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Plant architecture is one of the most important factors that determines crop yield potential and productivity. In apple (Malus), genetic improvement of tree architecture has been challenging due to a long juvenile phase and their growth as complex trees composed of a distinct scion and a rootstock. To better understand the genetic control of apple tree architecture, the dominant weeping growth phenotype was investigated. We report the identification ofMdLAZY1A(MD13G1122400) as the genetic determinant underpinning theWeeping(W)locus that largely controls weeping growth inMalus.MdLAZY1Ais one of the four paralogs in apple that are most closely related toAtLAZY1involved in gravitropism inArabidopsis. The weeping allele (MdLAZY1A-W) contains a single nucleotide mutation c.584T>C that leads to a leucine to proline (L195P) substitution within a predicted transmembrane domain that co-localizes with Region III, one of the five conserved regions in LAZY1-like proteins. Subcellular localization revealed that MdLAZY1A localizes to the plasma membrane and nucleus in plant cells. Over-expressing the weeping allele in apple cultivar Royal Gala (RG) with standard growth habit impaired its gravitropic response and altered the growth to weeping-like. Suppressing the standard allele (MdLAZY1A-S) by RNA interference (RNAi) in RG similarly changed the branch growth direction to downward. Overall, the L195P mutation in MdLAZY1A is genetically causal for weeping growth, underscoring not only the crucial roles of residue L195 and Region III in MdLAZY1A-mediated gravitropic response, but also a potential DNA base editing target for tree architecture improvement inMalusand other crops.

The organic acid concentration in apple (Malus ×domestica) juice is a major component of hard cider flavor. The goal of this study was to determine if the malic acid markers, Ma1 and Q8, could classify the titratable acidity concentration in cider apple accessions from the United States Department of Agriculture Malus germplasm collection into descriptive classifications. Our results indicate that for diploid genotypes, the Ma1 marker alone and the Ma1 and Q8 markers analyzed together could be used to predict cider apple acidity (P < 0.0001). Alone, the Ma1 marker categorized acidity into low (−1), medium (2.4–5.8 g⋅L−1), and high (>5.8 g⋅L−1) groups. The combination of Ma1 and Q8 markers provided more specificity, which would be useful for plant breeding applications. This work also identified a significant difference (P = 0.0132) in acidity associated with ploidy. On average, the triploids accessions had 0.33 g⋅L−1 higher titratable acidity than the diploid accessions. Based on the results of this work, we propose a genetics-based classification system for cider apples with the acidity component defined by the Ma1 and Q8 markers.

Somatic mutations may alter important traits in tree fruits, such as fruit color, size and maturation date. Autumn Gala (AGala), a somatic mutation from apple cultivar Gala, matures 4 weeks later than Gala. To understand the mechanisms underlying the delayed maturation, RNA-seq analyses were conducted with fruit sampled at 13 (Gala) and 16 (AGala) time-points during their growth and development. Weighted gene co-expression network analysis (WGCNA) of 23 372 differentially expressed genes resulted in 25 WGCNA modules. Of these, modules 1 (r = -0.98, P = 2E-21) and 2 (r = -0.52, P = 0.004), which were suppressed in AGala, were correlated with fruit maturation date. Surprisingly, 77 of the 152 member genes in module 1 were harbored in a 2.8-Mb genomic region on chromosome 6 that was deleted and replaced by a 10.7-kb gypsy-like retrotransposon (Gy-36) from chromosome 7 in AGala. Among the 77 member genes, MdACT7 was the most suppressed (by 10.5-fold) in AGala due to a disruptive 2.5-kb insertion in coding sequence. Moreover, MdACT7 is the exclusive apple counterpart of Arabidopsis ACT7 known of essential roles in plant development, and the functional allele MdACT7, which was lost to the deletion in AGala, was associated with early fruit maturation in 268 apple accessions. Overexpressing alleles MdACT7 and Mdact7 in an Arabidopsis act7 line showed that MdACT7 largely rescued its stunted growth and delayed initial flowering while Mdact7 did not. Therefore, the 2.8-Mb hemizygous deletion is largely genetically causal for fruit maturation delay in AGala, and the total loss of MdACT7 might have contributed to the phenotype.

Domestication of the apple was mainly driven by interspecific hybridization. In the present study, we report the haplotype-resolved genomes of the cultivated apple (Malus domestica cv. Gala) and its two major wild progenitors, M. sieversii and M. sylvestris. Substantial variations are identified between the two haplotypes of each genome. Inference of genome ancestry identifies ~23% of the Gala genome as of hybrid origin. Deep sequencing of 91 accessions identifies selective sweeps in cultivated apples that originated from either of the two progenitors and are associated with important domestication traits. Construction and analyses of apple pan-genomes uncover thousands of new genes, with hundreds of them being selected from one of the progenitors and largely fixed in cultivated apples, revealing that introgression of new genes/alleles is a hallmark of apple domestication through hybridization. Finally, transcriptome profiles of Gala fruits at 13 developmental stages unravel ~19% of genes displaying allele-specific expression, including many associated with fruit quality., Phased diploid genomes of the cultivated apple Malus domestica cv. Gala and its two major wild progenitors M. sieversii and M. sylvestris, as well as pan-genome analyses, provide insights into the genetic history of apple domestication.

