threonine protein kinase in regulating plasma membrane H+ ATPase activity and the NTA1 N-terminal amidase in protein degradation via the ubiquitin-proteasome system machinery, which affects tolerance to heat stress in S. cerevisiae.

Mapping of quantitative trait loci (QTL) in Brassica napus L. for tolerance to water stress.

Brassica napus L. plants are sensitive to water stress conditions throughout their life cycle from seed germination to seed setting. This study aims at identifying quantitative trait loci (QTL) linked to B. napus tolerance to water stress mimicked by applications of 10% polyethylene glycol-6000 (PEG-6000). Two doubled haploid populations, each consisting of 150 genotypes, were used for this research. Plants at the two true leaf stage of development were grown in the absence (control) or presence (stress) of PEG-6000 under controlled environmental conditions for 48 h, and the drought stress index was calculated for each genotype. All genotypes, along with their parents, were genotyped using the Brassica Infinium 90K SNP BeadChip Array. Inclusive composite interval mapping was used to identify QTL. Six QTL and 12 putative QTL associated with water stress tolerance were identified across six chromosomes (A2, A3, A4, A9, C3, and C7). Collectively, 2154 candidate genes for water stress tolerance were identified for all the identified QTL. Among them, 213 genes were identified as being directly associated with water stress (imposed by PEG-6000) tolerance based on nine functional annotations. These results can be incorporated into future breeding initiatives to select plant material with the ability to cope effectively with water stress.

QTL detection and candidate gene identification of qCTB1 for cold tolerance in the Yunnan plateau landrace rice.

Cold stress is one of the main abiotic stresses that affects rice growth and production worldwide. Dissection of the genetic basis is important for genetic improvement of cold tolerance in rice. In this study, a new source of cold-tolerant accession from the Yunnan plateau, Lijiangxiaoheigu, was used as the donor parent and crossed with a cold-sensitive cultivar, Deyou17, to develop recombinant inbred lines (RILs) for quantitative trait locus (QTL) analysis for cold tolerance at the early seedling and booting stages in rice. In total, three QTLs for cold tolerance at the early seedling stage on chromosomes 2 and 7, and four QTLs at the booting stage on chromosomes 1, 3, 5, and 7, were identified. Haplotype and linear regression analyses showed that QTL pyramiding based on the additive effect of these favorable loci has good potential for cold tolerance breeding. Effect assessment in the RIL and BC3F3 populations demonstrated that qCTB1 had a stable effect on cold tolerance at the booting stage in the genetic segregation populations. Under different cold stress conditions, qCTB1 was fine-mapped to a 341-kb interval between markers M3 and M4. Through the combination of parental sequence comparison, candidate gene-based association analysis, and tissue and cold-induced expression analyses, eight important candidate genes for qCTB1 were identified. This study will provide genetic resources for molecular breeding and gene cloning to improve cold tolerance in rice. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-024-01488-3.

Genes controlling hydrolysate toxin tolerance identified by QTL analysis of the natural Saccharomyces cerevisiae BCC39850.

Lignocellulosic material can be converted to valorized products such as fuels. Pretreatment is an essential step in conversion, which is needed to increase the digestibility of the raw material for microbial fermentation. However, pretreatment generates by-products (hydrolysate toxins) that are detrimental to microbial growth. In this study, natural Saccharomyces strains isolated from habitats in Thailand were screened for their tolerance to synthetic hydrolysate toxins (synHTs). The Saccharomyces cerevisiae natural strain BCC39850 (toxin-tolerant) was crossed with the laboratory strain CEN.PK2-1C (toxin-sensitive), and quantitative trait locus (QTL) analysis was performed on the segregants using phenotypic scores of growth (OD600) and glucose consumption. VMS1, DET1, KCS1, MRH1, YOS9, SYO1, and YDR042C were identified from QTLs as candidate genes associated with the tolerance trait. CEN.PK2-1C knockouts of the VMS1, YOS9, KCS1, and MRH1 genes exhibited significantly greater hydrolysate toxin sensitivity to growth, whereas CEN.PK2-1C knock-ins with replacement of VMS1 and MRH1 genes from the BCC39850 alleles showed significant increased ethanol production titers compared with the CEN.PK2-1C parental strain in the presence of synHTs. The discovery of VMS1, YOS9, MRH1, and KCS1 genes associated with hydrolysate toxin tolerance in S. cerevisiae indicates the roles of the endoplasmic-reticulum-associated protein degradation pathway, plasma membrane protein association, and the phosphatidylinositol signaling system in this trait. KEY POINTS: • QTL analysis was conducted using a hydrolysate toxin-tolerant S. cerevisiae natural strain • Deletion of VMS1, YOS9, MRH1, and KCS1 genes associated with hydrolysate toxin-sensitivity • Replacement of VMS1 and MRH1 with natural strain alleles increased ethanol production titers in the presence of hydrolysate toxins.

