Apple Germplasm Research Articles

Combinatorial approach based on conventional and molecular methods for identification of scab resistance genes in apple germplasm of Jammu and Kashmir, India

Management of scab through resistant varieties represents a cost effective and eco-friendly alternative to chemical control. The accurate identification of resistant genotypes is carried out both at phenotypic as well as genotypic levels and most of the previous studies on resistance screening have been carried out using only one of these strategies. We used a combination of conventional and molecular marker based approaches for screening of 75 apple genotypes, in Kashmir region of India, to get more reliable results. We identified 17 genotypes carrying major scab resistance genes Rvi2, Rvi6, Rvi8 and Rvi11 with genotypes like Shalimar 2nd, Liberty and Sangerwan Delicious carrying multiple scab resistance genes, a finding that bypasses the use of tedious and time consuming procedure of gene pyramiding and could save researchers invaluable resources and time. We also identified two genotypes of Indigenous Ambri cultivars carrying Rvi11 scab resistance gene, a finding that may play an important role for revival and commercialization of Ambri cultivars which is considered as native of Kashmir valley of Jammu and Kashmir but are under serious threat from other commercially viable cultivars. Earlier scales used for resistance screening lack a separate category for identification of quantitative resistance. We introduce a separate class for genotypes carrying quantitative resistance because we believe it can be very important for disease management especially when present alongside a major scab resistance gene. We also conclude that genetic background in which a scab resistance gene is embedded is the major factor for the phenotypic response it shows upon inoculation with the pathogen.

Abscisic acid and regulation of the sugar transporter gene MdSWEET9b promote apple sugar accumulation.

Enhancing fruit sugar contents, especially for high-flavonoid apples with a sour taste, is one of the main goals of horticultural crop breeders. This study analyzed sugar accumulation and the underlying mechanisms in the F2 progenies of a hybridization between the high-sugar apple (Malus × domestica) variety "Gala" and high-flavonoid apple germplasm "CSR6R6". We revealed that MdSWEET9b (sugars will eventually be exported transporter) helps mediate sugar accumulation in fruits. Functional characterization of MdSWEET9b in yeast mutants lacking sugar transport as well as in overexpressing and CRISPR/Cas9 knockdown apple calli revealed MdSWEET9b could transport sucrose specifically, ultimately promoting normal yeast growth and accumulation of total sugar contents. Moreover, MdWRKY9 bound to the MdSWEET9b promoter and regulated its activity, which responded to abscisic acid (ABA) signaling. Furthermore, MdWRKY9 interacted with MdbZIP23 (basic leucine zipper) and MdbZIP46, key ABA signal transducers, at the protein and DNA levels to enhance its regulatory effect on MdSWEET9b expression, thereby influencing sugar accumulation. Based on the contents of ABA in lines with differing sugar contents and the effects of ABA treatments on fruits and calli, we revealed ABA as one of the main factors responsible for the diversity in apple fruit sugar content. The results of this study have clarified how MdSWEET9b influences fruit sugar accumulation, while also further elucidating the regulatory effects of the ABA-signaling network on fruit sugar accumulation. This work provides a basis for future explorations of the crosstalk between hormone and sugar metabolism pathways.

Allelic variation of MdMYB123 controls malic acid content by regulating MdMa1 and MdMa11 expression in apple.

Acidity is a key determinant of fruit organoleptic quality. Here, a candidate gene for fruit acidity, designated MdMYB123, was identified from a comparative transcriptome study of two Ma1Ma1 apple (Malus domestica) varieties, "Qinguan (QG)" and "Honeycrisp (HC)" with different malic acid content. Sequence analysis identified an A→T SNP, which was located in the last exon, resulting in a truncating mutation, designated mdmyb123. This SNP was significantly associated with fruit malic acid content, accounting for 9.5% of the observed phenotypic variation in apple germplasm. Differential MdMYB123- and mdmyb123-mediated regulation of malic acid accumulation was observed in transgenic apple calli, fruits, and plantlets. Two genes, MdMa1 and MdMa11, were up- and down-regulated in transgenic apple plantlets overexpressing MdMYB123 and mdmyb123, respectively. MdMYB123 could directly bind to the promoter of MdMa1 and MdMa11, and induce their expression. In contrast, mdmyb123 could directly bind to the promoters of MdMa1 and MdMa11, but with no transcriptional activation of both genes. In addition, gene expression analysis in 20 different apple genotypes based on SNP locus from "QG" × "HC" hybrid population confirmed a correlation between A/T SNP with expression levels of MdMa1 and MdMa11. Our finding provides valuable functional validation of MdMYB123 and its role in the transcriptional regulation of both MdMa1 and MdMa11, and apple fruit malic acid accumulation.

