Selection and Validation of Reference Genes for Normalization of RT-qPCR Analysis in Developing or Abiotic-Stressed Tissues of Loquat (Eriobotrya japonica)

Loquat(Eriobotrya japonica Lindl.)is a subtropical evergreen fruit tree that produces fruits with abundant nutrients and medicinal components.Confirming suitable reference genes for a set of loquat samples before qRT-PCR experiments is essential for the accurate quantification of gene expression.In this study,eight candidate reference genes were selected from our previously published RNA-seq data,and primers for each candidate reference gene were designed and evaluated.The Cq values of the candidate reference genes were calculated by RT-qPCR in 31 different loquat samples,including 12 subgroups of developing or abiotic-stressed tissues.Different combinations of stable reference genes were screened according to a comprehensive rank,which was synthesized from the results of four algorithms,including the geNorm,NormFinder,BestKeeper andΔCt methods.The screened reference genes were verified by normalizing EjLGA1 in each subgroup.The obtained suitable combinations of reference genes for accurate normalization were GAPDH,EF1αand ACT for floral development;GAPDH,UBCE and ACT for fruit setting;EF1α,GAPDH and eIF2B for fruit ripening;ACT,EF1αand UBCE for leaves under heat stress;eIF2B,UBCE and EF1αfor leaves under freezing stress;EF1α,TUA and UBCE for leaves under salt stress;ACT,EF1αand eIF2B for immature pulp under freezing stress;ACT,UBCE and eIF2B for immature seeds under freezing stress;EF1α,eIF2B and UBCE for both immature pulp and seeds under freezing stress;UBCE,TUB and TUA for red-fleshed fruits under cold-storage stress;eIF2B,RPS3 and TUB for white-fleshed fruits under coldstorage stress;and eIF2B,UBCE and RPS3 for both red-and white-fleshed fruits under cold-storage stress.This study obtained different combinations of stable reference genes for accurate normalization in twelve subgroups of developing or abiotic-stressed tissues in loquat.To our knowledge,this is the first report to obtain stable reference genes for normalizing gene expression of abiotic-stressed tissues in E.japonica.The use of the three most stable reference genes could increase the reliability of future quantification experiments.

Taxonomic Status of Daduhe Loquat （Eriobotrya prinoides var. dadunensis）

The taxonomic status of Daduhe loquat （E. prinoides var. dadunensis） was studied through analyzing genetic relationships among Oakleaf loquat （E. prinoides）, Daduhe loquat and Common loquat （E. japonica） using inter simple sequence repeats （ISSR） molecular marker and morphologic marker in this paper. Based on ISSR marker research, the similarity coefficient between Oakleaf loquat and Common loquat was lower than the similarity coefficient between Oakleaf loquat and Daduhe loquat while the similarity coefficient between Daduhe loquat and Common loquat was intermediate. The highest additivity was obtained when Daduhe loquat was regarded as the undetermined hybrid （45.8%）. The specific bands of Oakleaf loquat and Common loquat were present in Daduhe loquat. Based on morphologic traits research, Daduhe loquat was also between Oakleaf loquat and Common loquat but a little leaning to Oakleaf loquat. All the results support that Daduhe loquat was hybrid of Oakleaf loquat and Common loquat.

Study on Fruit Quality, Phenological Phases and Shoot Histomorphology of a New Bud Mutant Line, ‘Chuanzao Loquat’

[Objective] This study was to explore the growth characteristics and fruit quality of a new bud mutant line, ＇Chuanzao Loquat＇. [Method] Paraffin section technique combined with field investigation method were adopted to conduct com- parative analysis of shoot histomorphology and phenological phases between two Io- quat varieties, ＇Chuanzao Loquat＇ and ＇Zaozhong 6＇. [Result] ＇Chuanzao Loquat＇ branched out and unfolded leaves about half to a month earlier than ＇Zaozhong 6＇; both the flowering and fruiting phases of ＇Chuanzao Loquat＇ were three months earlier than a precocious variety, ＇Zaozhong 6＇; the proportions of epidermis, cortex parenchyma, vascular tissue and medulla were 3.7%, 14.5%, 15.9% and 65.9%, re- spectively, in spdng shoots of ＇Chuanzao Loquat＇, and 3.1%, 42.5%, 6.9% and 47.5%, respectively, in ＇Zaozhong 6＇. [Conclusion] In terms of phenological phases, ＇Chuanzao Loqua＇ is earlier than ＇Zaozhong 6＇, a currently widely planted precocious variety, and thus is an important germplasm resource of Ioquats.

