Segregation distortion in Arabidopsis C24/Col-0 and Col-0/C24 recombinant inbred line populations is due to reduced fertility caused by epistatic interaction of two loci

A new large set of reciprocal recombinant inbred lines (RILs) was created between the Arabidopsis accessions Col-0 and C24 for quantitative trait mapping approaches, consisting of 209 Col-0 x C24 and 214 C24 x Col-0 F(7 )RI lines. Genotyping was performed using 110 evenly distributed framework single nucleotide polymorphism markers, yielding a genetic map of 425.70 cM, with an average interval of 3.87 cM. Segregation distortion (SD) was observed in several genomic regions during the construction of the genetic map. Linkage disequilibrium analysis revealed an association between a distorted region at the bottom of chromosome V and a non-distorted region on chromosome IV. A detailed analysis of the RILs for these two regions showed that an SD occurred when homozygous Col-0 alleles on chromosome IV coincided with homozygous C24 alleles at the bottom of chromosome V. Using nearly isogenic lines segregating for the distorted region we confirmed that this genotypic composition leads to reduced fertility and fitness.     

A collection of INDEL markers for map-based cloning in seven Arabidopsis accessions

The availability of a comprehensive set of resources including an entire annotated reference genome, sequenced alternative accessions, and a multitude of marker systems makes Arabidopsis thaliana an ideal platform for genetic mapping. PCR markers based on INsertions/DELetions (INDELs) are currently the most frequently used polymorphisms. For the most commonly used mapping combination, Columbia×Landsberg erecta (Col-0×Ler-0), the Cereon polymorphism database is a valuable resource for the generation of polymorphic markers. However, because the number of markers available in public databases for accessions other than Col-0 and Ler-0 is extremely low, mapping using other accessions is far from straightforward. This issue arose while cloning mutations in the Wassilewskija (Ws-4) background. In this work, approaches are described for marker generation in Ws-4 x Col-0. Complementary strategies were employed to generate 229 INDEL markers. Firstly, existing Col-0/Ler-0 Cereon predicted polymorphisms were mined for transferability to Ws-4. Secondly, Ws-0 ecotype Illumina sequence data were analyzed to identify INDELs that could be used for the development of PCR-based markers for Col-0 and Ws-4. Finally, shotgun sequencing allowed the identification of INDELs directly between Col-0 and Ws-4. The polymorphism of the 229 markers was assessed in seven widely used Arabidopsis accessions, and PCR markers that allow a clear distinction between the diverged Ws-0 and Ws-4 accessions are detailed. The utility of the markers was demonstrated by mapping more than 35 mutations in a Col-0×Ws-4 combination, an example of which is presented here. The potential contribution of next generation sequencing technologies to more traditional map-based cloning is discussed.     

Establishment of a high-efficiency SNP-based framework marker set for Arabidopsis

The major goal of this project was the establishment of a tool for rapid mapping of new mutations and genotyping in Arabidopsis consisting of at least 100 evenly spaced framework markers. We assembled a single nucleotide polymorphism (SNP)-based marker set consisting of 112 polymorphic sites with average spacing of 1.15 Mbp derived from an SNP database that we recently developed. This information was used to set up efficient SNP detection reactions based on multiplexed primer extension assays. The 112 Columbia (Col-0)/C24 framework markers were used to assemble 18 multiplexed SNaPshot assays with which up to eight separate loci can be genotyped in a single-tube/single-capillary format. In addition, for 110 framework markers matrix-assisted laser desorption/ionization time of flight (MALDI-ToF) assays have been established for high throughput analyses. We demonstrated the usefulness and the robustness of both procedures of this tool by genotyping 48 BC3F1 individuals created between the accessions Col-0 and C24. Subsets of 10-62 of the established markers discriminate between various combinations of the accessions Col-0, C24, Landsberg erecta (Ler), Cape Verdi Islands (Cvi) and Niederzenz (Nd). Using a subset of 17 evenly distributed and established SNP markers that are also polymorphic between Ler and Col-0, we were able to rapidly map a mutant gene (tbr1) to an interval of 2.3 Mbp in an Ler (tbr1) x Col-0 cross.     

