Genomic asymmetry in allopolyploid plants: wheat as a model

The evolvement of duplicated gene loci in allopolyploid plants has become the subject of intensive studies. Most duplicated genes remain active in neoallopolyploids contributing either to a favourable effect of an extra gene dosage or to the build-up of positive inter-genomic interactions when genes or regulation factors on homoeologous chromosomes are divergent. However, in a small number of loci (about 10%), genes of only one genome are active, while the homoeoalleles on the other genome(s) are either eliminated or partially or completely suppressed by genetic or epigenetic means. For several traits, the retention of controlling genes is not random, favouring one genome over the other(s). Such genomic asymmetry is manifested in allopolyploid wheat by the control of various morphological and agronomical traits, in the production of rRNA and storage proteins, and in interaction with pathogens. It is suggested that the process of cytological diploidization leading to exclusive intra-genomic meiotic pairing and, consequently, to complete avoidance of inter-genomic recombination, has two contrasting effects. Firstly, it provides a means for the fixation of positive heterotic inter-genomic interactions and also maintains genomic asymmetry resulting from loss or silencing of genes. The possible mechanisms and evolutionary advantages of genomic asymmetry are discussed.     

Variation for homoeologous gene silencing in hexaploid wheat

The absence of expression of individual members of a homoeologous set of genes in a polyploid is a well-established phenomenon. However, the extent to which such 'homoeologous silencing' can vary between individual genotypes within a species is unexplored. We have used the single-strand conformation polymorphism assay to identify homoeologue non-expression at 15 single-copy genes across a panel of 16 wheat varieties, representative of the genetic diversity present in modern northern European winter wheat (Triticum aestivum). There was no evidence for any homoeologous silencing at seven of the fifteen genes, but in the remaining eight, at least one of the three homoeologues varied qualitatively for expression in either the root or the seedling leaf. The identity of the non-expressed homoeologue was generally consistent, but when the expression profiles of eight informative genes were compared, only two varieties shared the same pattern of silencing. A small-scale study suggested that silencing patterns were largely inherited across self-pollinated generations, and some evidence is presented for the epigenetic segregation of these patterns in a population bred from parents having contrasting silencing profiles. Epigenetic variation exerts a significant effect on phenotype, so given the ubiquity and variability in homoeologous silencing observed in wheat, we suggest that it is likely to play a considerable role in generating phenotypic variation. Thus epigenetic profiling may need to be incorporated as part of the analytical tool kit for predictive wheat breeding.     

TaDEP1基因在普通六倍体小麦及其供体种中的保守与分化

根据已知小麦正源基因TaDEP1 cDNA序列设计引物,成功克隆了小麦TaDEP1基因组序列,发现该基因包含5个外显子,4个内含子.通过比较该基因在六倍体普通小麦A、B、D基因组中的差异,筛选出可以区分A、B、D基因组的分子标记Ta956.以中国春缺体-四体系为材料,利用该标记将TaDEP1基因定位于小麦5A、5B和5...     

A screening method to identify genetic variation in root growth response to a salinity gradient

Salinity as well as drought are increasing problems in agriculture. Durum wheat (Triticum turgidum L. ssp. durum Desf.) is relatively salt sensitive compared with bread wheat (Triticum aestivum L.), and yields poorly on saline soil. Field studies indicate that roots of durum wheat do not proliferate as extensively as bread wheat in saline soil. In order to look for genetic diversity in root growth within durum wheat, a screening method was developed to identify genetic variation in rates of root growth in a saline solution gradient similar to that found in many saline fields. Seedlings were grown in rolls of germination paper in plastic tubes 37 cm tall, with a gradient of salt concentration increasing towards the bottom of the tubes which contained from 50-200 mM NaCl with complete nutrients. Seedlings were grown in the light to the two leaf stage, and transpiration and evaporation were minimized so that the salinity gradient was maintained. An NaCl concentration of 150 mM at the bottom was found suitable to identify genetic variation. This corresponds to a level of salinity in the field that reduces shoot growth by 50% or more. The screen inhibited seminal axile root length more than branch root length in three out of four genotypes, highlighting changes in root system architecture caused by a saline gradient that is genotype dependent. This method can be extended to other species to identify variation in root elongation in response to gradients in salt, nutrients, or toxic elements.     