Effects of Peracetic Acid(PAA) Combined with Calcium Treatments on Storage Quality of Loquat Fruit

[Objective] The paper was to evaluate effects of peracetic acid （PAA） combined with calcium treatments on storage quality of Ioquat fruits, so as to pro- vide practical techniques to solve the problems of postharvest rot and quality deteri- oration for Ioquat fruits. [Method] With Ioquat fruits of Qingzhong variety as materi- als, 0.2%, 0.4% and 0.8% PAA combined with 0.8% CaCI2 was used to soak Ioquat fruits for 4 min, 0.8% CaCl2 and water treatments were set as two controls; the fruits were dried and packaged by 0.02 mm PE bags, then stored under non-chilling low temperature of （7±1） ℃. The indicators related to storage quality of Ioquat fruits were randomly tested once every 3 d, and their variation situations were analyzed. [Result] Compared with two control treatments and 0.2% PAA, 0.8% PAA combined with calcium treatments, the treatment of 0.4% PAA combined with 0.8% CaCl2 could significantly inhibit rot index, weight loss rate, firmness and cell membrane permeability of Ioquat fruits during storage period, which could also effectively delay the reduction of titratable acid, vitamin C, soluble solid content and juice yield, and maintain respiration intensity of fruits at a low level; the appearance and flavor qual- ity of fruits were good after stored for 25 d. [Conclusion] 0.4% PAA combined with 0.8% CaCl2 treatment is an efficient, safe and economical practice technology in an- ti-corrosion and quality preservation for postharvest Ioquat fruits .

Molecular Cloning and Expression of Carotenogenic Genes in Yellowish and Mutant Whitish Loquat (Eriobotrya japonica) Fruits

ObjectiveThe study aimed to explore the factors regulating carotenoid accumulation in flesh color. MethodA loquat mutation （red-or orange-fleshed plant emerged a bud mutation of white-flesh in trunk） was used as material; HPLC analysis of β-carotene content was conducted. ResultThe β-carotene concentration in the flesh of wild and mutant types was 60.9 and 4.6 μg/g fresh weight, respectively. According to the conserved regions of genes from rose family genome, carotenogenic gene fragments in wild and mutant types were obtained. No nucleotide variation of the carotenogenic gene fragments was observed between wild and mutant genome. Real-time quantitative polymerase chain reaction （Q-PCR） was compared and one carotenogenic gene, β-ring hydroxylase （HYB） were considerably suppressed in mature mutant loquat fruits compared with that in wild. The other six carotenogenic genes were also expressed but the expression patterns appeared to be not correlated with the amount of β-carotene concentration in wild loquat flesh. ConclusionThe mutant whitish loquat lacks the ability to synthesize β-carotene because of the transcriptional down-regulation of carotenogenic gene HYB.

"One Brand for One Village"Planning of Qingzhong Loquat Sightseeing Park in Jinting Township of Suzhou City,China

The planning and design of Qingzhong Loquat Sightseeing Park in Jinting Township, Wuzhong District, Suzhou City was taken for an example, industrial development, advantages and disadvantages of the study area were analyzed. Design concepts and techniques of"One Brand for One Village"for Qingzhong Loquat Sightseeing Park were introduced from the perspectives of project orientation, planning concepts, functional division, scenic spot design, infrastructure planning and so on. Six ancient relics and three grotesque trees were introduced to expound the planning scheme of typical landscapes. Finally, the architectural control planning from the perspectives of height and style was stressed. The planning scheme broke common restraints of other similar parks, such as poor infrastructure and disordered garden constructions, proposed the planning concept of"building a modern eco-agricultural sightseeing fruit park mainly for production but also integrating functions of tourism, ecological picking, recreation and entertainment", providing a basis for building "One Brand for One Village" and realizing the high-efficiency scale-development of agriculture.

