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Vacuolar malic acid accumulation largely determines fruit acidity, a key trait for the taste and flavor of apple and other fleshy fruits. Aluminum‐activated malate transporter 9 (ALMT9/Ma1) underlies a major genetic locus, Ma, for fruit acidity in apple, but how the protein transports malate across the tonoplast is unclear. Here, it is shown that overexpression of the coding sequence of Ma1 (Ma1α) drastically decreases fruit acidity in "Royal Gala" apple, leading to uncovering alternative splicing underpins Ma1's function. Alternative splicing generates two isoforms: Ma1β is 68 amino acids shorter with much lower expression than the full‐length protein Ma1α. Ma1β does not transport malate itself but interacts with the functional Ma1α to form heterodimers, creating synergy with Ma1α for malate transport in a threshold manner (When Ma1β/Ma1α ≥ 1/8). Overexpression of Ma1α triggers feedback inhibition on the native Ma1 expression via transcription factor MYB73, decreasing the Ma1β level well below the threshold that leads to significant reductions in Ma1 function and malic acid accumulation in fruit. Overexpression of Ma1α and Ma1β or genomic Ma1 increases both isoforms proportionally and enhances fruit malic acid accumulation. These findings reveal an essential role of alternative splicing in ALMT9‐mediated malate transport underlying apple fruit acidity. [ABSTRACT FROM AUTHOR]

Abstract Background A five-dimensional (5-D) clone pooling strategy for screening of bacterial artificial chromosome (BAC) clones with molecular markers utilizing highly-parallel Illumina GoldenGate assays and PCR facilitates high-throughput BAC clone and BAC contig anchoring on a genetic map. However, this strategy occasionally needs manual PCR to deconvolute pools and identify truly positive clones. Results A new implementation is reported here for our previously reported clone pooling strategy. Row and column pools of BAC clones are divided into sub-pools with 1~2× genome coverage. All BAC pools are screened with Illumina's GoldenGate assay and the BAC pools are deconvoluted to identify individual positive clones. Putative positive BAC clones are then further analyzed to find positive clones on the basis of them being neighbours in a contig. An exhaustive search or brute force algorithm was designed for this deconvolution and integrated into a newly developed software tool, FPCBrowser, for analyzing clone pooling data. This algorithm was used with empirical data for 55 Illumina GoldenGate SNP assays detecting SNP markers mapped on Aegilops tauschii chromosome 2D and Ae. tauschii contig maps. Clones in single contigs were successfully assigned to 48 (87%) specific SNP markers on the map with 91% precision. Conclusion A new implementation of 5-D BAC clone pooling strategy employing both GoldenGate assay screening and assembled BAC contigs is shown here to be a high-throughput, low cost, rapid, and feasible approach to screening BAC libraries and anchoring BAC clones and contigs on genetic maps. The software FPCBrowser with the integrated clone deconvolution algorithm has been developed and is downloadable at http://avena.pw.usda.gov/wheatD/fpcbrowser.shtml.

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.

SUMMARY: 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 ArabidopsisACT7 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. Significance Statement: Fruit maturation date is a developmental trait of great horticultural importance. However, the trait remains poorly understood in apple. By sequencing the fruit mRNA of Gala and its somatic mutant, Autumn Gala (AGala), we revealed that a large deletion that occurred concurrently with a retrotransposon insertion is the primary genetic cause for fruit maturation delay in AGala. We also provide evidence that MdACT7, an actin encoding gene, is likely an important factor. [ABSTRACT FROM AUTHOR]

Author(s): Kenong Xu [1]; Xia Xu [1]; Takeshi Fukao [2]; Patrick Canlas [1]; Reycel Maghirang-Rodriguez [3]; Sigrid Heuer [3]; Abdelbagi M. Ismail [3]; Julia Bailey-Serres [2]; Pamela C. Ronald (corresponding [...]

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 Sub1. 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 Sub1 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 Sub1 gene in a wide range of backgrounds, whereas RM464A was more useful in selection for the Sub1 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 Sub1, 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., IN ADDITION to being the major food crop grown in tropical climates, rice is also widely grown in temperate regions, where the japonica subspecies is predominant. Most highly productive rice [...]

