The transcriptional landscape of the developmental switch from regular pollen maturation towards microspore-derived plant regeneration in barley

Plant formation from in vitro-cultivated microspores involves a complex network of internal and environmental factors.Haploids/doubled haploids(DHs)derived from in vitro-cultured microspores are widely used in plant breeding and genetic engineering.However,the mechanism underlying the developmental switch from regular pollen maturation towards microspore-derived plant regeneration remains poorly defined.Here,RNA-sequencing was employed to elucidate the transcriptional landscapes of four early stages of microspore embryogenesis(ME)in barley cultivars Golden Promise and Igri,which exhibit contrasting responsiveness to microspore-derived plant formation.Our experiments revealed fundamental regulatory networks,specific groups of genes,and transcription factor(TF)families potentially regulating the developmental switch.We identified a set of candidate genes crucial for genotype-dependent responsiveness/recalcitrance to ME.Our high-resolution temporal transcriptome atlas provides an important resource for future functional studies on the genetic control of microspore developmental transition.

Leveraging the potential of big genomic and phenotypic data for genome-wide association mapping in wheat

Genome-wide association mapping studies(GWAS)based on Big Data are a potential approach to improve marker-assisted selection in plant breeding.The number of available phenotypic and genomic data sets in which medium-sized populations of several hundred individuals have been studied is rapidly increasing.Combining these data and using them in GWAS could increase both the power of QTL discovery and the accuracy of estimation of underlying genetic effects,but is hindered by data heterogeneity and lack of interoperability.In this study,we used genomic and phenotypic data sets,focusing on Central European winter wheat populations evaluated for heading date.We explored strategies for integrating these data and subsequently the resulting potential for GWAS.Establishing interoperability between data sets was greatly aided by some overlapping genotypes and a linear relationship between the different phenotyping protocols,resulting in high quality integrated phenotypic data.In this context,genomic prediction proved to be a suitable tool to study relevance of interactions between genotypes and experimental series,which was low in our case.Contrary to expectations,fewer associations between markers and traits were found in the larger combined data than in the individual experimental series.However,the predictive power based on the marker-trait associations of the integrated data set was higher across data sets.Therefore,the results show that the integration of medium-sized to Big Data is an approach to increase the power to detect QTL in GWAS.The results encourage further efforts to standardize and share data in the plant breeding community.

Discriminating ability of molecular markers and morphological characterization in the establishment of genetic relationships in cultivated genotypes of almond and related wild species

A total 23 morphological traits, 19 AFLP-primer combinations, 80 RAPD primers and 32 SSR primer pair were used to compare the informativeness and efficiency of random amplified polymorphic DNA （RAPD）, amplified fragment length polymorphism （AFLP） and simple sequence repeat （SSR） markers in establishing genetic relationships among 29 almond cultivars and three related wild species. SSRs presented a high level of polymorphism and greater information content, as assessed by the expected hetrozygosity, compared to AFLPs and RAPDs. The lowest values of expected hetrozygosity were obtained for AFLPs; however AFLPs showed the highest efficiency, owing to their capacity to reveal large numbers of bands per reaction, which led to high values for various types of indices of diversity. All the three techniques discriminated almond genotypes very effectively, except that SSRs failed to discriminate between ＇Monagha＇ and ＇Sefied＇ almond genotypes. The correlation coefficients of similarity were statistically significant for all the three marker systems, but were lower for the SSR data than for RAPDs and AFLPs. For all the markers, high similarity in dendrogram topologies was obtained, although some differences were observed. All the dendrograms, including that obtained by the combined use of all the marker data, reflect relationships for most of cultivars according to their geographic diffusion. AMOVA detected more variation among cultivated and related wild species of almond within each geographic group. Bootstrap analysis revealed that the number of markers used was sufficient for reliable estimation of genetic similarity and for meaningful comparisons of marker types.

