Population Genetic Diversity in Chinese Pomegranate (Punica granatum L.) Cultivars Revealed by Fluorescent-AFLP Markers

Eighty-five pomegranate （Punica granatum L.） cultivars from six geographical populations located at Shandong, Anhui, Shaanxi, Henan, Yunnan, and Xinjiang Provinces were studied for its population genetic diversity by means of fluorescent-AFLP markers. The results indicated that 135-185 polymorphic loci were amplified by eight pairs of primers at species level. An average of 158.25 polymorphic loci was amplified for each primer combination. The polymorphism percentage ranged from 62.5% to 86.11%, and the average polymorphism percentage was 73.26%. This indicated that there was plentiful genetic diversity in pomegranate cultivars. The genetic diversity at the species level was higher than that at the population level. The order of the genetic diversity was Henan population 〉 Xinjiang population 〉 Shaanxi population 〉 Anhui population 〉 Shandong population 〉 Yunnan population. Variance analysis showed that there was significant difference between populations in genetic diversity. The genetic differentiation coefficient between populations （Gsr） was 0.2018, which indicated that gene differentiation was mainly within the population, and between populations, it accounted for 20.18% of the total variation. Gene flow （Nm） between the populations measured was 1.9027 based on the genetic differentiation coefficient between populations, indicating that there was mild gene flow between populations. The UPGMA cluster analysis showed that most accessions from the same population were clustered together, but there was partly gene exchange. All genetic parameters indicated that there was plentiful genetic diversity in pomegranate cultivars in China, of which Henan population was significantly higher than the other populations, and it had wide application foreground in pomegranate breeding in China.

Population Genetic Structure in Apricot (Prunus armeniaca L.) Cultivars Revealed by Fluorescent-AFLP Markers in Southern Xinjiang,China

Population-wide genetic structure was studied using fluorescent-AFLP markers on 85 apricot （Prunus armeniaca L.） cultivars collected from Kuche, Kashi, Hetian in the Tarim Basin, southern Xinjiang Uygur Autonomous Region of China. The purpose of this study was to determine the genetic structure and genotypic diversity among the different eco-geographical populations. Based on the results from this study, 8 pairs of fluorescent-AFLP primers showed clear electrophoregram and high polymorphism amongst the 64 pairs of EcoR Ⅰ/Mse Ⅰ （Mse Ⅰ - a FAM fluorescent marked primer） primers screened. There was a significant polymorphic difference for the same primer pair in different populations and for the same population with different primer pairs. The percentage of polymorphic loci （P） at species level was higher than Kuche, Hetian, Kashi population levels, respectively. The Nei＇s gene diversity index （H） and Shannon＇s information index （I） at species level were higher than those of Kuche, Hetian, and Kashi at population level, respectively. H and I of Kuche population were the highest amongst the three populations. Apricot population genetic diversity was found mainly within the population, Genetic differentiation coefficient between populations （GST） was 0.0882. Gene flow Nm between the populations was 5.1689. Population genetic identity was between 0.9772-0.9811 and genetic distance was between 0.0191-0.0232. These results further indicated that the similarity between populations was higher and the genetic distance between populations was smaller. The UPGMA cluster analysis indicates that the geographical populations at Kuche, Kashi, Hetian were relatively independent Mendelian populations. Concurrently, there was also partial gene exchange between the populations. All the evidences indicated that the genetic diversity in Kuche population was the highest, suggesting that it could be a transition population from wild apricot to cultivated apricot. There were abundant genetic diversities in apricot cultivar populations in southern Xinjiang, China, which provide promising germplasm for further breeding and theoretical basis for biodiversity conservation and utilization for apricot population in this area.

