Cassava Brown Streak Virus Infection Genome

Genome-abundant prophecy and connection partition ce sensitivity to cassava brown streak bane contamination in Cassava

  • Siraj Ismail Kayondo, Dunia Pino Del Carpio, Roberto Lozano, Alfred Ozimati, Marnin Wolfe, Yona Baguma, Vernon Gracen, Offei Samuel, Robert Kawuki and Jean-Luc Jannink

 

ABSTRACT

Cassava (maniburning esculenta Crantz), a pilot carbohydrebuke fount faces rare dare of viral illnesss mainly, cassava brown streak illness (CBSD) and cassava mosaic illness (CMD). The economic competency of the quenchedenlargement are rendered unmarketable by these viral illnesss fruiting into mega fiscal determinedbacks. The singular substance of the cassava genome connote equips cassava breeders with further sharp-ended choice strategies to adduce surpassing varieties with twain laborer and activity preferred touchs.

This proviso reports genomic segments companiond to foliar and origin CBSV sensitivity meted at unanalogous enlargement stages and environmental stipulations.

We attested expressive solely nucleotide polymorphisms (SNPs) companiond to CBSV sensitivity in cassava on chromosome 4 and 11. The expressively companiond territorys on chromosome 4 co-localises with a Maniburning glaziovii introgression from the disorderly progenitors. While expressive SNPs markers on chromosome 11 are in linkage disequilibrium (LD) with a bunch of nucleotide-binding footing leucine-rich relate (NBS-LRR) proteins encoded by illness hindrance genes in introduces. Genotype by environmental interactions were expressive past SNP marker propertys differed oppostanding environments and years.

Pilot words: Genome-abundant connection studies (GWAS), bane sensitivity, augmented schemes, de-regressed best rectirectirectidirect unjaundiced Prophecys (dr-BLUPs), NBS-LRR proteins, QTLs  

INTRODUCTION

Cassava (Maniburning esculenta crantz), is a greater fount of pay and dietary calories ce aggravate a billion lives oppostanding the sphere in-particular in Sub Saharan Africa (SSA). Edge bitter technologies are ahead turning cassava into an indussuffering quenchedenlargement in-particular tapping into it’s sole lugubriousness qualities future commencement stconcatenate pay opportunities ce the bald (Pérez et al., 2011). Cassava brown streak bane illness (CBSD), a innate viral employment limiting genesis oppostanding SSA is chargeable on ce mega fiscal determinedbacks estimated at 100 US pet dollars per annum at physiological ripeness (ASARECA:, 2013; Ndunguru et al., 2015). As a connote of CBSVs, cassava succumbs were recorded to be eight seasons inferior than the expected succumb virtual in Uganda(). Span greater strains; Cassava brown streak bane (CBSV) and Uganda Cassava brown streak bane (UCBSV), bear successeasily colonized twain the lowland and elevatedland altitudes oppostanding East Africa though strangeer strains are substance reputed (Winter et al., 2010; Ndunguru et al., 2015; Alicai et al., 2016a). In attention to stormy exfluctuate of contaminated cassava steaks inaccomplished cassava laborers oppostanding percolable borders, the African whitefly (Besimia tobaci) stands quenched as the celebrated semi-persistent bane transmitter lower arena stipulations (Legg et al., 2014; McQuaid et al., 2015). Upon initiation, the bane exploits the introduce’s rapture assortment to obstruct the meek cassava introduce fruiting into yellow chlorotic temper defecation patterns concurrently puerileer tempers of the leaves. Prominent brown elongated lesions are cemed on the radix arrangementatically referred to as “brown streaks”. While the brown necrotic hard-corky layers are accidentally cemed in the origin cortex of most meek cassava clones.

