Elucidating the molecular responses of apple rootstock resistant to ARD pathogens:challenges and opportunities for development of genomics-assisted breeding tools

Apple replant disease(ARD)is a major limitation to the establishment of economically viable orchards on replant sites due to the buildup and long-term survival of pathogen inoculum.Several soilborne necrotrophic fungi and oomycetes are primarily responsible for ARD,and symptoms range from serious inhibition of growth to the death of young trees.Chemical fumigation has been the primary method used for control of ARD,and manipulating soil microbial ecology to reduce pathogen density and aggressiveness is being investigated.To date,innate resistance of apple rootstocks as a means to control this disease has not been carefully explored,partly due to the complex etiology and the difficulty in phenotyping the disease resistance.Molecular defense responses of plant roots to soilborne necrotrophic pathogens are largely elusive,although considerable progress has been achieved using foliar disease systems.Plant defense responses to necrotrophic pathogens consist of several interacting modules and operate as a network.Upon pathogen detection by plants,cellular signals such as the oscillation of Ca^(2+)concentration,reactive oxygen species(ROS)burst and protein kinase activity,lead to plant hormone biosynthesis and signaling.Jasmonic acid(JA)and ethylene(ET)are known to be fundamental to the induction and regulation of defense mechanisms toward invading necrotrophic pathogens.Complicated hormone crosstalk modulates the fine-tuning of transcriptional reprogramming and metabolic redirection,resulting in production of antimicrobial metabolites,enzyme inhibitors and cell wall refortification to restrict further pathogenesis.Transcriptome profiling of apple roots in response to inoculation with Pythium ultimum demonstrated that there is a high degree of conservation regarding the molecular framework of defense responses compared with those observed with foliar tissues.It is conceivable that the timing and intensity of genotype-specific defense responses may lead to different outcomes between rootstocks in response to invasion by necrotrophic pathogens.Elucidation of host defense mechanisms is critical in developing molecular tools for genomics-assisted breeding of resistant apple rootstocks.Due to their perennial nature,use of resistant rootstocks as a component for disease management might offer a durable and cost-effective benefit to tree performance than the standard practice of soil fumigation for control of ARD.

Genetic diversity and a population structure analysis of accessions in the Chinese cowpea [Vigna unguiculata(L.)Walp.]germplasm collection

Cowpea(Vigna imguicuiata) is an important legume crop with diverse uses. The species is presently a minor crop, and evaluation of its genetic diversity has been very limited. In this study, a total of 200 genic and 100 genomic simple sequence repeat(SSR) markers were developed from cowpea unigene and genome sequences, respectively. Among them, 27 genic and 27 genomic SSR markers were polymorphic and were used for assessment of genetic diversity and population structure in 105 selected cowpea accessions. A total of 155 alleles and 2.9 alleles per marker were identified, and the average polymorphic information content(PIC) value was 0.3615. The average PIC of genomic SSRs(0.3996) was higher than that of genic SSRs(0.3235), and most of the polymorphic genomic SSRs were composed of di-and trinucleotide repeats(51.9% and 37.0% of all loci, respectively). The low level of detected genetic diversity may be attributed to a severe genetic bottleneck that occurred during the cowpea domestication process. The accessions were classified by structure and cluster analysis into four subgroups that correlated well with their geographic origins or collection sites. The classification results were also consistent with the results from principal coordinate analysis and can be used as a guide during future germplasm collection and selection of accessions as breeding materials for cultivar improvement. The newly developed genic and genomic SSR markers described in this study will be valuable genomic resources for the assessment of genetic diversity, population structure, evaluation of germplasm accessions, construction of genetic maps, identification of genes of interest,and application of marker-assisted selection in cowpea breeding programs.