QTL Mapping of Adult Plant Resistance to Powdery Mildew in Chinese Wheat Landrace Baidatou.

Wheat powdery mildew, caused by the biotrophic fungus Blumeria graminis f. sp. tritici (Bgt), is one of the most devastating diseases affecting wheat throughout the world. Breeding and growing resistant wheat cultivars is one of the most economic and effective methods to control the disease, and as such, identifying and mapping the new and effective resistance genes is critical. Baidatou, a Chinese wheat landrace, shows excellent field resistance to powdery mildew. To identify the resistance gene(s) in Baidatou, 170 F7:8 recombinant inbred lines (RILs) derived from the cross Mingxian 169/Baidatou were evaluated for powdery mildew response at the adult-plant stage in the experimental fields in Yangling (YL) of Shaanxi Province and Tianshui (TS) in Gansu Province in 2019, 2020, and 2021. The relative area under disease progress curve (rAUDPC) of Mingxian 169/Baidatou F7:8 RILs indicated that the resistance of Baidatou to powdery mildew was controlled by quantitative trait loci (QTLs). Based on bulk segregation analysis combined with the 660K single nucleotide polymorphism (SNP) array and genotyping by target sequencing (16K SNP) of the entire RIL population, two QTLs, QPmbdt.nwafu-2AS and QPmbdt.nwafu-3AS, were identified, and these accounted for up to 44.5% of the phenotypic variation. One of the QTLs was located on the 3.32 cM genetic interval on wheat chromosome 2AS between the kompetitive allele-specific PCR markers AX-111012288 and AX_174233809, and another was located on the 9.6 cM genetic interval on chromosome 3AS between the SNP markers 3A_684044820 and 3A_686681822. These markers could be useful for successful breeding of powdery mildew resistance in wheat.

Morphological Analyses and QTL Mapping of Mottled Leaf in Zucchini (Cucurbita pepo L.).

The mottled leaf is one of the agronomic traits of zucchini and can be applied as a marker trait in aggregation breeding. However, the genetic mechanism responsible for mottled leaf has yet to be elucidated. In the present study, we used two inbred lines (line '19': silver mottled leaf; line '113': normal leaf) as parents for the physiological and genetic analysis of mottled leaf. The synthesis and net photosynthetic rate of chlorophyll were not significantly affected in the mottled areas of leaves. However, we detected a large space between the palisade parenchyma in the leaf mottle area of line '19', which may have caused the mottled leaf phenotype. Light also plays an important role in the formation of mottled leaf, and receiving light during the early stages of leaf development is a necessary factor. Genetic analysis has previously demonstrated that mottled leaf is a quantitative trait that is controlled by multiple genes. Based on the strategy of quantitative trait locus sequencing (QTL-seq), two QTLs were identified on chromosomes 1 and 17, named CpML1.1 and CpML17.1, respectively. Two major loci were identified using R/qtl software version 1.66 under greenhouse conditions in April 2019 (2019A) and April 2020 (2020A) and under open cultivation conditions in May 2020 (2020M). The major QTL, CpML1.1, was located in a 925.2-kb interval on chromosome 1 and explained 10.51%-24.15% of the phenotypic variation. The CpML17.1 was located in a 719.7-kb interval on chromosome 17 and explained 16.25%-38.68% of the phenotypic variation. Based on gene annotation, gene sequence alignment, and qRT-PCR analysis, the Cp4.1LG01g23790 at the CpML1.1 locus encoding a protein of the TPX2 family (target protein of Xklp2) may be a candidate gene for mottled leaf in zucchini. Our findings may provide a theoretical basis for the formation of mottled leaf and provide a foundation for the fine mapping of genes associated with mottled leaf. Molecular markers closely linked to mottled leaf can be used in molecular-assisted selection for the zucchini mottled leaf breeding.

Development of a 135K SNP genotyping array for Actinidia arguta and its applications for genetic mapping and QTL analysis in kiwifruit.

