PbrARF4 contributes to calyx shedding of fruitlets in ‘Dangshan Suli’ pear by partly regulating the expression of abscission genes

Fruitlet calyx shedding in pear plants is apparently regulated via numerous pathways that involve both environmental triggers and phytohormones cues such as auxin. In this study, we found at 10 days after full bloom (DAFB) higher levels of indoleacetic acid (IAA) and tryptophan (Trp) in calyx persistence fruitlet (CPF) than calyx shedding fruitlet (CSF) ofDanshan Suli’ pear (Pyrus bretschneideri Rhed.). Consisting with this, the activity of indolealdehyde oxidase (IAAIdO), which promotes IAA synthesis, was remarkably increased, and that of peroxidase(POD), which degrades IAA, dropped markedly in CPF but not in CSF. Further, qRT-PCR results revealed that most of 31 PbrARFs (encoding auxin response factors) in Pyrus bretschneideri were highly expressed in CPF, whereas PbrARF4, PbrARF24 and PbrARF26 were significantly downregulated in CPF vis-a-vis CSF. Phylogenetic analysis revealed that 6 PbrARFs clustered in the group III, where PbrARF4 showed the closest affinity with AtARF1 that promotes organ abscission, indicating a putative role of PbrARF4 in mediating the process of calyx shedding in pear. In fact, the ectopic overexpression of PbrARF4 in Solanum lycopersicum resulted in an earlier-formed and deeper abscission layer (AL) in the transgenic plants, whose calyxes were more prone to wilt at the mature red stage (MR) compared with the control plants (wild-type). More importantly, expression levels of the abscission genes SILS and Sl Cel2 in transgenic plants overexpressing PbrARF4 were significantly upregulated in comparation with the WT, whereas those of Sl BI and Sl TAPG2 were considerably inhibited. Further, PbrJOINTLESS and PbrIDA,the two genes related to calyx shedding in pear, were up-regulated more in CSF than CPF. The findings contribute to a better understanding of PbrARFs involved in fruitlet calyx shedding of pear, which could prove beneficial to improving the quality of pear fruit.

Genomic selection of eight fruit traits in pear

Genomic selection (GS) has the potential to improve selection efficiency and shorten the breeding cycle in fruit tree breeding. In this study,we evaluated the effect of prediction methods, marker density and the training population (TP) size on pear GS for improving its performance and reducing cost. We evaluated GS under two scenarios:(1) five-fold cross-validation in an interspecific pear family;(2) independent validation. Based on the cross-validation scheme, the prediction accuracy (PA) of eight fruit traits varied between 0.33 (fruit core vertical diameter)and 0.65 (stone cell content). Except for single fruit weight, a slightly better prediction accuracy (PA) was observed for the five parametrical methods compared with the two non-parametrical methods. In our TP of 310 individuals, 2 000 single nucleotide polymorphism (SNP) markers were sufficient to make reasonably accurate predictions. PAs for different traits increased by 18.21%-46.98%when the TP size increased from 50to 100, but the increment was smaller (-4.13%-33.91%) when the TP size increased from 200 to 250. For independent validation, the PAs ranged from 0.11 to 0.45 using rrBLUP method. In summary, our results showed that the TP size and SNP numbers had a greater impact on the PA than prediction methods. Furthermore, relatedness among the training and validation sets, and the complexity of traits should be considered when designing a TP to predict the test panel.