Transcriptional regulation of ethylene and jasmonate mediated defense response in apple(Malus domestica)root during Pythium ultimum infection

Apple replant disease(ARD)is a significant economic restraint to the successful re-establishment of new apple orchards on sites previously planted to the same crop.Pythium ultimum,an oomycete,is a significant component of the ARD pathogen complex.Although ethylene(ET)-and jasmonic acid(JA)-mediated defense responses are intensively studied in the foliar pathosystem,the transferability of this knowledge to the interaction between a perennial root system and soilborne pathogens is unknown.The aim of this study was to test the hypothesis that the ET/JA-mediated defense response is conserved in roots of tree crops in response to infection by P.ultimum.Apple genes with the annotated function of ET/JA biosynthesis,MdERF(ethylene response factor)for signaling transduction and a gene encoding a pathogenesis-related(PR)protein(b-chitinase,the target of ERF)were identified from the apple genome sequences.The transcriptional profiles of these genes during P.ultimum infection and after exogenous ET and/or JA treatment were characterized using qRT-PCR.Several genes showed a 10-to 60-fold upregulation in apple root tissue 24-48 h post inoculation(hpi).Exogenous ET and JA treatment exhibited either a positive or negative influence on expression of ET or JA biosynthesis genes,depending upon gene isoforms and the tissue types,while the expression of MdERF and the PR protein encoding gene was upregulated by both ET and JA treatment.Our data are consistent with the hypothesis that ET/JA-mediated defense pathways are functional in the root system of perennial tree species defending soilborne pathogens.

A key‘foxy’aroma gene is regulated by homologyinduced promoter indels in the iconic juice grape‘Concord’

‘Concord’,the most well-known juice grape with a parentage of the North American grape species Vitis labrusca L.,possesses a special‘foxy’aroma predominantly resulted from the accumulation of methyl anthranilate(MA)in berries.This aroma,however,is often perceived as an undesirable attribute by wine consumers and rarely noticeable in the common table and wine grape species V.vinifera.Here we discovered homology-induced promoter indels as a major genetic mechanism for species-specific regulation of a key‘foxy’aroma gene,anthraniloyl-CoA:methanol acyltransferase(AMAT),that is responsible for MA biosynthesis.We found the absence of a 426-bp and/or a 42-bp sequence in AMAT promoters highly associated with high levels of AMAT expression and MA accumulation in‘Concord’and other V.labrusca-derived grapes.These promoter variants,all with direct and inverted repeats,were further confirmed in more than 1,300 Vitis germplasm.Moreover,functional impact of these indels was validated in transgenic Arabidopsis.Superimposed on the promoter regulation,large structural changes including exonic insertion of a retrotransposon were present at the AMAT locus in some V.vinifera grapes.Elucidation of the AMAT genetic regulation advances our understanding of the‘foxy’aroma trait and makes it genetically trackable and amenable in grapevine breeding.

Patterns of genomic and phenomic diversity in wine and table grapes

Grapes are one of the most economically and culturally important crops worldwide,and they have been bred for both winemaking and fresh consumption.Here we evaluate patterns of diversity across 33 phenotypes collected over a 17-year period from 580 table and wine grape accessions that belong to one of the world’s largest grape gene banks,the grape germplasm collection of the United States Department of Agriculture.We find that phenological events throughout the growing season are correlated,and quantify the marked difference in size between table and wine grapes.By pairing publicly available historical phenotype data with genome-wide polymorphism data,we identify large effect loci controlling traits that have been targeted during domestication and breeding,including hermaphroditism,lighter skin pigmentation and muscat aroma.Breeding for larger berries in table grapes was traditionally concentrated in geographic regions where Islam predominates and alcohol was prohibited,whereas wine grapes retained the ancestral smaller size that is more desirable for winemaking in predominantly Christian regions.We uncover a novel locus with a suggestive association with berry size that harbors a signature of positive selection for larger berries.Our results suggest that religious rules concerning alcohol consumption have had a marked impact on patterns of phenomic and genomic diversity in grapes.