Identification of quantitative trait loci in rye introgression lines carrying multiple donor chromosome segments

Introgression libraries can be used to make favorable genetic variation of exotic donor genotypes available in the genetic background of elite breeding material. Our objective was to employ a combination of the Dunnett test and a linear model analysis to identify favorable donor alleles in introgression lines (ILs) that carry long or multiple donor chromosome segments (DCS). We reanalyzed a dataset of two rye introgression libraries that consisted of ILs carrying on average about four donor segments. After identifying ILs that had a significantly better per se or testcross performance than the recipient line with the Dunnett test, the linear model analysis was in most instances able to clearly identify the donor regions that were responsible for the superior performance. The precise localization of the favorable DCS allowed a detailed analysis of pleiotropic effects and the study of the consistency of effects for per se and testcross performance. We conclude that in many cases the linear model analysis allows the assignment of donor effects to individual DCS even for ILs with long or multiple donor segments. This may considerably increase the efficiency of producing sub-ILs, because only such segments need to be isolated that are known to have a significant effect on the phenotype.     

Construction of introgression lines carrying wild rice (Oryza rufipogon Griff.) segments in cultivated rice (Oryza sativa L.) background and characterization of introgressed segments associated with yield-related traits

Introgression lines (ILs) are useful tools for precise mapping of quantitative trait loci (QTLs) and the evaluation of gene action or interaction in theoretical studies. A set of 159 ILs carrying variant introgressed segments from Chinese common wild rice (Oryza rufipogon Griff.), collected from Dongxiang county, Jiangxi Province, in the background of Indica cultivar (Oryza sativa L.), Guichao 2, was developed using 126 polymorphic simple sequence repeats (SSR) loci. The 159 ILs represented 67.5% of the genome of O. rufipogon. All the ILs have the proportions of the recurrent parent ranging from 92.4 to 99.9%, with an average of 97.4%. The average proportion of the donor genome for the BC₄F₄ population was about 2.2%. The mean numbers of homozygous and heterozygous donor segments were 2 (ranging 0–8) and 1 (ranging 0–7), respectively, and the majority of these segments had sizes less than 10 cM. QTL analysis was conducted based on evaluation of yield-related traits of the 159 ILs at two sites, in Beijing and Hainan. For 6 out of 17 QTLs identified at two sites corresponding to three traits (panicles per plant, grains per panicle and filled grains per plant, respectively), the QTLs derived from O. rufipogon were usually associated with an improvement of the target trait, although the overall phenotypic characters of O. rufipogon were inferior to that of the recurrent parent. Of the 17 QTLs, 5 specific QTLs strongly associated with more than one trait were observed. Further analysis of the high-yielding and low-yielding ILs revealed that the high-yielding ILs contained relatively less introgressed segments than the low-yielding ILs, and that the yield increase or decrease was mainly due to the number of grain. On the other hand, low-yielding ILs contained more negative QTLs or disharmonious interactions between QTLs which masked trait-enchancing QTLs. These ILs will be useful in identifying the traits of yield, tolerance to low temperature and drought stress, and detecting favorable genes of common wild rice.     

Identification of salt-tolerant QTLs with strong genetic background effect using two sets of reciprocal introgression lines in rice

Effect of genetic background on detection of quantitative trait locus (QTL) governing salinity tolerance (ST) was studied using two sets of reciprocal introgression lines (ILs) derived from a cross between a moderately salinity tolerant japonica variety, Xiushui09 from China, and a drought tolerant but salinity susceptible indica breeding line, IR2061-520-6-9 from the Philippines. Salt toxicity symptoms (SST) on leaves, days to seedling survival (DSS), and sodium and potassium uptake by shoots were measured under salinity stress of 140?mmol/L of NaCl. A total of 47 QTLs, including 26 main-effect QTLs (M-QTLs) and 21 epistatic QTLs (E-QTLs), were identified from the two sets of reciprocal ILs. Among the 26?M-QTLs, only four (15.4%) were shared in the reciprocal backgrounds while no shared E-QTLs were detected, indicating that ST QTLs, especially E-QTLs, were very specific to the genetic background. Further, 78.6% of the M-QTLs for SST and DSS identified in the reciprocal ILs were also detected in the recombinant inbred lines (RILs) from the same cross, which clearly brings out the background effect on ST QTL detection and its utilization in ST breeding. The detection of ILs with various levels of pyramiding of nonallelic M-QTL alleles for ST from Xiushui09 into IR2061-520-6-9 allowed us to further improve the ST in rice.     

