Efficient identification of genomic insertions and flanking regions through whole-genome sequencing in three transgenic soybean events

Transgenic Research - Genomic insertions and flanking regions of transgenes in host genomes constitute a critical component of precise molecular characterization and event-specific detection, which...     

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.     

An informative linkage map of soybean reveals QTLs for flowering time, leaflet morphology and regions of segregation distortion.

A genetic linkage map covering a large region of the genome with informative markers is essential for plant genome analysis, including identification of quantitative trait loci (QTLs), map-based cloning, and construction of a physical map. We constructed a soybean genetic linkage map using 190 F2 plants derived from a single cross between the soybean varieties Misuzudaizu and Moshidou Gong 503, based on restriction-fragment-length polymorphisms (RFLPs) and simple-sequence-repeat polymorphisms (SSRPs). This linkage map has 503 markers, including 189 RFLP markers derived from expressed sequence tag (EST) clones, and consists of 20 major linkage groups that may correspond to the 20 pairs of soybean chromosomes, covering 2908.7 cM of the soybean genome in the Kosambi function. Using this linkage map, we identified 4 QTLs--FT1, FT2, FT3, and FT4--for flowering time, the QTLs for the 5 largest principal components determining leaflet shape, 6 QTLs for single leaflet area, and 18 regions of segregation distortion. All 503 analyzed markers identified were located on the map, and almost all phenotypic variations in flowering time were explained by the detected QTLs. These results indicate that this map covers a large region of the soybean genome.     

Changes in twelve homoeologous genomic regions in soybean following three rounds of polyploidy

With the advent of high-throughput sequencing, the availability of genomic sequence for comparative genomics is increasing exponentially. Numerous completed plant genome sequences enable characterization of patterns of the retention and evolution of genes within gene families due to multiple polyploidy events, gene loss and fractionation, and differential evolutionary pressures over time and across different gene families. In this report, we trace the changes that have occurred in 12 surviving homoeologous genomic regions from three rounds of polyploidy that contributed to the current Glycine max genome: a genome triplication before the origin of the rosids (~130 to 240 million years ago), a genome duplication early in the legumes (~58 million years ago), and a duplication in the Glycine lineage (~13 million years ago). Patterns of gene retention following the genome triplication event generally support predictions of the Gene Balance Hypothesis. Finally, we find that genes in networks with a high level of connectivity are more strongly conserved than those with low connectivity and that the enrichment of these highly connected genes in the 12 highly conserved homoeologous segments may in part explain their retention over more than 100 million years and repeated polyploidy events.     

An augmented Arabidopsis phenology model reveals seasonal temperature control of flowering time

? In this study, we used a combination of theoretical (models) and experimental (field data) approaches to investigate the interaction between light and temperature signalling in the control of Arabidopsis flowering. ? We utilised our recently published phenology model that describes the flowering time of Arabidopsis grown under a range of field conditions. We first examined the ability of the model to predict the flowering time of field plantings at different sites and seasons in light of the specific meteorological conditions that pertained. ? Our analysis suggested that the synchrony of temperature and light cycles is important in promoting floral initiation. New features were incorporated into the model that improved its predictive accuracy across seasons. Using both laboratory and field data, our study has revealed an important seasonal effect of night temperatures on flowering time. Further model adjustments to describe phytochrome (phy) mutants supported our findings and implicated phyB in the temporal gating of temperature-induced flowering. ? Our study suggests that different molecular pathways interact and predominate in natural environments that change seasonally. Temperature effects are mediated largely during the photoperiod during spring/summer (long days) but, as days shorten in the autumn, night temperatures become increasingly important.     