Pool-seq of diverse apple germplasm reveals candidate loci underlying ripening time, phenolic content, and softening

Pool-seq of diverse apple germplasm reveals candidate loci underlying ripening time, phenolic content, and softening

EVALUATION OF SCAB AND POWDERY MILDEW RESISTANCE OF APPLE GERMPLASM COLECTTED AT RIFG PITESTI

The rising incidence of biotic and abiotic stresses makes commercial fruit production increasingly difficult in Europe, especially apple orchards which are affected by fungal diseases, like scab and powdery mildew, caused by Venturia inaequalis Cooke Winter and Podosphaera leucotricha Ellis & Everh. Salmon. Releasing and planting of apple resistant cultivars is a promising strategy. A set of 527 apple cultivars from the apple gene bank collected at Research Institute for Fruit Growing (RIFG) Pitești was evaluated for the incidence of scab (on leaves and fruits) and powdery mildew (on leaves and top shoots) in three consecutive years (2018, 2019 and 2020). Phenotypic results were compared to data reported at the evaluation made in 2006. Unfortunately, the incidence of both scab and powdery mildew increased significantly in the last years. In 2021, 48 Romanian apple cultivars were analyzed with molecular markers linked to Rvi2, Rvi4, Rvi5, Rvi6, Rvi8 and Plw. The markers linked to Rvi6 was detected in 31 cultivars. Alleles linked to Plw were expressed from three cultivars. The information on resistance / susceptibility of apple genetic resources towards economically important diseases is important for breeding. Using a systematic screening of the apple cultivar collection, several cultivars with high value for resistance breeding and sustainable growing were identified.

Phytochemical Variation of Native Apple Germplasm Resources from the Eastern Black Sea Region, Turkey

Phytochemical Variation of Native Apple Germplasm Resources from the Eastern Black Sea Region, Turkey

Morphological characterization of 23 Malus domestica Borkh cultivars from central Spain

The purpose of this work was to morphologically characterize an apple tree collection composed of 67 individuals from 41 accessions belonging to 23 old Spanish apple cultivars (Malus domestica Borkh) alongside 9 reference cultivars. The studied germplasm was collected previously in rural areas of central Spain (Sierra Norte de Madrid and Tagus river basin) and it was analyzed through 67 descriptors mainly from IBPGR and UPOV. We found a very high morphological diversity in the studied old apple cultivars, as 48% of the descriptors (most of them devoted to fruit traits) were significantly different between types of cultivars. In addition, the sample cultivars resulted clearly distinct from reference cultivars in multivariate analysis. In general, no particular structure was found in old cultivars, but a strong differentiation of ‘Agridulce’ and ‘Hojancas’ is reported due to their bigger fruits. Our results support the molecular analysis and call for further analysis of the local apple germplasm and long-term conservation actions.