小麦基因组研究进展

本文从小麦遗传图谱、物理图谱、比较基因组、基因组测序和EST 5个方面,介绍国内外小麦基因组的研究进展。我们利用W7984×Opata重组近交系的RFLP作图群体,对33个与小麦水分利用效率有关的性状进行了QTL遗传图谱比较分析,结果显明：在第一部分同源群染色体（1A,1B）上的着丝粒周围,分布有控制光合作用和根系特性的基因簇。在第二部分同源染色体上,有控制单株水分利用效率、根冠形态和生长发育的基因簇存在。在第六部分同源染色体上,6A和6B上都分别有由控制根系多个QTL组成的基因簇,6D染色体着丝粒周围有一个大的基因簇,由7个控制叶片和单株水分利用效率的QTL组成,说明第六部分同源染色体在小麦水分利用效率遗传方面起重要作用。 Abstract:Research development of genetic mapping,physics mapping,genome sequencing and expressed sequence tags in wheat have been reviewed in this paper.RFLP genetic linkage map of wheat recombinant inbred lines derived from W7984×Opata,was used to study QTL of 33 traits associated with water use efficiency.Compared with QTL map of 7 group homeologues chromosomes,the results were showed as follows:nearby the centromeric region of 1A and 1B chromosome,the gene cluster of controlling photosynthetic and root traits were located.The gene clusters of controlling water use efficiency per plant,root and plant height and growth rate were located on the 2 group chromosomes.The gene clusters of controlling root traits were located on the 6A an 6B chromosome,there was a big gene cluster mad up by 7 QTLs controlling water use efficiency of wheat leaf and per plant nearby the centromeric region of 6D chromosome.It showed that 6th homeologous chromosomes play an important role in controlling water use efficiency in wheat.     

Current applications of wheat and wheat–alien precise genetic stocks

A comprehensive collection of wheat aneuploids, whole chromosome substitutions (both intervarietal and interspecific) and wheat–alien addition lines, along with various introgression and near-isogenic lines, has been created over a period of years, primarily to provide the means of localizing the genes underpinning traits and to introduce novel genes into the bread wheat genome. For a time, interest in this class of genetic material was on the wane, but more recently it has revived in the context, for example, of localizing DNA-based markers, designing chromosome-specific bacterial artificial chromosome libraries, and establishing functional differences between alleles and homoeoalleles. Here, a brief review is provided of recent applications of precise genetic stocks in the field of molecular genetics, functional genetics and genomics of the Triticeae species.     

CRISPR/Cas9-mediated disruption of TaNP1 genes results in complete male sterility in bread wheat

Male sterile genes and mutants are valuable resources in hybrid seed production for monoclinous crops.High genetic redundancy due to allohexaploidy makes it difficult to obtain the nuclear recessive male sterile mutants through spontaneous mutation or chemical or physical mutagenesis methods in wheat.The emerging effective genome editing tool,CRISPR/Cas9 system,makes it possible to achieve simultaneous mutagenesis in multiple homoeoalleles.To improve the genome modification efficiency of the CRISPR/Cas9 system in wheat,we compared four different RNA polymerase(Pol) Ⅲ promoters(TaU3 p,TaU6 p,OsU3 p,and OsU6 p) and three types of sgRNA scaffold in the protoplast system.We show that the TaU3 promoter-driven optimized sgRNA scaffold was most effective.The optimized CRISPR/Cas9 system was used to edit three TaNP1 homoeoalleles,whose orthologs,OsNP1 in rice and ZmIPE1 in maize,encode a putative glucose-methanol-choline oxidoreductase and are required for male sterility.Triple homozygous mutations in TaNP1 genes result in complete male sterility.We further demonstrated that anyone wild-type copy of the three TaNP1 genes is sufficient for maintenance of male fertility.Taken together,this study provides an optimized CRISPR/Cas9 vector for wheat genome editing and a complete male sterile mutant for development of a commercially viable hybrid wheat seed production system.     

CRISPR/Cas9技术在小麦育种中的应用进展

小麦（Triticum aestivum L.）是世界上主要的农作物之一,在粮食安全供应中发挥重要作用。在过去的几十年,由于小麦基因组复杂和遗传转化困难,导致小麦的基础和应用研究落后于其他谷类作物。2014年小麦基因组编辑取得了显著进展,进而促进了小麦生物技术的发展。综述了CRISPR/Cas9技术在小麦育种中的研究进展,简单介绍了CRISPR/Cas9基因编辑技术的发现、原理和优缺点,指出小麦基因编辑过程中农杆菌介导的遗传转化较粒子轰击法可降低转基因沉默频率,未来将成为基因编辑过程中主流的遗传转化方式;优化sgRNA的启动子、选择同源保守序列做为靶点可以提高基因编辑效率;新开发的碱基编辑器和prime editor需引入更多突变类型。展望了进一步提高小麦基因编辑效率和安全性的可行性,以期为未来小麦育种工作提供参考。     

