裸燕麦EMS突变体库筛选与分析

燕麦是重要的粮饲兼用作物,构建燕麦EMS突变体库对燕麦功能基因组学研究和遗传改良有重要意义。本试验利用化学诱变剂甲基磺酸乙酯(EMS,ethyl methane sulfonate)处理燕麦品种花早2号,获得了4083株M1材料;对其中2000个单株种植了M2株行,进行全生育期调查,鉴定其表型变化;对2份黄化苗突变材料种植了M3家系,进行相关突变性状的稳定性验证。结果表明,燕麦经EMS处理后代变异巨大,在M2发现表型突变材料196份,变异率为9.8%,变异类型非常丰富,包括幼苗习性、叶片性状、分蘖、株高、穗部形态及成熟期等突变株系。M3证实突变的黄化苗特性可以稳定遗传。本研究建立了燕麦EMS诱变体系,获得的燕麦变异类型丰富,为燕麦功能基因组学研究和燕麦遗传改良奠定了材料基础。     

Induction,rapid fixation and retention of mutations in vegetatively propagated banana

Mutation discovery technologies have enabled the development of reverse genetics for many plant species and allowed sophisticated evaluation of the consequences of mutagenesis. Such methods are relatively straightforward for seed‐propagated plants. To develop a platform suitable for vegetatively propagated species, we treated isolated banana shoot apical meristems with the chemical mutagen ethyl methanesulphonate, recovered plantlets and screened for induced mutations. A high density of GC‐AT transition mutations were recovered, similar to that reported in seed‐propagated polyploids. Through analysis of the inheritance of mutations, we observed that genotypically heterogeneous stem cells resulting from mutagenic treatment are rapidly sorted to fix a single genotype in the meristem. Further, mutant genotypes are stably inherited in subsequent generations. Evaluation of natural nucleotide variation showed the accumulation of potentially deleterious heterozygous alleles, suggesting that mutation induction may uncover recessive traits. This work therefore provides genotypic insights into the fate of totipotent cells after mutagenesis and suggests rapid approaches for mutation‐based functional genomics and improvement of vegetatively propagated crops.     

Marker-assisted introgression (MAI) of almond genes into the peach background: a fast method to mine and integrate novel variation from exotic sources in long intergeneration species

This paper proposes a new breeding strategy, marker-assisted introgression (MAI), to obtain lines of perennial species with a single introgressed fragment from a compatible species two generations after the interspecific hybrid. MAI allows enrichment of the genome of a species with genes from a wild or exotic relative in a short timeframe and with an intermediate step that allows a first exploration of genes/QTLs that the donor species can provide to the target crop. This method has three phases: (1) creating a large backcross one (BC1) population to select, with markers, a reduced number of individuals (15–30, called the prIL set) with a low number of introgressions; (2) phenotyping the prIL set for the traits of interest and inferring the inheritance and map position of segregating major genes/QTLs based on the known genotypes of the prILs; and (3) advancing selected lines carrying the traits of interest to a next generation of backcross or selfing to obtain individuals with a single introgression in the background of the elite commercial germplasm. The proof of concept of this strategy was implemented by using peach as the recurrent species and almond as the donor. The whole process can be done in 9–10 years as the identification of the first line with one introgression was after 5 years (2006–2011), and 4–5 additional years are needed for phenotypic evaluation of selected lines. The expansion of this method to other perennial clonally propagated crops and to other species of Prunus compatible with peach is discussed.     

The unmanaged reproductive ecology of domesticated plants in traditional agroecosystems: An example involving cassavaand a call for data

Although cassava is a strictly vegetatively propagated crop, in many traditional Amazonian agroecosystems, Amerindian farmers recognise volunteer seedlings of cassava and allow them to grow. If their properties are deemed desirable, plants originating from seedlings are included in the harvest of tuberous roots, and their stems are used to prepare cuttings for propagation. Incorporation of these products of spontaneous sexual reproduction appears to be important in origin and maintenance of genetic diversity in this clonally propagated plant. Our observations conducted in an Amerindian village in Guyana suggest that volunteer seedlings arise from a bank of viable seeds stored in soil, and that dispersal and burial of seeds by ants may be important in its constitution. Future investigations of the dynamics of genetic diversity in this crop in traditional agroecosystems must consider the role of the ‘wild’ sexual reproduction that occurs in parallel with vegetative propagation. We suggest that unmanaged processes of sexual reproduction play important but neglected roles in the evolutionary ecology of many domesticated plants in traditional agroecosystems.     