QTLs influencing panicle size detected in two reciprocal introgressive line (IL) populations in rice (Oryza sativa L.)

Two sets of reciprocal introgression line (IL) populations, i.e., ILs with Lemont as recurrent parent (IL_LT) and ILs with Teqing as recurrent parent (IL_TQ), were developed and evaluated for traits representing panicle size, including primary branch number (PBN), secondary branch number (SBN), and spikelet number per panicle (SNP). Together with the regression to recurrent parent by advanced backcross, transgressive segregations were observed for all traits. Correlation and regression analysis showed that SBN had much higher contribution to SNP than PBN. It was confirmed by the QTL analysis that many common loci were detected between SBN and SNP, in comparison with single common locus between PBN and SNP. One and three main effect QTLs (M-QTLs) were detected for PBN in IL_LT and IL_TQ, respectively. Six M-QTLs per trait per populations were associated with SBN and SNP. Less number and lower contribution of epistasis were detected in IL populations in comparison with mapping result from F ₂ or RI population. There were only four QTLs in fourteen loci (near 30%) commonly detected in both reciprocal IL populations implying the large impact of genetic background on QTLs expression. H. W. Mei and J. L. Xu contributed equally to this work.     

Development of Oryza rufipogon and O. sativa Introgression Lines and Assessment for Yield-related Quantitative Trait Loci

Introgression lines population was effectively used in mapping quantitative trait loci （QTLs）, identifying favorable genes, discovering hidden genetic variation, evaluating the action or interaction of QTLs in multiple conditions and providing the favorable experimental materials for plant breeding and genetic research. In this study, an advanced backcross and consecutive selfing strategy was used to develop introgression lines （ILs）, which derived from an accession of Oryza rufipogon Griff. collected from Yuanjiang County, Yunnan Province of China, as the donor, and an elite indica cultivar Teqing （O. sativa L.）, as the recipient. Introgression segments from O. rufipogon were screened using 179 polymorphic simple sequence repeats （SSR） markers in the genome of each IL. Introgressed segments carried by the introgression lines population contained 120 ILs covering the whole O. rufipogon genome. The mean number of homozygous O. rufipogon segments per introgression line was about 3.88. The average length of introgressed segments was approximate 25.5 cM, and about 20.8% of these segments had sizes less than 10 cM. The genome of each IL harbored the chromosomal fragments of O. rufipogon ranging from 0.54% to 23.7%, with an overall average of 5.79%. At each locus, the ratio of substitution of O. rufipogon alleles had a range of 1.67-9.33, with an average of 5.50. A wide range of alterations in morphological and yield-related traits were also found in the introgression lines population. Using single-point analysis, a total of 37 putative QTLs for yield and yield components were detected at two sites with 7%-20% explaining the phenotypic variance. Nineteen QTLs （51.4%） were detected at both sites, and the alleles from O. rufipogon at fifteen loci （40.5%） improved the yield and yield components in the Teqing background. These O. rufipogon-O, sativa introgression lines will serve as genetic materials for identifying and using favorable genes from common wild rice.     

A genome-wide library of CB4856/N2 introgression lines of Caenorhabditis elegans

Recombinant inbred lines (RILs) derived from Caenorhabditis elegans wild-type N2 and CB4856 are increasingly being used for mapping genes underlying complex traits. To speed up mapping and gene discovery, introgression lines (ILs) offer a powerful tool for more efficient QTL identification. We constructed a library of 90 ILs, each carrying a single homozygous CB4856 genomic segment introgressed into the genetic background of N2. The ILs were genotyped by 123 single-nucleotide polymorphism (SNP) markers. The proportion of the CB4856 segments in most lines does not exceed 3%, and together the introgressions cover 96% of the CB4856 genome. The value of the IL library was demonstrated by identifying novel loci underlying natural variation in two ageing-related traits, i.e. lifespan and pharyngeal pumping rate. Bin mapping of lifespan resulted in six QTLs, which all have a lifespan-shortening effect on the CB4856 allele. We found five QTLs for the decrease in pumping rate, of which four colocated with QTLs found for average lifespan. This suggests pleiotropic or closely linked QTL associated with lifespan and pumping rate. Overall, the presented IL library provides a versatile resource toward easier and efficient fine mapping and functional analyses of loci and genes underlying complex traits in C. elegans.     