Evaluation of Apple Scab and Occurrence of Venturia Inaequalis Races on Differential Malus Genotypes in Latvia

Abstract Apple scab caused by the heterothallic ascomycetous fungus Venturia inaequalis is one of the most severe diseases in apple-growing regions worldwide. Annual sexual reproduction of the pathogen ensures the formation of populations with high variability and better adaptability to resistant cultivars. The present study aimed to assess the virulence of the V. inaequalis population in the experimental orchard where introduction, evaluation and breeding of apple cultivars are carried out, and the apple germplasm collection of Latvia is located. Fifteen differential Malus genotypes, obtained within the international Vinquest initiative, were planted in 2016 in the germplasm collection sector of the orchard in five blocks, each containing one tree of each geno-type. In 2017–2021, apple scab symptoms on the leaves were scored according to the methodology used in the Vinquest network. The disease severity increased with each vegetation season, and more genotypes were affected, reaching the maximum in 2021 and clearly indicating the infection source build-up and pathogen’s adaptation on particular genotypes due to the absence of scab control. At least four races of V. inaequalis were found to be established in Latvia, and the resistance genes Rvi1, Rvi3, Rvi4, and Rvi8 have been overcome. On h(9) and h(13), the first symptoms appeared in 2021, indicating the first signs of Rvi9 and Rvi13 gene breakdown.

Genetic Identity and Diversity of Apple Accessions within a Candidate Collection for the Norwegian National Clonal Germplasm Repository

In order to best conserve, as well as utilize, traditional apple germplasm in Norway, an apple heritage cultivar collection was established in Ullensvang, western Norway, which aims to become the National Clonal Germplasm Repository. The establishment of the apple heritage cultivar collection was preceded by a molecular study that aimed to genotype a large number of apple accessions maintained in various ex situ sites in western and south-eastern Norway, using a rather small set of eight SSR markers. However limited, the marker set managed to identify synonyms, homonyms, and duplicates within and among the investigated collections. In this study, 171 apple accessions from the Ullensvang apple heritage cultivar collection were genotyped using a set of 20 different SSR markers. Approximately half of the accessions have been previously genotyped using eight SSR markers, enabling an assessment of whether the use of a larger marker set would yield a more accurate characterization. Based on the obtained molecular data, the apple heritage cultivar collection was determined to hold a key part of the overall genetic diversity of the Norwegian apple germplasm. Furthermore, the twelve additional SSR markers were able to differentiate several accessions groups originally thought to be synonyms, as well as to provide a more detailed insight into the genetic structure of this germplasm.

Integer programming for selecting set of informative markers in paternity inference

BackgroundParentage information is fundamental to various life sciences. Recent advances in sequencing technologies have made it possible to accurately infer parentage even in non-model species. The optimization of sets of genome-wide markers is valuable for cost-effective applications but requires extremely large amounts of computation, which presses for the development of new efficient algorithms.ResultsHere, for a closed half-sib population, we generalized the process of marker loci selection as a binary integer programming problem. The proposed systematic formulation considered marker localization and the family structure of the potential parental population, resulting in an accurate assignment with a small set of markers. We also proposed an efficient heuristic approach, which effectively improved the number of markers, localization, and tolerance to missing data of the set. Applying this method to the actual genotypes of apple (Malus × domestica) germplasm, we identified a set of 34 SNP markers that distinguished 300 potential parents crossed to a particular cultivar with a greater than 99% accuracy.ConclusionsWe present a novel approach for selecting informative markers based on binary integer programming. Since the data generated by high-throughput sequencing technology far exceeds the requirement for parentage assignment, a combination of the systematic marker selection with targeted SNP genotyping, such as KASP, allows flexibly enlarging the analysis up to a scale that has been unrealistic in various species. The method developed in this study can be directly applied to unsolved large-scale problems in breeding, reproduction, and ecological research, and is expected to lead to novel knowledge in various biological fields. The implementation is available at https://github.com/SoNishiyama/IP-SIMPAT.