AFLP Fingerprint and SCAR Marker of Watermelon Core Collection

The identification of germplasm is an important step for effective utilization of the available germplasm. In previous studies, isoenzyme, RAPD and SSR techniques had been used to conduct the genetic identification of watermelon ( Citrullus lanatus (Thunb.) Mansf.), but their effectiveness was limited due to the extremely narrow genetic background among watermelon genotypes. In this research, amplified fragment length polymorphism (AFLP), which was reported as a reliable technique with high efficiency in detecting polymorphism, was used to conduct genetic analysis and variety identification of thirty genotypes of watermelon core collection that represent a wide range of breeding and commercially available germplasm. As a result, a DNA fingerprint based on 15 bands amplified with four primer combinations was developed. In this fingerprint, each genotype has its unique fingerprint pattern and can be distinguished from each other. Furthermore, in or der to facilitate the utilization of AFLP marker in practice, one specific AFLP band of genotype 'PI296341' coming from fragment amplified by primer combination E-AT/M-CAT was successfully converted into a sequence characterized amplified region (SCAR) marker.

Segregation of AFLP Markers in A ( Populus tomentosa× P. bolleana) ×P. tomentosa Carr. BC_1 Family

To investigate the levels of polymorphisms and Mendelian segregation ratio in clone “TB01” (P. tomentosa×P. bolleana) ×clone “LM50” (P. tomentosa) BC 1 population at the entire genome level, amplified fragment length polymorphisms (AFLPs) analysis was conducted for both parents and 120 progenies. Forty one pairs of selective primers were used to detect 2?707 bands, of which 712 (26.4%) were polymorphic. Chi\|square tests were performed to examine if the observed genotypic frequencies of AFLP loci deviated from expected 1∶1 Mendelian segregation ratio ( P <0.01) in BC 1 population. Among the 712 loci 571 (80.2%) fit to Mendelian 1∶1 segregation ratio, corresponding to DNA polymorphisms heterozygous in one parent and a null in the other. The result shows that the AFLP markers are very suitable for fingerprinting and genetic mapping in the Chinese white poplar (Populus tomentosa Carr.).

基于AFLP遗传多样性和功能成分含量的葛资源特性研究

目的:研究葛资源的遗传多样性和品质特征。方法:提取葛幼嫩叶片基因组DNA,使用扩增片段长度多态性(AFLP)分子标记技术对各样品进行扩增,并应用GeneMapper分析软件对AFLP扩增结果进行分析;采用高效液相色谱(HPLC)对葛根中异黄酮类化合物葛根素、大豆苷、大豆甙元等主要成分进行含量测定,并利用SPSS软件进行成分含量的聚类分析。结果:5对AFLP荧光引物扩增获得1113条多态性条带,多态性比率达到100%。非加权组平均法(UPGMA)聚类分析结果显示,20号样品(湖南茶陵野葛)与其它葛资源差异较大,在相关系数为0.35时与其它样品分开,剩余样品在0.48的相关系数处分为两大类群,其中一类主要为野葛(云南、重庆),但有17号(湖南武冈粉葛)、18号样品(湖南江背粉葛)与该类聚集为一群,其遗传背景与野葛相似;而另一个类群主要为粉葛(广东、广西、湖南)和湖南的野葛资源,湖南野葛资源与云南等地区野葛差异较大。成分含量聚类分析结果表明所有样品可分属野葛与粉葛2个群,其中野葛的成分含量比粉葛高,但17号样品(湖南武冈粉葛)聚集在野葛群。结论:综合分析发现AFLP分子标记技术能揭示葛的遗传多样性,野葛及粉葛在成分含量方面存在明显差异,其中17号样品(湖南武冈粉葛)成分含量与野葛相近,为优异的粉葛资源。

牛膝化学成分和药理作用研究进展及其质量标志物(Q-Marker)预测分析

牛膝在《神农本草经》中被列为上品,近几年研究证实牛膝的化学成分主要包括甾体、三萜皂苷、多糖、挥发油等化合物,具有抗炎镇痛、调节免疫、抗骨质疏松、保护心血管等药效。本文从牛膝的植物亲缘性,牛膝特有性、有效性、可测性化学成分和炮制后化学成分等方面,对牛膝的质量标志物(quality marker,Q-Marker)进行预测分析,牛膝甾酮A、25 S-牛膝甾酮、25 R-牛膝甾酮、齐墩果酸-3-O-β-D-(6′-丁酯)-吡喃葡萄糖醛酸苷、齐墩果酸-3-O-β-D-(6′-甲酯)-吡喃葡萄糖醛酸苷、β-蜕皮甾酮等可作为牛膝质量标志物,为建立牛膝质量评价体系和深入研究其药理作用提供依据。