Comparative population genomics reveals the domestication history of the peach,Prunus persica,and human influences on perennial fruit crops

Background

Recently, many studies utilizing next generation sequencing have investigated plant evolution and domestication in annual crops. Peach, Prunus persica, is a typical perennial fruit crop that has ornamental and edible varieties. Unlike other fruit crops, cultivated peach includes a large number of phenotypes but few polymorphisms. In this study, we explore the genetic basis of domestication in peach and the influence of humans on its evolution.

Results

We perform large-scale resequencing of 10 wild and 74 cultivated peach varieties, including 9 ornamental, 23 breeding, and 42 landrace lines. We identify 4.6 million SNPs, a large number of which could explain the phenotypic variation in cultivated peach. Population analysis shows a single domestication event, the speciation of P. persica from wild peach. Ornamental and edible peach both belong to P. persica, along with another geographically separated subgroup, Prunus ferganensis.We identify 147 and 262 genes under edible and ornamental selection, respectively. Some of these genes are associated with important biological features. We perform a population heterozygosity analysis in different plants that indicates that free recombination effects could affect domestication history. By applying artificial selection during the domestication of the peach and facilitating its asexual propagation, humans have caused a sharp decline of the heterozygote ratio of SNPs.

Conclusions

Our analyses enhance our knowledge of the domestication history of perennial fruit crops, and the dataset we generated could be useful for future research on comparative population genomics.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0415-1) contains supplementary material, which is available to authorized users.     

An efficient viral vector for functional genomic studies of Prunus fruit trees and its induced resistance to Plum pox virus via silencing of a host factor gene

RNA silencing is a powerful technology for molecular characterization of gene functions in plants. A commonly used approach to the induction of RNA silencing is through genetic transformation. A potent alternative is to use a modified viral vector for virus‐induced gene silencing (VIGS) to degrade RNA molecules sharing similar nucleotide sequence. Unfortunately, genomic studies in many allogamous woody perennials such as peach are severely hindered because they have a long juvenile period and are recalcitrant to genetic transformation. Here, we report the development of a viral vector derived from Prunus necrotic ringspot virus (PNRSV), a widespread fruit tree virus that is endemic in all Prunus fruit production countries and regions in the world. We show that the modified PNRSV vector, harbouring the sense‐orientated target gene sequence of 100‐200 bp in length in genomic RNA3, could efficiently trigger the silencing of a transgene or an endogenous gene in the model plant Nicotiana benthamiana. We further demonstrate that the PNRSV‐based vector could be manipulated to silence endogenous genes in peach such as eukaryotic translation initiation factor 4E isoform (eIF(iso)4E), a host factor of many potyviruses including Plum pox virus (PPV). Moreover, the eIF(iso)4E‐knocked down peach plants were resistant to PPV. This work opens a potential avenue for the control of virus diseases in perennial trees via viral vector‐mediated silencing of host factors, and the PNRSV vector may serve as a powerful molecular tool for functional genomic studies of Prunus fruit trees.     

EMS mutagenesis and qPCR-HRM prescreening for point mutations in an embryogenic cell suspension of grapevine

Key message

Embryogenic suspension cultures are suitable for EMS mutagenesis in grapevine, and HRM prescreening of EMS-treated somatic embryo clusters allows rapid detection of point mutations before plant regeneration.