Applying the latest advances in genomics and phenomics for trait discovery in polyploid wheat

Improving traits in wheat has historically been challenging due to its large and polyploid genome, limited genetic diversity and in‐field phenotyping constraints. However, within recent years many of these barriers have been lowered. The availability of a chromosome‐level assembly of the wheat genome now facilitates a step‐change in wheat genetics and provides a common platform for resources, including variation data, gene expression data and genetic markers. The development of sequenced mutant populations and gene‐editing techniques now enables the rapid assessment of gene function in wheat directly. The ability to alter gene function in a targeted manner will unmask the effects of homoeolog redundancy and allow the hidden potential of this polyploid genome to be discovered. New techniques to identify and exploit the genetic diversity within wheat wild relatives now enable wheat breeders to take advantage of these additional sources of variation to address challenges facing food production. Finally, advances in phenomics have unlocked rapid screening of populations for many traits of interest both in greenhouses and in the field. Looking forwards, integrating diverse data types, including genomic, epigenetic and phenomics data, will take advantage of big data approaches including machine learning to understand trait biology in wheat in unprecedented detail.     

High‐efficiency gene targeting in hexaploid wheat using DNA replicons and CRISPR/Cas9

The ability to edit plant genomes through gene targeting (GT) requires efficient methods to deliver both sequence‐specific nucleases (SSNs) and repair templates to plant cells. This is typically achieved using Agrobacterium T‐DNA, biolistics or by stably integrating nuclease‐encoding cassettes and repair templates into the plant genome. In dicotyledonous plants, such as Nicotinana tabacum (tobacco) and Solanum lycopersicum (tomato), greater than 10‐fold enhancements in GT frequencies have been achieved using DNA virus‐based replicons. These replicons transiently amplify to high copy numbers in plant cells to deliver abundant SSNs and repair templates to achieve targeted gene modification. In the present work, we developed a replicon‐based system for genome engineering of cereal crops using a deconstructed version of the wheat dwarf virus (WDV). In wheat cells, the replicons achieve a 110‐fold increase in expression of a reporter gene relative to non‐replicating controls. Furthermore, replicons carrying CRISPR/Cas9 nucleases and repair templates achieved GT at an endogenous ubiquitin locus at frequencies 12‐fold greater than non‐viral delivery methods. The use of a strong promoter to express Cas9 was critical to attain these high GT frequencies. We also demonstrate gene‐targeted integration by homologous recombination (HR) in all three of the homoeoalleles (A, B and D) of the hexaploid wheat genome, and we show that with the WDV replicons, multiplexed GT within the same wheat cell can be achieved at frequencies of ~1%. In conclusion, high frequencies of GT using WDV‐based DNA replicons will make it possible to edit complex cereal genomes without the need to integrate GT reagents into the genome.     

QTL analysis to study the association between leaf size and abscisic acid accumulation in droughted rice leaves and comparisons across cereals

Plants accumulate abscisic acid (ABA) under droughted conditions. Genetic variation in the accumulation of ABA in detached and partially dehydrated leaves of rice has previously been reported, and this was found to be associated with variation in leaf size (smaller leaves made more ABA). Correlation analysis failed to distinguish clearly between a causal relationship between the two traits and close genetic linkage between loci controlling the traits. Here we present a detailed genetic analysis of ABA accumulation in detached and partially dehydrated rice leaves, using a population of F₂ plants generated from the lowland × upland cross IR20 (high-ABA) × 63-83 (low-ABA) which was mapped with RFLP and AFLP markers. Several highly significant quantitative trait loci (QTLs) for ABA accumulation and leaf weight were identified. Only one of the minor QTLs for ABA accumulation (accounting for only 4% of the phenotypic variance) was coincident with any QTLs for leaf size such that the high-ABA allele was associated with smaller leaves. This analysis, therefore, showed that the association previously found between ABA accumulation and leaf size was probably largely due to genetic linkage and not to a direct effect of leaf size on ABA accumulation or vice versa. Because of the importance of ABA accumulation in regulating responses of plants to drought stress and the effects of plant size on the rate of development of stress, QTLs for drought-induced ABA accumulation, leaf size and tiller number were compared between rice and wheat. In particular, a possible location in rice was sought for a homoeologue of the major wheat vernalization responsive gene, Vrn1, as this gene is also associated with major effects on leaf size, tiller number and ABA accumulation in wheat. The likelihood of homoeologous loci regulating ABA accumulation, leaf size and tiller number in the two crops is discussed.     