Population structure and association mapping to detect QTL controlling tomato spotted wilt virus resistance in cultivated peanuts

Tomato spotted wilt(TSW)is a serious virus disease of peanut in the United States.Breeding for TSWV resistance would be facilitated by the implementation of marker-assisted selection in breeding programs;however,genes associated with resistance have not been identified.Association mapping is a type of genetic mapping that can exploit relationships between markers and traits in many lineages.The objectives of this study were to examine genetic diversity and population structure in the U.S.peanut mini-core collection using simple sequence repeat(SSR)markers,and to conduct association mapping between SSR markers and TSWV resistance in cultivated peanuts.One hundred and thirty-three SSR markers were used for genotyping 104 accessions.Four subpopulations,generally corresponding to botanical varieties,were classified by population structure analysis.Association mapping analysis indicated that five markers:pP GPseq5D5,GM1135,GM1991,TC23C08,and TC24C06,were consistently associated with TSW resistance by the Q,PCA,Q+K,and PCA+K models.These markers together explained 36.4%of the phenotypic variance.Moreover,pP GPseq5D5 and GM1991 were associated with both visual symptoms of TSWV and ELISA values with a high R^2.The potential of these markers for use in a marker-assisted selection program to breed peanut for resistance to TSWV is discussed.

A comparison of methods used to determine the oleic/linoleic acid ratio in cultivated peanut (Arachis hypogaea L.)

There is an increasing demand in the peanut industry for high oleic peanuts and also for the incorporation of the high oleate trait into newly released varieties. Early generation screening of breeding lines for high oleic acid content greatly increases the efficiency of developing new peanut varieties. The objective of this study was to compare the accuracy of methods used to classify individual peanut seed as high oleic or not high oleic. Results from capillary electrophoresis (CE), two variations of near-infrared reflectance spectroscopy (NIRS), and real-time polymerase chain reaction (RT-PCR) genotyping, were compared with the traditionally accepted reference standard results from gas chromatography (GC). Three hundred and seventy-four (374) seeds, spanning twenty-three (23) genotypes and all four peanut market-types (runner, Spanish, Valencia and Virginia), were individually tested by each method. Percent accuracy levels for rating individual seed as high oleic (H) ranged from 97.4% (NIRS) to 99.5% (CE). All of the methods examined in this study carry only a minor risk for miss-classification (loss of material) and are suitable for use by peanut breeding programs in early generation breeding line screening.

Apple whole genome sequences:recent advances and new prospects

In 2010,a major scientific milestone was achieved for tree fruit crops:publication of the first draft whole genome sequence(WGS)for apple(Malus domestica).This WGS,v1.0,was valuable as the initial reference for sequence information,fine mapping,gene discovery,variant discovery,and tool development.A new,high quality apple WGS,GDDH13 v1.1,was released in 2017 and now serves as the reference genome for apple.Over the past decade,these apple WGSs have had an enormous impact on our understanding of apple biological functioning,trait physiology and inheritance,leading to practical applications for improving this highly valued crop.Causal gene identities for phenotypes of fundamental and practical interest can today be discovered much more rapidly.Genome-wide polymorphisms at high genetic resolution are screened efficiently over hundreds to thousands of individuals with new insights into genetic relationships and pedigrees.High-density genetic maps are constructed efficiently and quantitative trait loci for valuable traits are readily associated with positional candidate genes and/or converted into diagnostic tests for breeders.We understand the species,geographical,and genomic origins of domesticated apple more precisely,as well as its relationship to wild relatives.The WGS has turbo-charged application of these classical research steps to crop improvement and drives innovative methods to achieve more durable,environmentally sound,productive,and consumer-desirable apple production.This review includes examples of basic and practical breakthroughs and challenges in using the apple WGSs.Recommendations for“what’s next”focus on necessary upgrades to the genome sequence data pool,as well as for use of the data,to reach new frontiers in genomics-based scientific understanding of apple.

Comparative anatomical studies of Artocarpus altilis(Parkinson) Fosberg and Artocarpus communis(J.R.& G.Forster) in Nigeria