Development of upland rice introgression lines and identification of QTLs for basal root thickness under different water regimes

Introgression lines (ILs) are valuable materials for identifying quantitative trait loci (QTLs),evaluating genetic interactions,and marker assisted breeding.A set of 430 ILs (BC5F3) containing segments from upland tropical japonica cultivar IRAT109 in a lowland temperate japonica cultivar Yuefu background were developed.One hundred and seventy-six polymorphic markers were used to identify introgressed segments.No segment from IRAT 109 was found in 160 lines.Introgressed segments of the other 270 lines covered 99.1％ of the donor genome.The mean number of introgressed donor segments per individual was 3.3 with an average length of 14.4 cM.QTL analysis was conducted on basal root thickness (BRT) of the 270 ILs grown under irrigated lowland,upland and hydroponic conditions.A total of 22 QTLs affecting BRT were identified,six QTLs (qBRT3.1,qBRT3.2,qBRT6.1,qBRT8.2,qBRT9.1,and qBRT9.2) were consistently expressed under at least two environments (location and water regime),and qBRT7.2 was a new BRT QTL identified under lowland conditions.IL255 containing qBRT9.1 showed an increase of 10.09％ and 7.07％ BRT over cultivar Yuefu when grown under upland and lowland conditions,respectively.Using a population of 304 F2:3 lines derived from the cross IL255 × Yuefu,qBRT9.1 was validated and mapped to a 1.2 cM interval between RM24271 and RM566.The presence of qBRT9.1 explained 12％ of BRT variation.The results provide upland rice ILs and BRT QTLs for analyzing the genetic basis of drought resistance,detecting favorable genes from upland rice,and rice drought resistance breeding.     

Identification of heterotic metabolite QTL in Arabidopsis thaliana RIL and IL populations

Two mapping populations of a cross between the Arabidopsis thaliana accessions Col-0 and C24 were cultivated and analyzed with respect to the levels of 181 metabolites to elucidate the biological phenomenon of heterosis at the metabolic level. The relative mid-parent heterosis in the F₁ hybrids was <20% for most metabolic traits. The first mapping population consisting of 369 recombinant inbred lines (RILs) and their test cross progeny with both parents allowed us to determine the position and effect of 147 quantitative trait loci (QTL) for metabolite absolute mid-parent heterosis (aMPH). Furthermore, we identified 153 and 83 QTL for augmented additive (Z₁) and dominance effects (Z₂), respectively. We identified putative candidate genes for these QTL using the aracyc database ( http://www.arabidopsis.org/biocyc ), and calculated the average degree of dominance, which was within the dominance and over-dominance range for most metabolites. Analyzing a second population of 41 introgression lines (ILs) and their test crosses with the recurrent parent, we identified 634 significant differences in metabolite levels. Nine per cent of these effects were classified as over-dominant, according to the mode of inheritance. A comparison of both approaches suggested epistasis as a major contributor to metabolite heterosis in Arabidopsis. A linear combination of metabolite levels was shown to significantly correlate with biomass heterosis ( r  = 0.62).     

Fine-structure mapping by transformation in the tryptophan region of Bacillus subtilis

Carlton, Bruce C. (Yale University, New Haven, Conn.). Fine-structure mapping by transformation in the tryptophan region of Bacillus subtilis. J. Bacteriol. 91:1795-1803. 1966.-The arrangements of eight mutational sites within a segment of the tryptophan cluster of Bacillus subtilis have been determined by three-point reciprocal crosses with a mutation in the same gene cluster used as an outside marker. Estimates of distances between sites were made by two-point crosses and normalization to recombination values for an unlinked marker to correct for physiological variabilities between recipient strains. In general, the additivities of map distances determined by this procedure were reasonable, although some discrepancies were noted in reciprocal crosses between widely separated sites within the gene region examined. No definitive polarity effect on recombination was evident, and it appears that the variations observed in reciprocal crosses may be an effect of the probability of recovery of crossovers between exogenotic and endogenotic segments. It is concluded that fine-structure mapping is feasible for the transformation system in this organism and that a high degree of resolution can be attained.     