Discriminating ability of molecular markers and morphological characterization in the establishment of genetic relationships in cultivated genotypes of almond and related wild species

A total 23 morphological traits, 19 AFLP-primer combinations, 80 RAPD primers and 32 SSR primer pair were used to compare the informativeness and efficiency of random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers in establishing genetic relationships among 29 almond cultivars and three related wild species. SSRs presented a high level of polymorphism and greater information content, as assessed by the expected hetrozygosity, compared to AFLPs and RAPDs. The lowest values of expected hetrozygosity were obtained for AFLPs; however AFLPs showed the highest efficiency, owing to their capacity to reveal large numbers of bands per reaction, which led to high values for various types of indices of diversity. All the three techniques discriminated almond genotypes very effectively, except that SSRs failed to discriminate between ‘Monagha’ and ‘Sefied’ almond genotypes. The correlation coefficients of similarity were statistically significant for all the three marker systems, but were lower for the SSR data than for RAPDs and AFLPs. For all the markers, high similarity in dendrogram topologies was obtained, although some differences were observed. All the dendrograms, including that obtained by the combined use of all the marker data, reflect relationships for most of cultivars according to their geographic diffusion. AMOVA detected more variation among cultivated and related wild species of almond within each geographic group. Bootstrap analysis revealed that the number of markers used was sufficient for reliable estimation of genetic similarity and for meaningful comparisons of marker types.

Assessment of genetic variation in tomato (Solanum lycopersicum L.) inbred lines using SSR molecular markers

A study was conducted to determine the genetic diversity of 39 determinate and indeterminate tomato inbred lines collected from China, Japan, S. Korea, and USA. Using 35 SSR polymorphic markers, a total of 150 alleles were found with moderate levels of diversity, and a high number of unique alleles existing in these tomato lines. The mean number of alleles per locus was 4.3 and the average polymorphism information content （PIC） was 0.31. Unweighted Pair Group Method with Arithmetic Mean （UPGMA） clustering at genetic similarity value of 0.85 grouped the inbred lines into four groups, where one USA cultivar formed a separate and more distant cluster. The most similar inbred lines are from USA, both with determinate type, whereas the most different lines are from USA （Us-16） and Japan （Ja-2） with determinate and indeterminate growth habit, respectively. Clustering was consistent with the known information regarding geographical location and growth habit. The genetic distance information reported in this study might be used by breeders when planning future crosses among these inbred lines.

Deep genotyping of the gene GmSNAP facilitates pyramiding resistance to cyst nematode in soybean

Soybean cyst nematode(SCN)is a highly destructive pathogen.The soybean host genome harbors at least two major genes for resistance(rhg1 and Rhg4),as well as a minor locus(SCN3-11).In the present study,a splicing site in GmSNAP11,the potential causal gene of SCN3-11,was identified by comparison of the GmSNAP11 cDNA sequences generated from resistant and susceptible soybean accessions.The sequence information was used to design a codominant CAPS marker,GmSNAP11-2565,which was used to genotype a panel of 209 soybean accessions varying with respect to SCN resistance.Analyses of the effect of the haplotypes formed by GmSNAP11-2565 and another large-effect(nonsynonymous)locus,GmSNAP11-2307,previously identified in GmSNAP11,revealed linkage disequilibrium(P<0.0001)between the two loci,suggesting that GmSNAP11-2565 could be used as a marker for GmSNAP11.GmSNAP11-2565 was accordingly used,along with established markers for GmSNAP18(rhg1)and GmSHMT(Rhg4),to characterize the panel accessions.The mean SCN female index of accessions carrying only the GmSNAP11 allele associated with resistance(20.3%)was higher than that associated with accessions carrying alleles for resistance at both GmSNAP11 and GmSNAP18(12.4%),while the index for accessions carrying alleles for resistance at all of GmSNAP11,GmSNAP18,and GmSHMT was very low(1.9%).Selection on all three markers was effective for maintaining a high level of resistance to SCN race 3.

QTL mapping of qSCN3-1 for resistance to soybean cyst nematode in soybean line Zhongpin 03-5373