Jujube (Ziziphus jujuba) was domesticated from wild jujube (Z. jujuba var. spinosa). Here, integrative physiological, metabolomic, and comparative proteomic analyses were performed to investigate the fruit expansion and fruit taste components in a jujube cultivar 'Junzao' and a wild jujube 'Qingjiansuanzao' with contrasting fruit size and taste. We revealed that the duration of cell division and expansion largely determined the final fruit size, while the intercellular space in the mesocarp dictated the ratio of mesocarp volume in mature fruits. The high levels of endogenous gibbereline3 (GA) and zeatin in the growing fruit of 'Junzao' were associated with their increased fruit expansion. Compared with 'Junzao,' wild jujube accumulated lower sugars and higher organic acids. Furthermore, several protein co-expression modules and important member proteins correlated with fruit expansion, sugar synthesis, and ascorbic acid metabolism were identified. Among them, GA20OX involved in GA biosynthesis was identified as a key protein regulating fruit expansion, whereas sucrose-6-phosphate synthase (SPS) and neutral invertase (NINV) were considered as key enzymes promoting sugar accumulation and as major factors regulating the ratio of sucrose to hexose in jujube fruits, respectively. Moreover, the increase of Nicotinamide adenine dinucleotide-Malate dehydrogenase (NAD-MDH) activity and protein abundance were associated with the malic acid accumulation, and the high accumulation of ascorbic acid in wild jujube was correlated with the elevated abundance of GalDH, ZjAPXs, and MDHAR1, which are involved in the ascorbic acid biosynthesis and recycling pathways. Overall, these results deepened the understanding of mechanisms regulating fruit expansion and sugar/acids metabolisms in jujube fruit. [ABSTRACT FROM AUTHOR]

Abstract Background A five-dimensional (5-D) clone pooling strategy for screening of bacterial artificial chromosome (BAC) clones with molecular markers utilizing highly-parallel Illumina GoldenGate assays and PCR facilitates high-throughput BAC clone and BAC contig anchoring on a genetic map. However, this strategy occasionally needs manual PCR to deconvolute pools and identify truly positive clones. Results A new implementation is reported here for our previously reported clone pooling strategy. Row and column pools of BAC clones are divided into sub-pools with 1~2× genome coverage. All BAC pools are screened with Illumina's GoldenGate assay and the BAC pools are deconvoluted to identify individual positive clones. Putative positive BAC clones are then further analyzed to find positive clones on the basis of them being neighbours in a contig. An exhaustive search or brute force algorithm was designed for this deconvolution and integrated into a newly developed software tool, FPCBrowser, for analyzing clone pooling data. This algorithm was used with empirical data for 55 Illumina GoldenGate SNP assays detecting SNP markers mapped on Aegilops tauschii chromosome 2D and Ae. tauschii contig maps. Clones in single contigs were successfully assigned to 48 (87%) specific SNP markers on the map with 91% precision. Conclusion A new implementation of 5-D BAC clone pooling strategy employing both GoldenGate assay screening and assembled BAC contigs is shown here to be a high-throughput, low cost, rapid, and feasible approach to screening BAC libraries and anchoring BAC clones and contigs on genetic maps. The software FPCBrowser with the integrated clone deconvolution algorithm has been developed and is downloadable at http://avena.pw.usda.gov/wheatD/fpcbrowser.shtml.