Kiwifruit (Actinidia spp) is a woody, perennial and deciduous vine. In this genus, there are multiple ploidy levels but the main cultivated cultivars are polyploid. Despite the availability of many genomic resources in kiwifruit, SNP genotyping is still a challenge given these different levels of polyploidy. Recent advances in SNP array technologies have offered a high-throughput genotyping platform for genome-wide DNA polymorphisms. In this study, we developed a high-density SNP genotyping array to facilitate genetic studies and breeding applications in kiwifruit. SNP discovery was performed by genome-wide DNA sequencing of 40 kiwifruit genotypes. The identified SNPs were stringently filtered for sequence quality, predicted conversion performance and distribution over the available Actinidia chinensis genome. A total of 134 729 unique SNPs were put on the array. The array was evaluated by genotyping 400 kiwifruit individuals. We performed a multidimensional scaling analysis to assess the diversity of kiwifruit germplasm, showing that the array was effective to distinguish kiwifruit accessions. Using a tetraploid F1 population, we constructed an integrated linkage map covering 3060.9 cM across 29 linkage groups and performed QTL analysis for the sex locus that has been identified on Linkage Group 3 (LG3) in Actinidia arguta. Finally, our dataset presented evidence of tetrasomic inheritance with partial preferential pairing in A. arguta. In conclusion, we developed and evaluated a 135K SNP genotyping array for kiwifruit. It has the advantage of a comprehensive design that can be an effective tool in genetic studies and breeding applications in this high-value crop.

Allelic to genome wide perspectives of swine genetic variation to litter size and its component traits.

Litter size is a complex and sex limited trait that depends on various biological, managemental and environmental factors. Owing to its low heritability it is inefficaciously selected by traditional methods. However, due to higher heritability of ovulation rate and embryo survival, selection based on component traits of litter size is advocated. QTL analysis and candidate gene approach are among the various supplementary/alternate strategies for selection of litter size. QTL analysis is aimed at identifying genomic regions affecting trait of interest significantly. Candidate gene approach necessitates identification of genes potentially affecting the trait. There are various genes that significantly affect litter size and its component traits viz. ESR, LEP, BF, IGFBP, RBP4, PRLR, CTNNAL1, WNT10B, TCF12, DAZ, and RNF4. These genes affect litter size in a complex interacting manner. Lately, genome wide association study (GWAS) have been utilized to unveil the genetic and biological background of litter traits, and elucidate the genes governing litter size. Favorable SNPs in these genes have been identified and offers a scope for inclusion in selection programs thereby increasing breeding efficiency and profit in pigs. The review provides a comprehensive coverage of investigations carried out globally to unravel the genetic variation in litter size and its component traits in pigs, both at allelic and genome wide level. It offers a current perspective on different strategies including the profiling of candidate genes, QTLs, and genome wide association studies as an aid to efficient selection for litter size and its component traits.

Wild and cultivated allele effects on rice phenotypic traits in reciprocal backcross populations between Oryza rufipogon and two cultivars, O. sativa Nipponbare and IR36.

A total of four populations of reciprocal backcross recombinant inbred lines were produced from a cross between a wild accession of Oryza rufipogon W630 and two major cultivars, O. sativa Japonica Nipponbare and Indica IR36. Using these populations, quantitative trait locus (QTL) analysis for eight morphological traits (culm length, panicle length, days to heading, panicle shape, pericarp color, hull color, seed shattering and seed awning) was carried out, and the putative QTL regions were compared among the populations. The QTLs with strong allele effects were commonly detected for culm length, panicle shape, pericarp color and hull color in all four populations, and their peak locations were close to the major genes of sd1, Spr3, Rc and Bh4, respectively. For panicle length and days to heading, some QTL regions overlapped between two or three populations. In the case of seed shattering and seed awning, strong wild allele effects at major loci were observed only in the populations with cultivated backgrounds. Since the wild and cultivated alleles have never been evaluated in the reciprocal genetic backgrounds, the present results provide new information on gene effects in breeding and domestication studies.

QTL Mapping Reveals Both All-Stage and Adult-Plant Resistance to Powdery Mildew in Chinese Elite Wheat Cultivars.