Preliminary Identification of Loquat Leaf Mould Pathogenic Fungus in Mengzi City of Yunnan Province

[Objective] The study aimed to investigate the Ioquat leaf mould disease in Mengzi City of Yunnan Province and lay the foundation for determination of effective prevention and control methods.[Method] Loquat leaf mould pathogenic fungus was isolated by tissue separation method and inoculated with conidial suspension.The pathogenicity of Ioquat leaf mould pathogen was verified by Koch＇s postulate.Under a microscope,mycelial morphology and conidial fructification were observed,spore sizes were measured,and Ioquat leaf mould pathogen was identified according to the morphological characteristics.[Result] Velvet-like,olive green fungal colonies were generated on PDA medium.Conidiophores erect,apex curved,dark brown,smooth,with obvious spore marks and no diaphragm,（33.0-152.8） μm×（2.6-4.0）μm.Cladosporium was brown or pale olive with spore marks,monocelled or with one diaphragm,（7.1-19.0） μm × （1.9-5.9） μm.Conidia concatenate （2-4）,oval or spherical,with no spore mark,light olive,monocelled,smooth,（2.1-9.4） μm × （1.2-2.6） μm.[Conclusion] Loquat leaf mold disease began to occur in the germination period of spring shoots and summer shoots and became serious in the germination period of autumn shoots.Sooty mold-like layer grew on both front and back surfaces and densely covered the whole leaves,thus seriously affecting the photosynthesis of plants.The pathogen was preliminarily identified as Cladosporium eriobotryae Pass.＆ Beltrani.

Monilinia fructicola on loquat:An old pathogen invading a new host

Monilinia fructicola has been widely reported as the causal agent of brown rot disease on many Rosaceae family fruits worldwide.It has been reported on stone fruits,e.g.,peach,plum,cherry,apricot and mume;as well as pome fruits,e.g.,apple,pear and hawthorn.Loquat is a member of the Eriobotrya genus in subfamily Maloideae along with apple,pear and hawthorn.So far,loquat has not been reported as the host of any Monilia species.In June 2019,brown rot symptoms were observed on loquat fruits in an orchard in Wuhan,Hubei Province,China.Thirty single spore isolates were obtained and identified as M.fructicola based on morphological characteristics and molecular analysis.This is the first report of loquat brown rot disease caused by Monilia species in the world.Furthermore,upon artificial inoculation,all three Monilia species from peach in China,i.e.,M.fructicola,M.mumecola and M.yunnanensis,could cause typical brown rot disease on loquat fruits.At the same time,M.fructicola isolates from loquat showed virulence similar to those isolates from peach when the pathogenicity test was conducted on peach fruits.These results suggested that loquat could be infected by other Monilia species and that isolates from loquat also have potential to damage other Rosaceae family fruits in practice.

Endogenous phytohormones and the expression of flowering genes synergistically induce flowering in loquat

Flowering is an important process for the reproduction of higher plants.Up to this point,the studies on flowering have mostly focused on the model plant Arabidopsis thaliana,and the flowering mechanism of fruit trees remains mostly unknown.The diversity of the flowering time of loquat(Eriobotrya japonica Lindl.)makes it an ideal material to study the regulation of flowering.In this study,we first observed the inflorescence bud differentiation in two varieties of loquat that had different blooming times(cv.Dawuxing(E.japonica),that blooms in the fall and cv.Chunhua(E.japonica×Eriobotrya bengalensis Hook.f.)that blooms in the spring)and found that the starting time of inflorescence bud differentiation and the speed of inflorescence development were responsible for the difference in blooming times.The determination of endogenous phytohormones by high performance liquid chromatography(HPLC)indicated that abscisic acid(ABA),zeatin(ZT),and gibberellin(GA3)promoted flowering in loquat,while indole-3-acetic acid(IAA)was mainly involved in inflorescence bud differentiation in Chunhua.A transcription level analysis illustrated that multiple flowering-related genes could respond to different signals,integrate to the TFL1,AP1 and FT genes,and then synergistically regulate flowering in loquat.Thus,this study provides a new insight into flowering regulation mechanisms in loquat.

