Evidence of gene introgression in apple using RAPD markers

Summary A genomic remnant of Malus floribunda clone 821 introgressed into the cultivated apple M. x domestica Borkh. was identified using randomly amplified polymorphic DNA (RAPD) markers obtained by the polymerase chain reaction (PCR). Using a set of 59 oligonucleotide decamer primers, polymorphic DNA markers were identified among three pooled DNA samples. Based on the presence or absence of bands among bulked apple scab-resistant selections and cultivars, bulked scab-susceptible cultivars, and a M. floribunda clone 821 sample, one primer, A 15, identified amplified fragments in the scab-resistant bulked sample that was also unique to the M. floribunda clone 821. The unique band from M. floribunda clone 821 was amplified in four out of 17 scab-resistant selections/cultivars. This RAPD, designated OA15₉₀₀, identifies an introgressed fragment that has as yet no known function.     

DNA fingerprinting of apple (Malus spp.) rootstocks using Simple Sequence Repeats

Identification of apple clonal rootstocks based on visual morphological characteristics is often difficult because of the genetic similarity of many rootstocks and the restriction of observable characteristics at the nursery stage. In this study, a total of 66 rootstock clones representing a sub‐sample of a collection of apple rootstocks from different countries maintained at NIFTS, Morioka, Japan, was screened with seven simple sequence repeat (SSR) markers to establish genetic identities, estimate genetic diversity, verify parentage and examine genetic relationships. All seven SSR loci produced one or two amplified fragments and made it possible to differentiate among the rootstocks, except between virus‐free subclones and their original parents as well as between ‘Aotea 40’ and ‘Aotea 106’. The parentage of all but one rootstock clone was confirmed. The placement of individual rootstock clones into subgroups in a phenogram generated from un‐weighted pair group average cluster analysis was generally based on parentage or genetic origin. Based upon the ability of the SSR data to differentiate these apple rootstocks and the high level of gene diversity, it is proposed that all commercial apple rootstocks be fingerprinted to provide a mechanism to verify the identity of individual clones.     

Comparison of tree architecture using tree edit distances: application to 2-year-old apple hybrids

In fruit trees, understanding genetic determinisms of architectural traits is considered as a promising manner to control vegetative development and yield regularity. In this context, our study aimed to classify 2-year-old apple hybrids on the basis of their architectural traits. From a fine phenotyping, trees were described as tree graphs, including topological and geometric information. To evaluate the similarity between trees, comparison methods based on edit operations (substitution, insertion and deletion) were carried out. Distance between two tree graphs was computed by minimising the sum of the costs of the edit operations applied to transform one tree into another. Two algorithms for the comparison of unordered and partially ordered tree graphs were applied to a sub-sample of the population, taking into account several geometric attributes. For each comparison, a dissimilarity matrix was computed, and subsequently trees were clustered. A local interpretation of the matched entities was proposed through schematic representations of the trees, and similarities between trees were analysed within and between clusters. The tree graphs, both unordered or partially ordered and whether the attributes were considered or not, were grouped, by clustering, according to the number of entities per tree. The robustness of the unordered comparison was demonstrated by its application to the whole population, since it provided results similar to those obtained on the sub-sample. Further developments towards a higher relative weight of geometric versus topological information are discussed in the perspective to define an architectural ideotype in apple.     

A practical method for apple cultivar identification and parent-offspring analysis using simple sequence repeat markers

Differentiation of cultivars with simple sequence repeat (SSR) markers is a very useful technique for the true-to-type characterization of cultivars and clarification of parent-offspring relationships. We developed an SSR marker set for cultivar identification comprising 15 markers that were screened from 46 previously published SSRs. This marker set could be used for apple varieties including Malus × domestica and/or other Malus species. These SSRs successfully characterized 95 apples, including the leading and major founding cultivars used worldwide for modern apple breeding. Therefore, this marker set could be applied to almost all apple cultivars. We also analyzed the parent-offspring relationships of 69 cultivars by considering allele transmissions. This analysis revealed the true parentage of the following seven cultivars: ‘Kizashi’, ‘Chinatsu’, ‘Honey Queen’, ‘Haruka’, ‘Seirin’, ‘Ozenokurenai’, and Morioka #48. This analysis also revealed a parentage discrepancy for ‘Hacnine’. From the parent-offspring analysis, two microsatellite mutation events at alleles inherited from pollen parents were observed.     