Comparative anatomy of two Artocarpus species was carried out to identify and describe anatomical characters in search of distinctive characters that could possibly be used to delimit the two taxa. Transverse, tangential and radial longitudinal sections and macerated samples of the stem and root wood were prepared onto microscopic slides. Epidermal peels and cleared leaves of the two species were made. Characteristic similarity disparity in the tissues arrangement as well as cell inclusions were noted for description and delimitation. The two Artocarpus species studied had essentially the same an- atomical features; however, there were characters that seem to be taxon specific. The study revealed that at the transverse plane of the root, A. communis （J. R. ＆ G. Forster） have predominantly solitary vessel, whereas pore multiple was pre- dominant in A. altilis （Parkinson） Fosberg. Tyloses in vessels of the root were more frequent in A. communis than in A. altilis. In the cleared leaves, venation pattem also revealed some differences in the two species. The veins ofA. communis were more or less straight and that ofA. altilis were undulating especially in the secondary veins. Prismatic crystals were found in the cortex of the petiole in A. communis but not in A. altilis. Similarly, tannins were found in the root bark of A. communis but not in A. altilis. Trichomes and scales were more abundant in A. communis than in A. altilis. In the transverse section of the leaves, abaxial and adaxial epidermis were uniseriate in A. cornmunis but only the abaxial epidermis was uniseriate in A. altilis, the adaxial epidermis was made up of 2 to 3 layers of cells. The epidermal cells in A. communis were predominantly short cylindrical shaped cells but were not so in A. altilis.

Development of Root Phenotyping Platforms for Identification of Root Architecture Mutations in EMS-Induced and Low-Path-Sequenced Sorghum Mutant Population

Sorghum’s natural adaptation to a wide range of abiotic stresses provides diverse genetic reserves for potential improvement in crop stress tolerance. Growing interest in sorghum research has led to the expansion of genetic resources though establishment of the sorghum association panel (SAP), generation of mutagenized populations, and recombinant inbred line (RIL) populations, etc. Despite rapid improvement in biotechnological tools, lack of efficient phenotyping platforms remains one of the major obstacles in utilizing these genetic resources. Scarcity of efforts in root system phenotyping hinders identification and integration of the superior root traits advantageous to stress tolerance. Here, we explored multiple approaches in root phenotyping of an ethyl methanesulfonate (EMS)-mutagenized sorghum population. Paper-based growth pouches (PGP) and hydroponics were employed to analyze root system architecture (RSA) variations induced by mutations and to test root development flexibility in response to phosphorus deficiency in early growing stages. PGP method had improved capabilities compared to hydroponics providing inexpensive, space-saving, and high-throughput phenotyping of sorghum roots. Preliminary observation revealed distinct phenotypic variations which were qualitatively and quantitatively systemized for association analysis. Phenotypes/ideotypes with root architecture variations potentially correlated with Pi acquisition were selected to evaluate their contribution to P-efficiency (PE). Sand mixed with P-loaded activated alumina substrate (SAS) provided closely to natural but still controlled single-variable conditions with regulated Pi availability. Due to higher labor and cost input we propose SAS to be used for evaluating selected sorghum candidates for PE. The ability of rapidly screening root phenotypes holds great potential for discovering genes responsible for relevant root traits and utilizing mutations to improve nutrient efficiency and crop productivity.

Comparison of Stem Damage and Carbohydrate Composition in the Stem Juice between Sugarcane and Sweet Sorghum Harvested before and after Late Fall Frost

A late fall frost may significantly affect sugar crops’ stem sugar composition, yield and juice quality for biofuel and bioproduct manufacture. Research on the effects of late fall frost in sugarcane is well documented, but information is lacking for sweet sorghum. Three and six commercial cultivars of sugarcane and sweet sorghum, respectively, were selected and evaluated for exposure to a late fall frost (-2.8&degC) in Griffin, Georgia, USA. Under the same controlled environmental conditions in a screen house, the late fall frost induced more damage to sugarcane than sweet sorghum stems. The frost caused damage to sugarcane tissue and for juice to exude from stems, whereas similar behavior was not observed for sweet sorghum. In both sugarcane and sweet sorghum, the glucose/fructose ratio was significantly reduced, but this change may not be totally directly related to the frost effect. Overall, these initial results suggest that sweet sorghum may have a better tolerance to fall frost than sugarcane. Two sweet sorghum cultivars, Grassl and M81E, responded well to the late fall frost, and they can possibly be used as feedstocks for biofuel/bioproduct manufacture in areas susceptible to frosts including northern regions of the Southeastern US.