Genome-wide identification of the mitogen-activated protein kinase kinases in pear and their functional analysis in response to black spot

The mitogen-activated protein kinase(MAPK)cascade is crucial to plant growth,development,and stress responses.MAPK kinases(MAPKK)play a vital role in linking upstream MAPKK kinases(MAPKKK)with the downstream MAPK.Black spot is one of the most serious fungal diseases of pear which is an important part of the fruit industry in China.The MAPKK genes have been identified in many plants,however,none has been reported in pear(Pyrus bretschneideri).In order to explore whether MAPK gene of pear is related to black spot disease,we designed this experiment.The present study investigated eight putative PbrMAPKK genes obtained from the Chinese white pear genome.The phylogenetic analysis revealed that PbrMAPKK genes were divided into A,B,C,and D groups.These PbrMAPKK genes are randomly distributed on 7 out of 17 chromosomes and mainly originated from the whole-genome duplication(WGD)event.The expression analysis of PbrMAPKK genes in seven pear tissues and the leaves of susceptible and resistant varieties after Alternaria alternata infection by quantitative real-time PCR(qRT-PCR)identified seven candidate genes associated with resistance.Furthermore,virus-induced gene silencing(VIGS)indicated that PbrMAPKK6 gene enhanced resistance to pear black spot disease in pear.

The HY5 transcription factor negatively regulates ethylene production by inhibiting ACS1 expression under blue light conditions in pear

Ethylene is the main factor controlling fruit ripening of pear(Pyrus ussuriensis).Ethylene production rate is negatively correlated with fruit shelf life;therefore,it is important to decrease the ethylene levels for optimal fruit storage.Here,we observed that blue light treatment could inhibit ethylene production and promote the expression of ELONGATED HYPOCOTYL 5(PuHY5),a basic leucine zipper domain(bZIP)transcription factor.The following studies showed that PuHY5 could bind to the promoter of ACC synthase 1(PuACS1),a rate-limiting enzyme in ethylene biosynthesis,and inhibit its expression.For pears in which Pu HY5 was silenced,the ethylene production and PuACS1 expression were much higher than those in the control fruit.These results demonstrated that blue light inhibited ethylene production through the induction of Pu HY5 in pear.Our finding provides a new method for prolonging fruit shelf life.

Isolation and Characterization of a Cinnamoyl CoA Reductase Gene(CCR) in Pear(Pyrus pyrifolia)

Pear is a popular and commercially important fresh fruit, and its texture is related to the presence of sclereid formatted by parenchyma cell with lignification in vascular plants. Previous studies have demonstrated that content of lignin may be regulated by cinnamoyl CoA reductase(CCR) in various plants. However, the function of CCR in pears remains very limited. In the present study, we isolated a cDNA encoding CCR(PpCCR, GenBank accession No. KF999958) and its promoter(proPpCCR) from Whangkeumbae pear to investigate the function of CCR in lignin biosynthesis. PpCCR-GFP expressed in rice mesophyll protoplast demonstrated that PpCCR-GFP was localized in the cytoplasm, indicating that CCR may function in cytoplasm without localization signals. In transgenic plants carrying PpCCR, we observed higher lignin content compared with that in wild type plants, further suggesting that PpCCR can affect the lignin contents through regulating lignin biosynthesis in Arabidopsis thaliana. More studies in other plants are needed to confirm our conclusion.

砂梨(Pyrus pyrifolia)过敏原蛋白基因(Ppmal)的克隆与表达分析

【目的】分离和克隆梨过敏原蛋白基因Ppmal(Gen Bank登录号为KP008110),探讨其在梨果实发育过程中的功能。【方法】以砂梨品种‘翠冠’[Pyrus pyrifolia(Burm.f.)Nakai]为试材,在盛花期后30 d对植株果柄进行涂抹赤霉素处理,利用同源克隆和RACE方法克隆目的基因全长序列,并通过实时荧光定量技术和半定量技术分析该基因在梨不同组织以及梨果实发育过程中的表达变化。【结果】Ppmal的全长是906 bp,含有480 bp的开放阅读框(ORF),编码159个氨基酸,预测的蛋白质相对分子质量为17.56 k D,等电点为5.62。生物信息学分析显示该基因没有信号肽序列,没有跨膜结构域,具亲水性。与其他物种的过敏原蛋白基因核苷酸序列同源性超过75%,与苹果和西洋梨编码的氨基酸序列同源性分别高达97.48%和96.23%,进化树分析表明该基因与苹果的进化关系最近。同时,定量结果显示经过赤霉素处理后的果实中该基因的表达量随着果实的生长发育呈现先降低后升高的趋势,并且具有组织差异表达的特点,其表达量在叶片中最大,其次是嫩叶,再次是花,枝条最低。【结论】获得梨Ppmal基因全长,对该基因在砂梨品种‘翠冠’发育过程中的表达分析显示,Ppmal受到赤霉素的调控,并在砂梨果实膨大期发生上调表达。