Soybean cyst nematode(SCN,Heterodera glycines Ichinohe)is one of the most economically destructive pathogens.The soybean line Zhongpin03-5373(ZP),which combines resistance genes from several donors,is highly resistant to SCN race 3(SCN3).In our previous study,two QTL(rhg1 and GmSNAP11)were identified in a population of recombinant inbred lines derived from a cross between ZP and the susceptible parent Zhonghuang 13.The two QTL explained around one-third of the resistance,suggesting the presence of further QTL contributing to SCN resistance.In the present study,we used an improved version of the geneticmap comprising the previously applied 1062 molecular markers and 47 newly developed InDel(insertion-deletion)markers.The improved map revealed a novel locus contributing to SCN3 resistance:qSCN3-1,flanked by InDelmarker InDel1-7 and SNPmarker Map-0047,explained 4.55%of the phenotypic variance for resistance to SCN3 and was not involved in digenic epistatic interaction with rhg1 and GmSNAP11.Haplotypes of Map-0047_CAPS(a CAPS marker developed for Map-0047)and InDel1-7 were significantly associated with SCN3 resistance in a panel of 209 resistant and susceptible accessions.Using further allele-combination analysis for three functional markers representing three cloned resistance genes(rhg1,Rhg4,andGmSNAP11)and twomarkers flanking qSCN3-1,we found that adding the resistance allele of qSCN3-1 greatly increased soybean resistance to SCN,even in diverse genetic backgrounds.The qSCN3-1 locus will be useful for marker-assisted polygene pyramid breeding and should be targeted for the future identification of candidate genes.

Binary Vector Construction for Site-Directed Mutagenesis of <i>Kafirin</i>Genes in Sorghum

Sorghum (Sorghum bicolor (L.) Moench) is one of the world’s leading cereal crops in agricultural production, which has a special importance in the arid regions. However, unlike other cereals, sorghum grain has a lower nutritional value, which is caused, inter alia, by the resistance of its seed storage proteins (kafirins) to protease digestion. One of the effective approaches to improve the nutritional value of sorghum grain is to obtain mutants with partially or completely suppressed synthesis or altered amino acid composition of kafirins. The employment of genome editing may allow to solve this problem by introducing mutations into the nucleotide sequences of the α- and γ-kafirin genes. In this study, genomic target motifs (23 bp sequences) were selected for the introduction of mutations into the α- and γ-KAFIRIN genes of sorghum. The design of the gRNAs was conducted using the online tools CRISPROR and CHOPCHOP. Two most suitable targets were chosen for α-KAFIRIN (k1C5) and two for γ-KAFIRIN (gKAF1) genes. The insertion of respective sequences in the generic vector pSH121 was performed at the BsaI (Eco31I) sites. Validation of the cloning procedure was performed by DNA sequencing. Subcloning of the resulting constructs was performed using the SfiI restriction sites into the compatible binary vector B479p7oUZm-LH. The correct assembly of binary vectors was confirmed by restriction analysis using the MluI and SfiI cleavage sites. The four vectors created (1C - 4C) were transferred by electroporation into the Agrobacterium tumefaciens strain AGL0. Currently, this vector series is used for stable transformation of sorghum using immature embryo explants.

Domestication and crop evolution of wheat andbarley: Genes, genomics, and future directions

Wheat and barley are two of the founder crops of the agricultural revolution that took place 10,000 years ago in the Fertile Crescent and both crops remain among the world's most important crops. Domestication of these crops from their wild ancestors required the evolution of traits useful to humans, rather than survival in their natural environment. Of these traits, grain retention and threshability, yield improvement, changes to photoperiod sensitivity and nutritional value are most pronounced between wild and domesticated forms. Knowledge about the geographical origins of these crops and the genes responsible for domestication traits largely pre-dates the era of nextgeneration sequencing, although sequencing will lead to new insights. Molecular markers were initially used to calculate distance(relatedness), genetic diversity and to generate genetic maps which were useful in cloning major domestication genes. Both crops are characterized by large,complex genomes which were long thought to be beyond the scope of whole-genome sequencing. However, advances in sequencing technologies have improved the state of genomic resources for both wheat and barley. The availability of reference genomes for wheat and some of its progenitors,as well as for barley, sets the stage for answering unresolved questions in domestication genomics of wheat and barley.

