Background Knowledge about genetic diversity and relationships among germplasms could be an invaluable aid in diospyros improvement strategies. Methods This study was designed to analyze the genetic diversity and relationship of local and natural varieties in Guangxi Zhuang Autonomous Region of China using start codon targeted polymorphism (SCoT) markers. The accessions of 95 diospyros germplasms belonging to four species Diospyros kaki Thunb, D. oleifera Cheng, D. kaki var. silverstris Mak, and D. lotus Linn were collected from different eco-climatic zones in Guangxi and were analyzed using SCoT markers. Results Results indicated that the accessions of 95 diospyros germplasms could be distinguished using SCoT markers, and were divided into three groups at similarity coefficient of 0.608; these germplasms that belong to the same species were clustered together; of these, the degree of genetic diversity of the natural D. kaki var. silverstris Mak population was richest among the four species; the geographical distance showed that the 12 natural populations of D. kaki var. silverstris Mak were divided into two groups at similarity coefficient of 0.19. Meanwhile, in order to further verify the stable and useful of SCoT markers in diospyros germplasms, SSR markers were also used in current research to analyze the genetic diversity and relationship in the same diospyros germplasms. Once again, majority of germplasms that belong to the same species were clustered together. Thus SCoT markers were stable and especially useful for analysis of the genetic diversity and relationship in diospyros germplasms. Discussion The molecular characterization and diversity assessment of diospyros were very important for conservation of diospyros germplasm resources, meanwhile for diospyros improvement.

•Sixteen candidate reference genes of kumquat was evaluated for normalization in real-time quantitative PCR studies, including two groups of homologous genes and six other genes also commonly used as reference genes in previous studies.•Popular statistical methods in this field, geNorm, Normfinder, and Bestkeeper, has been used flexibly aimed to get reliable results in this study.•Each ACT gene or UBQ gene performed differently in this study and homologous genes should be taken into account in other similar studies.•We confirmed the best reference gene and the best reference gene combinations for normalization under different experimental conditions.

The diversity and genetic relationship among and within thirty-six varieties of longan and one Dimocarpus confinis from China,Vietnam and Thailand were analyzed using SCoT(Start Codon Targeted) and ISSR(Inter Simple Sequence Repeat) markers.The results showed 127 strips were amplified by 12 SCoT primers while 117 strips by 15 ISSR primers.The average strips amplified by SCoT and ISSR primers were 10.58 and 7.8,respectively.The genetic relationships were analyzed using unweighted pair-group method of arithmetic average cluster analysis(UPGMA)and the genetic Jaccard similarity coefficients were calculated.The cluster analysis results showed that the thirty-seven materials could be clustered into 6 groups at the similarity coefficient level of 0.672 and 0.685.Comparison and mixture analysis of the two molecular markers demonstrated that both SCoT and ISSR were efficient approaches for genetic diversity analysis of longan germplasm.These results would provide theoretical basis for longan germplasm storage and utilization in future.

The protein expressions under low temperature stress were studied using proteomics method.The results showed that the expression of DLPIP1 protein was up-regulated. The full length of DLPIP1 eDNA obtained by RT-PCR was 1 132 bp with a 900 bp open read frame which encoded a putative DLPIP1 pro- tein with 299 amino acids. Comparison of the amino acid sequence homology among DLPIP1 proteins from 21 different species indicated that DLPIP1 protein had a range of 90% to 93% identity with homologues of other plants. Bioinformatics analysis demonstrated that DLPIP1 exhibited a typical structure with seven membrane-spanning domains and an internal symmetry showing two highly conserved NPA motifs and possessing the MIP family signal consensus sequence. The DLPIP1 amino acids showed high identity with the PIP plasmalemma subfamily of other 21 plant species by homology comparison analysis. Quantitative real-time PCR results showed that the DLPIP1 expressed in root, stem and leaf, while the amount of ex- pressions were different in different organs. The mRNA of DLPIP1 was the most abundant in root, the least in leaf and stem. Furthermore, the mRNA of DLPIP1 changed with time extension under low temperature stress. These results suggested that DLPIP1 might be involved in function of chilling stress.