Mangrove apple (Sonneratia caseolaris): A promising fruit in Patuakhali coast of Bangladesh

Mangrove apple (Sonneratia caseolaris) is a widespread underutilized fruit species of coastal region of Bangladesh. The present study was undertaken to find out the flowering, fruiting, nutritional quality and extent of variability among different mangrove apple germplasm. Twelve germplasm were selected from three locations - six (SC 02, SC 03, SC 04, SC 05, SC O6 and SC 07) from Dumki sadar, two (SC 01 and SC 08) from PSTU germplasm centre and four (SC 09, SC 10, SC 11 and SC 12) from Patabuniya, Dumki, Patuakhali. Flowering season started from May and continued up to November. Percentage of fruit setting ranged from 96.03 to 89.96% in SC 01 and SC 12, respectively. Required time for fruit maturation ranged from 106 days (SC 06) to 55 days (SC 05). Single fruit weight varied from 122.3 g (SC 01) to 54.33 g (SC 04). The length and diameter of fruit ranged from 2.9 cm (SC 05) to 2.20 cm (SC 07) and 5.20 cm (SC 05) to 3.60 cm (SC 04), respectively. The maximum edible portion (66.15%) was recorded in SC 01, and it was the lowest (51.45%) in SC 07. Chemical analysis was done with the pulp of selected germplasm where the germplasm SC 01 exhibited the best performance in all parameters of chemical analysis. This analysis was performed at four different ripening stages. The highest TSS (8.72%) was recorded in stage I. The highest pH level (3.51) was observed in stage III and the lowest pH level (2.83) was found in stage I. The highest ascorbic acid content (37.50 mg/100g) was noted in stage IV. Flower of mangrove apple produces a kind of honey. Fresh fruits and seeds have high nutritional and medicinal value. Wood of this plants provides great livelihood support to the local residents for fetching good price in the markets. Proper utilization of S. caseolaris may contributes the nutritional security of the women and children of this area by fulfilling the demand for micronutrients, especially vitamin C. Bangladesh J. Agri. 2019-2021, 44-46: 9-17

Malice at the Gates of Eden: current and future distribution of Agrilus mali threatening wild and domestic apples.

The apple buprestid, Agrilus mali Matsumura, that was widespread in north-eastern China, was accidently introduced to the wild apple forest ecosystem in mountainous areas of Xinjiang, China. This invasive beetle feeds on domesticated apples and many species of Malus and presents a serious threat to ancestral apple germplasm sources and apple production worldwide. Estimating the potential area at risk of colonization by A. mali is crucial for instigating appropriate preventative management strategies, especially under global warming. We developed a CLIMEX model of A. mali to project this pest's potential distribution under current and future climatic scenarios in 2100 using CSIRO-Mk 3.0 GCM running the SRES A1B emissions scenario. Under current climate, A. mali could potentially invade neighbouring central Asia and eventually the mid-latitude temperate zone, and some subtropical areas and Pampas Steppe in the Southern Hemisphere. This potential distribution encompasses wild apples species, the ancestral germplasm for domesticated apples. With global warming, the potential distribution shifts to higher latitudes, with the potential range expanding slightly, though the overall suitability could decline in both hemispheres. In 2100, the length of the growing season of this pest in the mid-latitude temperature zone could increase by 1-2 weeks, with higher growth rates in most sites compared with current climate in mid-latitudes, at least in China. Our work highlights the need for strategies to prevent the spread of this pest, managing the threats to wild apples in Tian Shan Mountain forests in Central Asia, and commercial apple production globally. We discuss practical management tactics to reduce the spread of this pest and mitigate its impacts.

Meta‐analysis of apple (Malus × domestica Borkh.) fruit and juice quality traits for potential use in hard cider production