Abstract Background Current techniques of screening bacterial artificial chromosome (BAC) libraries for molecular markers during the construction of physical maps are slow, laborious and often assign multiple BAC contigs to a single locus on a genetic map. These limitations are the principal impediment in the construction of physical maps of large eukaryotic genomes. It is hypothesized that this impediment can be overcome by screening multidimensional pools of BAC clones using the highly parallel Illumina GoldenGate™ assay. Results To test the efficacy of the Golden Gate assay in BAC library screening, multidimensional pools involving 302976 Aegilops tauschii BAC clones were genotyped for the presence/absence of specific gene sequences with multiplexed Illumina GoldenGate oligonucleotide assays previously used to place single nucleotide polymorphisms on an Ae. tauschii genetic map. Of 1384 allele-informative oligonucleotide assays, 87.6% successfully clustered BAC pools into those positive for a BAC clone harboring a specific gene locus and those negative for it. The location of the positive BAC clones within contigs assembled from 199190 fingerprinted Ae. tauschii BAC clones was used to evaluate the precision of anchoring of BAC clones and contigs on the Ae. tauschii genetic map. For 41 (95%) assays, positive BAC clones were neighbors in single contigs. Those contigs could be unequivocally assigned to loci on the genetic map. For two (5%) assays, positive clones were in two different contigs and the relationships of these contigs to loci on the Ae. tauschii genetic map were equivocal. Screening of BAC libraries with a simple five-dimensional BAC pooling strategy was evaluated and shown to allow direct detection of positive BAC clones without the need for manual deconvolution of BAC clone pools. Conclusion The highly parallel Illumina oligonucleotide assay is shown here to be an efficient tool for screening BAC libraries and a strategy for high-throughput anchoring of BAC contigs on genetic maps during the construction of physical maps of eukaryotic genomes. In most cases, screening of BAC libraries with Illumina oligonucleotide assays results in the unequivocal relationship of BAC clones with loci on the genetic map.

Summary: 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. Significance Statement: Fruit softening is a hallmark of ripening in apple fruit, but the degree of softening varies among cultivars, with corresponding variation in their quality and consumer preference. Much attention has been paid to the molecular characterization of fruit softening; however, the underlying genetic mechanisms are still not well understood. Here, we show that a mutation in the seqeucene encoding the EAR motif of ERF4 results in a decrease in its repression activity, thereby promoting fruit softening and ripening. [ABSTRACT FROM AUTHOR]

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 C o 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 < h k × h k>, < l m × ll >, < nn × n p>, < l m × mm>, 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. [ABSTRACT FROM AUTHOR]

Background: Acidity is a major contributor to fruit quality. Several organic acids are present in apple fruit, but malic acid is predominant and determines fruit acidity. The trait is largely controlled by the Malic acid (Ma) locus, underpinning which Ma1 that putatively encodes a vacuolar aluminum-activated malate transporter1 (ALMT1)-like protein is a strong candidate gene. We hypothesize that fruit acidity is governed by a gene network in which Ma1 is key member. The goal of this study is to identify the gene network and the potential mechanisms through which the network operates. Results: Guided by Ma1, we analyzed the transcriptomes of mature fruit of contrasting acidity from six apple accessions of genotype Ma_ (MaMa or Mama) and four of mama using RNA-seq and identified 1301 fruit acidity associated genes, among which 18 were most significant acidity genes (MSAGs). Network inferring using weighted gene co-expression network analysis (WGCNA) revealed five co-expression gene network modules of significant (P < 0.001) correlation with malate. Of these, the Ma1 containing module (Turquoise) of 336 genes showed the highest correlation (0.79). We also identified 12 intramodular hub genes from each of the five modules and 18 enriched gene ontology (GO) terms and MapMan sub-bines, including two GO terms (GO:0015979 and GO:0009765) and two MapMap sub-bins (1.3.4 and 1.1.1.1) related to photosynthesis in module Turquoise. Using Lemon-Tree algorithms, we identified 12 regulator genes of probabilistic scores 35.5–81.0, including MDP0000525602 (a LLR receptor kinase), MDP0000319170 (an IQD2-like CaM binding protein) and MDP0000190273 (an EIN3-like transcription factor) of greater interest for being one of the 18 MSAGs or one of the 12 intramodular hub genes in Turquoise, and/or a regulator to the cluster containing Ma1. Conclusions: The most relevant finding of this study is the identification of the MSAGs, intramodular hub genes, enriched photosynthesis related processes, and regulator genes in a WGCNA module Turquoise that not only encompasses Ma1 but also shows the highest modular correlation with acidity. Overall, this study provides important insight into the Ma1-mediated gene network controlling acidity in mature apple fruit of diverse genetic background. [ABSTRACT FROM AUTHOR]