Powdery mildew caused by Blumeria graminis f. sp. tritici is a threat to wheat production in China. Mapping quantitative trait loci (QTL) for resistance to powdery mildew and developing breeder-friendly markers are important initial steps in breeding resistant cultivars. An all-stage resistance gene and several QTL were identified using a population of 254 recombinant inbred lines developed from a Jingdong 8/Aikang 58 cross. The population was evaluated for powdery mildew resistance across six field environments over three consecutive growing seasons utilizing two different mixtures of B. graminis f. sp. tritici isolates, named #Bgt-HB and #Bgt-BJ. Using genotypic data obtained from the Wheat TraitBreed 50K single-nucleotide polymorphism array, seven stable QTL were identified on chromosome arms 1DL, 2AL, 2DS, 4DL, 5AL, 6BL.1, and 6BL.2. The QTL on 2AL conferred all-stage resistance to B. graminis f. sp. tritici race E20 in greenhouse tests and explained up to 52% of the phenotypic variance in field trials but was resistant only against #Bgt-HB. The gene involved in this QTL was predicted to be Pm4a based on genome location and gene sequence. QPmja.caas-1DL, QPmja.caas-4DL, and QPmja.caas-6BL.1 were identified as potentially new QTL for powdery mildew resistance. QPmja.caas-2DS and QPmja.caas-6BL.1 were effective against both B. graminis f. sp. tritici mixtures, indicating their probable broad-spectrum resistance. A Kompetitive allele-specific PCR marker closely linked to QPmja.caas-2DS was developed and validated in a panel of 286 wheat cultivars. Because both Jingdong 8 and Aikang 58 have been leading cultivars and breeding parents, the QTL and marker reported represent valuable resources for wheat researchers and breeders.

Combining Genetic and Transcriptomic Approaches to Identify Transporter-Coding Genes as Likely Responsible for a Repeatable Salt Tolerance QTL in Citrus.

The excessive accumulation of chloride (Cl-) in leaves due to salinity is frequently related to decreased yield in citrus. Two salt tolerance experiments to detect quantitative trait loci (QTLs) for leaf concentrations of Cl-, Na+, and other traits using the same reference progeny derived from the salt-tolerant Cleopatra mandarin (Citrus reshni) and the disease-resistant donor Poncirus trifoliata were performed with the aim to identify repeatable QTLs that regulate leaf Cl- (and/or Na+) exclusion across independent experiments in citrus, as well as potential candidate genes involved. A repeatable QTL controlling leaf Cl- was detected in chromosome 6 (LCl-6), where 23 potential candidate genes coding for transporters were identified using the C. clementina genome as reference. Transcriptomic analysis revealed two important candidate genes coding for a member of the nitrate transporter 1/peptide transporter family (NPF5.9) and a major facilitator superfamily (MFS) protein. Cell wall biosynthesis- and secondary metabolism-related processes appeared to play a significant role in differential gene expression in LCl-6. Six likely gene candidates were mapped in LCl-6, showing conserved synteny in C. reshni. In conclusion, markers to select beneficial Cleopatra mandarin alleles of likely candidate genes in LCl-6 to improve salt tolerance in citrus rootstock breeding programs are provided.

Genetic architecture of berry aroma compounds in a QTL (quantitative trait loci) mapping population of interspecific hybrid grapes (Vitis labruscana × Vitis vinifera).

BACKGROUND: Although grapes accumulate diverse groups of volatile compounds, their genetic regulation in different cultivars remains unelucidated. Therefore, this study investigated the volatile composition in the berries of an interspecific hybrid population from a Vitis labruscana 'Campbell Early' (CE) × Vitis vinifera 'Muscat of Alexandria' (MA) cross to understand the relationship among volatile compounds and their genetic regulation. Then, a quantitative trait locus (QTL) analysis of its volatile compounds was conducted. RESULTS: While MA contained higher concentrations of monoterpenes and norisoprenoids, CE contained higher concentrations of C6 compounds, lactones and shikimic acid derivatives, including volatiles characteristic to American hybrids, i.e., methyl anthranilate, o-aminoacetophenone and mesifurane. Furthermore, a cluster analysis of volatile profiles in the hybrid population discovered ten coordinately modulated free and bound volatile clusters. QTL analysis identified a major QTL on linkage group (LG) 5 in the MA map for 14 monoterpene concentrations, consistent with a previously reported locus. Additionally, several QTLs detected in the CE map affected the concentrations of specific monoterpenes, such as linalool, citronellol and 1,8-cineol, modifying the monoterpene composition in the berries. As for the concentrations of five norisoprenoids, a major common QTL on LG2 was discovered first in this study. Several QTLs with minor effects were also discovered in various volatile groups, such as lactones, alcohols and shikimic acid derivatives. CONCLUSIONS: An overview of the profiles of aroma compounds and their underlying QTLs in a population of interspecific hybrid grapes in which muscat flavor compounds and many other aroma compounds were mixed variously were elucidated. Coordinate modulation of the volatile clusters in the hybrid population suggested an independent mechanism for controlling the volatiles of each group. Accordingly, specific QTLs with significant effects were observed for terpenoids, norisoprenoids and some volatiles highly contained in CE berries.