Societal Impact StatementThe production of hard cider is a burgeoning industry in North America. However, traditional European cider‐specific apples are largely unsuitable for production in modern systems and have not been widely adopted. Evaluation of the biochemical variability in apples can provide a better characterization of varieties for use in hard cider production. Many varieties exhibited extensive variability in biochemical traits. Varieties with high phenolics and acidity displayed greater plasticity of these traits between seasons, which supports the concept of “vintage quality” and opens new marketing opportunities. This study provides valuable insight for apple producers to integrate cider‐specific and nonspecific varieties into their production systems.Summary The North American fermented beverage market has undergone extensive growth in the fermented apple juice (hard cider) sector over the last decade. Traditional hard cider producing regions such as England, France, and Spain have historically utilized cider‐specific apples that have desirable balances between sugar, organic acid, and phenolics. However, in North America, culinary apples (lower acidity and phenolics) are predominantly used, and information distinguishing the varieties with novel use for hard cider production is lacking. In this study, we evaluated variation of four major apple biochemical traits (total soluble solids, pH, titratable acidity, and total phenolics) as they relate to use for hard cider production by using a meta‐analysis approach. We analyzed the variation in cider quality traits across 47 published works and more than 800 unique apple varieties, including an additional analysis of 142 varieties from cider apple germplasms in the United States. The biochemical traits of many varieties exhibited plasticity, and the characterization of a subset of varieties using two cider classification system revealed that most belonged to the “sweet” or “sharp” (acidic) categories rather than “bitter” (high phenolic content). We also compared the classification of a subset of varieties from two US germplasm resources across two seasons and found varieties that have high percent phenolics and/or malic acid experienced greater biochemical plasticity and potential classification change across vintages. Ultimately, this work provides information regarding the variability in apple biochemical traits and also suggests a unique opportunity for producers to market “vintage” quality.

Development of multiplex RT-PCR assay for simultaneous detection of four viruses infecting apple (Malus domestica).

The major viruses infecting apple cultivars throughout the world including India are apple mosaic virus (ApMV), apple stem pitting virus (ASPV), apple stem grooving virus (ASGV), apple chlorotic leaf spot virus (ACLSV), and recently, a new virus, apple necrotic mosaic virus (ApNMV), was reported from mosaic-infected apple cultivars in India. The aim of this study was to detect the ApNMV virus along with the other three viruses (ApMV, ASPV and ASGV) simultaneously by multiplex RT-PCR. Four primer-pair-produced amplicons of 670, 550, 350 and 210 bp corresponding to ApNMV, ApMV, ASPV and ASGV, respectively, were found to be specific for these viruses when tested individually. The annealing temperature (55°C), primer concentration (0·8 µl) and other components of the master mix were standardized for the development of one-step m-RT-PCR assay. The m-RT-PCR protocol developed was further validated with 30 samples from seven symptomatic or asymptomatic apple cultivars, which revealed the presence of more than one virus in these cultivars. Most of the viruses were found to be present either alone or in mixed infection; however, ASPV was more common in tested cultivars. An easy, cost-effective and rapid multiplex RT-RCR protocol was developed to detect the four viruses, which infect apple plants either in individually or together in the field. This assay will help in the surveying and indexing of apple germplasm and the distribution of all four viruses in the apple growing regions of India.

Apple cultivars and rootstocks assay for the identification of diverse viruses and healthy genotypes for breeding

The prevalence of harmful viruses, viz., apple stem grooving virus (ASGV), apple stem pitting virus (ASPV), apple chlorotic leaf spot virus (ACLSV), apple mosaic virus (ApMV), and tomato ringspot virus (ToRSV) in apple tree plantations in the Ryazan, Yaroslavl, and Moscow regions, Russian Federation, based on genotype features, planting type (industrial, collection, and repository), and tree age was studied during 2018–2021. The prevalence of harmful and latent viruses ranged from 49.6% to 53.8% in apple cultivars and from 8.3% to 100% in apple clonal rootstocks. The most common virus in the Moscow and Yaroslavl regions was ACLSV (34.7% and 53.8%) and that in the Ryazan region was ApMV (33.3%). Relative to that in young apple trees, the virus influence in older tree tissues was higher by 20% to 43% depending on virus type. Monoviral infection prevailed (59% of all trees were infected with one virus) in the studied apple tree cultivars. Of the cultivars, 25% were infected by a complex of two viruses (ASPV + ACLSV), 10% were infected by three viruses, and 6% were infected by four viruses. The highest prevalence of latent viruses was observed in old Russian cultivars (53.5%) and selections from old foreign cultivars (57.2%). Virus occurrence was slightly lower (51.6%) in new Russian cultivars than in other cultivars. Columnar apple tree cultivars had the lowest virus occurrence (30%) and were found to be more tolerant than other genotypes. The highest virus incidence was recorded in industrial orchards (63.4%), followed by that recorded in collection (20.8%) and repository (18.3%) plantations. By using ELISA, the apple genotypes that were free from harmful viruses were identified as source plants, i.e., 183 plants from 18 apple tree cultivars and 131 plants from clonal rootstocks. Results indicated that virus-free apple germplasm is highly effective for the successful implementation of breeding and genetic improvement.