Mango (Mangifera indica L.) is one of the most economically grown fruits in the tropical and subtropical areas around the world. In the present study, start codon targeted (SCoT) markers were employed to investigate the genetic diversity of 73 mango accessions obtained from Guangxi province, China. A total of 275 bands were amplified by thirty-four SCoT primers, of which 203 (73.82%) were polymorphic. Genetic similarity between accessions was in the range of 66.2–94.2% with an average of 78.8%. The observed highest genetic similarity value (94.2%) was found between ‘Ren Mian Mango’ and ‘Hong Hua Mango’, the observed lowest genetic similarity value (66.2%) was found between‘Xia Mao Xiang Mango’ and ‘India No. 15’. These coefficients were utilized to construct a dendrogram using the unweighted pair group of arithmetic means. All the accessions were grouped into four (A, B, C, D) clusters and correspond well with their geographical origin and their known history. These results have an important implication for mango’s rue germplasm characterization, improvement, management and conservation.

The genetic diversity of 8 podocarpus germplasms was analyzed by SCoT markers.The results showed that a total of 136 bands were produced by 10 primers,of which 122 bands(88.97%) were polymorphic,and the genetic similarity coefficients were between 0.39-0.80.So there were comparatively great genetic diversity among the podocarpus germplasms.A cluster analysis conducted with UPGMA also showed that the 8 podocarpus germplasms could be classified into 2 groups.The result of principal component analysis(PCA) was the same as the results of UPGMA cluster analysis.SCoT markers can be effectively used to evaluate the genetic diversity of podocarpus germplasms,thus providing a theoretical foundation for the identification and classification of podocarpus germplasms.

【Objective】The aim of this study was to clone full-length cDNA of a key enzyme gene DLCCoAOMT related to lignin metabolism in longan,investigate its sequence characteristics and analyze its expression in different organs under low temperature stress,as well as expression in the prokaryote.【Method】 The DLCCoAOMT gene cDNA sequence was cloned from longan leaf using RT-PCR and RACE techniques.The bioinformatics methods were used to analyze putative amino acid sequence and real-time PCR method were used to study the expression of DLCCoAOMT gene in different tissues.【Result】 The full-length cDNA of DLCCoAOMT(GenBank accession number: JN093023) in longan was cloned.The sequence consists of 993 bp with an open reading frame of 744 bp,encoding a polypeptide of 247 amino acids.Homology analysis showed that the deduced DLCCoAOMT protein was highly homologous to other CCoAOMT proteins from different species.Phylogenetic analysis also indicated that DLCCoAOMT was very closely related to DLCCoAOMT of Betula.Real-time PCR results showed that the DLCCoAOMT expressed in root,stem and leaf,and its expression was different among three organs.The mRNA of DLCCoAOMT was abundant in root and stem,but less in leaf.Furthermore,DLCCoAOMT transcription level was significantly different among root,stem and leaf with time courses of chilling treatment.Prokaryotic expression showed that recombinant plasmid was efficiently expressed in Escherichia coli BL21.【Conclusion】 Gene DLCCoAOMT was firstly isolated and characterized from longan,which may be involved in chilling stress.

The protein expressions of longan(Dimocarpus longan Lour.)under low temperature stress were studied using proteomics method.The results showed that the expression of carbonic anhydras(eCA) protein was down-regulated.The CA(GenBank accession number:JN033201)cDNA obtained by RT-PCR was 1 119 bp of full length with a 966 bp open read frame which encodes a putative CA gene with 321 amino acids.Comparison of the amino acids sequences homology in CA from 12 different species indicated that CA had a range of 81%to 88%identity in amino acids sequence with homologues of other plants.The deduced amino acids sequence not only contained a typical CA domain,but also was very conservative.Quantitative real-time PCR results showed that the CA expressed in root,stem and leaf,while the amount of expressions were different in different organs.The mRNA of CA was the most abundant in leaf,the least in root and stem.Furthermore,the mRNA of CA was changed with time extension under low temperature stress.A 40.5 kD heterologous protein was obtained when CA gene was expressed in E.coli.These results suggested that CA might be involved in function of chilling stress.