Strategies for timing nitrogen fertilization of pear trees based on the distribution, storage, and remobilization of ^(15)N from seasonal application of (^(15)NH_4)_2SO_4

In order to improve the management of nitrogen(N) fertilization in pear orchards, we investigated the effects of application timing on the distribution, storage, and remobilization of N in mature pear trees in a field experiment at Jingtai County, Gansu Province, China. Nine trees were selected for the experiment and each received equal aliquots of 83.33 g N in the autumn, spring, and summer, with ^(15)N-labeled(NH_4)_2SO_4 used in one of the aliquots each season. Results showed that the(^(15)NH_4)_2SO_4 applied in the autumn remained in the soil during the winter. In the following spring this N was absorbed and rapidly remobilized into each organ, especially new organs(leaves, fruit and new shoots). The ^(15)N supplied in spring was rapidly transported to developing fruit between the young fruit and fruit enlargement stages. ^(15)N from the summer application of fertilizer was mainly stored in the coarse roots over the winter, then was mobilized to support growth of new organs in spring. In conclusion, for pear trees we recommend that the autumn application of N-fertilizer be soon after fruit harvest in order to increase N stores in fine roots. Spring application should be between full bloom and the young fruit stages to meet the high N demands of developing fruit. Summer application of fertilizer at the fruit enlargement stage does not contemporaneously affect the growth of pears, but increases the N stored in coarse roots, and in turn the amount available for remobilization in spring.

Genome-wide identification and co-expression analysis of GDSL genes related to suberin formation during fruit russeting in pear

Glycine-aspartic acid–serine-leucine(GDSL)type lipases/esterases genes play critical roles in plant development and are related to the responses to abiotic and biotic stress.However,little is known about the GDSL family in pear(Pyrus spp.).Studies have shown GDSL-domain proteins play key roles in suberin deposition.Suberin deposition in the fruit epidermis,also called russeting,is an important defect that negatively affects consumer's appeal in some fruit species,such as pear,apple and grapevine.Fruit russeting is mainly associated with cuticle microcracking and suberin accumulation in the inner part of the epidermal cell walls.To gain insight into the role of the GDSL gene family in suberin deposition and russet development in pear,we performed a genome-wide characterization of the GDSL family,including their identification,chromosomal localization,phylogenetic relationships,and expression patterns,in different tissues/organs in pear.One hundred and thirteen GDSL-type lipases/esterases genes were identified in the pear genome,and a phylogenetic analysis revealed that GDSL family can be classified into four distinct groups.Thirty GDSL genes were co-expressed with five homolog pear genes of three well-known suberin biosynthesis Arabidopsis genes(AtGPAT5,AtASFT,and AtCYP86B1)in the transcriptional co-expression network during pear fruit development.Among the 30 co-expressed GDSL genes,twelve genes were further analyzed by quantitative Real-time PCR,and the results showed the expression levels of the 12 genes were different between the russet exocarp and green exocarp of sand pear at different fruit development stages.Our study provides a detailed overview of the GDSL gene family and lays the foundation for future functional characterization of GDSL genes in P.bretschneideri.