QTL mapping of naturally-occurring variation in flowering time of Arabidopsis thaliana

A segregating F₂ population of Arabidopsis thaliana derived from a cross between the late-flowering ecotype Hannover/Münden (HM) and the early-flowering ecotype Wassilewskija (WS) was analyzed for flowering time and other morphological traits. Two unlinked quantitative trait loci (QTLs) affecting days to first flower (DFF-a and DFF-b) mapped to chromosome 5. QTLs which affect node number (NN), leaf length at flowering (LLF), and leaf length at 35 days (LL35) also mapped to chromosome 5; LLF-a, LL35-a, NN-a map to the same region of chromosome 5 as DFF-a; LLF-b and LL35-bmap to the same region of chromosome 5 as DFF-b. Another QTL affecting leaf length at flowering (LLF-c) maps to chromosome 3. The proximity of DFF-a, LLF-a, LL35-a and NN-a, as well as the similarity in gene action among these QTLs (additivity), suggest that they may be pleiotropic consequences of a single gene at this locus. Similarly, LL35-b and LLF-b map near each other and both display recessive gene action, again suggesting the possibility of pleiotropy. DFF-b, which also maps near LL35-b and LLF-b, displays largely additive gene action (although recessive gene action could not be ruled out). This suggests that DFF-b may represent a different gene from LL35-b and/or LLF-b. DFF-a maps near two previously identified mutants: co (which also affects flowering time and displays gene action consistent with additivity) and flc. Similar map locations and gene actions of QTLs affecting the correlated traits DFF, LLF, LL35 and NN suggest that these genomic regions harbor naturally occurring allelic variants involved in the general transition of the plant from vegetative to reproductive growth.     

QTL mapping of root traits in phosphorus-deficient soils reveals important genomic regions for improving NDVI and grain yield in barley

Key message

Major QTLs for root rhizosheath size are not correlated with grain yield or yield response to phosphorus. Important QTLs were found to improve phosphorus efficiency.

Abstract

Root traits are important for phosphorus (P) acquisition, but they are often difficult to characterize and their breeding values are seldom assessed under field conditions. This has shed doubts on using seedling-based criteria of root traits to select and breed for P efficiency. Eight root traits were assessed under controlled conditions in a barley doubled-haploid population in soils differing in P levels. The population was also phenotyped for grain yield, normalized difference vegetation index (NDVI), grain P uptake and P utilization efficiency at maturity (PutE_GY) under field conditions. Several quantitative traits loci (QTLs) from the root screening and the field trials were co-incident. QTLs for root rhizosheath size and root diameter explained the highest phenotypic variation in comparison to QTLs for other root traits. Shared QTLs were found between root diameter and grain yield, and total root length and PutE_GY. A common major QTL for rhizosheath size and NDVI was mapped to the HvMATE gene marker on chromosome 4H. Collocations between major QTLs for NDVI and grain yield were detected on chromosomes 6H and 7H. When results from BIP and MET were combined, QTLs detected for grain yield were also those QTLs found for NDVI. QTLs qGY5H, qGY6H and qGY7Hb on 7H were robust QTLs in improving P efficiency. A selection of multiple loci may be needed to optimize the breeding outcomes due to the QTL x Environment interaction. We suggest that rhizosheath size alone is not a reliable trait to predict P efficiency or grain yield.

     

Natural variation in GmGBP1 promoter affects photoperiod control of flowering time and maturity in soybean

The present study screened for polymorphisms in coding and non‐coding regions of the GmGBP1 gene in 278 soybean accessions with variable maturity and growth habit characteristics under natural field conditions in three different latitudes in China. The results showed that the promoter region was highly diversified compared with the coding sequence of GmGBP1. Five polymorphisms and four haplotypes were closely related to soybean flowering time and maturity through association and linkage disequilibrium analyses. Varieties with the polymorphisms SNP_‐796G, SNP_‐770G, SNP_‐307T, InDel_‐242normal, SNP_353A, or haplotypes Hap‐3 and Hap‐4 showed earlier flowering time and maturity in different environments. The shorter growth period might be largely due to higher GmGBP1 expression levels in soybean that were caused by the TCT‐motif with SNP_‐796G in the promoter. In contrast, the lower expression level of GmGBP1 in soybean caused by RNAi interference of GmGBP1 resulted in a longer growth period under different day lengths. Furthermore, the gene interference of GmGBP1 also caused a reduction in photoperiod response sensitivity (PRS) before flowering in soybean. RNA‐seq analysis on GmGBP1 underexpression in soybean showed that 94 and 30 predicted genes were significantly upregulated and downregulated, respectively. Of these, the diurnal photoperiod‐specific expression pattern of three significant flowering time genes GmFT2a, GmFT5a, and GmFULc also showed constantly lower mRNA levels in GmGBP1‐i soybean than in wild type, especially under short day conditions. Together, the results showed that GmGBP1 functioned as a positive regulator upstream of GmFT2a and GmFT5a to activate the expression of GmFULc to promote flowering on short days.     