Discussion on AFLP Molecular Markers in Piper methysticum and Pepper

The aim of the research was to discuss the genetic relationships between Piper methysticum, Pepper and other wild species in Pepper genus. DNA was extracted from leaves which belonged to 28 germplasms including 6 materials of P. methysticum, 21 maerials of cultivated and wild Pepper, 1 material of Peperomia pellucida belonged to different genus. Premiers with good band-type and high polymorphism and resolution were selected from 64 pairs of primers for AFLP amplification and the clustering analysis was conducted with MVSP3.13f software. 191 bands were amplified by 4 pairs of premiers, 189 of which had polymorphism, being 98.6%. 28 germplasms were classified into 6 different groups at the genetic similarity coefficient of 0.52 by silver staining AFLP, in which 6 materials of Piper methysticum were clustered into a single group, indicating that P. methysticum belonged to Pepper family of Pepper genus but were distantly related to the others. The research provided the basis for selecting rootstocks for P. methysticum graft, molecular identification of P. methysticum and the fingerprint construction of P. methysticum.

Genetic Diversity Within a Jackfruit (Artocarpus heterophyllus Lam.) Germplasm Collection in China Using AFLP Markers

Jackfruit is cross-pollinated and mostly seed propagated, a wide range of variation exists in fruit quality. With the development of efficient vegetative propagation methods, excellent genotypes selected from these seed propagated seedlings will gradually replace other genotypes in jackfruit producing areas. In this study, genetic diversity of 50 jackfruit accessions from three provinces in China was analyzed based on amplified fragment length polymorphic （AFLP） markers. A total of 320 unambiguous bands were produced by eight primer combinations, and 65 （20.3%） of them were polymorphic. Genetic similarity coefficients ranged from 0 to 0.9841, with an average of 0.5000, indicating a moderate genetic diversity in this collection. The dendrogram derived by unweighted pair group method with arithmetic mean algorithm （UPGMA） analysis revealed five groups, and no correlation between genetic relationship and geographical origin were found. Accessions of soft and firm flesh type were not clustered into distinct groups, neither could yearly bearing once, or twice fruit accessions. This study has provided useful information for collection and preservation of jackfruit germplasm worldwide.

Inheritance of Powdery Mildew Resistance in Cucumber (Cucumis sativus L.) and Development of an AFLP Marker for Resistance Detection

Cucumber powdery mildew is one of the most destructive diseases of cucumber throughout the world. In the present study, inheritance of powdery mildew resistance in three crosses, and linkage of resistance with amplified fragment length polymorphism （AFLP） markers are studied to formulate efficient strategies for breeding cultivars resistant to powdery mildew. The joint analysis of multiple generations and AFLP technique has been applied in this study. The best model is the one with two major genes, additive, dominant, and epistatic effects, plus polygenes with additive, dominant, and epistatic effects （E-l-0 model）. The heritabilities of the major genes varied from 64.26% to 97.82%, and susceptibility was incompletely dominant for the two major genes in the three crosses studied. The additive effects of the two major genes and the dominant effect of the second major gene were high, and the epistatic effect of the additive-dominant between the two major genes was the highest in cross I . In cross II, the absolute value of the additive effect, dominant effect, and potential ratio of the first major gene were far higher than those of the second major gene, and the epistatic effect of the additive-additive was the highest. The genetic parameters of the two major genes in cross III were similar to those in cross II. Correlation and regression analyses showed that marker E25/M63-103 was linked to a susceptible gene controlling powdery mildew resistance. The marker could account for 19.98% of the phenotypic variation. When the marker was tested on a diverse set of 29 cucumber lines, the correlation between phenotype and genotype was not significant, which suggested cultivar specialty of gene expression or different methods of resistance to powdery mildew. The target DNA fragment was 103 bp in length, and only a small part was found to be homologous to DNA in the other species evaluated, which indicated that it was unique to the cucumber genome.