Abstract

Somatic embryogenesis is an excellent system for induced mutagenesis and clonal propagation in woody plants. Our work was focused on establishing a procedure for inducing ethyl methanesulfonate (EMS) mutagenesis in grapevine. Embryogenic cell aggregates (ECAs) growing in liquid medium were treated with increasing concentrations of EMS. We found that EMS dramatically affects the viability of ECAs at concentrations above 20 mM (25.5 ± 2.9 % survival), whereas concentrations above 10 mM affect embryogenic potential (22.1 ± 1.7 % of ECAs gave rise to embryos). Embryo masses generated from EMS-treated embryogenic cell aggregates were prescreened by quantitative PCR-High Resolution Melting (qPCR-HRM) to detect single nucleotide polymorphisms (SNPs) in a 1,000-bp VvNCED1-encoding DNA fragment, which served as the target gene. Detected mutations were verified in regenerated plants by PCR and sequencing. qPCR-HRM analysis of the difference plots for the fluorescence signals allowed detection of a mutation in a sample from an embryogenic aggregate treated with 10 mM EMS. To confirm the nature of the mutation, embryos from this aggregate were recovered and germinated, and leaves were collected for PCR and sequencing analysis. The alignment of sequences from regenerated plants with the wild-type sequence revealed a transitional mutation (G/C to A/T) in the 1,000-bp VvNCED1-encoding region. To our knowledge, this is the first time that EMS mutagenesis has been performed using an embryogenic cell suspension of grapevine.     

Ethyl methanesulfonate mutant library construction in Gossypium hirsutum L. for allotetraploid functional genomics and germplasm innovation

As the gene pool is exposed to both strain on land resources and a lack of diversity in elite allotetraploid cotton, the acquisition and identification of novel alleles has taken on epic importance in facilitating cotton genetic improvement and functional genomics research. Ethyl methanesulfonate (EMS) is an excellent mutagen that induces genome‐wide efficient mutations to activate the mutagenic potential of plants with many advantages. The present study established, determined and verified the experimental procedure suitable for EMS‐based mutant library construction as the general reference guide in allotetraploid upland cotton. This optimized method and procedure are efficient, and abundant EMS mutant libraries (approximately 12 000) in allotetraploid cotton were successfully obtained. More than 20 mutant phenotypes were observed and screened, including phenotypes of the leaf, flower, fruit, fiber and plant architecture. Through the plants mutant library, high‐throughput and high‐resolution melting technology‐based variation evaluation detected the EMS‐induced site mutation. Additionally, based on overall genome‐wide mutation analyses by re‐sequencing and mutant library assessment, the examination results demonstrated the ideal quality of the cotton EMS‐treated mutant library constructed in this study with appropriate high mutation density and saturated genome. What is more, the collection is composed of a broad repertoire of mutants, which is the valuable resource for basic genetic research and functional genomics underlying complex allotetraploid traits, as well as cotton breeding.     

60Co γ射线辐照和EMS处理对火龙果成活率及幼苗生长的影响

采用不同剂量的60Coγ射线辐照火龙果枝条,结果表明,枝条的成活率随辐照剂量的增加而降低,白肉火龙果枝条的适宜辐照范围为5 446.6～6 060.1 Rad,红肉火龙果枝条的适宜辐照范围为4 444.1～4 981.7 Rad,粉红火龙果枝条的适宜辐照范围为5 069.1～5 601.0 Rad;辐照处理抑制了火龙果...     

Evolution of the Rdr1 TNL-cluster in roses and other Rosaceous species

ABSTRACT: BACKGROUND: The resistance of plants to pathogens relies on two lines of defense: a basal defense response and a pathogen-specific system, in which resistance (R) genes induce defense reactions after detection of pathogen-associated molecular patterns (PAMPS). In the specific system, a so-called arms race has developed in which the emergence of new races of a pathogen leads to the diversification of plant resistance genes to counteract the pathogens' effect. The mechanism of resistance gene diversification has been elucidated well for short-lived annual species, but data are mostly lacking for long-lived perennial and clonally propagated plants, such as roses. We analyzed the rose black spot resistance gene, Rdr1, in five members of the Rosaceae: Rosa multiflora, Rosa rugosa, Fragaria vesca (strawberry), Malus x domestica (apple) and Prunus persica (peach), and we present the deduced possible mechanism of R-gene diversification. RESULTS: We sequenced a 340.4-kb region from R. rugosa orthologous to the Rdr1 locus in R. multiflora. Apart from some deletions and rearrangements, the two loci display a high degree of synteny. Additionally, less pronounced synteny is found with an orthologous locus in strawberry but is absent in peach and apple, where genes from the Rdr1 locus are distributed on two different chromosomes. An analysis of 20 TIR-NBS-LRR (TNL) genes obtained from R. rugosa and R. multiflora revealed illegitimate recombination, gene conversion, unequal crossing over, indels, point mutations and transposable elements as mechanisms of diversification.A phylogenetic analysis of 53 complete TNL genes from the five Rosaceae species revealed that with the exception of some genes from apple and peach, most of the genes occur in species-specific clusters, indicating that recent TNL gene diversification began prior to the split of Rosa from Fragaria in the Rosoideae and peach from apple in the Spiraeoideae and continued after the split in individual species. Sequence similarity of up to 99% is obtained between two R. multiflora TNL paralogs, indicating a very recent duplication. CONCLUSIONS: The mechanisms by which TNL genes from perennial Rosaceae diversify are mainly similar to those from annual plant species. However, most TNL genes appear to be of recent origin, likely due to recent duplications, supporting the hypothesis that TNL genes in woody perennials are generally younger than those from annuals. This recent origin might facilitate the development of new resistance specificities, compensating for longer generation times in woody perennials.     