Occurrence, Prevention and Control of Cryphalus eriobotryae, a New Devastating Pest of Loquat

A devastating trunk beetle has been found in Baisha loquat in Dongshan Town, Wuzhong District, Suzhou City in recent years. It is iden-tified as a new insect species, Cryphalus eriobotryae, belonging to Scolytinae, Curculionidae, Coleoptera. So far, loquat is known as the unique host of C. eriobotryae, and trees of 20-30 years old or 3-4 years old are all attacked by the insect. Holes drilled by adults with the diameter of about0.8 mm and sores(ate by adults) with the diameter of 1 mm are found on bark and trunk of damaged trees. Three-dimensional channels in 3-4 layers are formed in cortex, which are filled with insect dung. The phloem turns black-brown in late stage, and loses transport function, resulting in plant death and a great impact on loquat production and industrial development. During the occurrence period of C. eriobotryae from April to November,the damaged trunks and branches should be sprayed with 48% chlorpyrifos EC or 5% imidacloprid EC 100 times dilution once every 10 d for con-tinuous 2-3 times, and various states of the insect could be killed. If the phloem is damaged in the early and middle stages and does not turn black and brown completely, the plant could grow new cortex to realize recovery.

The variation of NAD^+-SDH gene in mutant white-fleshed loquat

supported by the Key Laboratory Program of the Ministry of Agriculture of China （2013JCYJ-004） and its supplementary items （2015JSCX-036, 2015LWJJ-010）;the Program of Modern Agriculture Technology Innovation and Demonstration of Provincial Finance Department, China （2014CXSF-015）

Polyphenol Oxidase Inactivation by Microwave Oven and Its Effect on Phenolic Profile of Loquat (<i>Eriobotrya japonica</i>) Fruit

The objective of this research was investigated the effect of polyphenol oxidase microwave treatment on phenolic composition, antioxidant activity and microstructure of loquat fruit. Phenolic profile of methanolic extracts prepared from fresh, and microwave-treated samples were analyzed. Antioxidant activity was also evaluated by 2,2’-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS?+) and 1,1-diphenyl-2-picrylhydrazyl (DPPH+) methods. In addition, polyphenol oxidase inactivation was carried out using a response surface methodology to establish the optimal conditions of treatment. The phenolic content of fresh mesocarp was 311 ± 0.60 mg gallic acid equivalents (GAE)/100g dry weight (DW) and that of microwave-treated mesocarp was 1230 ± 0.36 mg GAE/100g DW. Total phenolic content of water/ methanol extract significantly increases after microwave treatment rather than methanolic extract of fresh loquat. Five glycoside phenolics were identified by HPLC-DAD-MS as 3-caffeoylquinic acid, 3-p-coumaroylquinic acid, 5-caffeoylquinic acid and quercetin-3-O-sambubioside. Methanolic extract of microwave-treated mesocarp showed higher antioxidant activity than that of fresh mesocarp. Thus, polyphenol oxidase inactivation by microwave energy preserved the integrity of phenolic compounds as well as antioxidant activity in mesocarp extracts prepared from loquat fruit. It was also noted that phenolics were more abundant in the microwaved samples than in the fresh samples.

Inoculation of Loquat Endophytic Trichoderma Strain P3.9 into Loquat Rhizosphere Soil and Its Effect on Quantity of Indigenous Fungi

In order to explore whether the endophytic Trichoderma strain P3.9 of loquat has an adverse effect on indigenous fungi in loquat rhizosphere soil, the quantitative change of aboriginal fungi is determined by dish dilution and plate colony-counting method with time changing in one season. The results showed that after the inoculation of endophytic Trichoderma strain P3.9, the total number of indigenous fungi in loquat rhizosphere soil had an obviously downward trend in contrast with the control which was without inoculation. For treatment groups, 5~60 d after inoculation, the number of indigenous fungi showed a cyclic upward-downward trend at a 10-d interval except for the insignificant changes from 35 to 40 d;60~90 d after inoculation, the upward-downward trend repeated at a 30-d interval. For the control group, the number of indigenous fungi first presented a downward-upward trend every 15 d during the period of 5~50 d, and then an upward-downward trend from the period of 50~70 d and the 75~85 d, and lastly continuous growth from 85 to 90 d;particularly, it did not vary greatly from 70 to 75 d. In general, the quantity of indigenous fungi is unstable in the control group which fluctuates more significantly than in the treatment group. The number of indigenous fungi in the treatment group was significantly lower than that in the control group. The Trichoderma strain P3.9 can inhibit indigenous fungi in loquat rhizosphere soil effectively.