Low cost measurement setup based on a piezoelectric microphone for estimating apple bruising using Shannon entropy

This study presents a low cost measurement setup for estimating apple bruise volume during drop impact tests. The proposed measurement system consists of three main stages namely recording impact sound, signal processing, and estimating bruise volume. In the recording stage, the impact sound occurred when striking the apple on the surface was recorded by a piezoelectric microphone into computer. The impact sound was converted into Shannon entropy signals based on the Kernel density approach, and two measurable quantities namely entropy peak and pulse-width time were extracted from Shannon entropy signals in the signal processing stage. In the bruise volume estimating stage, the regression curves that map entropy peak and pulse-width features into bruise volume are estimated using measurements data. To evaluate the performance of the proposed method, we conducted several drop experiments with six different drop heights and two different temperatures (1 and 19 °C). Experimental results showed that bruise volume was exponentially proportional to the entropy peak and it was inversely proportional to pulse-width time as power functions, and all the curves were fitted with coefficients of determination of more than 0.98. Moreover, it was found that colder apples developed less bruise volume then warmer ones.     

Frost response in wheat and early detection using proximal sensors

Frost can significantly reduce production of field crops grown in Mediterranean‐type environments, where significant economic losses for Australian wheat occur annually. If non‐destructive sensors could make rapid, spatial assessment of frost damage, this could limit economic losses through timely management decisions. This paper reports on a methodology for imposing frost treatments to wheat under field conditions and the utility of canopy reflectance data for detecting early crop damage. Purpose‐built chambers using stepped additions of dry ice allowed for a range of frost scenarios to be tested when applied at wheat heading and anthesis. For frost treatments applied at anthesis, grain number and yield were reduced by 8.8% and 7.2%, respectively, for every degree Celsius below zero (down to ?4°C). This effect was additive over two consecutive nights. For cold load equivalent, there was a 2.2% and 1.9% reduction in grain number and yield, respectively, per °C hr (below 0°C). For wheat, spectral indices PRI and NDVI (reflectance) and FRF_G and SFR_G (fluorescence) showed significant relationships, with cold load applied for heading treatments. Next steps include targeting frost intensities equivalent to cold loads between 20 and 80°C hr and testing the utility of these proposed indices in a commercial paddock setting.     

Identification of potential gene‐associated major traits using GBS‐GWAS for Korean apple germplasm collections

Genomewide association study (GWAS), which queries the association between loci and a particular trait by examining single nucleotide polymorphisms (SNPs) of the entire genome, is used in many fields of study. The development of next‐generation sequencing techniques has facilitated GWASs by decreasing the sequencing costs and time. In particular, genotyping by sequencing (GBS) is useful for sequencing many samples simultaneously and at a moderate price. Herein, we describe a potential GWAS using GBS, focused on the apple germplasm, with the goal of developing an effective apple breeding strategy through the identification of useful markers. From 308 Korean apple germplasm, SNPs were selected after GBS, and major traits were investigated. Proprietary individuals were confirmed and grouped by association through genetic diversity and population structure analyses of the selected SNPs. Genes highly associated with the target traits were identified, respectively. As the first GWAS report on the apple germplasm, these results will be useful as base data for GWASs on other apple populations and traits.     

Two-dimensional NMR relaxation studies of apple quality

The internal sub-cellular physiological changes associated with ripening and mealiness in apples were investigated with novel two-dimensional NMR relaxation and diffusion techniques. It is shown that two-dimensional relaxometry reveals more subtle physiological changes than hitherto observed with conventional one-dimensional relaxometry. In particular it is shown that the T₁ of the peak associated with the cell wall in mealy apples is much longer than that of fresh apples. These changes could, perhaps, be exploited in the development of on-line NMR sensors of fruit quality.     

Detection of cuticle defects on cherry tomatoes using hyperspectral fluorescence imagery

Cherry tomatoes are one of the major vegetables consumed in the fresh-cut market. However, the quality evaluation process, which is dependent on simple size- or color-sorting techniques, is inadequate to meet increased consumer demands for high quality and safety. Among various quality evaluations, detection of cracking defects in cherry tomatoes is a critical process since this type of damage can harbor pathogenic microbes that may have detrimental consequences on consumer health. In this study, a multi-spectral fluorescence imaging technique has been presented as a diagnostic tool for non-destructive detection of defective cherry tomatoes. Fluorescence intensity in the area of cracked cuticle was significantly higher in the blue-green spectral region than that of the sound surfaces, suggesting the multi-spectral fluorescence imaging technique as an effective classification tool for detecting cracking defects on cherry tomatoes. Simple ANOVA classification analysis and principal component analysis were employed to investigate optimal fluorescence wavebands. The results illustrate that a multi-spectral fluorescence image in linear combination with a pair of selected wavebands based on the results of ANOVA analysis was able to detect defective cherry tomatoes with >99% accuracy. The detection algorithm investigated in this study is expected to be used to develop on-site and real-time multi-spectral systems for quality evaluation of cherry tomatoes in postharvest processing plants.     