Malate accumulation in the vacuole largely determines apple (Malus domestica) fruit acidity, and low fruit acidity is strongly associated with truncation of Ma1, an ortholog of ALUMINUM-ACTIVATED MALATE TRANSPORTER9 (ALMT9) in Arabidopsis (Arabidopsis thaliana). A mutation at base 1,455 in the open reading frame of Ma1 leads to a premature stop codon that truncates the protein by 84 amino acids at its C-terminal end. Here, we report that both the full-length protein, Ma1, and its naturally occurring truncated protein, ma1, localize to the tonoplast; when expressed in Xenopus laevis oocytes and Nicotiana benthamiana cells, Ma1 mediates a malate-dependent inward-rectifying current, whereas the ma1-mediated transmembrane current is much weaker, indicating that ma1 has significantly lower malate transport activity than Ma1. RNA interference suppression of Ma1 expression in ‘McIntosh’ apple leaves, ‘Empire’ apple fruit, and ‘Orin’ apple calli results in a significant decrease in malate level. Genotyping and phenotyping of 186 apple accessions from a diverse genetic background of 17 Malus species combined with the functional analyses described above indicate that Ma1 plays a key role in determining fruit acidity and that the truncation of Ma1 to ma1 is genetically responsible for low fruit acidity in apple. Furthermore, we identified a C-terminal domain conserved in all tonoplast-localized ALMTs essential for Ma1 function; protein truncations into this conserved domain significantly lower Ma1 transport activity. We conclude that the truncation of Ma1 to ma1 reduces its malate transport function by removing a conserved C-terminal domain, leading to low fruit acidity in apple.

Columnar apples trees, originated from a bud mutation ‘Wijcik McIntosh,’ develop a simple canopy and set fruit on spurs. These characteristics make them an important genetic resource for improvement of tree architecture. Genetic studies have uncovered that columnar growth habit is a dominant trait and is caused by a retroposon insertion that induces the expression of the neighboring gene Co encoding a 2OG-Fe(II) oxygenase. Here we report the genetic mapping of two loci of recessive suppressors (genes) c2 (on Chr10) and c3 (on Chr9) that are linked to repression of the retroposon-induced Co gene expression and associated columnar phenotype in 275 F1 seedlings, which were developed from a reciprocal cross between two columnar selections heterozygous at the Co locus. The mapping was accomplished by sequencing a genomic pool comprising 18 columnar seedlings and another pool of 16 standard seedlings that also carry the retroposon insertion, and by exploring DNA variants of segregation types that are informative for mapping recessive traits in apple. The informative segregation types include , , , , and , where each letter denotes one of the four DNA bases and the letters in bold represent variants in relation to the reference genome. The alleles in each first and third positions are assumed in linkage with the recessive suppressors’ allele in the two parents, respectively. Using RNA-seq analysis, we further revealed that the Co gene together with the differentially expressed genes under loci c2 and c3 formed a co-expression gene-network module associated with growth habit, in which 12 MapMan Bins were enriched.

The firmness of fleshy fruit crops has a significant effect on their quality, consumer preference, shelf life and transportability. In a combined quantitative trait locus and genome-wide association studies study of apple fruit texture, we identified a mutation (C-G) in the ethylene response factor-associated amphiphilic repression (EAR) motif in the coding region of the apple ETHYLENE RESPONSE FACTOR4 (ERF4) gene. Chromatin immunoprecipitation sequencing showed that ERF4 binds to the promoter of ERF3, which is involved in regulation of ethylene biosynthesis. The EAR mutation in ERF4 results in reduced repression of ERF3 expression, which is turn promotes ethylene production and loss of fruit firmness. ERF4 acts as a transcriptional repressor whose activity is modulated by a TOPLESS co-repressor 4 (TPL4)-binding EAR repression motif. Biolayer interferometry analysis showed that the mutation in the EAR motif causes a reduction in the interaction with TPL4. Suppression of ERF4 or TPL4 promoted fruit ripening and ethylene production. Taken together, our results provide insights into how ERF4 allelic variation underlies an important fruit quality trait.