Effects of Neodymium on Nitrogen Metabolism of Plantlet Loquat in Vitro

The effect of NdCl_3 on the rooting and nitrogen metabolism of loquat in vitro was studied when NdCl_3 was added to the rooted medium. The results show that 0.4 μmol·L^(-1) NdCl_3 in the rooted medium can obviously increase the rooting rate, length of root and fresh weight of roots, and enhance the activities of nitrate reductase, glutamine synthetase, glutamate dehydrogenase in the root system and in the leaves. The transformation of NO_3^- to NH_4^+ in root system and leaves are promoted and the nitrogen metabolism is accelerated with 0.4 μmol·L^(-1) NdCl_3 treatment.

Polysaccharide from Loquat Leaves to Prepare Hierarchical Porous Carbon as a Matrix for Active Sulfur

Fossil fuel exhaustion and overdevelopment usually lead to a recession,which is worsened by the environmental pollution.So it is of high priority to develop high-efficiency energy storage device.Here,agreen and environment-friendly strategy is devised to fabricate carbon materials from biomass.By water extraction and alcohol precipitation,polysaccharide is extracted from loquat leaves.After calcining under high temperature,hierarchical porous carbon materials(HPCM)are obtained,possessing a variety of macropores,mesopores and micropores.Such ample and hierarchical pores enable the electrolyte infiltration and the buffering of the volume expansion of sulfur in repeated electrochemical reactions.The structure stability of the entire electrode can thus be well maintained.When evaluated as the scaffold for sulfur,the electrochemical performance of carbon/sulfur composite was tested.Even after 500 cycles,the reversible capacity is retained as high as 485.4 mA·h/g at the current density of 1.6 A/g.It also offers a notable rate capability,attaining the discharge capacity of 700.7 mA·h/g at 2 C.All the electrochemical performance results prove the feasibility of the proposed strategy.

Fingerprinting Patterns of SSR Markers Constructed for 33 Loquat[Eriobotrya japonica (Thunb. ) Lindl. ]Germplasms

[ Objective ] This study aimed to explore the ways of using SSR technology to construct the fingerprinting maps of the loquat. [ Method ] Fingerprinting patterns of the SSR markers for 33 loquat varieties in national loquat germplasm resource garden were constructed by specifically amplifying palymorphism from 7 pairs of primers screened from 51 pairs of primers of the relatives Mains and Pyrus. [ Result ] The results showed that the uses of 7 primers, namely, Hi01d05F, Hil2iO4F, CH02B10F, Hi03a03, Hi03e04, Hi04a05 and Hi02c07 could distinguish 33 loquat germplasms from one another completely. [ Conclusion] This study provides an essential criterion for classification and identification of the loquat resources.

Sorbitol induces flower bud formation via the MADS-box transcription factor EjCAL in loquat

Sorbitol is an important signaling molecule in fruit trees. Here, we observed that sorbitol increased during flower bud differentiation(FBD) in loquat(Eriobotrya japonica Lindl.). Transcriptomic analysis suggested that bud formation was associated with the expression of the MADS-box transcription factor(TF) family gene, EjCAL. RNA fluorescence in situ hybridization showed that EjCAL was enriched in flower primordia but hardly detected in the shoot apical meristem. Heterologous expression of EjCAL in Nicotiana benthamiana plants resulted in early FBD. Yeast-one-hybrid analysis identified the ERF12 TF as a binding partner of the EjCAL promoter. Chromatin immunoprecipitation-PCR confirmed that EjERF12 binds to the EjCAL promoter, and β-glucuronidase activity assays indicated that EjERF12 regulates EjCAL expression.Spraying loquat trees with sorbitol promoted flower bud formation and was associated with increased expression of EjERF12 and EjCAL. Furthermore, we identified EjUF3GaT1 as a target gene of EjCAL and its expression was activated by EjCAL. Function characterization via overexpression and RNAi reveals that EjUF3GaT1 is a biosynthetic gene of flavonoid hyperoside. The concentration of the flavonoid hyperoside mirrored that of sorbitol during FBD and exogenous hyperoside treatment also promoted loquat bud formation. We identified a mechanism whereby EjCAL might regulate hyperoside biosynthesis and confirmed the involvement of EjCAL in flower bud formation in planta. Together,these results provide insight into bud formation in loquat and may be used in efforts to increase yield.