In light of the speedy barring steadily bane disconnection rebukes and the absence of dependable bane cue machines (Alicai et al., 2016b), air ce persistent CBSD hindrance emerges as a prompt and economically viable liberty. Earlier CBSD hindrance air initiatives bear elevatedlighted it’s polygenic barring recessive kind of heritage in twain intraspecific and interspecific cassava hybrids (Nichols, 1947; Hillocks and Jennings, 2003; Munga, 2008; Kulembeka, 2010). The rebuke of advancement to genetic increase in a transmitted cassava air pipeline has been sinferior imputable to sundry biology-kindred opportunities like; bashful puerile, prolixity of air cycle, scant genetic dissimilarity and lazy rebuke of reiter-ation of introduceing materials. Most of the advantageous aristocracy cassava lines bear exhibited some flatten of sensitivity to CBSVs ranging from placid sensitivity entirety excitability.

Therefore, a terse barring then targeted question ce virtual founts of hindrance using the advantageous biotechnology machines could be a calm diplomacy. The singular substance of the cassava genome connote equips cassava breeders with further sharp-ended choice strategies to adduce surpassing varieties with twain laborer and activity preferred touchs. A con-balance by Bredeson et al., (2016) reports the intercourse of introgressions segments from the disorderly progenitors into the aristocracy air lines exposed by the Amani air program in Tanzania.

Hence, hindrance founts to CBSD insist barring may bear been reshuffled aggravate generations of returning choice thus referable easily agricultural and insufficiency to be exploited.

Moving ceward, a genome abundant superintend ce insisting consistent alterations as explained by the observed phenotypes ce a attached course of agronomic touchs could facilitate identification of causal loci companiond with the heritage of a touch of cause. This machine, arrangementatically referred to as genome-abundant connection con-balance (GWAS) exploits the propertyiveness of statistical analyses to establish such unromantic recombination events that bear occurred aggravate season (Jannink and Walsh, 2002; Hamblin, Buckler and Jannink, 2011). Future, GWA-studies conquer fulfilment bi-parental mapping efforts that bear been abundantly applied in cassava air in the coercionegoing decade (Ferguson et al., 2012; Ceballos et al., 2015). GWA-studies bear been abundantly lowertaken by lewd, anthropological and introduce geneticists to establish leading touch loci (QTLs) in end connection to sundry main touchs. However, GWAS has been thinly applied in cassava air in-particular in the specification of the genetic architecture of cassava mosaic illness (Wolfe et al., 2016) and beta carotene (unpublished). In this con-over, we exploited the cheap genotyping costs using genotyping by sequencing (GBS) to genotype axioms ce our connection mapping panel.

The scheme of this con-balance was to establish genomic territorys endly companiond with sensitivity to CBSV contamination in a separeprimand territoryal cassava air panel. Fine mapping about the attested territorys would pilot in marker solution as courteous-mannered-mannered as identification of franking genes ce CBSV sensitivity ce marker assisted air.

MATERIALS AND METHODS

Introduce material

The axioms determined intervening of arena illness evaluations lowertaken oppostanding five dregss; Namulonge, Kamuli, Serere, Ngetta and Kasese in Uganda. Span unanalogous barring endly kindred GWAS panels were evaluated oppostanding environments.

Betwixt 2012 and 2013, GWAS panel 1 consisted of betwixt 308 to 429 entries that were replicated twice oppostanding three dregss. Each suffering was planned as a accidentalized accomplished fill (RCB) with span-grade plots of five introduces each at a spacing of 1 meter by 1 meter. In 2015, GWAS panel 2 consisting of entries ranging from 715 to 872 clones was evaluated in three dregss barring contrasting footings ce CBSD rule. These entries were evaluated as solely entries per footing substance alike by six contemptible checks in an augmented accomplishedly accidentalized fill scheme with 38 fills per footing (Federer, Nguyen and others, 2002; Federer and Crossa, 2012).

The span GWAS panels had particular dregs in contemptible; Namulonge that is treasured as the CBSD burning dishonor with the elevatedest CBSD rule.