Identifying and exploiting three informative DNA variant segregation types in pooled genome sequencing analysis of the weeping phenotype in Malus empowers an effective genetic mapping strategy in out-crossing woody species., To unlock the power of next generation sequencing-based bulked segregant analysis in allele discovery in out-crossing woody species, and to understand the genetic control of the weeping trait, an F1 population from the cross ‘Cheal’s Weeping’ × ‘Evereste’ was used to create two genomic DNA pools ‘weeping’ (17 progeny) and ‘standard’ (16 progeny). Illumina pair-end (2 × 151 bp) sequencing of the pools to a 27.1× (weeping) and a 30.4× (standard) genome (742.3 Mb) coverage allowed detection of 84562 DNA variants specific to ‘weeping’, 92148 specific to ‘standard’, and 173169 common to both pools. A detailed analysis of the DNA variant genotypes in the pools predicted three informative segregation types of variants: (type I) in weeping pool-specific variants, and (type II) and (type III) in variants common to both pools, where the first allele is assumed to be weeping linked and the allele shown in bold is a variant in relation to the reference genome. Conducting variant allele frequency and density-based mappings revealed four genomic regions with a significant association with weeping: a major locus, Weeping (W), on chromosome 13 and others on chromosomes 10 (W2), 16 (W3), and 5 (W4). The results from type I variants were noisier and less certain than those from type II and type III variants, demonstrating that although type I variants are often the first choice, type II and type III variants represent an important source of DNA variants that can be exploited for genetic mapping in out-crossing woody species. Confirmation of the mapping of W and W2, investigation into their genetic interactions, and identification of expressed genes in the W and W2 regions provided insight into the genetic control of weeping and its expressivity in Malus.

Human selection has reshaped crop genomes. Here we report an apple genome variation map generated through genome sequencing of 117 diverse accessions. A comprehensive model of apple speciation and domestication along the Silk Road is proposed based on evidence from diverse genomic analyses. Cultivated apples likely originate from Malus sieversii in Kazakhstan, followed by intensive introgressions from M. sylvestris. M. sieversii in Xinjiang of China turns out to be an “ancient” isolated ecotype not directly contributing to apple domestication. We have identified selective sweeps underlying quantitative trait loci/genes of important fruit quality traits including fruit texture and flavor, and provide evidences supporting a model of apple fruit size evolution comprising two major events with one occurring prior to domestication and the other during domestication. This study outlines the genetic basis of apple domestication and evolution, and provides valuable information for facilitating marker-assisted breeding and apple improvement., Apple is one of the most important fruit crops. Here, the authors perform deep genome resequencing of 117 diverse accessions and reveal comprehensive models of apple origin, speciation, domestication, and fruit size evolution as well as candidate genes associated with important agronomic traits.

Sorbitol is a major product of photosynthesis in apple (Malus domestica) that is involved in carbohydrate metabolism and stress tolerance. However, little is known about how the global transcript levels in apple leaves respond to decreased sorbitol synthesis. In this study we used RNA sequencing (RNA-seq) profiling to characterize the transcriptome of leaves from transgenic lines of the apple cultivar 'Greensleeves' exhibiting suppressed expression of aldose-6-phosphate reductase (A6PR) to gain insights into sorbitol function and the consequences of decreased sorbitol synthesis on gene expression. We observed that, although the leaves of the low sorbitol transgenic lines accumulate higher levels of various primary metabolites, only very limited changes were found in the levels of transcripts associated with primary metabolism. We suggest that this is indicative of post-transcriptional and/or post-translational regulation of primary metabolite accumulation and central carbon metabolism. However, we identified significantly enriched gene ontology terms belonging to the 'stress related process' category in the antisense lines (P-value < 0.05). These include genes involved in the synthesis/degradation of abscisic acid, salicylic acid and jasmonic acid, nucleotide-binding site leucine-rich repeat (NBS-LRR) disease resistance genes and ATP-binding cassette (ABC) transporter genes. This suggests that sorbitol plays a role in the responses of apple trees to abiotic and biotic stresses.

Apple fruit acidity, which affects the fruit's overall taste and flavor to a large extent, is primarily determined by the concentration of malic acid. Previous studies demonstrated that the major QTL malic acid (Ma) on chromosome 16 is largely responsible for fruit acidity variations in apple. Recent advances suggested that a natural mutation that gives rise to a premature stop codon in one of the two aluminum-activated malate transporter (ALMT)-like genes (called Ma1) is the genetic causal element underlying Ma. However, the natural mutation does not explain the developmental changes of fruit malate levels in a given genotype. Using RNA-seq data from the fruit of 'Golden Delicious' taken at 14 developmental stages from 1 week after full-bloom (WAF01) to harvest (WAF20), we characterized their transcriptomes in groups of high (12.2 ± 1.6 mg/g fw, WAF03-WAF08), mid (7.4 ± 0.5 mg/g fw, WAF01-WAF02 and WAF10-WAF14) and low (5.4 ± 0.4 mg/g fw, WAF16-WAF20) malate concentrations. Detailed analyses showed that a set of 3,066 genes (including Ma1) were expressed not only differentially (P FDR < 0.05) between the high and low malate groups (or between the early and late developmental stages) but also in significant (P < 0.05) correlation with malate concentrations. The 3,066 genes fell in 648 MapMan (sub-) bins or functional classes, and 19 of them were significantly (P FDR < 0.05) co-enriched or co-suppressed in a malate dependent manner. Network inferring using the 363 genes encompassed in the 19 (sub-) bins, identified a major co-expression network of 239 genes. Since the 239 genes were also differentially expressed between the early (WAF03-WAF08) and late (WAF16-WAF20) developmental stages, the major network was considered to be associated with developmental regulation of apple fruit acidity in 'Golden Delicious'.