RACE technique is a rapid and effective method for cloning gene 5' end and 3' end,and one of main methods to obtain full-length genes.However,RACE technique has its own shortcomings.We further improved the previous RACE protocols and obtained a modified RACE technique which is simple,rapid,high-efficient and low-cost.Our modified RACE technique is suitable for obtaining large number of genes 5' end and can be used in ordinary laboratories.

The technique of two-dimensional electrophoresis in Nanfeng mandarin peel proteomics was established and optimized by phenol-extraction method.The two dimension electrophoresis profiles of Nanfeng mandarin peel proteins were good repeatability and high resolution.The differential expression proteins of Nanfeng mandarine peel during storage were separated with 2-DE and then analyzed with Imagemaster 2D PDQuest.The result showed that the number of detected differential expression protein spots in Nanfeng mandarin peel under storage was 45,33 proteins were up-regulated,and 12 proteins were down-regulated.It was presumed that these proteins were related to the storability of Nanfeng mandarin peel.

In order to make good use and innovate germplasm resources of kumquat,the genetic relationships of 25 Fortunella Swingle germplasm resources from China and Vietnam and the variation accessions of F.crassifolia Swingle were examined by ISSR markers in this study.Of the 100 ISSR primers screened,only 10 primers gave reproducible,polymorphic DNA amplification patterns with a total of 120 amplified bands.The percentage of polymorphic band was 85%.By banding patterns obtained from these 10 primers,each accession could be distinguished from the others.The genetic similarity ranged from 0.48 to 0.99 UPGMA relationship tree was established based on the similarity coefficients.At the coefficient level of 0.85,the 25 germplasm resources were divided into 8 groups.The classification results were discussed in this paper.Shanju（Fortunella Swingle） found along the border between China and Vietnam could be a new variety or species of Fortunella Swingle.

Two molecular marker systems, SCoT and ISSR were used for identification and genetic comparison analysis of 23 mango germplasm accessions collected within Guangxi province of China. Using 18 selected SCoT primers 158 bands were generated, of which 104 (65.82%) were polymorphic. Eighteen selected ISSR primers amplified 156 bands with 87 (55.77%) being polymorphic. The cultivars of Xiang Ya Mango type and their progeny have high genetic similarity with each other. The 23 cultivars were clustered into two major groups based on the SCoT analysis and three major groups based on the ISSR analysis with UPGMA. These clusters are in accordance with their known origins and main phenotypic characteristics. Our results indicated that the SCoT analysis better represents the actual relationships than ISSR analysis, although both analyses give similar results. The results also demonstrate that the SCoT marker system is useful for identification and genetic diversity analysis of mango cultivars.

A full-length cDNA sequence of a homologous Cab gene was cloned by employing RT-PCR, which was named as Mcab. The accession code in GenBank is FJ907952. The Mcab is 915 bp in length, encoding a protein of 264 amino acids, with an estimated molecular weight and an isoelectric point of 28.19 ku and 5.35 respectively. A comparison of the nucleotide sequences of homologous Cab genes from different species indicated that Mcab gene had a range of 72% to 85% identity in nucleotide sequence with homologues of other plants. The results indicated that Mcab protein had one chlorophyll a/b binding domain between the 63 st to 232 st amino acid and very conservative of the position between woody plants. Protein subcellular localization prediction showed that Mcab protein was located in the chloroplast and the gene was one of important genes in photosynthesis system of green plants. Prediction of the secondary structure of the protein showed that Mcab protein had 8 α helices, 5 β sheets and 21 β turns. The research was beneficial to the further understanding of the molecular mechanism of photosynthesis in mango.

The present experiment was conducted to study the physiological and biochemical parameters related to low-temperature resistance in the leaves of two longan cultivars,viz.,Shixia and Chuliang.The plants were treated with 10,6,3 and 0℃.The results showed that,compared with the normal temperature 25℃(control),the activities of SOD in longan seedlings with low-temperature treatments increased initially,then decreased followed by an increase at last,while a reverse trend was observed in case of POD activity.The content of MDA in longan seedlings with low-temperature treatments decreased initially and then increased,and relative conductivity and soluble protein content increased slightly and then decreased with decreasing temperatures.Soluble sugar content did not vary much.It was concluded that the cold resistance of variety Chuliang was higher than that of variety Shixia,which showed consistent with external injuries of two varieties’ leaves under low-temperature stress.