The CRISPR/Cas revolution continues: From efficient gene editing for crop breeding to plant synthetic biology

Since the discovery that nucleases of the bacterial CRISPR(clustered regularly interspaced palindromic repeat)-associated(Cas) system can be used as easily programmable tools for genome engineering,their application massively transformed different areas of plant biology. In this review, we assess the current state of their use for crop breeding to incorporate attractive new agronomical traits into specific cultivars of various crop plants. This can be achieved by the use of Cas9/12 nucleases for double-strand break induction,resulting in mutations by non-homologous recombinatr e-tion. Strategies for performing such experiments à from Rthe design of guide RNA to the use of different transformation technologies à are evaluated. Furtherweive-more, we sum up recent developments regarding the use of nuclease-deficient Cas9/12 proteins, as DNAbinding moieties for targeting different kinds of enzyme activities to specific sites within the genome. Progress in base deamination, transcriptional induction and transcriptional repression, as well as in imaging in plants, is also discussed. As different Cas9/12 enzymes are at hand, the simultaneous application of various enzyme activities, to multiple genomic sites, is now in reach to redirect plant metabolism in a multifunctional manner and pave the way for a new level of plant synthetic biology.

TOPLESS promotes plant immunity by repressing auxin signaling and is targeted by the fungal effector Naked1

In plants,the antagonism between growth and defense is hardwired by hormonal signaling.The perception of pathogen-associatedmolecularpatterns(PAMPs)frominvadingmicroorganismsinhibits auxin signalingand plant growth.Conversely,pathogens manipulate auxin signaling to promote disease,but how this hormone inhibits immunity is not fully understood.Ustilago maydis is a maize pathogen that induces auxin signaling in its host.We characterized a U.maydis effector protein,Naked1(Nkd1),that is translocated into the host nucleus.Through its native ethylene-responsive element binding factor-associated amphiphilic repression(EAR)motif,Nkd1 binds to the transcriptional co-repressors TOPLESS/TOPLESS-related(TPL/TPRs)and prevents the recruitment of a transcriptional repressor involved in hormonal signaling,leading to the derepression of auxin and jasmonate signaling and thereby promoting susceptibility to(hemi)biotrophic pathogens.A moderate upregulation of auxin signaling inhibits the PAMP-triggered reactive oxygen species(ROS)burst,an early defense response.Thus,our findings establish a clear mechanism for auxin-induced pathogen susceptibility.Engineered Nkd1 variants with increased expression or increased EAR-mediated TPL/TPR binding trigger typical salicylic-acid-mediated defense reactions,leading to pathogen resistance.This implies that moderate binding of Nkd1 to TPL is a result of a balancing evolutionary selection process to enable TPL manipulation while avoiding host recognition.

Evolution-aided engineering of plant specialized metabolism

The evolution of new traits in living organisms occurs via the processes of mutation,recombination,genetic drift,and selection.These processes that have resulted in the immense biol ogical diversity on our planet are also being employed in metabolic engineering to optimize enzymes and pathways,create new-to-nature reactions,and synthesize complex natural products in heterologous systems.In this review,we discuss two evolution-aided strategies for metabolic engineering-directed evolution,which improves upon existing genetic templates using the evolutionary process,and combinatorial pathway reconstruction,which brings together genes evolved in different organisms into a single heterol ogous host.We discuss the general principles of these strategies,describe the technologies involved and the molecular traits they influence,provide examples of their use,and discuss the roadblocks that need to be addressed for their wider adoption.A better understanding of these strategies can provide an impetus to research on gene function discovery and biochemical evolution,which is foundational for improved metabolic engineering.These evolution-aided approaches thus have a substantial potential for improving our understanding of plant metabolism in general,for enhancing the production of plant metabolites,and in sustainable agriculture.

用基因芯片技术分析干旱胁迫下生殖生长期大麦的基因表达(英文)

耐旱性是干旱地区稳定和增加大麦产量的一个关键因素。鉴定出与耐旱性相关的功能基因,一方面可了解大麦的耐旱机理,同时还可以促进利用生物技术来改良大麦的耐旱性。在研究中,2个在耐旱性上具有明显差异的大麦品种Tadmor(耐旱)和WI2291(干旱敏感)被选作材料,采用22000个ESTs(基因表达序列标签)的Affymetrix大麦基因芯片Barley1来分析生殖生长期干旱胁迫下2个大麦材料的差异表达基因。研究结果表明,干旱胁迫下2个大麦材料中有77个共调节基因,其中部分基因已被报道过可能与抗旱性相关。这些基因中已有功能注释的基因按其生物学功能被分为14组,猜测它们是干旱胁迫的响应基因,在抗旱性上可能不起重要作用,或者是必需的但单独不足以提高大麦的抗旱性。进一步比较2个材料差异表达的基因,发现二材料之间有372个受干旱调节基因的差异。这些基因中有功能注释基因的生物学功能中可分为15组,其中一些已被认为与抗旱性相关;而对那些未知功能的基因,推测可能亦在大麦的抗旱性上扮演一定的角色。研究所得结果可为阐述生殖生长期大麦的耐旱性机理提供新的认识。