A strategy for developing representative germplasm sets for systematic QTL validation,demonstrated for apple,peach, and sweet cherry

Horticultural crop improvement would benefit from a standardized, systematic, and statistically robust procedure for validating quantitative trait loci (QTLs) in germplasm relevant to breeding programs. Here, we describe and demonstrate a strategy for developing reference germplasm sets of perennial, clonally propagated crops, especially those with long juvenile periods. Germplasm is chosen to efficiently represent important members of larger pedigree-connected genepools. To facilitate validation of multiple QTLs, genome-wide representation of alleles is optimized for designated important breeding parents (IBPs) by estimating average allelic representation in relatives. The strategy and arising principles were demonstrated in a simulated germplasm set. Strong statistical power can be achieved with a carefully chosen germplasm set composed of IBPs, their numerous unselected progenies and close relatives, and all available founders and intermediate ancestors. Crop Reference Sets were developed in the marker-assisted breeding (MAB)-enabling “RosBREED” project as a base resource for QTL validation in US breeding germplasm of apple (Malus × domestica), peach (Prunus persica), and sweet cherry (Prunus avium) consisting of 467, 452, and 268 individuals, respectively. These sets adequately represent the most designated IBPs, have distinct advantages for QTL validation over other germplasm arrangements of equal size, and are recommended as a base resource for QTL validation by breeders of these US crops. The strategy described here can be used to develop efficient reference germplasm sets suiting other breeding genepools or to calculate the statistical power for QTL validation of germplasm sets already established.     

Recent advances in genetic engineering for improvement of fruit crops

Fruits are one of the major sources of vitamins, essential nutrients, antioxidants and fibers in human diet. During the last two–three decades, genetic engineering methods based on the use of transgenes have been successfully adopted to improve fruit plants and focused mainly on enhanced tolerance to biotic and abiotic stresses, increased fruit yield, improved post harvest shelf life of fruit, reduced generation time and production of fruit with higher nutritional value. However, the development of transgenic fruit plants and their commercialization are hindered by many regulatory and social hurdles. Nowadays, new genetic engineering approaches i.e. cisgenesis or intragenesis receive increasing interest for genetic modification of plants. The absence of selectable marker gene in the final product and the introduced gene(s) derived from the same plant or plants sexually compatible with the target crop should increase consumer’s acceptance. In this article, we attempt to summarize the recent progress achieved on the genetic engineering in fruit plants and their applications in crop improvement. Challenges and opportunities for the deployment of genetic engineering in crop improvement programs of fruit plants are also discussed.     

Validating a QTL region characterized by multiple haplotypes

Validation of a quantitative trait locus (QTL) for outcrossing perennial plants is rarely reported due to complexity of plausible genetic models and reliance on field designs already available. Here, a particular marker-QTL haplotype exerted a large, positive effect on height for Pinus taeda and its origin could be traced to a founder, GP₃, in a three-generation QTL pedigree. To validate this QTL effect, we used an extended GP₃-based pedigree. In the validation cross, each of the 46 offspring was clonally propagated from developing seeds using somatic embryogenesis technology. Subsequent analyses were conducted separately for seedlings and for other somatic emblings. For seedlings, the original QTL effect could not be fully validated. For somatic emblings, a strong negative QTL effect was detected in the validation cross; some evidence from another cross-supported the original positive QTL effect. From this part of the analysis, three distinct marker-QTL haplotypes at a single locus could be inferred. Validating QTL haplotypes in readily available field tests was feasible despite the genetic model complexity inherent to outcrossing long-lived perennials.     