The axioms was generated from 1281 cassava clones exposed through three cycles of genetic recombination with extremeical aristocracy lines by the National origin quenchedgrowths air program at NaCRRI. These cassava clones had a separeprimand genetic beting whose genealogy could be traced tail to introductions from interpolitical induct ce rhetorical husbandry (IITA), Interpolitical disposition ce rhetorical Husbandry (CIAT) and Tanzania[KI1] air program (sup.fig1).

Phenotyping protocol ce CBSV sensitivity   

The pilot touchs were CBSD injustice and stroke jawd at 3, 6, and 9 months subjoined introduceing (MAP) ce foliar and 12 MAP ce origin symptoms respectively. CBSD injustice was meted invetereprimand on a 5 sharp-end flake with a jaw of 1 implying asymptomatic stipulations and a jaw 5 implying aggravate 50% leaf unreal defecation lower foliar symptoms. However, at 12 MAP a jaw of 5 implies aggravate 50% of origin-core substance dressed by a necrotic corky layer. (fig.1)

Clones were classified with a jaw of 5 if pronounced temper defecation at greater leaf tempers were jointly displayed with brown streaks on the radixs and spray die-tail that appeared as a candle-stick. Clones with 31 – 40% leaf temper defecation conjointly with brown steaks at the radixs were classified lower jaw 4. A Jaw of 3 was assigned to clones with 21 – 30% leaf temper defecation with emerging brown streaks on the radixs. While a jaw of 2 was assigned to clones that solely displayed 1 – 20% leaf temper defecation withquenched any conspicuous brown streak symptoms on the radixs. Introduces classified with a jaw of 1 showed no conspicuous memorial of leaf necrosis and brown streaks on the radixs. On the other rule, origin symptoms were to-boot classified into 5 unanalogous categories invetereprimand on a 5 – sharp-end touchstone flake.

Two-stage genomic analyses

Ce the panel 1 which was planned as a accidentalized accomplished fill (RCB) we serve the gauge: , using the lmer office from the lme4 R bundle (Bates et al., 2015).In this gauge, β comprised a agricultural property ce the population medium and dregs. The stroke matrix Zclone and the vector c indicate a accidental property ce clparticular and I indicate the particularness matrix. The concatenate mutable, which is the grade or support concurrently which plots are arrayed, is nested in dregs-rep and is indicateed by the stroke matrix Zrange(loc.) and accidental propertys vector .Fill propertys were nested in concatenates and incorporated as accidental with stroke matrix Zblock(range) and propertys vector . Residuals were serve as accidental, with .

Ce panel 2, which followed an augmented scheme, we serve the gauge Where y was the vector of unfinished phenotypes, β comprised a agricultural property ce the population medium and dregs with checks comprised as a covariate, The stroke matrix Zclone and the vector c are the selfselfsame as aggravate and the fills were to-boot gaugeed with stroke matrix and b indicates the accidental property ce fill. The best rectirectirectidirect predictors (BLUPs) of the clparticular property (ĉ) were extracted as de-regressed BLUPS subjoined the cemula:

Unreserved feeling heritability was pleaseted using hostility contents extracted from the span trudge lmer output. SNP-invetereprimand heritability was pleaseted by extracting the hostility contents from the quenchedput obtained by serveting the SNPs as a kinship covariate pleaseted using the A.mat office from the rrBLUP R bundle and comprised in a particular trudge gauge using the emmreml office from the EMMREML R bundle (Akdemir and Okeke, 2015).

DNA provision and Genotyping by sequencing (GBS)

Aggregate cassava clones comprised in the phenotypic axioms determined had their entirety genomic DNA extracted from puerile meek leaves according to touchstone procedures using the DNAeasy introduce mini lineage kit (Qiagen, 2012).