Callus and Adventitious Buds Formation from Stem Explants of Balsam Pear （Momordica charantia L.）

The effects of sterilization pretreatment, sampling time and growth regulators on callus formation, explant contamination and adventitious bud induction from stems of balsam pear （Momordica charantia L.） were investigated. Sterilized for 6 rain with 0.1% （w/v） mercuric chloride solution is an essential sterilization method for stems. June and July proved to be better for material taking, followed by May. The callus formation rate of balsam pears reached the highest when medium added with IBA 1.0mg/L and BA 2.0 mg/L, while the optimum medium for adventitious bud induction was MS added with IBA 0.5mg/L and BA 4.0 mg/L. And it will provide theoretical and technical basis for rapid propagation and breeding.

Morphological Characteristics of Floral Organ at Different Microspore Developmental Stage in Balsam Pear （Momordica Charantia L.）

The relationship between microspore developmental stages and morphological characteristics of floral organ in balsam pear was studied. The results showed that flower buds developed a series of morphological changes at different microspore developmental stages, and morphological characteristics of floral organ were significantly different when sampling from different part or cultivar of balsam pear. Anther at late-uninucleate stage was best for culture, in which stage flower buds swelled obviously, and white floss on the surface reduced. In addition, calyxes were obvious but did not spread, and the most of anthers were light green.

Effect of Different Treatment on Anther Callus Formation and Browning in Balsam Pear （Mornordica charantia L.）

Brown callus derived from anther limited the application of anther culture in balsam pear. In order to establish a more perfect regeneration system from anther cultuer, the effects of low temperature pretreatment, 2,4-dichlorophenoxyacetic acid （2,4-D）, vitamin C （Vc） and silver nitrate （AgNO3） on callus induction and browning in anther culture of balsam pear （Momordiea charantia L.） were investigated. The results showed that after pretreatment at 4 ℃ for 1 day, callus induction rate was the highest and browning rate was the lowest. Anthers on MS medium supplemented with 2,4-D 0.5 mg/L formed more and better callus. The medium supplemented with Vc or AgNO3 was advantageous to the induction of callus and reduction of browning. When cultured on medium supplemented with 50 mg/L Vc or 5 mg/L AgNO3, callus induction rate was the highest and browning rate was rather low.

Advances in Origin,Evolution and Classification of Pyrus L.

China is not only one of the origin centers of Pyrus L.,but also the earliest birthplace of Pyrus L.in the world.This paper reviews the evolution of Pyrus L.from the aspects of leaf edge morphology,inflorescence and fruit type,and summarizes the research progress of classification and species distribution of Pyrus L.,which is of great significance for the protection,evaluation and utilization of germplasm resources.

Primary study on anther culture of balsum pear for callus and organ formation

The balsum pear （Momordica charantia L. ） anthers in the monokaryotic stage of microspore development were cultured in this experiment. Different Plant growth regulators＇ combinations, base media and carbon sources were studied for callus formation and organ differentiation from balsum pear anthers. The result showed that the best media for callus inducement was： MS＋BA 0.5 mg/L＋NAA 0.2 mg/L＋ 2, 4-D 0.5 mg/L＋KT 2.0 mg/L, with 3% sugar and 0.8% agar. The best media to induce roots from balsum pear anther callus was： MS＋NAA 0.05 rag/L＋ KT 0.5 rag/L, with 3% sugar and 0.8% agar. Most of adventitious roots from callus were triploid（2N=3X=33）