群体构成方式对大豆百粒重全基因组选择预测准确度的影响

百粒重是大豆产量的重要构成因子,在一定条件下与产量呈显著正相关。百粒重是一个复杂的数量性状,用传统的育种方法其遗传增益不明显。本研究对280份大豆品种进行了多年多点田间鉴定,通过混合线性模型预测获得品种百粒重的最佳线性无偏预测值。同时利用分布在大豆全基因组的5361个SNP标记鉴定参试品种基因型,结合随机回归最佳线性无偏预测模型和交互验证方法,探讨了群体构成方式对大豆百粒重的全基因组选择预测准确度的影响。结果表明,大豆百粒重的全基因组选择预测准确度变化范围为–0.15^+0.75;群体构成方式对百粒重的预测准确度影响明显;亚群内的预测准确度(+0.24^+0.75)高于亚群间(-0.15^+0.29);当群体间遗传距离由0.1566增加到0.2201时,预测准确度下降27.87%;相比随机构建的训练群体,基于群体遗传结构构建的训练群体能将百粒重的预测准确度提高2.34%。本研究明确了大豆百粒重的全基因组选择预测准确度,阐明了群体结构对大豆百粒重的全基因组选择预测准确度的影响,为大豆分子育种提供了新的思路和方法。

Nutrient-hormone relations:Driving root plasticity in plants

Optimal plant development requires root uptake of 14 essential mineral elements from the soil.Since the bioavailability of these nutrients underlies large variation in space and time,plants must dynamically adjust their root architecture to optimize nutrient access and acquisition.The information on external nutrient availability and whole-plant demand is translated into cellular signals that often involve phytohormones as intermediates to trigger a systemic or locally restricted developmental response.Timing and extent of such local root responses depend on the overall nutritional status of the plant that is transmitted from shoots to roots in the form of phytohormones or other systemic long-distance signals.The integration of these systemic and local signals then determines cell division or elongation rates in primary and lateral roots,the initiation,emergence,or elongation of lateral roots,as well as the formation of root hairs.Here,we review the cascades of nutrient-related sensing and signaling events that involve hormones and highlight nutrient-hormone relations that coordinate root developmental plasticity in plants.

Genome-wide prediction for hybrids between parents with distinguished difference on exotic introgressions in Brassica napus

Extensive exotic introgression could significantly enlarge the genetic distance of hybrid parental populations to promote strong heterosis.The goal of this study was to investigate whether genome-wide prediction can support pre-breeding in populations with exotic introgressions.We evaluated seed yield,seed yield related traits and seed quality traits of 363 hybrids of Brassica napus (AACC) derived from two parental populations divergent on massive exotic introgression of related species in three environments.The hybrids presented strong heterosis on seed yield,which was much higher than other investigated traits.Five genomic best linear unbiased prediction models considering the exotic introgression and different marker effects (additive,dominance,and epistatic effects) were constructed to test the prediction ability for different traits of the hybrids.The analysis showed that the trait complexity,exotic introgression,genetic relationship between the training set and testing set,training set size,and environments affected the prediction ability.The models with best prediction ability for different traits varied.However,relatively high prediction ability (e.g.,0.728 for seed yield) was also observed when the simplest models were used,excluding the effects of the special exotic introgression and epistasis effect by5-fold cross validation,which would simplify the prediction for the trait with complex architecture for hybrids with exotic introgression.The results provide novel insights and strategies for genome-wide prediction of hybrids between genetically distinct parent groups with exotic introgressions.