AFLP Marker Linked to Turnip Mosaic Virus Susceptible Gene in Chinese Cabbage(Brassica rapa L.ssp.pekinensis)

Turnip mosaic virus (TuMV) which has several strains causes the most important virusdisease in Chinese cabbage in terms of crop damage. In China, Chinese cabbage is infectedby a mixture of strains, breeding of cultivar for the TuMV resistance has become themajor aim. Screening the molecular marker linked to the TuMV-resistance gene formolecular assisted selection is the major method to improve the breeding efficiency. Inthis study, we used AFLP technique and the method of bulked segregant analysis(BSA) tostudy the progeny of Brp0058Brp0108, and identified two DNA molecular marker linked toTurnip mosaic virus-resistance gene with a recombination frequency 7.5 cM and 8.4 cM.

AFLP and SCAR Markers Associated with Peel Color in Eggplant (Solanum melongena)

Peel color is an important breeding objective for eggplant. Dark purple and purplish red are the most common colors in commercial eggplant cultivars. A co-dominant amplified fragment length polymorphism （AFLP） marker which was associated with the peel color （each in coupling phase to dark purple and purplish red） was found in studying the genetic diversity in 58 eggplant accessions （contained cultivars and wild relatives）. The maker bands were sequenced and converted to SCAR marker, this polymorphism in sequence was from an inserted/deleted （indels） mutation. And DNA from 136 eggplant materials （inbred lines, F1, and wild relatives） were amplified with the designed SCAR primers as template, high correlation between the SCAR marker and peel color （dark purple and purplish red） was found. Then, bulked line analysis （BLA） combined with AFLP was further used to identify polymorphic fragments, and another six AFLP markers were tested and verified to be associated with peel color, which demonstrated that BLA was an useful method for identifying molecular markers of interested traits. In conclusion, these markers will facilitate the MAS （maker-assisted selection） of eggplant breeding for peel color.

Sharpie marker在试管上标记对荧光PCR检测Ct值的影响

为了探明Sharpie marker在试管上标记对荧光PCR检测Ct值的影响,本试验模拟现实工作中可能遇到的各类情形,从Sharpie marker标记试管的各种方式,以对照组、全黑管、管壁全黑管、管壁和盖半黑管、管壁半黑管、盖半黑管和盖全黑管等为对象,通过荧光PCR检测,比较分析Ct值。结果显示,全黑管和盖全黑管对Ct值的影响最大,管壁和盖半黑管与盖半黑管对Ct值的影响较大,而管壁全黑管和管壁半黑管对反应Ct值没有影响。本研究为正确使用Sharpie marker,确保荧光PCR检测结果的准确提供了参考。

Application of AFLP markers for population genetic study on half-smooth tongue sole Cynoglossus semilaevis

The genetic diversity of wild and hatchery populations of half-smooth tongue sole Cynoglossus semilaevis, based on observation of amplified fragment length polymorphism (AFLP) was described. Two hundred individuals from four wild populations, Laizhou (LZ), Weihai (WH), Qingdao (QD), Rizhao (RZ), and one hatchery population, Mingbo (MB), were screened using eight different AFLP primer combinations. A total of 384 loci were screened in the five studied populations. 48.4%, 51.3%, 50.7%, 49.3% and 45.8% of these loci were polymorphic among the individuals tested in the LZ, WH, QD, RZ and MB populations, respectively. The number of polymorphic loci detected by single primer combinations ranged from 17 to 35. The average heterozygosity of the LZ, WH, QD, RZ and MB populations was 0.072, 0.093, 0.092, 0.090 and 0.063, respectively. The WH population showed the highest genetic diversity in terms of total number of AFLP bands, total number of polymorphic bands, average heterozygosity and percentage of low frequency (0-0.2) polymorphic loci among all the populations, while the LZ population was the lowest among the wild populations. Compared with the wild populations, the hatchery population showed a low genetic viability.