Size asymmetry in intraspecific competition and the density-dependence of inbreeding depression in a natural plant population: a case study in cassava (Manihot esculenta Crantz, Euphorbiaceae)

The effects of competition on the genetic composition of natural populations are not well understood. We combined demography and molecular genetics to study how intraspecific competition affects microevolution in cohorts of volunteer plants of cassava (Manihot esculenta) originating from seeds in slash-and-burn fields of Palikur Amerindians in French Guiana. In this clonally propagated crop, genotypic diversity is enhanced by the incorporation of volunteer plants into farmers' stocks of clonal propagules. Mortality of volunteer plants was density-dependent. Furthermore, the size asymmetry of intraspecific competition increased with local clustering of plants. Size of plants was correlated with their multilocus heterozygosity, and stronger size-dependence of survival in clusters of plants, compared with solitary plants, increased the magnitude of inbreeding depression when competition was severe. The density-dependence of inbreeding depression of volunteer plants helps explain the high heterozygosity of volunteers that survive to harvest time and thus become candidates for clonal propagation. This effect could help favour the maintenance of sex in this 'vegetatively' propagated crop plant.     

苦荞突变体库构建与突变体中芦丁合成相关基因表达分析

苦荞是重要的小杂粮作物之一,营养物质丰富,是天然芦丁的重要来源。突破苦荞育种难题,创制苦荞新种质是目前研究的重要方面。本试验利用甲基磺酸乙酯(EMS)构建了黑丰1号苦荞突变体库,明确了当EMS浓度为1.2%时,诱变效果较好。通过对M1突变株表型观察统计,共获得叶色、叶型、株型、粒型变异单株102株,突变率为3.85%;高效液相色谱技术(HPLC)测定1000株M3材料,获得高芦丁含量突变株系2个和低芦丁突变体株系5个;qRT-PCR对芦丁含量突变体株系中芦丁代谢关键酶基因(CHS、F3H、4CL、FLS、UFGT)进行表达量分析,发现不同株系中上述基因的表达量与芦丁含量相关性不明显,但个别基因如FtFLS基因表达量在高芦丁含量突变体中达到对照的4.55倍。通过突变体的筛选丰富了苦荞基因资源,创新了苦荞新种质,也为苦荞芦丁代谢的分子基础研究提供了材料保证与技术支持。     

Mutagenesis of kiwifruit CENTRORADIALIS‐like genes transforms a climbing woody perennial with long juvenility and axillary flowering into a compact plant with rapid terminal flowering

Annualization of woody perennials has the potential to revolutionize the breeding and production of fruit crops and rapidly improve horticultural species. Kiwifruit (Actinidia chinensis) is a recently domesticated fruit crop with a short history of breeding and tremendous potential for improvement. Previously, multiple kiwifruit CENTRORADIALIS (CEN)‐like genes have been identified as potential repressors of flowering. In this study, CRISPR/Cas9‐ mediated manipulation enabled functional analysis of kiwifruit CEN‐like genes AcCEN4 and AcCEN. Mutation of these genes transformed a climbing woody perennial, which develops axillary inflorescences after many years of juvenility, into a compact plant with rapid terminal flower and fruit development. The number of affected genes and alleles and severity of detected mutations correlated with the precocity and change in plant stature, suggesting that a bi‐allelic mutation of either AcCEN4 or AcCEN may be sufficient for early flowering, whereas mutations affecting both genes further contributed to precocity and enhanced the compact growth habit. CRISPR/Cas9‐mediated mutagenesis of AcCEN4 and AcCEN may be a valuable means to engineer Actinidia amenable for accelerated breeding, indoor farming and cultivation as an annual crop.     