The dagger nematode, Xiphinema index, feeds aggressively on grape roots and in the process vectors grapevine fanleaf virus (GFLV) leading to the severe viral disease known as fanleaf degeneration. Disease symptoms consist of disrupted fruit set caused by GFLV and depressed plant growth caused by root damage from X. index. The use of fumigants to control X. index in vineyards is no longer recommended because of their high cost, detrimental environmental effects and lack of effective soil penetration to control nematodes on a deep perennial root system. Therefore, resistance to X. index has been an important objective in grape rootstock breeding programs. We previously demonstrated that resistance to X. index derived from a Vitis arizonica/girdiana hybrid b42-26 was largely controlled by a major quantitative trait locus, XiR1 (X. index Resistance 1) located on chromosome 19. Genetic studies leading to the isolation and characterization of the genes conferring resistance to X. index would further our understanding of resistance and assist molecular and classical breeding efforts to control X. index. In this report, we present the development of high resolution genetic and physical maps in the XiR1 region as well as the isolation of the XiR1 locus by a positional cloning approach. This study has identified the first locus responsible for ectoparasitic nematode resistance. The markers developed from this study are being used to expedite the breeding of resistant grape rootstocks.

The reference genome of apple (Malus × domestica) has been available since 2010. Despite being a milestone in apple genomics, the reference genome is difficult to be used as a reference in RNA-seq (RNA sequencing) analysis, a widespread technology in transcriptomic studies. One of the major limitations appears to be the low coverage of the reference transcriptome in RNA-seq mapping of reads. To improve the reference transcriptome, we obtained 14 sets of strand-specific RNA-seq data of 168.5 million reads in total from fruit of Golden Delicious (GD, the source of the reference genome) in varying growth and developmental stages. Using a combination of genome-guided assembly and de novo assembly, the apple reference transcriptome was improved to a collection of 71,178 genes or transcripts, which includes 53,654 genes predicted originally (with MDP prefixed in their IDs) and 17,524 novel transcripts. Of these novel transcripts, 8,144 were identified from reads directly mapped to the reference genome while the remaining 9,380 were extracted from de novo assemblies of reads that could not be initially mapped to the reference genome. Evaluating the improved apple reference transcriptome with reads from Golden Delicious and other genotypes used in this and other studies showed that it allowed 62.5 ± 9.3-82.3 ± 2.7 % of reads to be mapped, a marked increase from the low rates of 37.4 ± 7.7-46.6 ± 7.1 % offered by the original reference transcriptome. The improved reference transcriptome therefore represents a step forward towards a complete reference transcriptome in apple.

Phytohormone ethylene largely determines apple fruit shelf life and storability. Previous studies demonstrated that MdACS1 and MdACS3a, which encode 1-aminocyclopropane-1-carboxylic acid synthases (ACS), are crucial in apple fruit ethylene production. MdACS1 is well-known to be intimately involved in the climacteric ethylene burst in fruit ripening, while MdACS3a has been regarded a main regulator for ethylene production transition from system 1 (during fruit development) to system 2 (during fruit ripening). However, MdACS3a was also shown to have limited roles in initiating the ripening process lately. To better assess their roles, fruit ethylene production and softening were evaluated at five time points during a 20-day post-harvest period in 97 Malus accessions and in 34 progeny from 2 controlled crosses. Allelotyping was accomplished using an existing marker (ACS1) for MdACS1 and two markers (CAPS866 and CAPS870) developed here to specifically detect the two null alleles (ACS3a-G289V and Mdacs3a) of MdACS3a. In total, 952 Malus accessions were allelotyped with the three markers. The major findings included: The effect of MdACS1 was significant on fruit ethylene production and softening while that of MdACS3a was less detectable; allele MdACS1-2 was significantly associated with low ethylene and slow softening; under the same background of the MdACS1 allelotypes, null allele Mdacs3a (not ACS3a-G289V) could confer a significant delay of ethylene peak; alleles MdACS1-2 and Mdacs3a (excluding ACS3a-G289V) were highly enriched in M. domestica and M. hybrid when compared with those in M. sieversii. These findings are of practical implications in developing apples of low and delayed ethylene profiles by utilizing the beneficial alleles MdACS1-2 and Mdacs3a.

Acidity levels greatly affect the taste and flavor of fruit, and consequently its market value. In mature apple fruit, malic acid is the predominant organic acid. Several studies have confirmed that the major quantitative trait locus Ma largely controls the variation of fruit acidity levels. The Ma locus has recently been defined in a region of 150 kb that contains 44 predicted genes on chromosome 16 in the Golden Delicious genome. In this study, we identified two aluminum-activated malate transporter-like genes, designated Ma1 and Ma2, as strong candidates of Ma by narrowing down the Ma locus to 65-82 kb containing 12-19 predicted genes depending on the haplotypes. The Ma haplotypes were determined by sequencing two bacterial artificial chromosome clones from G.41 (an apple rootstock of genotype Mama) that cover the two distinct haplotypes at the Ma locus. Gene expression profiling in 18 apple germplasm accessions suggested that Ma1 is the major determinant at the Ma locus controlling fruit acidity as Ma1 is expressed at a much higher level than Ma2 and the Ma1 expression is significantly correlated with fruit titratable acidity (R (2) = 0.4543, P = 0.0021). In the coding sequences of low acidity alleles of Ma1 and Ma2, sequence variations at the amino acid level between Golden Delicious and G.41 were not detected. But the alleles for high acidity vary considerably between the two genotypes. The low acidity allele of Ma1, Ma1-1455A, is mainly characterized by a mutation at base 1455 in the open reading frame. The mutation leads to a premature stop codon that truncates the carboxyl terminus of Ma1-1455A by 84 amino acids compared with Ma1-1455G. A survey of 29 apple germplasm accessions using marker CAPS(1455) that targets the SNP(1455) in Ma1 showed that the CAPS(1455A) allele was associated completely with high pH and highly with low titratable acidity, suggesting that the natural mutation-led truncation is most likely responsible for the abolished function of Ma for low pH or high acidity in apple.