Genotyping-by-sequencing (GBS) (Elshire et al., 2011) libraries were deceptive using the ApeKI incapability enzyme as authenticationd precedently (Hamblin & Rabbi, 2014). Marker genotypes were overlookominated using TASSEL GBS pipeline V4 (Glaubitz et al., 2014) subjoined aligning the reads to the Cassava v6 regard genome (Phytozome 10.3; http://phytozome.jgi.doe.gov) (Interpolitical Cassava Genetic Map Consortium, 2014; Prochnik et al., 2012). Variant Calling Cemat (VCF) files were generated ce each chromosome. Markers with further than 60% detriment calls were removed. Genotypes with near than 5 reads were masked precedently befoulment. Attentionally, solely biallelic SNP markers were considered ce further trudges.

The marker axiomsdetermined consisted of a entirety of 173,647 SNP bi-allelic markers overlookominated ce 986 people. This modereprimand axiomsdetermined was imputed using Beagle 4.1 (Browning and Browning, 2016). Subjoined the befoulment 63,016 SNPs had an AR2 (Estimated Aggregateelic r-squared) better than 0.3 and were kept ce partition; from these, 41,530 had a puerileer aggregateele calculate (MAF) better than 0.01 in our population. Dosage files ce this last axiomsdetermined were generated and authenticationd ce twain GWAS and GS.

Constitution and Genetic stratification partition

The interval of phylogenetic kindred and class of family kindredness amid the cassava lines was assessed using coercionemost content partition (PCA) implemented in R.

Genome-abundant connection partition ce CBSV sensitivity

The input binary PED files were prompt from the genotype dosage files using PLINK representation 1.07 (Rentería, Cortes and Medland, 2013; Purcell et al., 2007). Mixed rectirectirectidirect modal connection partition (MLMA) implemented by GCTA representation 1.26.0 was authenticationd to generated GWAS fruits (Yang et al., 2011). MLMA was implemented such that in integral cycle of partition, the chromosome on which the aspirant SNPs insisted got outside from the GRM regard using the modal in equation 3.

Where y is the phenotype, a is substance the medium tidings, b substance the agricultural embracing propertys of the aspirant SNP substance touchstoneed ce connection, x substance the SNP genotype indicator mutable and gis the accumulated property of aggregate SNPs save those where the aspirant SNP is located making our partition gauge further propertyivenessful.

We estimated hostility contents using unpopular completion aspect (REML). Sub population stratification was corrected ce by preliminary GRM as a accidental property tidings in the gauge during partition. A further stationary Bonferroni emendation arrange was authenticationd to decide genome-abundant memorialificance buildation at P≤10-7 as a practice of correcting ce experimental-wise hallucination.

Manhattan and Quantile – Quantile plots ce aggregate the touchs were deceptive using R bundle qqman bundle implemented in R (Turner, 2014).

Genomic prophecy gauges

GBLUP. In this prophecy gauge the GEBVs are obtained by turgid , where is the embracing genetic hostility, and K is the symmetric genomic realized pleaseness matrix invetereprimand on GBS SNP marker dosages. The genomic kindred matrix authenticationd was deceptive using the office A.mat in the R bundle rrBLUP(Endelman, 2011) and follows the cemula of FaceRaoverlook (2008), arrange span. GBLUP prophecys were made with the office emmreml in the R bundle EMMREML (Akdemir and Okeke, 2015).

RKHS. Unlike GBLUP ce RKHS we authentication a Gaussian fruit office: , where Kij is the meted kindred betwixt span people, dij is their euclidean genetic interval invetereprimand on marker dosages and θ is a tuning (“bandwidth”) parameter that determines the rebuke of rottenness of corfitness inaccomplished people. This office is nonrectirectidirect hence the fruits authenticationd ce RKHS can withhold non-embracing as courteous-mannered-mannered as embracing genetic alteration. To serve a multiple-fruit gauge with six cohostility matrices we authenticationd the emmremlMultiKernel office in the EMMREML bundle, with the subjoined bandwidth parameters: 0.0000005, 0.00005, 0.0005, 0.005, 0.01, 0.05 (Multi-fruit RKHS) and aggregateowed REML to invent optimal weights ce each fruit.Ce the “optimal fruit RKHS” we authenticationd the fruit weights assigned by emmremlMultiKernel in the first trudge to coercionm a solely fruit that is the weighted mediocre of the coercionmer six. We then authenticationd this “optimal fruit” in solely-fruit prophecys.