Construction of a high-density genetic map using specific-length amplified fragment markers and identification of QTLs for branching angle in poplar.

Branching angle is a critical factor that determines the morphological establishment and is a typical quantitative trait controlled by multiple genes. In this study, we used SLAF-seq to construct a high-density genetic map, to investigate the genetic architecture of branching angle in poplar (Populus leucopyramidalis). A total of 240,672 SLAF tags were obtained, including 103,691 polymorphic SLAF tags. After filtering, 53,407 polymorphic markers were sorted into eight segregation types, and 11,162 of them were used to construct the genetic map. 8447 were on the female parent map, 8532 were on the male parent map, and 11,162 were on the integrated map. The marker coverage was 4820.84 and 5044.80 cM for the female and male maps, and 3142.61 cM for the integrated map. The average intervals between two adjacent mapped markers were 0.55, 0.59, and 0.28 cM for the three maps, respectively. Two quantitative trait loci (QTLs) were detected. Seven markers that exceeded the threshold in these two regions were considered as being associated with branching angle and the phenotypic variance explained by each of these marker was 10.64-11.66%. After functional annotation, we identified 15 candidate genes and analyzed the expression of candidate genes in narrow and wide crown progenies by qRT-PCR. These results show that the combination of QTL and SLAF-seq will contribute to future breeding plans in poplar breeding, especially in narrow crown poplar breeding.

Polygenic Analysis of Tolerance to Carbon Dioxide Inhibition of Isoamyl Acetate "Banana" Flavor Production in Yeast Reveals MDS3 as Major Causative Gene.

The introduction in modern breweries of tall cylindroconical fermentors, replacing the traditional open fermentation vats, unexpectedly revealed strong inhibition of flavor production by the high CO2 pressure in the fermentors. We have screened our collection of Saccharomyces cerevisiae strains for strains displaying elevated tolerance to inhibition of flavor production by +0.65 bar CO2, using a laboratory scale CO2 pressurized fermentation system. We focused on the production of isoamyl acetate, a highly desirable flavor compound conferring fruity banana flavor in beer and other alcoholic beverages, from its precursor isoamyl alcohol (IAAc/Alc ratio). We selected the most tolerant Saccharomyces cerevisiae strain, saké yeast Kyokai no. 1, isolated a stable haploid segregant seg63 with the same high IAAc/Alc ratio under CO2 pressure, crossed seg63 with the unrelated inferior strain ER7A and phenotyped 185 haploid segregants, of which 28 displaying a high IAAc/Alc ratio were pooled. Mapping of Quantitative Trait Loci (QTLs) by whole-genome sequence analysis based on SNP variant frequency revealed two QTLs. In the major QTL, reciprocal hemizygosity analysis identified MDS3 as the causative mutant gene, a putative member of the TOR signaling pathway. The MDS3Seg.63 allele was dominant and contained a single causative point mutation, T2171C, resulting in the F274S substitution. Introduction of MDS3Seg.63 in an industrial tetraploid lager yeast with CRISPR/Cas9 enhanced isoamyl acetate production by 145% under CO2 pressure. This work shows the strong potential of polygenic analysis and targeted genetic modification for creation of cisgenic industrial brewer's yeast strains with specifically improved traits. IMPORTANCE The upscaling of fermentation to very tall cylindroconical tanks is known to negatively impact beer flavor. Most notably, the increased CO2 pressure in such tanks compromises production by the yeast of the desirable fruity "banana" flavor (isoamyl acetate). The cause of the CO2 inhibition of yeast flavor production has always remained enigmatic. Our work has brought the first insight into its molecular-genetic basis and provides a specific gene tool for yeast strain improvement. We first identified a yeast strain with superior tolerance to CO2 inhibition of flavor production, and applied polygenic analysis to identify the responsible gene. We narrowed down the causative element to a single nucleotide difference, MDS3T2171C, and showed that it can be engineered into brewing yeast to obtain strains with superior flavor production in high CO2 pressure conditions, apparently without affecting other traits relevant for beer brewing. Alternatively, such a strain could be obtained through marker-assisted breeding.