Dissection of a QTL reveals an adaptive, interacting gene complex associated with transgressive variation for flowering time in rice

A days to heading QTL (dth1.1) located on the short arm of rice chromosome 1 was sub-divided into eight sub-introgression lines (SILs) to analyze the genetic basis of transgressive variation for flowering time. Each SIL contained one or more introgression(s) from O. rufipogon in the genetic background of the elite Oryza sativa cultivar, Jefferson. Each introgression was defined at high resolution using molecular markers and those in the dth1.1 region were associated with the presence of one or more flowering time genes (GI, SOC1, FT-L8, EMF1, and PNZIP). SILs and controls were evaluated for flowering time under both short- and long-day growing conditions. Under short-day lengths, lines with introgressions carrying combinations of linked flowering time genes (GI/SOC1, SOC1/FT-L8, GI/SOC1/FT-L8 and EMF1/PNZIP) from the late parent, O. rufipogon, flowered earlier than the recurrent parent, Jefferson, while recombinant lines carrying smaller introgressions marked by the presence of GI, SOC1, EMF1 or PNZIP alone no longer flowered early. Under long-day length, lines carrying SOC1/FT-L8, SOC1 or PNZIP flowered early, while those carrying GI or EMF1 delayed flowering. Across all experiments and in the field, only SIL_SOC1/FT-L8 was consistently early. A preliminary yield evaluation indicated that the transgressive early flowering observed in several of the SILs was also associated with a measurable and positive effect on yield. These SILs represent a new source of variation that can be used in breeding programs to manipulate flowering time in rice cultivars without the reduction in yield that is often associated with early maturing phenotypes.     

QTL analysis of flowering time and ripening traits suggests an impact of a genomic region on linkage group 1 in Vitis

In the recent past, genetic analyses of grapevine focused mainly on the identification of resistance loci for major diseases such as powdery and downy mildew. Currently, breeding programs make intensive use of these results by applying molecular markers linked to the resistance traits. However, modern genetics also allows to address additional agronomic traits that have considerable impact on the selection of grapevine cultivars. In this study, we have used linkage mapping for the identification and characterization of flowering time and ripening traits in a mapping population from a cross of V3125 (‘Schiava Grossa’ × ‘Riesling’) and the interspecific rootstock cultivar ‘Börner’ (Vitis riparia × Vitis cinerea). Comparison of the flowering time QTL mapping with data derived from a second independent segregating population identified several common QTLs. Especially a large region on linkage group 1 proved to be of special interest given the genetic divergence of the parents of the two populations. The proximity of the QTL region contains two CONSTANS-like genes. In accordance with data from other plants such as Arabidopsis thaliana and Oryza sativa, we hypothesize that these genes are major contributors to control the time of flowering in Vitis.     