Physiological and transcriptional responses in the iron–sulphur cluster assembly pathway under abiotic stress in peach (Prunus persica L.) seedlings

As one of the most indispensable element in mineral nutrition of plants, iron (Fe) is closely related to fruits quality and yield. However, molecular mechanisms towards Fe metabolism in fruit trees is largely unclear. In higher plants, iron–sulphur (Fe–S) cluster assembly occurs in chloroplasts, mitochondria and cytosol involving dozens of genes. In this study, we identified 44 putative Fe–S cluster assembly genes in peach (Prunus persica cv. ‘Xiahui6’), and analyzed Fe–S cluster assembly gene expression profiles in response to abiotic stresses. Peach seedlings were more sensitive to iron deficiency, drought and salinity stress, evidenced in reduced photosynthetic performance and altered activity of nitrite reductase, succinate dehydrogenase and aconitase. In addition, Fe–S cluster assembly genes are differentially regulated by abiotic stresses. Iron depletion and drought stress are likely to affect Fe–S cluster assembly genes in leaves. Excess iron toxicity mainly induces Fe–S cluster assembly gene expression in roots, whereas salinity stress massively inhibits Fe–S cluster assembly gene expression in roots. Interestingly, we found that un-functional scaffolds are more prone to disappear during the long-term evolution in perennial woody plants. Our findings directly provide molecular basis for Fe metabolism in peach, and favorably reveal potential candidate genes for further functional determination.     

Genome assembly of the Chinese maize elite inbred line RP125 and its EMS mutant collection provide new resources for maize genetics research and crop improvement

Maize is an important crop worldwide, as well as a valuable model with vast genetic diversity. Accurate genome and annotation information for a wide range of inbred lines would provide valuable resources for crop improvement and pan-genome characterization. In this study, we generated a high-quality de novo genome assembly (contig N50 of 15.43 Mb) of the Chinese elite inbred line RP125 using Nanopore long-read sequencing and Hi-C scaffolding, which yield highly contiguous, chromosome-length scaffolds. Global comparison of the RP125 genome with those of B73, W22, and Mo17 revealed a large number of structural variations. To create new germplasm for maize research and crop improvement, we carried out an EMS mutagenesis screen on RP125. In total, we obtained 5818 independent M2 families, with 946 mutants showing heritable phenotypes. Taking advantage of the high-quality RP125 genome, we successfully cloned 10 mutants from the EMS library, including the novel kernel mutant qk1 (quekou: “missing a small part” in Chinese), which exhibited partial loss of endosperm and a starch accumulation defect. QK1 encodes a predicted metal tolerance protein, which is specifically required for Fe transport. Increased accumulation of Fe and reactive oxygen species as well as ferroptosis-like cell death were detected in qk1 endosperm. Our study provides the community with a high-quality genome sequence and a large collection of mutant germplasm.     

Comparison of the Frequencies of Spontaneous and Chemically-Induced 5-Bromodeoxyuridine-Resistance Mutations in Wild-Type and Revertant Bhk-21/13 Cells

5-bromodeoxyuridine resistance mutations induced by mutagenesis were studied. The average expression time for induced mutations varied with the concentration of the mutagen ethyl methanesulfonate (EMS). However, a constant number of two generation times was necessary for half maximal expression of induced mutations. Also, induced mutation rates were compared under optimal expression conditions for bromodeoxyuridine, fluorodeoxyuridine and azaguanine resistance markers. Ten independent bromodeoxy-uridine-resistant clones were tested for reversion. Two clones reverted-one spontaneously and the other after mutagenesis. The spontaneous rate of mutation to bromodeoxyuridine resistance, estimated by the fluctuation test, was high in revertant clones (4 x 10(-6) / cell / generation) and low in the wild-type cells (< 3.5 x 10(-8) / cell / generation). A comparison of induced mutation frequencies at variable EMS concentrations showed a single-hit curve for revertant clones and a multihit curve for the wild-type cells. Thymidine kinase activities of resistant clones were usually less than 2% of that of the wild-type clone. Inducibility, thermal stability and intracellular localization of the thymidine kinases of the wild-type cells and of a revertant clone were identical. A low, but significant (P < 0.10), Km discrepancy was observed between enzyme extracts of these lines. The genetic implications of these results are discussed.