Apple fruit flavor is greatly affected by the level of malic acid, which is the major organic acid in mature apple fruit. To understand the genetic and molecular basis of apple fruit acidity, fruit juice pH and/or titratable acidity (TA) were measured in two half-sib populations GMAL 4595 [Royal Gala × PI (Plant Introduction) 613988] and GMAL 4590 (Royal Gala × PI 613971) of 438 trees in total. The maternal parent Royal Gala is a commercial variety and the paternal parents are two M. sieversii (the progenitor species of domestic apple) elite accessions. The low-acid trait segregates recessively and the overall acidity variations in the two populations were primarily controlled by the Ma (malic acid) locus, a major gene discovered in the 1950s (Nybom in Hereditas 45:332–350, 1959) and later mapped to linkage group 16 (Maliepaard et al. in Theor Appl Genet 97:60–73, 1998). The allele Ma has a strong additive effect in increasing fruit acidity and is incompletely dominant over ma. QTL (quantitative trait locus) analyses in GMAL 4595 mapped the major QTL Ma in both Royal Gala and PI 613988, the effects of which explained 17.0–42.3% of the variation in fruit pH and TA. In addition, two minor QTL, tentatively designated M2 and M3, were also detected for fruit acidity, with M2 on linkage group 6 of Royal Gala and M3 on linkage group 1 of PI 613988. By exploring the genome sequences of apple, eight new simple sequence repeat markers tightly linked to Ma were developed, leading to construction of a fine genetic map of the Ma locus that defines it to a physical region no larger than 150 kb in the Golden Delicious genome.

Malus sieversii is a progenitor species of domestic apple M. × domestica. Using population “GMAL 4595” of 188 individuals derived from a cross of Royal Gala × PI 613988 (apple scab resistant, M. sieversii), 287 SSR (simple sequence repeats) loci were mapped. Of these SSRs, 80 are published anchors and 207 are newly developed EST (expressed sequence tag) contig-based SSRs, representing 1,630 Malus EST accessions in GenBank. Putative gene functions of these EST contigs are diverse, including regulating plant growth, development and response to environmental stresses. Among the 80 published SSRs, 18 are PI 613988 specific, 38 are common and 24 are Royal Gala specific. Out of the 207 newly developed EST contig-based SSRs, 79 are PI 613988 specific, 45 are common and 83 are Royal Gala specific. These results led to the construction of a M. sieversii map (1,387.0 cM) of 180 SSR markers and a Royal Gala map (1,283.4 cM) of 190 SSR markers. Mapping of scab resistance was independently conducted in two subsets of population “GMAL 4595” that were inoculated with Ventura inaequalis races (1) and (2), respectively. In combination with the two major resistance reactions Chl (chlorotic lesions) and SN (stellate necrosis) to each race, four subsets of resistance data, i.e., Chl/race (1), SN/race (1), Chl/race (2) and SN/race (2), were constituted and analyzed, leading to four resistance loci mapped to the linkage group 2 of PI 613988; SNR1 (stellate necrosis resistance to race (1)) and SNR2 are tightly linked in a region of known scab resistance genes, and ChlR1 (Chlorotic lesion resistance to race (1)) and ChlR2 are also linked tightly but in a region without known scab resistance genes. The utility of the two linkage maps, the new EST contig-based markers and M. sieversii as sources of apple scab resistance are discussed.

The dagger nematode, Xiphinema index, feeds aggressively on grape roots and in the process, vectors grapevine fanleaf virus (GFLV) leading to the severe viral disease known as fanleaf degeneration. Resistance to X. index and GFLV has been the key objective of grape rootstock breeding programs. A previous study found that resistance to X. index derived from Vitis arizonica was largely controlled by a major quantitative trait locus, XiR1 (X. index Resistance 1), located on chromosome 19. The study presented here develops high-resolution genetic and physical maps in an effort to identify the XiR1 gene(s). The mapping was carried out with 1,375 genotypes in three populations derived from D8909-15, a resistant selection from a cross of V. rupestris A. de Serres (susceptible) × V. arizonica b42-26 (resistant). Resistance to X. index was evaluated on 99 informative recombinants that were identified by screening the three populations with two markers flanking the XiR1 locus. The high-resolution genetic map of XiR1 was primarily constructed with seven DNA markers developed in this study. Physical mapping of XiR1 was accomplished by screening three bacterial artificial chromosome (BAC) libraries constructed from D8909-15, V. vinifera Cabernet Sauvignon and V. arizonica b42-26. A total of 32 BAC clones were identified and the XiR1 locus was delineated within a 115 kb region. Sequence analysis of three BAC clones identified putative nucleotide binding/leucine-rich repeat (NB-LRR) genes. This is the first report of a closely linked major gene locus responsible for ectoparasitic nematode resistance. The markers developed from this study are being used to expedite the breeding of resistant grape rootstocks.