Cloning,characterization of two female-specific AFLP markers and development of PCR-based sex identification method for the half-smooth tongue sole Cynoglossus semilaevis

Two female-specific AFLP(amplified fragment length polymorphism)markers(named CseF464 and CseF136)were isolated by using one selective primer combination(E-AGC/M-CTG)from the genomic DNA of 20 females and 20 males of the half-smooth tongue sole Cynoglossus semilaevis.Both the markers were re-amplified,recovered from the agarose gels,cloned and sequenced.Bioinformatics analysis indicated that the length of the two markers were 468 bp and 134 bp,respectively,and the sequences showed no similarity to each other,as well as to the known sequences deposited in the GenBank database using BLASTn.Two pairs of SCAR(sequence characterized amplified regions)primers were designed based on the sequences of the two female-specific markers.Furthermore,PCR-based genetic sex identification method was developed in Cynoglossus semilaevis.A specific fragment was amplified in all females but not in any males by using these SCAR primers on the initial 20 female and 20 male individuals of Cynoglossus semilaevis.The feasibility of the two SCAR primer pairs was confirmed in additional 100 individuals(50 females and 50 males).This allowed for reliable,rapid molecular identification of genetic sex of the species,genetic mapping on the sex chromosomes and better understanding of the sex determination and sex differentiation in the half-smooth tongue sole.

Identification of AFLP Markers Linked to Lr19 Resistance to Wheat Leaf Rust

AFLP analyses were carried out on Thatcher, 23 near-isogenic lines and F2 generation of TcLr19 × Thatcher, to develop molecular markers for gene Lr19 resistance to wheat leaf rust. Seven markers linked to Lr19 resistance trait were obtained, which were P-AGT/M-GAG289 bp （3.3 cM）, P-ACA/M-GGT102 bp （4.1 cM）, P-ACA/M-GGT106 bp （4.1 cM）, P-AAC/M-CAG123 bp （4.9 cM）, P-AAC/M-GGT203bp （5.0 cM）, P-ACA/M-GGT290bp （5.7 cM）, and P-ATC/M-GAG293bp （9.6 cM）. All of these specific fragments were isolated from the polyacrylamide gels, reamplified, cloned, and sequenced. The research may facilitate genetic mapping, physical mapping, and the eventual cloning of Lr19.

Isolation, cloning and sequencing of AFLP markers related to disease-resistance traits in Fenneropenaeus chinensis

Amplified fragment length polymorphisms (AFLP) technique was used to analyze the fingerprint- ing of four successive generations of Fenneropenaeus chinensis to reveal their disease-resistance traits. Some loci showed quite different genetic frequencies due to artificial selection, which implied that these fragments were putative markers related to the disease-resistance trait. We developed a simple and effective method to fur- ther characterize these AFLP fragments. Specific AFLP bands were cut directly from polyacrylamide gels, re-amplified, cloned and sequenced. Eight putative genetic markers were sequenced and their sizes ranged from 63 to 209 bp. The sequences were submitted to dbGSS (database of Genome Sequence Survey); and the BLAST analysis showed low similarity to the function genes, indicating these markers were tightly linked to a dis- ease-resistance trait but were not functional genes.