Potential roles of melatonin and ABA on apple dwarfing in semi-arid area of Xinjiang China.

Dwarfing is a typic breeding trait for mechanical strengthening and relatively high yield in modern apple orchards. Clarification of the mechanisms associated with dwarfing is important for use of molecular technology to breed apple. Herein, we identified four dwarfing apple germplasms in semi-arid area of Xinjiang, China. The internodal distance of these four germplasms were significantly shorter than non-dwarfing control. Their high melatonin (MT) contents are negatively associated with their malondialdehyde (MDA) levels and oxidative damage. In addition, among the detected hormones including auxin (IAA), gibberellin (GA), brassinolide (BR), zeatin-riboside (ZR), and abscisic acid (ABA), only ABA and ZR levels were in good correlation with the dwarfing phenotype. The qPCR results showed that the expression of melatonin synthetic enzyme genes MdASMT1 and MdSNAT5, ABA synthetic enzyme gene MdAAO3 and degradative gene MdCYP707A, ZR synthetic enzyme gene MdIPT5 all correlated well with the enhanced levels of MT, ABA and the reduced level of of ZR in the dwarfing germplasms. Furthermore, the significantly higher expression of ABA marker genes (MdRD22 and MdRD29) and the lower expression of ZR marker genes (MdRR1 and MdRR2) in all the four dwarf germplasms were consistent with the ABA and ZR levels. Considering the yearly long-term drought occurring in Xinjiang, China, it seems that dwarfing with high contents of MT and ABA may be a good strategy for these germplasms to survive against drought stress. This trait of dwarfing may also benefit apple production and breeding in this semi-arid area.

Thermotherapy Followed by Shoot Tip Cryotherapy Eradicates Latent Viruses and Apple Hammerhead Viroid from In Vitro Apple Rootstocks.

Virus and viroid-free apple rootstocks are necessary for large-scale nursery propagation of apple (Malus domestica) trees. Apple stem grooving virus (ASGV) and Apple chlorotic leaf spot virus (ACLSV) are among the most serious apple viruses that are prevalent in most apple growing regions. In addition to these viruses, a new infectious agent named Apple hammerhead viroid (AHVd) has been identified. We investigated whether thermotherapy or cryotherapy alone or a combination of both could effectively eradicate ACLSV, ASGV, and AHVd from in vitro cultures of four apple rootstocks developed in the Cornell-Geneva apple rootstock breeding program (CG 2034, CG 4213, CG 5257, and CG 6006). For thermotherapy treatments, in vitro plants were treated for four weeks at 36 °C (day) and 32 °C (night). Plant vitrification solution 2 (PVS2) and cryotherapy treatments included a shoot tip preculture in 2 M glycerol + 0.8 M sucrose for one day followed by exposure to PVS2 for 60 or 75 min at 22 °C, either without or with liquid nitrogen (LN, cryotherapy) exposure. Combinations of thermotherapy and PVS2/cryotherapy treatments were also performed. Following treatments, shoot tips were warmed, recovered on growth medium, transferred to the greenhouse, grown, placed in dormancy inducing conditions, and then grown again prior to sampling leaves for the presence of viruses and viroids. Overall, thermotherapy combined with cryotherapy treatment resulted in the highest percentage of virus- and viroid-free plants, suggesting great potential for producing virus- and viroid-free planting materials for the apple industry. Furthermore, it could also be a valuable tool to support the global exchange of apple germplasm.