不同苹果产区壁蜂授粉生物学特性及授粉效应研究

为了解决苹果座果率低、品质不高的问题,以不同苹果产区的苹果树为试验材料,采用田间试验的方法对不同苹果产区壁蜂授粉生物学特性及授粉效应进行了研究。结果表明：壁蜂始飞温度低（10-11℃）,日工作时间长（10h）,日访花数量多（4200朵）,授粉能力强,访花一次授粉率高达40%。壁蜂授粉的有效距离为60m;壁蜂授粉有方位选择性,西边和南边向阳果树座果率明显大于北边和东边;壁蜂授粉的果个、单果重比对照都有所增加,壁蜂授粉增加了果园产量,平均增产7012.5 kg/ hm2,同时,壁蜂授粉能够提高果品品质,增加苹果的商品价值,壁蜂授粉处理园的果实品质各指标均大于对照园。投放巢管口颜色以红色营巢回收比例最高,营巢比例苇管最高,其次是纸管,最后是塑料管。     

Some methodological aspects of apple transformation by Agrobacterium

Summary Leaf explants of apple cvs Gala and Golden Delicious were infected with the Agrobacterium tumefaciens strain AGL0(pMOG410). The effects of a 2 d preculture of the explants before infection and the addition of 5-azacytidine to the selection medium were studied. The percentages of GUS-positive explants after 5 w did not significantly alter due to these treatments. One of the Gala shoots, which was removed from a leaf explant cultured for 8 w on selection medium, proved to be GUS-positive and will be analyzed further. In general, however, it should be concluded that regeneration of transgenic shoots directly from leaf tissue was not very effective.Abbreviations MS Murashige and Skoog medium - GD Golden Delicious     

Optical coherence tomography visualizes microstructure of apple peel

Optical coherence tomography (OCT) has emerged recently as a non-destructive technique to visualize subsurface structures of materials. The objective of this work was to investigate whether OCT is an appropriate method to non-destructively assess the peel structural properties of apple fruit and to compare OCT to confocal microscopy and micro-CT. Peel structural properties of apples from different cultivars (‘Braeburn’, ‘Arlet’, ‘Royal Gala’ and ‘Ida Red’) were measured. The effects of preharvest fertilization treatments and storage duration on calculated structural properties of apple peel from OCT images were investigated. It was found that OCT is a fast, high resolution and high field of view technique that renders 2D and 3D images of apple peel. En-face OCT allows the visualization of the surface topology including cracks in the wax, surface roughness and lenticels. Depth scans with OCT reveal the cellular structure down to a few hundreds of micrometre into the fruit, superior to confocal imaging. Spectral-domain OCT resulted in higher quality images than time-domain OCT. Cuticle, epidermis and hypodermis cells can be resolved in a superior way compared to micro-CT. It was concluded that OCT can be used to visualize peel structural differences between apples, as well as to measure structural changes that occur during storage. OCT is a fast method and allows for combining a high depth resolution with a wide lateral image size.     

Limited application of homozygous genotypes in apple breeding

Apple is a temperate fruit species with several breeding disadvantages such as the highly heterozygous nature of the genome and a long reproductive cycle. It is expected that homozygous apple genotypes can increase breeding efficiency, but inbreeding is prevented by the gametophytic self‐incompatibility mechanism. In this paper, the production and characterization of homozygous genotypes, generated from anther culture in Malus × domestica cv. ‘Braeburn’, is described as well as their potential for use in breeding programmes. After determination of large scale anther culture efficiency, anther culture‐derived genotypes were simple sequence repeat (SSR)‐fingerprinted to determine their homozygous nature and confirm their androgenic origin. Their value for breeding was estimated based on growth and fertility measurements. The use of homozygous androgenic genotypes from ‘Braeburn’ in apple breeding programmes is currently not a realistic approach, partly because of the low efficiency of anther culture, but mainly because of the reduced vigour and severe sterility of the androgenic genotypes produced.     