Bayesian creator retrogradations.We touchstoneed immodest Bayesian prophecy gauges: BayesCpi (Habier et al., 2011), the Bayesian LASSO (BL; Park and Occurrencella, 2008), BayesA, and BayesB (Meuwissen, Hayes and Goddard, 2001). The Bayesian gauges we touchstoneed aggregateow ce reorigin genetic architectures by unanalogousial shrinkage of marker propertys. We executed Bayesian prophecys with the R bundle BGLR (Pérez and De Los Campos, 2014)

Accidental Ceest. Accidental ceest (RF) is a machine enlightenment arrange authenticationd ce retrogradation and assortment (Strobl et al. 2009, Breiman 2001). Accidental ceest retrogradation with marker axioms has been shown to withhold epistatic propertys and has been successeasily authenticationd ce prophecy (Sakar et al 2015, Heslot et al 2012, Charmet et al 2014, Spindel et al 2015, Breiman, et al 2001, Michaelson et al 2010, Motsinger-Reif et al 2008). We implemented RF using the accidentalJungle bundle in R (Liaw and Wiener 2002) with the parameter, ntree determined to 500 and the calculate of mutables specimend at each divide (mtry) similar to 300.

Multifruit GBLUP

We followed a multifruit admission by serveting three fruits deceptive with SNPs with MAF> 0.01 from chromosomes 4,11 and the SNPs from the other chromosomes. We separated chromosomes 4 and 11 becaauthentication they contained QTLs ce foliar injustice 3 and 6 MAP. Multifruit GBLUP prophecys were made with the office emmremlMultiFruit in the R bundle EMMREML (Akdemir and Okeke, 2015)

Introgression Segment Detection

To establish the genome segments in our germplasm, we followed the admission of Bredeson et al. (2015). We authenticationd the M. glaziovii cue markers attested Supplementary Axiomsdetermined 2 of Bredeson et al. (2015). These house cue (AI) SNPs were attested as substance agricultural ce unanalogous aggregateeles in a specimen of span innocent M. esculenta (Albert and CM33064) and span innocent M. glaziovii (GLA XXX-8 and M. glaziovii(S)).

Quenched of 173,647 SNP in our imputed axiomsset, 12,502 matched published AI SNPs. Ce these AI SNPs, we separated each chromosome into non-overlapping windows of 20 SNP. Amid each window, ce each particular, we pleaseted the relation of genotypes that were homozygous (G/G) or heterozygous (G/E) ce M. glaziovii aggregateele and the relation that were homozygous ce the M. esculenta aggregateele (E/E). We assigned G/G, G/E or E/E house to each window, ce each particular solely when the relation of the most contemptible genotype in that window was at last twice the relation of the assist most contemptible genotype. We assigned windows a “No Call” status otherwise.

We to-boot authenticationd this admission on six whole-genome referableed specimens from the cassava HapMap II (Punna et al. lower Revisal). These comprised the span “innocent cassava” and M. glaziovii(S) from Bredeson et al. (2015), plus an attentional M. glaziovii, and span specimens labeled Namikonga. Becaauthentication these specimens came from a unanalogous fount from the greaterity of our specimens, we were able to invent solely 11,686 SNPs that matched twain the footings in the peace of our con-balance specimen and the roll of house informative footings ce partition.

Linkage disequilibrium plots

LD jaws were pleaseted ce integral SNP in chromosome 4 with a window of 1Mb using the GCTA Software (Yang et al., 2011). Briefly, LD jaw ce a attached marker is the unite of R2 adjusted betwixt the protouchstone marker and aggregate markers amid a certain window. The adjusted R2 is an unjaundiced mete of LD:

where “n” is the population expandedness and R2 is the common estimator of the squared Pearson’s corfitness (Bulik-Sulliface et al 2015).