The cold resistance of longan was comprehensively summarized from the aspects of the influencing factors of cold injury on longan,classification of cold injury,the resistance mechanism of cold injury and the management measures of longan before and after cold injury.The study provided theoretical basis for exploring the mechanism of cold resistance of longan further.

Genetic variation and relationships among 47 mango germplasm and 3 relative species from Guangxi province in China, were analyzed using Start Codon Targeted (SCoT) markers. Using 33 selected SCoT primers 273 bands were generated with an average of 8.27 bands per primer among the 50 accessions, of which 208 (76.19%) were polymorphic. Genetic relationships estimated using the SM similarity coefficient generated values between different pairs of accessions that varied from 0.531 to 0.923 with an average of 0.782. These coefficients were utilized to construct a dendrogram using the UPGMA. All 50 accessions were basically classified into six clusters and correspond well with their recorded pedigrees. The results will provide much more useful information for the management of germplasm and will also be useful to improve the current breeding strategies. The results also demonstrate that the SCoT marker system is useful for identification and genetic diversity analysis of mango cultivars.

本研究利用ISSR技术对37份龙眼种质资源进行遗传多样性检测。研究结果表明,从100条ISSR引物中筛选出7条重复性好,条带清晰的引物对37份龙眼品种基因组DNA进行扩增,共扩增出54条带,其中43条具有多态性,比率为79.6%。不同龙眼品种间遗传相似系数变幅为0.69～0.97,平均达0.83,说明ISSR标记能够揭示材料间较高的遗传多样性。UPGMA聚类结果表明,ISSR标记能将37份龙眼品种完全区分开,并能将来源于中国、越南和泰国的37份龙眼品种分别聚类到中国、越南和泰国三大品种群,说明龙眼品种资源的亲缘关系与地理因素有关,三个国家的龙眼品种之间存在较大的遗传差异。本研究结果将为为龙眼品种资源的研究利用提供参考。

SCoT markers were used to study genetic diversity and relationship of 24 longan accessions.Twenty-four primers were screened from 80 SCoT primers and then used to amplify the genome DNAs of the accessions.Totally 211 SCoT loci were produced,including 181 polymorphic loci,with a polymorphism rate of 85.8%.The Jaccard genetic similarity coefficients among the accessions were calculated with NTSYS-pc2.10e software and the values were between 0.65 and 0.86.The results showed that there was relatively high genetic diversity among the accessions.The cluster analysis conducted with UPGMA showed that 24 accessions could be distinguished from each other and divided into two groups.

近年来分子技术的快速发展,使其在龙眼中也得到应用.由于分子标记具有诸多优点,因此它的发展和应用为龙眼研究提供了一条有效的途径.此文就RAPD,AFLP,ISSR,SRAP等几种分子标记在龙眼中应用进行了详细阐述,同时分析了分子标记在龙眼研究中存在的问题和今后研究工作的重点.

我们采用RT-PCR方法克隆了2个AP1同源基因全长cDNA,分别命名为MAP1-1（GenBank accession No.FJ529206）和MAP1-2（GenBank accession No.FJ529207）。MAP1-1编码247个氨基酸,开放阅读框长度为741bp,蛋白质分子量为28.54kD,等电点为8.31;MAP1-2编码248个氨基酸,开放阅读框长度为744bp,蛋白质分子量为28.78kD,等电点为8.70。同源性分析表明,它们的核苷酸序列与其它木本植物AP1同源基因的一致性为72%~81%。实验分析表明,MAP1-1和MAP1-2第1至第61个氨基酸含有一个MADS盒结构域,第88至第178个为K盒结构域;两个基因均定位于细胞核,且功能位点分布存在着不同,推测这两个基因在花器官发育过程中的功能存在差异。蛋白二级结构预测显示,MAP1-1蛋白有12个α-螺旋,4个β折叠区,14个β-转角;而MAP1-2蛋白有11个α-螺旋,5个β折叠区,15个β-转角;其大多数氨基酸具有亲水性。本研究有助于进一步了解芒果的开花分子机理及成花的生物学发育阶段。