Detection of QTL for flowering time in multiple families of elite maize

Flowering time is a fundamental quantitative trait in maize that has played a key role in the postdomestication process and the adaptation to a wide range of climatic conditions. Flowering time has been intensively studied and recent QTL mapping results based on diverse founders suggest that the genetic architecture underlying this trait is mainly based on numerous small-effect QTL. Here, we used a population of 684 progenies from five connected families to investigate the genetic architecture of flowering time in elite maize. We used a joint analysis and identified nine main effect QTL explaining approximately 50?% of the genotypic variation of the trait. The QTL effects were small compared with the observed phenotypic variation and showed strong differences between families. We detected no epistasis with the genetic background but four digenic epistatic interactions in a full 2-dimensional genome scan. Our results suggest that flowering time in elite maize is mainly controlled by main effect QTL with rather small effects but that epistasis may also contribute to the genetic architecture of the trait.     

QTL mapping reveals a striking coincidence in the positions of genomic regions associated with adaptive variation in body size in parallel clines of Drosophila melanogaster on different continents

Latitudinal genetic clines in body size are common in many ectotherm species and are attributed to climatic adaptation. Here, we use Quantitative Trait Loci (QTL) mapping to identify genomic regions associated with adaptive variation in body size in natural populations of Drosophila melanogaster from extreme ends of a cline in South America. Our results show that there is a significant association between the positions of QTL with strong effects on wing area in South America and those previously reported in a QTL mapping study of Australian cline end populations (P < 0.05). In both continents, the right arm of the third chromosome is associated with QTL with the strongest effect on wing area. We also show that QTL peaks for wing area and thorax length are associated with the same genomic regions, indicating that the clinal variation in the body size traits may have a similar genetic basis. The consistency of the results found for the South American and Australian cline end populations indicate that the genetic basis of the two clines may be similar and future efforts to identify the genes producing the response to selection should be focused on the genomic regions highlighted by the present work.     

Population-based resequencing reveals that the flowering time adaptation of cultivated barley originated east of the Fertile Crescent

Gene resequencing and association analysis present new opportunities to study the evolution of adaptive traits in crop plants. Here we apply these tools to an extensive set of barley accessions to identify a component of the molecular basis of the flowering time adaptation, a trait critical to plant survival. Using an association-based study to relate variation in flowering time to sequence-based polymorphisms in the Ppd-H1 gene, we identify a causative polymorphism (SNP48) that accounts for the observed variation in barley flowering time. This polymorphism also shows latitude-dependent geographical distribution, consistent with the expected clinal variation in phenotype with the nonresponsive form predominating in the north. Networks, genealogies, and phylogenetic trees drawn for the Ppd-H1 haplotypes reveal population structure both in wild barley and in domesticated barley landraces. The spatial distribution of these population groups indicates that phylogeographical analysis of European landraces can provide information relevant to the Neolithic spread of barley cultivation and also has implications for the origins of domesticated barley, including those with the nonresponsive ppd-H1 phenotype. Haplotypes containing the nonresponsive version of SNP48 are present in wild barley accessions, indicating that the nonresponsive phenotype of European landraces originated in wild barley. The wild accessions whose nonresponsive haplotypes are most closely similar to those of landraces are found in Iran, within a region suggested as an area for domestication of barley east of the Fertile Crescent but which has previously been thought to have contributed relatively little to the diversity of European cultivars.     

QTL mapping of naturally-occurring variation in flowering time of Arabidopsis thaliana

A segregating F₂ population of Arabidopsis thaliana derived from a cross between the late-flowering ecotype Hannover/Münden (HM) and the early-flowering ecotype Wassilewskija (WS) was analyzed for flowering time and other morphological traits. Two unlinked quantitative trait loci (QTLs) affecting days to first flower (DFF-a and DFF-b) mapped to chromosome 5. QTLs which affect node number (NN), leaf length at flowering (LLF), and leaf length at 35 days (LL35) also mapped to chromosome 5; LLF-a, LL35-a, NN-a map to the same region of chromosome 5 as DFF-a; LLF-b and LL35-bmap to the same region of chromosome 5 as DFF-b. Another QTL affecting leaf length at flowering (LLF-c) maps to chromosome 3. The proximity of DFF-a, LLF-a, LL35-a and NN-a, as well as the similarity in gene action among these QTLs (additivity), suggest that they may be pleiotropic consequences of a single gene at this locus. Similarly, LL35-b and LLF-b map near each other and both display recessive gene action, again suggesting the possibility of pleiotropy. DFF-b, which also maps near LL35-b and LLF-b, displays largely additive gene action (although recessive gene action could not be ruled out). This suggests that DFF-b may represent a different gene from LL35-b and/or LLF-b. DFF-a maps near two previously identified mutants: co (which also affects flowering time and displays gene action consistent with additivity) and flc. Similar map locations and gene actions of QTLs affecting the correlated traits DFF, LLF, LL35 and NN suggest that these genomic regions harbor naturally occurring allelic variants involved in the general transition of the plant from vegetative to reproductive growth.     