The self-incompatible RNase S-alleles of Brazilian apple cultivars

Apple plants are self-incompatible because a genetic mechanism allows the female reproductive organ to recognize and reject self-pollen or pollen from genetic related individuals and allows non-self pollen to effect fertilization. Thus, there are implications to both breeding strategies and orchard management for fruit production. The purpose of this study was to identify and to characterize the S-RNase alleles of the gametophytic incompatibility among apple cultivars developed in Brazil, seeking to give support for choosing right combinations of parent in the apple breeding programs. It also sought to identify correct combinations of scion/pollinator cultivars of commercial apple orchards. A total of 16 specific S-RNase alleles primers were tested against DNA extracted from 12 Brazilian cultivars and their parents. The molecular analysis confronted to the reference cultivars, showed that the cultivars Daiane, Imperatriz and Princesa have the same incompatibility S3 and S5 alleles, while ‘Lisgala’ showed the alleles S2 and S5; ‘Suprema’, S1 and S9; ‘Catarina’, S1 and S19; ‘Joaquina’ and ‘Fred Hough’, S5 and S19; ‘Baronesa’, S3 and S9; ‘Duquesa’, S2 and S3. For ‘Primícia’ and ‘Condessa’ it was only possible to identify one of the S-alleles, namely S24 and S2, respectively, with the second allele remaining to be identified. Progeny test indicated the Mendelian inheritance for RNase alleles. Results of this study will be helpful to judiciously choose parents in apple breeding programs to improve compatibility.     

兰花上凤仙花坏死斑病毒和建兰花叶病毒双重RT-PCR检测方法的建立

根据凤仙花坏死斑病毒(Impatiens necrotic spot vjrus,INSV)和建兰花叶病毒(Cymbidium mosaic virus,CyMV)的保守序列设计特异性引物,建立了双重RT-PCR同时检测两种病毒的方法.用该方法对兰花基地的可疑发病兰株进行检测,结果从田间采取的12株可疑兰花中有6株检出INSV,1株检出CyMV,2株同时检出两种病毒.     

Screening methods of apple trees for resistance toValsa canker

Summary Valsa canker is the most serious wood rot disease of apple in Japan. Dormant, excised twig assay in vitro was used to determine resistance to Valsa canker. ‘Fuji’, ‘Starking Delicious’, ‘M.26 EMLA’, and ‘MM.106’ were considered to be highly susceptible, while Malus sieboldii showed moderate resistance. Several cultivars belonging toM. prunifolia, M. baccata, andM. × domestica showed comparative resistance. The phloridzin content in the bark ofMalus species was studied in relation toValsa canker resistance. Phloridzin content ofM. sieboldii was lower than inM. × domestica. Comparatively resistant cultivars had similar or higher phloridzin content compared to susceptible cultivars. No clear correlations were found between resistance and phloridzin contents in the bark ofMalus species.     

NIR spectroscopy for the optimization of postharvest apple management

Apples can be stored for long time under controlled temperature and atmosphere conditions, and therefore, non-destructive and rapid tools are required to assess fruit quality and to monitor changes during the postharvest period. The aim of this study was to evaluate the feasibility of NIR spectroscopy to optimize postharvest apple management and to follow changes in fruit quality during storage. An FT-NIR system operating in diffuse reflectance in the range 12,500–3600 cm⁻¹ was used to evaluate the physico-chemical (dry matter, soluble solids, colour and firmness) and some nutraceutical characteristics (total phenolics, total flavonoids and antioxidant activity) of ‘Golden Delicious’ apples, which were stored for about six months at 1 °C in controlled atmosphere, over two subsequent years. Spectral data were elaborated by PLS regression and LDA classification techniques. Good correlation models between spectral data and chemical and physical parameters were obtained for soluble solids, a* colour coordinate and firmness (0.81 < R² < 0.90 in calibration and 0.79 < R² < 0.89 in cross validation). Even higher correlation values (0.89 < R² < 0.95 in calibration and 0.86 < R² < 0.92 in cross validation) were obtained for indexes correlated to the antioxidant capacity of apples. The classification technique Linear Discriminant Analysis was applied to spectral data, in order to discriminate apples on the basis of storage time. Average correct classification was higher than 93% in validation and close to 100% in calibration, indicating high potential of NIR spectroscopy for the estimation of storage time of apple lots.