Resistance to the dagger nematode Xiphinema index has been an important objective in grape rootstock breeding programs. This nematode not only causes severe feeding damage to the root system, but it also vectors grapevine fanleaf virus (GFLV), the causal agent of fanleaf degeneration and one of the most severe viral diseases of grape. The established screening procedures for dagger nematode resistance are time consuming and can produce inconsistent results. A fast and reliable greenhouse-based system for screening resistance to X. index that is suitable for genetic studies and capable of evaluating breeding populations is needed. In this report, the dynamics of nematode numbers, gall formation, and root weight loss were investigated using a variety of soil mixes and pot sizes over a 52-week period. Results indicated that the number of galls formed was correlated with the size of the nematode population and with the degree of root weight loss. After inoculation with 100 nematodes, gall formation could be reliably evaluated in 4-8 weeks in most plant growth conditions and results were obtained 6 months more rapidly than past evaluation methods. This modified X. index resistance screening method was successfully applied to 185 of the 188 F(1) progeny from a cross of D8909-15 x F8909-17 (the 9621 population), which segregates for a form of X. index resistance originally derived from Vitis arizonica. Quantitative trait loci (QTL) analysis was carried out on both parental genetic maps of 255 markers using MapQTL 4.0. Results revealed that X. index resistance is controlled by a major QTL, designated Xiphinema index Resistance 1 (XiR1), near marker VMC5a10 on chromosome 19. The XiR1 QTL was supported by a LOD score of 36.9 and explained 59.9% of the resistance variance in the mapping population.

Submergence stress regularly affects 15 million hectares or more of rainfed lowland rice areas in South and Southeast Asia. A major QTL on chromosome 9, Sub1, has provided the opportunity to apply marker assisted backcrossing (MAB) to develop submergence tolerant versions of rice cultivars that are widely grown in the region. In the present study, molecular markers that were tightly linked with Sub1, flanking Sub1, and unlinked to Sub1 were used to apply foreground, recombinant, and background selection, respectively, in backcrosses between a submergence-tolerant donor and the widely grown recurrent parent Swarna. By the BC(2)F(2) generation a submergence tolerant plant was identified that possessed Swarna type simple sequence repeat (SSR) alleles on all fragments analyzed except the tip segment of rice chromosome 9 that possessed the Sub1 locus. A BC(3)F(2) double recombinant plant was identified that was homozygous for all Swarna type alleles except for an approximately 2.3-3.4 Mb region surrounding the Sub1 locus. The results showed that the mega variety Swarna could be efficiently converted to a submergence tolerant variety in three backcross generations, involving a time of two to three years. Polymorphic markers for foreground and recombinant selection were identified for four other mega varieties to develop a wider range of submergence tolerant varieties to meet the needs of farmers in the flood-prone regions. This approach demonstrates the effective use of marker assisted selection for a major QTL in a molecular breeding program.

Overview of RNA-seq reads mapping. (DOCX 31Â kb)

Other regulators from modules Turquoise and Brown and their assigned tight clusters. Elements and their contents, formats and messages are same as those noted in Fig. 8a. (A) Regulator M239684 and Cluster 41 of 68 genes. (B) Regulator M239684 and Cluster 5 of 14 genes. (C) Regulator M239684 and Cluster 7 of 14 genes. (D) Regulator M753318 and Cluster 23 of 11 genes. (E) Regulator M753318 and Cluster 32 of 11 genes. (F) Regulator M175481 and Cluster 2 of 16 genes. (G) Regulator M134341 and Cluster 42 of 12 genes. (PPTX 213 kb)

Regulator genes identified by Lemon-Tree. (DOCX 31Â kb)

Regulator M190273 and its other five assigned tight clusters. Elements and their contents, formats and messages are same as those noted in Fig. 8a. (A) Cluster 8 of 31 genes. (B) Cluster 9 of 28 genes. (C) Cluster 40 of 55 genes. (D) Cluster 21 of 11 genes. (E) Cluster 45 of 10 genes. (PPTX 238 kb)

Analysis of modules Black, Brown, Blue and Yellow. (A) Module eigengene values across the 29 samples, including 17 in Ma_ on left and 12 in mama on right. Samples are represented by the combination of a letter (abbreviated cultivar name) and a number (replicate) (see legends in Fig. 1, 4 for keys). (B) Correlation between module membership (MM) and gene significance (GS) for malate. (PPTX 75 kb)

List of primers used in qRT-PCR. (DOCX 29Â kb)