两个秋子梨果实品质分析

为探究两个秋子梨品种差异,更好地对其进行开发利用,以红丰和早黄成熟期果实为试材,利用显著性分析、相关性分析、主成分分析和聚类分析等方法对两个不同品种秋子梨果实品质进行分析。结果表明,两种梨的果实在果实外观、果肉风味、果实香气、果汁含量上有所不同,在外观品质上红丰梨单果重为212.96 g、横径为75.49 mm、纵径为70.25 mm、果梗长度为36.48 mm,这4个指标分别达到了早黄梨的171.13%、119.33%、121.29%和113.19%;红丰梨的果实硬度显著低于早黄梨,为早黄梨的79.37%。因此红丰梨相较于早黄梨果形较大、果实较重、果肉较软,适合喜好大果、软果的消费者选择;在营养成分上红丰梨的可溶性固形物、可溶性糖、矿质元素含量均显著高于早黄梨,其中可溶性固形物含量为11.10%、矿质元素含量为3748.49 mg·kg^(-1),而可溶性糖含量为124.61 mg·g^(-1),达到了早黄梨可溶性糖含量的206.89%;果实的单果重、果实横径、果实纵径、果实硬度4项形态指标与可溶性糖、可溶性固形物、维生素C、矿质元素呈现出显著相关性,与可滴定酸呈极显著相关;在矿质元素含量上,红丰梨果肉和果皮中矿质元素Mg、P、K、Ca、Fe的含量以及12种矿质元素的总含量均高于早黄梨。对两个品种果实品质综合比较得出,红丰梨果实品质优于早黄梨,更适合在生产上开发利用。

‘巴梨’和‘早红考密斯’梨汁品质特性及其抗氧化活性分析

该研究以冷藏的‘巴梨’和‘早红考密斯’梨为材料,采用去皮和带皮的果实为原料制备梨汁,测定梨汁的理化性质、抗氧化活性和香气品质等指标。结果表明,同冷藏15 d梨果所制梨汁相比,冷藏90 d梨果所制梨汁的可溶性固形物含量、果糖和葡萄糖含量增加,可滴定酸含量、柠檬酸和苹果酸含量降低;主要多酚组分——熊果苷和绿原酸含量、总酚含量(total phenolics content,TPC)和总黄酮含量(total flavonoids content,TFC)以及DPPH自由基和羟自由基清除活性降低;酯类、醇类、酮类和烯类化合物的含量增加,醛类物质含量降低。同去皮榨汁相比,带皮压榨的梨汁中熊果苷和绿原酸含量、TPC和TFC以及抗氧化活性显著增加;糖类、有机酸化合物以及香气物质的变化规律不明显。电子鼻技术可以有效区分不同梨汁,W5S和W1W传感器响应值有明显区别;电子舌分析结果显示,不同梨汁的甜味和酸味强度值差异较明显,并分别与梨汁的糖类和有机酸含量呈显著正相关。因此,较长时间冷藏和带皮榨汁可改善梨汁的理化性质、抗氧化性能和风味品质。

梨AFLP分子连锁图谱的构建与分析

遗传图谱是数量性状基因定位、基因图位克隆及分子辅助育种等研究的基础,而目前国内在梨树上这方面的研究尚属空白。试验以早美酥×八月红的杂交F192株实生苗为作图群体,采用了扩增稳定、多态性丰富的16对MseⅠ/EcoRⅠ引物组合,对分离群体进行AFLP多态性检测,共获得208条多态性带,其中偏离孟德尔遗传的比率29.8%（P〈0.01）。应用Joinmap 3.0软件且结合“双假测交”策略,对208个AFLP分子标记进行了遗传连锁分析,构建了一张包含145个AFLP标记,分属20个连锁群的梨分子遗传连锁图谱,每个连锁群包含4-20个标记,平均为9.06个标记,图谱总长度覆盖梨基因组618.7 cM,标记间平均图距为6.58 cM。此图谱为梨树基因定位、比较基因组学和重要农艺性状的QTL定位等研究奠定了基础。

不同西洋梨品种果实糖酸组分及含量分析

为了给梨果实综合评价及产业品种结构调整提供理论依据,以烟台市十个代表性果园三个西洋梨品种(‘巴梨’‘三季梨’和‘红茄梨’)为研究对象,采用高效液相色谱法(HPLC)测定果实中可溶性糖和有机酸的组分及含量。研究结果表明:在三个西洋梨品种中,总糖含量和甜度值最高的是‘红茄梨’,‘三季梨’次之,‘巴梨’的总糖含量最少;三种西洋梨成熟果实可溶性糖积累以葡萄糖和果糖为主,可溶性糖含量由高到低为葡萄糖>果糖>山梨醇>蔗糖;另外,三种西洋梨果实总酸含量差异不显著,有机酸积累以柠檬酸和苹果酸为主,这两种酸占有机酸总量的90%左右;‘红茄梨’甜酸比和糖酸比最高,显著高于‘巴梨’,与‘三季梨’差异不显著。综上所述,‘红茄梨’糖酸风味优异,可作为烟台地区西洋梨产业结构调整的备选推广品种。