The architecture of apple trees plays a pivotal role in shaping their growth and fruit-bearing potential, forming the foundation for precision apple management. Traditionally, 2D imaging technologies were employed to delineate the architectural traits of apple trees, but their accuracy was hampered by occlusion and perspective ambiguities. This study aimed to surmount these constraints by devising a 3D geometry-based processing pipeline for apple tree structure segmentation and architectural trait characterization, utilizing point clouds collected by a terrestrial laser scanner (TLS). The pipeline consisted of four modules: (a) data preprocessing module, (b) tree instance segmentation module, (c) tree structure segmentation module, and (d) architectural trait extraction module. The developed pipeline was used to analyze 84 trees of two representative apple cultivars, characterizing architectural traits such as tree height, trunk diameter, branch count, branch diameter, and branch angle. Experimental results indicated that the established pipeline attained an R 2 of 0.92 and 0.83, and a mean absolute error (MAE) of 6.1 cm and 4.71 mm for tree height and trunk diameter at the tree level, respectively. Additionally, at the branch level, it achieved an R 2 of 0.77 and 0.69, and a MAE of 6.86 mm and 7.48° for branch diameter and angle, respectively. The accurate measurement of these architectural traits can enable precision management in high-density apple orchards and bolster phenotyping endeavors in breeding programs. Moreover, bottlenecks of 3D tree characterization in general were comprehensively analyzed to reveal future development., Competing Interests: Competing interests: The authors declare that they have no competing interests., (Copyright © 2024 Tian Qiu et al.)

Plant architecture is 1 of the most important factors that determines crop yield potential and productivity. In apple (Malus domestica), genetic improvement of tree architecture has been challenging due to a long juvenile phase and 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 of MdLAZY1A (MD13G1122400) as the genetic determinant underpinning the Weeping (W) locus that largely controls weeping growth in Malus. MdLAZY1A is 1 of the 4 paralogs in apple that are most closely related to AtLAZY1 involved in gravitropism in Arabidopsis (Arabidopsis thaliana). 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 colocalizes with Region III, 1 of the 5 conserved regions in LAZY1-like proteins. Subcellular localization revealed that MdLAZY1A localizes to the plasma membrane and nucleus in plant cells. Overexpressing 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 in Malus and other crops., Competing Interests: Conflict of interest statement. The authors declare that they have no conflict of interests., (© American Society of Plant Biologists 2023. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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., (© 2020 American Society of Plant Biologists. All Rights Reserved.)

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.

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.

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) x 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.

Background: Current techniques of screening bacterial artificial chromosome (BAC) libraries for molecular markers during the construction of physical maps are slow, laborious and often assign multiple BAC contigs to a single locus on a genetic map. These limitations are the principal impediment in the construction of physical maps of large eukaryotic genomes. It is hypothesized that this impediment can be overcome by screening multidimensional pools of BAC clones using the highly parallel Illumina GoldenGate assay., Results: To test the efficacy of the Golden Gate assay in BAC library screening, multidimensional pools involving 302976 Aegilops tauschii BAC clones were genotyped for the presence/absence of specific gene sequences with multiplexed Illumina GoldenGate oligonucleotide assays previously used to place single nucleotide polymorphisms on an Ae. tauschii genetic map. Of 1384 allele-informative oligonucleotide assays, 87.6% successfully clustered BAC pools into those positive for a BAC clone harboring a specific gene locus and those negative for it. The location of the positive BAC clones within contigs assembled from 199190 fingerprinted Ae. tauschii BAC clones was used to evaluate the precision of anchoring of BAC clones and contigs on the Ae. tauschii genetic map. For 41 (95%) assays, positive BAC clones were neighbors in single contigs. Those contigs could be unequivocally assigned to loci on the genetic map. For two (5%) assays, positive clones were in two different contigs and the relationships of these contigs to loci on the Ae. tauschii genetic map were equivocal. Screening of BAC libraries with a simple five-dimensional BAC pooling strategy was evaluated and shown to allow direct detection of positive BAC clones without the need for manual deconvolution of BAC clone pools., Conclusion: The highly parallel Illumina oligonucleotide assay is shown here to be an efficient tool for screening BAC libraries and a strategy for high-throughput anchoring of BAC contigs on genetic maps during the construction of physical maps of eukaryotic genomes. In most cases, screening of BAC libraries with Illumina oligonucleotide assays results in the unequivocal relationship of BAC clones with loci on the genetic map.