A comprehensive and precise set of intervarietal substitution lines to identify candidate genes and quantitative trait loci in oilseed rape (<Emphasis Type="Italic">Brassica napus</Emphasis> L.)

Key message

A set of intervarietal substitution lines were developed in rapeseed by recurrent backcrossing and marker-assisted selection and employed for mapping both qualitative and quantitative traits.

Abstract

Intervarietal substitution lines (ISLs) may be assembled into advanced secondary mapping populations that have remarkable potential for resolving trait loci and mapping candidate genes. To facilitate the identification of important genes in oilseed rape (canola, Brassica napus), we developed 89 ISLs using an elite cultivar ‘Zhongyou 821’ (ZY821) as the recipient and a re-synthesized line ‘No.2127’ as the donor. In the whole process of ISLs development, the target chromosome segments were selected based on the genotypes of 300 microsatellite markers evenly distributed across the genome. Eighty-nine ISLs fixed at BC₅F₄ were genotyped by sequencing using double digestion to survey the lengths of target substitution segments from the donor parent and the background segments from the recurrent parent. The total length of the substituted chromosome segments was 3030.27 Mb, representing 3.56?×?of the Darmor-bzh reference genome sequence (version 4.1). Gene mapping was conducted for two qualitative traits, flower colour and seed-coat colour, and nine quantitative traits including yield- and quality-related traits, with 19 QTLs identified for the latter. Overlapping substitution segments were identified for flower colour and seed-coat colour loci, as well as for QTLs consistently detected in 2 or 3 years. These results demonstrate the value of these ISLs for locus resolution and subsequent cloning, targeted mutation or editing of genes controlling important traits in oilseed rape.

     

Somatic embryogenesis in cultured immature zygotic embryos and leaf protoplasts of Arabidopsis thaliana ecotypes

Immature zygotic embryos of six ecotypes (Nd-0, Ler, C24, Col-0, Nossen, Ws-2) of Arabidopsis thaliana (L.) Heynh. were cultured in vitro. The same ecotypes, except Nossen, were used for studies on leaf protoplast culture. Experimental conditions for the induction of somatic embryos were established in both culture systems. In the case of immature zygotic embryos, the parameters investigated were the influence of developmental stage of the explant, the ecotypes used, and various concentrations and combinations of growth regulatory substances (phytohormones). In the ecotype Ler, structures were discovered which were very similar to those found in the early stages of zygotic embryogenesis: globular structures at the end of a suspensor-like single file of cells were frequently observed. In the case of leaf protoplasts, high efficiencies of colony formation and plant regeneration occurred in Ws-2 and C24. A novel type of cell division pattern was found in Col-0 and C24, again highly reminiscent of the early division patterns in zygotic embryos. Similarities and differences between zygotic and somatic embryogenesis are discussed. Received: 2 August 1996 / Accepted: 4 February 1997     

水稻红莲型CMS育性恢复QTL分析

红莲型CMS是在我国杂交水稻生产中被广泛利用的雄性不育细胞质之一。为了同时定位红莲型CMS育性恢复主效和微效QTL,利用红莲型CMS不育系粤泰A(YTA)与“Lemont／特青”RIL群体测交,结合1张含有198个DNA分子标记的高密度遗传图谱,对测交F1群体的小穗育性和花粉育性进行复合区间作图。在对YTA的育性恢复性方面,该。RIL群体的2个亲本之间具有明显差异,特青的恢复性较强,其测交F1的小穗育性和花粉育性分别为72％和51％;而Lemont测交F1的小穗育性和花粉育性分别为32％和9％。复合区间作图定位到4个育性恢复QTL,分别位于水稻第1、2和10号染色体上,单个QTL的贡献率在5％～24％之间。其中,除1个QTL的增效基因来源于Lemont外,其余3个QTL的增效基因均来源于特青。效应最大的QTL为qRF-10-1,该QTL位于10号染色体RM258-C16标记区间,对小穗育性表型变异的贡献率为24％,对花粉育性的贡献率为17％,且该QTL被检测到的LOD值显著较高,因此是1个主效QTL,其增效基因来源于特青。除了主效QTLqRF-10-1外,其它3个QTL对性状的贡献率均在10％以下(5％～8％)。由此表明,该RIL群体对红莲型CMS的育性恢复由1个主效QTL控制,并受其它几个微效QTL的影响。该QTL定位结果与小穗育性在测交F1群体中呈连续的双峰分布的结果相一致。与主效QTL qRF-10-1紧密连锁的SSR标记为RM258,该主效QTL可作为分子标记辅助育种的操作目标之一,用于杂交稻分子育种中培育红莲型CMS的强恢复系。     