We pleaseted the LD betwixt that marker and other markers in a window of 2Mb (1Mb upstream and 1Mb downstream) Ce the extreme expressive SNP reach in chromosome 11 ce the 6MAP GWAS fruit from panel 1 and panel 2. The LD was evaluated using squared Pearson’s corfitness coefficient (r2) as pleaseted with the −r2 -ld-snp commands in the software PLINK representation 1.9.

Aspirant gene identification

To establish aspirant genes ce CBSD injustice in leaves and CBSD origin necrosis we authenticationd the GCTA mlma GWAS quenchedput obtained ce each touch. We filtered the SNP markers invetereprimand on -log10 (P-value)> Bonf, substance these treasures better than the Bonferroni buildation (~ 5.9). The fruiting SNP markers were assigned onto genes using the SNP dregs and gene title from the Mesculenta_305_v6.1.gene.gff3 advantageous in Phytozome 11 (ref) ce Maniburning esculenta v6.1 using the intersect office from bedtools (ref).

RESULTS

Phenotypic assessment of cassava ce sensitivity to cassava brown streak bane contamination

Most clones showed multiplied responses to CBSV contamination spanning from super excitability that indicateed candle-like die-tail of the spray to tolerance (Fig.). Foliar phenotyping clumped these introduce responses into five greater classes invetereprimand on a 1 to 5 flake. The unreserved feeling heritability of the elaboscold touchs concatenated 0.17 to 0.72 ce twain GWAS panels (Table 1). Partition of the phenotypic axioms showed very expressive GxE interactions (P<0.001) future justifying the relation of solely environment axioms partition.

Genetic appositions and heritability estimates

We build moderebuke heritability estimates ce CBSV sensitivity ce foliar phenotypes at 3, 6 and 9 MAP as courteous-mannered-mannered as origin phenotypes lower five environments (fig..) . Genetic corfitness on the touchs assayed were executed and biblical that concatenated from moderebuke to elevated decisive appositions inaccomplished touchs elaborate.

Assessment of linkage disequilibrium

Genome-abundant connection mapping repeatedly explores the beneserve of insistence of sundry recorded recombination events aggravate season to companion observed phenotypic alteration with genome.

Detection of aspirant QTLs ce CBSV sensitivity in cassava

To efficiently a rush GWAS, we authenticationd SNP axioms to search the interval of genetic interrelatedness and sub-population constitution of the cassava clones. A coercionemost content partition (PCA) to representation ce constitution showed no sepascold bunchs implying that the separated clones were referable elevatedly constitutiond (Fig.1). Hence, we did referable apprehend PCs in our GWAS rectirectirectidirect modal partition.

The Bonferroni entreative buildation (α = 0.05) was authenticationd to establish loci companiond to CBSV sensitivity on twain chromosome 4 and 11 that had serene peak memorialals at the unanalogous stages of phenotyping (Fig. 2). The observed P-values abstinently aligned courteous-mannered-mannered with the expected P-values barring later differed really imputable the expanded introgression fill on chromosome 4 presumably from the disorderly progenitors of cassava traceable from the AMANI air program (Jennings, 1959).

The expressive memorialals on chromosome 11 contained loci with pungent-muscular connection with CBSV sensitivity in a 2 Mb territory that annotated courteous-mannered-mannered with sundry aspirant genes.

Genome-abundant prophecy ce CBSV sensitivity in cassava

We did genome abundant prophecy ce CBSV sensitivity invetereprimand on the attested SNPs with the elevatedest propertys build on twain chromosome 4 and 11 in arrange to withhold most of the genetic alteration. We explored sundry genomic prophecy gauge arranges; GBLUP, RR-BLUP, B-LASSO, accidental ceest, BayesA, BayesB, and BayesC.

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