Fruit harvest date and weight. (DOCX 29Â kb)

List of the most significant acidity genes (MSAGs). (DOCX 31Â kb)

Most Oryza sativa cultivars die within a week of complete submergence--a major constraint to rice production in south and southeast Asia that causes annual losses of over US 1 billion dollars and affects disproportionately the poorest farmers in the world. A few cultivars, such as the O. sativa ssp. indica cultivar FR13A, are highly tolerant and survive up to two weeks of complete submergence owing to a major quantitative trait locus designated Submergence 1 (Sub1) near the centromere of chromosome 9 (refs 3, 4, 5-6). Here we describe the identification of a cluster of three genes at the Sub1 locus, encoding putative ethylene response factors. Two of these genes, Sub1B and Sub1C, are invariably present in the Sub1 region of all rice accessions analysed. In contrast, the presence of Sub1A is variable. A survey identified two alleles within those indica varieties that possess this gene: a tolerance-specific allele named Sub1A-1 and an intolerance-specific allele named Sub1A-2. Overexpression of Sub1A-1 in a submergence-intolerant O. sativa ssp. japonica conferred enhanced tolerance to the plants, downregulation of Sub1C and upregulation of Alcohol dehydrogenase 1 (Adh1), indicating that Sub1A-1 is a primary determinant of submergence tolerance. The FR13A Sub1 locus was introgressed into a widely grown Asian rice cultivar using marker-assisted selection. The new variety maintains the high yield and other agronomic properties of the recurrent parent and is tolerant to submergence. Cultivation of this variety is expected to provide protection against damaging floods and increase crop security for farmers.

Submergence tolerance is an important trait for rice (Oryza sativa L.) in rainfed lowland conditions and is being evaluated as a weed control strategy for rice seeded directly into standing water. The trait is largely controlled by a major gene designated Subl. The present study was conducted to assess the use of the microsatellite marker RM219 and the codominant PCR-based marker RM464A (derived from a microsatellite marker, RM464) to select for submergence tolerance. The two markers RM219 and RM464A were found to be linked to Subl by 3.4 and 0.7 cM, respectively. These two markers were further tested in 55 diverse indica and japonica rice cultivars and breeding lines. RM219 was highly polymorphic in this set of cultivars, showing 14 different alleles, and none of the 55 cultivars had the same allele as the submergence tolerance source IR40931-26. RM464A showed three different alleles in the 55 cultivars. The allele of RM464A of most japonica genotypes was 231 bp in size, 5 bp larger than that of IR40931-26, whereas the allele of most indica rice samples was the same as that of IR40931-26. This indicated that the marker RM219 could be used in breeding programs to select for the Subl gene in a wide range of backgrounds, whereas RM464A was more useful in selection for the Subl gene in japonica rice background. The two markers were used to identify lines from backcrossing a submergence tolerance donor to the temperate japonica cultivar M-202. Several lines were identified that were homozygous for RM219, RM464A, and Subl, and were otherwise genetically similar to M-202. Near isogenic lines (NILs) developed from these sources will be a valuable tool for genetic and physiological studies of submergence tolerance and will also be valuable for evaluating submergence tolerance as a useful trait in water-seeded temperate rice production.

Stem rot (Sclerotium oryzae) is an important disease constraint in Californian rice production. Measurement of resistance is laborious, and the low heritability of the trait limits the effectiveness of selection in breeding programs. Molecular markers linked to the trait would therefore provide a superior selection screen to assist in transferring resistance into improved cultivars. The genetics of resistance to stem rot was studied in the germplasm line 87-Y-550 (PI566666), which inherited its resistance from the wild species Oryza rufipogon. Four crosses of 87-Y-550 with susceptible lines were made and recombinant inbred lines of only the most-resistant and most-susceptible progeny within each cross were advanced for late-generation testing. Approximately 900 AFLP (amplified fragment length polymorphism) primer combinations were applied to resistant and susceptible bulks within each cross. One AFLP marker showed significant association with stem rot resistance and accounted for approximately 45.0% of the phenotypic variation in 59 progenies. This marker was mapped on rice chromosome 2 between the RFLP markers RZ166 and RG139 by using F2-reference population information. The accuracy of AFLP marker mapping was validated by size and sequence comparison of AFLP bands from 87-Y-550 and the reference population. With the strategy of selective genotyping combined with a parental survey, two microsatellite markers, RM232 and RM251, on chromosome 3 were also found associated with stem rot resistance and accounted for 41.1% and 37.9% of the phenotypic variation, respectively. The multiple linear regression model included TAA/GTA167 on chromosome 2 and RM232 on chromosome 3 and cumulatively explained 49.3% of total variation. The molecular markers linked to stem rot resistance should facilitate selection for this recalcitrant trait in rice breeding programs by eliminating the need for early generation screening.