Detection of skin defects on loquat using hyperspectral imaging combining both band radio and improved three-phase level set segmentation method

Background and objectives Skin defects are one of the primary problems that occur in post-harvest grading and processing of loquats.Skin defects lead to the loquat being easily destroyed during transportation and storage,which causes the risk of other loquats being infected,affecting the selling price.Materials and Methods In this paper,a method combining band radio image with an improved three-phase level set segmentation algorithm(ITPLSSM)is proposed to achieve high accuracy,rapid,and non-destructive detection of skin defects of loquats.Principal component analysis(PCA)was used to find the characteristic wavelength and PC images to distinguish four types of skin defects.The best band ratio image based on characteristic wavelength was determined.Results The band ratio image(Q782/944)based on PC2 image is the best segmented image.Based on pseudo-color image enhancement,morphological processing,and local clustering criteria,the band ratio image(Q782/944)has better contrast between defective and normal areas in loquat.Finally,the ITPLSSM was used to segment the processing band ratio image(Q782/944),with an accuracy of 95.28%.Conclusions The proposed ITPLSSM method is effective in distinguishing four types of skin defects.Meanwhile,it also effectively segments images with intensity inhomogeneities.

双水相协同超声法提取枇杷叶中绿原酸的工艺研究

试验采用响应面法优化双水相协同超声法提取枇杷叶中绿原酸的工艺,探讨提取液中乙醇体积分数、超声温度、超声时间、磷酸氢二钾用量和超声功率对绿原酸提取率的影响。结果显示,双水相协同超声法提取枇杷叶中绿原酸最佳提取工艺条件为乙醇体积分数61%、超声温度61℃、超声时间25 min、磷酸氢二钾用量4.6 g,此条件下绿原酸提取率1.53%,与预测值相接近。研究表明,双水相协同超声法提取枇杷叶中绿原酸的提取工艺稳定可行,可为枇杷叶的资源化开发与利用提供参考。

贵州枇杷种质资源分布及其产业发展概述

贵州立体气候突出,野生枇杷种质资源丰富,性状差异性明显。目前已找到普通枇杷、大花枇杷、小叶枇杷3个种类,普通枇杷在全省广泛分布,大花枇杷主要分布在黔东南州雷公山一带,小叶枇杷主要分布在黔西南州兴义市一带。贵州省枇杷商品化栽培始于1958年,由黔东南州率先从江苏、浙江、福建等省引进优良品种进行试验示范。进入20世纪80年代中后期,贵阳市、黔南州、黔西南州等各地州(市)相继开展引种试验及小规模推广应用。截至2010年,在贵阳市、黔东南州、毕节市等地区拥有了万亩以上的规模化种植。特别是2015年以来,贵州各地枇杷种植生产发展速度迅猛,据2022年贵州省农业农村厅统计数据,全省枇杷种植面积已达2.3万hm^(2),年产量16.3万t,产值达17.1亿元,鲜果采摘期超过2个月。但栽培技术与管理水平仍较低,面积上升与产量增长不成正比,全省平均每666.7 m^(2)商品果产量不足500 kg,提升空间很大。存在问题主要为,一是缺乏地方规范的引、试、推制度体系,盲目性引种,造成品种混杂,良莠不齐,商品性低;二是对遗传优良性状的种质资源挖掘不足,缺乏地方特色的主导品种,市场竞争力不强;三是枇杷品牌创建与营销策略意识不强,形成行业内竞争压价销售,影响效益;四是鲜果产后冷链及深加工产品技术研发滞后,产业附加值增长有限;五是旱涝寒暑等极端天气频繁出现,山地果园设施栽培配套条件滞后,抗风险能力不强等。主要围绕贵州枇杷种质资源分布特征、引种与育种、栽培技术演变、产业发展及其制约因子等方面进行了分析,尝试提出贵州枇杷产业当前与未来的主要任务,以推动枇杷产业高质量发展。