Environmental control of flowering time in Antirrhinum majus

The effect of different environmental conditions on flowering time and the number of leaves produced before the first flower is formed has been investigated in Antirrhinum majus L. The effect of light quality has been tested by decreasing the red/far‐red ratio, generally resulting in a reduced flowering time and leaf number. Furthermore, it could be shown that photoperiod, temperature and light intensity are inversely correlated with flowering time and leaf number. However, lowering the temperature from 15 to 12°C resulted in a reduction of flowering time. This observation shows that Antirrhinum can be vernalised.
Using defined combinations of the four environmental factors we have been able to reduce flowering time to only 42 days or to delay flowering for at least 2 years. The results obtained allow an optimisation of the screening conditions for identifying flowering time mutants in Antirrhinum .     

Identification of putative QTL that underlie yield in interspecific soybean backcross populations

Glycine soja, the wild progenitor of soybean, is a potential source of useful genetic variation in soybean improvement. The objective of our study was to map quantitative trait loci (QTL) from G. soja that could improve the crop. Five populations of BC₂F₄-derived lines were developed using the Glycine max cultivar IA2008 as a recurrent parent and the G. soja plant introduction (PI) 468916 as a donor parent. There were between 57 and 112 BC₂F₄-derived lines in each population and a total of 468 lines for the five populations. The lines were evaluated with simple sequence repeat markers and in field tests for yield, maturity, plant height, and lodging. The field testing was done over 2 years and at two locations each year. Marker data were analyzed for linkage and combined with field data to identify QTL. Using an experimentwise significance threshold of P=0.05, four yield QTL were identified across environments on linkage groups C2, E, K, and M. For these yield QTL, the IA2008 marker allele was associated with significantly greater yield than the marker allele from G. soja. In addition, one lodging QTL, four maturity QTL, and five QTL for plant height were identified across environments. Of the 14 QTL identified, eight mapped to regions where QTL with similar effects were previously mapped. Many regions carrying the yield QTL were also significant for other traits, such as plant height and lodging. When the significance threshold was reduced and the data were analyzed with simple linear regression, four QTL with a positive allele for yield from G. soja were mapped. One epistatic interaction between two genetic regions was identified for yield using an experimentwise significance threshold of P=0.05. Additional research is needed to establish whether multiple trait associations are the result of pleiotropy or genetic linkage and to retest QTL with a positive effect from G. soja.Communicated by H.C. Becker     

Extent and distribution of linkage disequilibrium in three genomic regions

The positional cloning of genes underlying common complex diseases relies on the identification of linkage disequilibrium (LD) between genetic markers and disease. We have examined 127 polymorphisms in three genomic regions in a sample of 575 chromosomes from unrelated individuals of British ancestry. To establish phase, 800 individuals were genotyped in 160 families. The fine structure of LD was found to be highly irregular. Forty-five percent of the variation in disequilibrium measures could be explained by physical distance. Additional factors, such as allele frequency, type of polymorphism, and genomic location, explained <5% of the variation. Nevertheless, disequilibrium was occasionally detectable at 500 kb and was present for over one-half of marker pairs separated by <50 kb. Although these findings are encouraging for the prospects of a genomewide LD map, they suggest caution in interpreting localization due to allelic association.