Genetic diversity and population structure of apple germplasm from Eastern Black Sea region of Turkey by SSRs

Turkey has a diverse apple germplasm and Northeastern Anatolia had great contributions to domestication of the Malus genus. Despite quite high rainfall (900–2300 mm) and humidity (70–85%) levels, several local apple cultivars have been successfully grown for centuries at the coastal zone of Eastern Black Sea region of Northeastern Anatolia without any pesticide applications. In this study, a set of 206 local genotypes, including 6 international cultivars as references was analyzed using 13 SSR primers with the aim of assessing the genetic diversity and population structure among the genotypes. Bayesian genetic structure analysis was used to reveal differentiation of the groups and the results were confirmed by factorial correspondence analysis (FCA). A total of 234 alleles (average 18) were generated by 13 SSR markers. CH02c06 was the most informative (PI: 0.017) and CH03g07 was the least informative (PI: 0.07) locus. The average He was 0.859 with a range of 0.775–0.929 and the average Ho was 0.760 with a range of 0.556–0.872. Considerable genetic variation was detected among the genotypes and genetic similarity varied between 11 and 96%. A Bayesian genetic structure analysis indicated three subpopulations (K) and admixture among the accessions. Several loci yielded three alleles in 39 accessions. No synonyms or identical cultivars were detected but several genotypes known by the same names formed 22 homonym groups. The present SSR data will have great contributions to future germplasm management efforts as well as to further comparative studies that investigate genetic relationships among the local apples of Northeastern Anatolia.

MdHB7-like confers drought tolerance and improves water-use efficiency through modulating stomatal density in apple (Malus domestica)

Improving plant drought tolerance and water-use efficiency (WUE) is important for plant survival and crop yield under conditions of water shortage. HD-Zip transcription factors (TFs) have been shown to help plants respond to drought. Here, MdHB7-like (an HD-Zip I TF) was identified as a drought-induced gene in apple plants. Transgenic apple with upregulated expression of MdHB7-like showed enhanced drought tolerance, whereas MdHB7-like RNA interference (RNAi) lines were more sensitive to drought. Further analysis showed that overexpression of MdHB7-like reduced stomatal density, but the stomatal density of MdHB7-like RNAi lines was increased. Transcriptome analysis showed that MdHB7-like overexpressing plants may reduce stomatal density by affecting the expression of stomatal development related genes, such as MdEPFL9.1 and MdEPFL9.2. The decrease in stomatal density reduced leaf water loss, helping to enhance the drought tolerance of overexpression lines. In addition, MdHB7-like overexpression enhanced drought tolerance of apple plants by promoting stomatal closure. Further study showed that the overexpression of MdHB7-like significantly increased WUE under long-term moderate drought conditions, which also related to the reduction of stomatal density. These results suggest that MdHB7-like is a valuable genetic resource for enhancing drought tolerance and increasing the WUE of apple germplasm.

Genome-wide association study (GWAS) of leaf wax components of apple

The wax layer of apple leaves plays an important role in improving stress resistance, but relatively little is known about the mechanisms of wax synthesis and transport in apple leaves. In this study, 17 wax components, including alcohols, alkanes, fatty acids and terpenes, were analyzed by gas chromatography-tandem mass spectrometry (GC-MS) from the leaves of 123 apple germplasms. Whole-genome sequencing of these apple accessions yielded 5.9 million high-quality single nucleotide polymorphisms (SNPs). We performed a genome-wide association study (GWAS) on 17 wax components and identified several genes related to wax synthesis and transport, including MdSHN1 (SHINE1), MdLTP4 (LIPID TRANSFER PROTEIN4), MdWSD1 (WAX ESTER SYNTHASE/ACYL-COA DIAC-YLGLYCEROL ACYLTRANSFERASE1), MdRDR1 (RNA-DEPENDENT RNA POLYMERASE1), MdACBP6 (ACYL-COA-BINDING PROTEIN6), MdNLE (NOTCHLESS) and MdABCG21 (ATP-BINDING CASSETTE G21). Moreover, we identified some prominent SNPs that may affect gene expression and protein function. These results provide insights into mechanisms of wax synthesis and transport in apple leaves and broaden the genetic resources and basis for facilitating resistance breeding.