Characterization of polyploid wheat genomic diversity using a high‐density 90 000 single nucleotide polymorphism array

High‐density single nucleotide polymorphism (SNP) genotyping arrays are a powerful tool for studying genomic patterns of diversity, inferring ancestral relationships between individuals in populations and studying marker–trait associations in mapping experiments. We developed a genotyping array including about 90 000 gene‐associated SNPs and used it to characterize genetic variation in allohexaploid and allotetraploid wheat populations. The array includes a significant fraction of common genome‐wide distributed SNPs that are represented in populations of diverse geographical origin. We used density‐based spatial clustering algorithms to enable high‐throughput genotype calling in complex data sets obtained for polyploid wheat. We show that these model‐free clustering algorithms provide accurate genotype calling in the presence of multiple clusters including clusters with low signal intensity resulting from significant sequence divergence at the target SNP site or gene deletions. Assays that detect low‐intensity clusters can provide insight into the distribution of presence–absence variation (PAV) in wheat populations. A total of 46 977 SNPs from the wheat 90K array were genetically mapped using a combination of eight mapping populations. The developed array and cluster identification algorithms provide an opportunity to infer detailed haplotype structure in polyploid wheat and will serve as an invaluable resource for diversity studies and investigating the genetic basis of trait variation in wheat.     

水稻葡萄糖-6-磷酸脱氢酶cDNA的电子克隆

电子克隆是基因克隆的新策略,以小麦胞质葡萄糖－6－磷酸脱氢酶cDNA(Tagpdl克隆）序列为信息探针,在GenBank水稻nr数据库中找到高度同源的水稻基因组序列,通过人工序列拼接及RT-PCR确认得到了水稻该基因的全长cDNA序列,命名为OsG6PDH,OsG6PDH与小麦Tagpdl克隆的DNA一致率为88％,推导的氨基酸序列与小麦,番茄,烟草的胞质葡萄糖－6－磷酸脱氢酶基因的一致率分别为89％,79％,80％,经RT－PCR表达谱分析,OsG6PDH在水稻幼穗,胚,根,叶中都有表达,在幼穗与根中表达略高,另外,讨论了利用水稻基因组信息的电子克隆方法克隆水稻功能基因的可行性。     

Molecular Polymorphism of the Wheat Leaf Rust Fungus in Morocco Using Amplified Fragment Length Polymorphism

The genetic variability and collection structure of the wheat leaf rust fungus Puccinia recondita collected from four agro‐ecological areas of Morocco, Abda‐doukala, Chaouia‐Tadla, Gharb and Tangérois were investigated by amplified fragment length polymorphism (AFLP) markers. A set of five AFLP primers combinations which generated 253 polymorphic loci were used. Hierarchical partitioning revealed that bread wheat collections of Puccinia recondita form a single collection. No significant variation was observed between durum wheat collections of Puccinia recondita; they maintained most of the genetic variability within rather among collections. The distribution pattern of genetic variation of Puccinia recondita collections seems to be the result of high gene flow and the mixed reproduction system.     

Antagonistic action of Streptomyces pratensis S10 on Fusarium graminearum and its complete genome sequence

Wheat scab, mainly caused by Fusarium graminearum, can decrease wheat yield and grain quality. Chemical pesticides are currently the main control method but have an inevitable negative consequence on the environment and in food safety. This research studies a promising substitute, Streptomyces pratensis S10, which was isolated from tomato leaf mould and shows a significant inhibition effect on F. graminearum based on antagonism assays. The biocontrol mechanism is studied by enhanced green fluorescent protein labelling, quantitative real-time PCR, the Doskochilova 8 solvents system test and complete genome sequencing. Strain S10 can colonize in the wheat root, control wheat scab and decrease deoxynivalenol (DON) content. The control effects in vitro, planta and the plot experiments were 92.86%, 68.67% and 40.87% to 86.62%, respectively. S10 decreased DON content by inhibiting the mycelium growth and DON synthesis gene expression. The active substances of the S10 secondary metabolites had a high-temperature resistance and 29 putative biosynthetic gene clusters in its genome. The S10 control mechanism is multivariate, which shows potential in controlling wheat scab.