Genome-wide prediction in a hybrid maize population adapted to Northwest China

Genome-wide prediction is a promising approach to boost selection gain in hybrid breeding.Our main objective was to evaluate the potential and limits of genome-wide prediction to identify superior hybrid combinations adapted to Northwest China.A total of 490 hybrids derived from crosses among 119 inbred lines from the Shaan A and Shaan B heterotic pattern were used for genome-wide prediction of ten agronomic traits.We tested eight different statistical prediction models considering additive(A)effects and in addition evaluated the impact of dominance(D)and epistasis(E)on the prediction ability.Employing five-fold cross validation,we show that the average prediction ability ranged from 0.386 to 0.794 across traits and models.Six parametric methods,i.e.ridge regression,LASSO,Elastic Net,Bayes B,Bayes C and reproducing kernel Hilbert space(RKHS)approach,displayed a very similar prediction ability for each trait and two non-parametric methods(random forest and support vector machine)had a higher prediction performance for the trait rind penetrometer resistance of the third internode above ground(RPR_TIAG).The models of A+D RKHS and A+D+E RKHS were slightly better for predicting traits with a relatively high non-additive variance.Integrating trait-specific markers into the A+D RKHS model improved the prediction ability of grain yield by 3%,from 0.528 to 0.558.Of all 6328 potential hybrids,selection of the top 44 hybrids would lead to a 6%increase in grain yield compared with Zhengdan 958,a commercially successful hybrid variety.In conclusion,our results substantiate the value of genome-wide prediction for hybrid breeding and suggest dozens of promising single crosses for developing high-yielding hybrids for Northwest China.

Genome-wide signatures of the geographic expansion and breeding of soybean

Soybean is a leguminous crop that provides oil and protein. Exploring the genomic signatures of soybean evolution is crucial for breeding varieties with improved adaptability to environmental extremes. We analyzed the genome sequences of 2,214 soybeans and proposed a soybean evolutionary route, i.e., the expansion of annual wild soybean(Glycine soja Sieb. & Zucc.) from southern China and its domestication in central China, followed by the expansion and local breeding selection of its landraces(G. max(L.) Merr.). We observed that the genetic introgression in soybean landraces was mostly derived from sympatric rather than allopatric wild populations during the geographic expansion. Soybean expansion and breeding were accompanied by the positive selection of flowering time genes, including GmSPA3c. Our study sheds light on the evolutionary history of soybean and provides valuable genetic resources for its future breeding.

A reference-guided TILLING by amplicon-sequencing platform supports forward and reverse genetics in barley

Barley is a diploid species with a genome smaller than those of other members of the Triticeae tribe,making it an attractive model for genetic studies in Triticeae crops.The recent development of barley genomics has created a need for a high-throughput platform to identify genetically uniform mutants for gene function investigations.In this study,we report an ethyl methanesulfonate(EMS)-mutagenized population consisting of 8525M_(3) lines in the barley landrace“Hatiexi”(HTX),which we complement with a high-quality de novo assembly of a reference genome for this genotype.The mutation rate within the population ranged from 1.51 to 4.09 mutations per megabase,depending on the treatment dosage of EMS and the mutation discrimination platform used for genotype analysis.We implemented a three-dimensional DNA pooling strategy combined with multiplexed amplicon sequencing to create a highly efficient and cost-effective TILLING(targeting induced locus lesion in genomes)platform in barley.Mutations were successfully identified from 72 mixed amplicons within a DNA pool containing 64 individual mutants and from 56 mixed amplicons within a pool containing 144 individuals.We discovered abundant allelic mutants for dozens of genes,including the barley Green Revolution contributor gene Brassinosteroid insensitive 1(BRI1).As a proof of concept,we rapidly determined the causal gene responsible for a chlorotic mutant by following the MutMap strategy,demonstrating the value of this resource to support forward and reverse genetic studies in barley.

Integration of genomic selection with doubled-haploid evaluation in hybrid breeding: From GS 1.0 to GS 4.0 and beyond

DOUBLED-HAPLOID TECHNOLOGY FACES A GREAT CHALLENGE FOR HYBRID BREEDING,Ensuring food security for the ever-growing population is a common mission and a great challenge for agricultural scientists worldwide.Historically,advances in crop breeding and management practices have contributed substantially to crop productivity.Indeed,the substantial increase in global grain yields over the last eight decades is largely due to the adoption of hybrids.However,the rate of increase of hybrid yields began to slow down in the early 2000s,and since then,it has reached a plateau for many crops and regions(https://faostat.fao.org).Therefore,we must find solutions to accelerating genetic gain and boost hybrid development,for which developing new breeding technologies provides novel creative opportunities.