Development,identification and utilization of introgression lines using Chinese endemic and synthetic wheat as donors

Chromosome segmental introgression lines (ILs) are an effective way to utilize germplasm resources in crops. To improve agronomic traits of wheat cultivar (Triticum aestivum) Shi 4185, four sets of ILs...     

Supplementary ultraviolet-B irradiation reveals differences in stress responses between Arabidopsis thaliana ecotypes

Irradiation of Arabidopsis thaliana ecotypes C24, Wassilewskija (Ws) and Columbia-0 (Col-0) with supplementary ultraviolet-A+B (UV-A+B) radiation revealed ecotype-specific differences in expression of the gene for the pathogenesis-related protein PR-5. C24 showed an increased expression level of PR-5 (5- and 20-fold higher compared with Col-0 and Ws, respectively). Expression of other molecular markers such as CHS (encoding chalcone synthase), MEB5.2 [encoding a gene strongly up-regulated by ultraviolet-B (UV-B)] and PYROA [encoding a pyridoxine (Vitamin B6) biosynthesis enzyme] only showed slight differences between ecotypes. Oxidative stress during UVA+B exposure was monitored by staining for H2O2. This analysis also revealed important ecotype-specific differences. 'H2O2 hot spots' were found in C24, whereas an even distribution of H2O2 was found in Ws and Col-0. Necrotic lesions also appeared on C24 leaves after prolonged UV-B exposure. There was a reverse correlation between the H2O2 steady-state concentration and the PR-5 gene expression; Ws showed the highest level of H2O2 accumulation but the lowest expression level of the PR-5 gene. Furthermore, application of paraquat on the rosettes led to similar PR-5 expression and H2O2 accumulation patterns as were found after UV-A+B irradiation. The observed ecotypic differences were also reflected in a statistically significant UV-B-dependent decrease in biomass, rosette size and leaf area for Ws, but not for C24 and Col-0. Our results show that a significant ecotype-specific genetic variability in general UV-B responses in Arabidopsis exists. Moreover, the signal transduction or gene regulation pathway for PR-5 differs from the other molecular markers used in this study.     

Heterosis of biomass production in Arabidopsis. Establishment during early development

Heterosis has been widely used in agriculture to increase yield and to broaden adaptability of hybrid varieties and is applied to an increasing number of crop species. We performed a systematic survey of the extent and degree of heterosis for dry biomass in 63 Arabidopsis accessions crossed to three reference lines (Col-0, C24, and Nd). We detected a high heritability (69%) for biomass production in Arabidopsis. Among the 169 crosses analyzed, 29 exhibited significant mid-parent-heterosis for shoot biomass. Furthermore, we analyzed two divergent accessions, C24 and Col-0, the F(1) hybrids of which were shown to exhibit hybrid vigor, in more detail. In the combination Col-0/C24, heterosis for biomass was enhanced at higher light intensities; we found 51% to 66% mid-parent-heterosis at low and intermediate light intensities (60 and 120 micromol m(-2) s(-1)), and 161% at high light intensity (240 micromol m(-2) s(-1)). While at the low and intermediate light intensities relative growth rates of the hybrids were higher only in the early developmental phase (0-15 d after sowing [DAS]), at high light intensity the hybrids showed increased relative growth rates over the entire vegetative phase (until 25 DAS). An important finding was the early onset of heterosis for biomass; in the cross Col-0/C24, differences between parental and hybrid lines in leaf size and dry shoot mass could be detected as early as 10 DAS. The widespread occurrence of heterosis in the model plant Arabidopsis opens the possibility to investigate the genetic basis of this phenomenon using the tools of genetical genomics.