巍山红雪梨PpPAL基因的克隆及生物信息学分析

为了进一步研究红皮梨花色素苷的合成调控机制，以巍山红雪梨为试验材料，采用同源克隆方法，克隆得到了苯丙氨酸解氨酶（PAL）基因的cDNA片段，名之为PpPAL，登录号为JQ749640，并利用相关生物软件对该基因及其相应蛋白质进行了生物信息学分析。结果表明：PpPAL长1919bp，编码628个氨基酸；其与西洋梨eyrus communis的PAL基因（DQ230992）、鸭梨Pyrus bretschneideri的PAL基因（GU906268、JQ247318）、甜樱桃Prunus avium的PAL基因（AF036948）和覆盆子Rubus idaeus的PAL基因（AF237955）在核苷酸水平上的同源性分别为98％、95％、95％、88％和83％。氨基酸序列比对中发现，PAL序列包含L180、V181、L230、A231等脱氨基氨基酸残基，其催化活性位点分别为N234、G235、N356、D357、N358、H370、HNQDV（460～464）。PpPAL基因不存在导肽和信号肽，无跨膜结构域，定位于细胞质中，肽链表现为亲水性；α-螺旋和无规则卷曲是其蛋白质二级结构中量最大的结构元件，β-转角和延伸链散布于整个蛋白质中。在对其编码的蛋白的预测中发现，该蛋白含有苯丙氨酸解氨酶．组氨酸解氨酶（PAL-HAL）和苯丙氨酸解氨酶（phe-am-lyase）。

基于参数L系统的梨树枝干模型三维可视化

为了真实的模拟果树枝干形态,提出形态特征信息和参数L系统相结合的果树枝干可视化方法。以梨树的树体结构为研究对象,提炼其相关的形态结构特征以及生态生理等特性变化;应用L系统建模方法对梨树建立包含树枝的粗细、分叉角度等细节信息的模型;最后使用OpenGL三维建模技术将梨树生长几何信息转化为直观的三维可视化图形信息,模拟出梨树常见树形(自然开心形、主干疏层形、小冠疏层形)的基本形态结构,为开发梨树整形修剪计算机仿真系统奠定基础。

近红外光谱法快速检测梨汁中柠檬酸和L苹果酸含量

利用近红外光谱技术对梨汁中柠檬酸和L-苹果酸含量进行快速定量检测,并通过6种光谱预处理及3种数学校正方法对柠檬酸和L-苹果酸含量预测模型进行优化。本研究采用近红外光谱仪在1 100~1 650 nm光谱范围内共采集70个梨汁样本的近红外光谱图,并利用高效液相色谱法测定柠檬酸和L-苹果酸的含量。首先对样本原始光谱图经过多元散射校正(MSC)、一阶导数、二阶导数等方法进行预处理,然后通过主成分分析(PCR)、修正的最小二乘法(MPLS)及偏最小二乘法(PLS)等分别建立梨汁中柠檬酸和L-苹果酸含量的预测模型,并对该模型进行优化。优化后,柠檬酸的最佳模型为PLS结合MSC; L-苹果酸的最佳模型为MPLS结合SNV且去散射;最佳预测模型的预测相关系数最高可达0.985,预测标准差最低为0.039%,相对分析误差最大为3.46。结果表明,近红外光谱可作为一种可靠、快速、无损、简单的方法用于梨汁中柠檬酸和L-苹果酸含量的测定。该方法为生产企业及监管部门开展梨汁的掺伪鉴别提供了依据。