Resistance to submergence stress is an important breeding objective in areas where rice cultivars are subjected to complete inundation for a week or more. The present study was conducted to develop a high-resolution map of the region surrounding the submergence tolerance gene Sub1 in rice, which derives from the Indian cultivar FR13A. Submergence screening of 8-day-old plants of F3 families kept for 14 days submerged in 60 cm of water allowed an accurate classification of Sub1 phenotypes. Bulked segregant analysis was used to identify AFLP markers linked to Sub1. A population of 2950 F2 plants segregating for Sub1 was screened with two RFLP markers flanking the Sub1 locus, 2.4 and 4.9 cM away. Submergence tolerance was measured in the recombinant plants, and AFLP markers closely linked to Sub1 were mapped. Two AFLP markers cosegregated with Sub1 in this large population, and other markers were localized within 0.2 cM of Sub1. The high-resolution map should serve as the basis for map-based cloning of this important locus, as it will permit the identification of BAC clones spanning the region.

Submergence stress is a widespread problem in rice-growing environments where drainage is impeded. A few cultivars can tolerate more than 10 days of submergence, but the genes conferring this tolerance have not been identified. We used randon-amplified polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP) markers to map submergence tolerance in 169 F2 plants and the resulting F3 families of a cross between a tolerant indica rice line, IR40931-26, and a susceptible japonica line, PI543851. IR40931-26 inherited strong submergence tolerance from the unimproved cultivar FR13A. Eight-day old F3 seedlings were submerged for 14–16 days in 55-cm deep tanks, and tolerance was scored after 7 days recovery on a scale of 1 (tolerant) to 9 (susceptible). The tolerant and susceptible parents scored 1.5 and 8.4, respectively, and the F3 means ranged from 1.6 to 8.9. Two bulks were formed with DNA from F2 plants corresponding to the nine most tolerant and the nine most susceptible F3 families. Of 624 RAPD primers used to screen the bulks, five produced bands associated with either tolerance or susceptibility. These markers were mapped to a region of chromosome 9 by linkage to RFLP markers. A submergence tolerance quantitative trait locus (QTL), here designatedSub1, was located ca. 4 cM from the RFLP marker C1232 and accounted for 69% of the phenotypic variance for the trait.

The Arabidopsis RTE1 (REVERSION-TO-ETHYLENE SENSITIVITY1) and its tomato ortholog GREEN RIPE (GR) are important in plant ethylene signaling, response and/or fruit ripening. To investigate the potential role of RTE1-like genes in apple (M. ×domestica Borkh.) fruit ripening, two orthologs of RTE1 were identified in the apple genome, designated MdRTE1a and MdRTE1b. Using yeast two-hybrid (Y2H) approach, a cDNA prey library was constructed from a pool of maturing/ripening fruit of ‘Golden Delicious’. Screening the library with MdRTE1a identified two putative NAC (NAM/ATAF1, 2/CUC2) proteins, designated MdNAC1 and MdNAC2, that interacted with MdRTE1a in yeast cells. MdRTE1b, a closely related paralog of MdRTE1a, was also confirmed to be interactive with MdNAC1 and MdNAC2 using the Y2H system. The expression of MdRTE1a and MdNAC1 was high in young fruit but low in more developed and ripening fruit. This pattern of low expression remained largely consistent and was not affected by treatment with 1-methylcyclopropene (1-MCP), an inhibitor of ethylene perception, or ethephon, an ethylene releasing compound. However, the expression of MdRTE1b and MdNAC2 rapidly increased during fruit ripening and was highly inhibited by 1-MCP. These data suggested that MdRTE1a, MdNAC1 and their possible interactions in apple cells may play roles in growth, whereas MdRTE1b, MdNAC2 and their putative interactions are involved in ethylene signaling, response and fruit ripening. The possible interactions between an RTE1-like protein and a NAC protein in plant cells have not been reported thus far. Its implications in ethylene signaling and response are discussed.

Submergence-1 (Sub1), a major quantitative trait locus affecting tolerance to complete submergence in lowland rice (Oryza sativa), contains two or three ethylene response factor (ERF)–like genes whose transcripts are regulated by submergence. In the submergence-intolerant japonica cultivar M202, this locus encodes two ERF genes, Sub1B and Sub1C. In the tolerant near-isogenic line containing the Sub1 locus from the indica FR13A, M202(Sub1), the locus additionally encodes the ERF gene Sub1A. During submergence, the tolerant M202(Sub1) displayed restrained leaf and internode elongation, chlorophyll degradation, and carbohydrate consumption, whereas the enzymatic activities of pyruvate decarboxylase and alcohol dehydrogenase were increased significantly compared with the intolerant M202. Transcript levels of genes associated with carbohydrate consumption, ethanolic fermentation, and cell expansion were distinctly regulated in the two lines. Sub1A and Sub1C transcript levels were shown to be upregulated by submergence and ethylene, with the Sub1C allele in M202 also upregulated by treatment with gibberellic acid (GA). These findings demonstrate that the Sub1 region haplotype determines ethylene- and GA-mediated metabolic and developmental responses to submergence through differential expression of Sub1A and Sub1C. Submergence tolerance in lowland rice is conferred by a specific allele variant of Sub1A that dampens ethylene production and GA responsiveness, causing quiescence in growth that correlates with the capacity for regrowth upon desubmergence.