The genetic architecture of flowering time changes in pea from wild to crop.

Change in phenology has been an important component in crop evolution, and selection for earlier flowering through a reduction in environmental sensitivity has helped broaden adaptation in many species. Natural variation for flowering in domesticated pea (Pisum sativum L.) has been noted and studied for decades, but there has been no clear account of change relative to its wild progenitor. Here we examined the genetic control of differences in flowering time between wild P. sativum ssp. humile and a typical late-flowering photoperiodic P. s. sativum accession in a recombinant inbred population under long and short photoperiods. Our results confirm the importance of the major photoperiod sensitivity locus Hr/PsELF3a and identify two other loci on chromosomes 1 (DTF1) and 3 (DTF3) that contribute to earlier flowering in the domesticated line under both photoperiods. The domesticated allele at a fourth locus on chromosome 6 (DTF6) delays flowering under long days only. Map positions, inheritance patterns, and expression analyses in near-isogenic comparisons imply that DTF1, DTF3, and DTF6 represent gain-of-function alleles of the florigen/antiflorigen genes FTa3, FTa1, and TFL1c/LF, respectively. This echoes similar variation in chickpea and lentil, and suggests a conserved route to reduced photoperiod sensitivity and early phenology in temperate pulses.

Genetic analysis of early phenology in lentil identifies distinct loci controlling component traits.

Modern-day domesticated lentil germplasm is generally considered to form three broad adaptation groups: Mediterranean, South Asian, and northern temperate, which correspond to the major global production environments. Reproductive phenology plays a key role in lentil adaptation to this diverse ecogeographic variation. Here, we dissect the characteristic earliness of the pilosae ecotype, suited to the typically short cropping season of South Asian environments. We identified two loci, DTF6a and DTF6b, at which dominant alleles confer early flowering, and we show that DTF6a alone is sufficient to confer early flowering under extremely short photoperiods. Genomic synteny confirmed the presence of a conserved cluster of three florigen (FT) gene orthologues among potential candidate genes, and expression analysis in near-isogenic material showed that the early allele is associated with a strong derepression of the FTa1 gene in particular. Sequence analysis revealed a 7.4 kb deletion in the FTa1-FTa2 intergenic region in the pilosae parent, and a wide survey of >350 accessions with diverse origin showed that the dtf6a allele is predominant in South Asian material. Collectively, these results contribute to understanding the molecular basis of global adaptation in lentil, and further emphasize the importance of this conserved genomic region for adaptation in temperate legumes generally.

Genetic control of the operculum and capsule morphology of Eucalyptus globulus.

BACKGROUND AND AIMS: The petaline operculum that covers the inner whorls until anthesis and the woody capsule that develops after fertilization are reproductive structures of eucalypts that protect the flower and seeds. Although they are distinct organs, they both develop from flower buds and this common ontogeny suggests shared genetic control. In Eucalyptus globulus their morphology is variable and we aimed to identify the quantitative trait loci (QTL) underlying this variation and determine whether there is common genetic control of these ecologically and taxonomically important reproductive structures. METHODS: Samples of opercula and capsules were collected from 206 trees that belong to a large outcrossed F2E. globulus mapping population. The morphological variation in these structures was characterized by measuring six operculum and five capsule traits. QTL analysis was performed using these data and a linkage map consisting of 480 markers. KEY RESULTS: A total of 27 QTL were detected for operculum traits and 28 for capsule traits, with the logarithm of odds ranging from 2.8 to 11.8. There were many co-located QTL associated with operculum or capsule traits, generally reflecting allometric relationships. A key finding was five genomic regions where co-located QTL affected both operculum and capsule morphology, and the overall trend for these QTL was to affect elongation of both organs. Some of these QTL appear to have a significant effect on the phenotype, with the strongest QTL explaining 26.4 % of the variation in operculum shape and 16.4 % in capsule shape. Flower bud measurements suggest the expression of these QTL starts during bud development. Several candidate genes were found associated with the QTL and their putative function is discussed. CONCLUSIONS: Variation in both operculum and capsule traits in E. globulus is under strong genetic control. Our results suggest that these reproductive structures share a common genetic pathway during flower bud development.