Assessing genetic diversity of wild populations of Japanese flounderusing AFLP markers

Amplified fragment length polymorphism （AFLP） analysis was used to evaluate the genetic diversity of four wild geographical populations of Japanese flounder （Paralichthys olivaceus）. A total of 775 loci （58.32% of which was polymorphic） in the range between 100 and 1 300 base pairs were detected from 110 individuals using seven primer combinations. The percentage of polymorphic loci detected by single primer combination for each population was calculated, ranging from 19.59% to 53.33%. Genetic similarities within and among the populations were calculated from the binary matrices of presence - absence. Phylogenetic tree of four populations was constructed by using the UPGMA method using PHYLIP Version 3.5. According to intrapopulation genetic similarities, CW population displayed the highest genetic diversity value and KY population had the lowest genetic diversity value. The distance between CW and CF populations was the farthest, which was possibly resulted from the farthest distance of Weihai of Shandong and Fujian of China compared with the geographical distance between other locations of populations. The subpopulation differentiation value （G.,） is 0. 356 5, showing a certain extent of differentiation among the four geographical populations. AFLP technology was confirmed to be an effective tool to assess within- and among-population genetic diversity of Japanese flounder. The present survey provided significant insights for research in the Japanese flounder breeding program.

Genetic Analysis of Floating Enteromorpha prolifera in the Yellow Sea with AFLP Marker

Extremely large accumulation of green algae Enteromorpha prolifera floated along China'coastal region of the Yellow Sea ever since the summer of 2008.Amplified Fragment Length Polymorphism (AFLP) analysis was applied to assess the genetic diversity and relationships among E. prolifera samples collected from 9 affected areas of the Yellow Sea.Two hundred reproducible fragments were generated with 8 AFLP primer combinations,of which 194 (97%) were polymorphic. The average Nei's genetic diversity, the coefficiency of genetic differentiation (Gst), and the average gene flow estimated from Gst in the 9 populations were 0.4018, 0.6404 and 0.2807 respectively. Cluster analysis based on the unweighed pair group method with arithmetic averages (UPGMA) showed that the genetic relationships within one population or among different populations were all related to their collecting locations and sampling time. Large genetic differentiation was detected among the populations.The E. prolifera originated from different areas and were undergoing a course of mixing.

Construction of a Genetic Linkage Map of Mango Based on SRAP, AFLP and ISSR Markers

A hybrid Ft population was obtained by crossing between mango cultivars ＇ Jinhuang＇ and ＇ Guifei＇. Among the hybrid population, 98 F1 plants were selected as the mapping population. The molecular genetic map of mango was constructed by linkage analysis by SRAP, AFLP and ISSR markers using Joinmap4. 0 software. The genetic linkage map consisted of 33 linkage groups, with a total genetic distance of 1 561.1 cM. Moreover, the genetic linkage map involved 245 polymorphic markers, including 149 SRAP markers, 90 AFLP markers and 6 ISSR markers, with an average genetic distance of 6.37 cM. This study laid a founda- tion for further investigation of the important agronomic traits of mango.

The Genetic Relationship of Two New Colorful Sweet Osmanthus fragrans Cultivars Revealed by AFLP and SSR Markers

Osmanthus fragrans is well-known as an ornamental and agricultural plant noted for its unique fragrance.We investigated the genetic relationships between two new colorful O.fragrans cultivars,Ziyan Gongzhu and Qiannan Guifei,and their relationships with other O.fragrans using amplified fragment length polymorphism(AFLP)and simple sequence repeat(SSR),compared to the Changye Muxi species.55 AFLP primer pairs and 17 SSRs were screened;1103 and 45 amplification products were produced,of which 92.29%of the AFLP and 62.20%of the SSRs were polymorphic.At the population level,the number of effective alleles varied from 0.76 to 1.11,and the Shannon index(I)ranged from 0 to 0.11,indicating a narrow genetic base of O.fragrans cultivars.The genetic similarity varied from 0.61 to 0.80 between Ziyan Gongzhu and other O.fragrans cultivars,and 0.57 to 0.67 in the Qiannan Guifei and other cultivars.The unweighted pair group method with arithmetic mean(UPGMA)clustering revealed that Ziyan Gongzhu may belong to the O.fragrans Albus group.However,the Qiannan Guifei cultivar was clustered into a group,showing that it has great genetic variation.These results provide new molecular evidence for germplasm resources protection,and new cultivars for breeding.