农林高校《分子遗传学》教学体系的改革与创新

全国农林高校应重视创新性人才的培养,而课程改革又是创新性人才培养的重中之重。《分子遗传学》是生命科学中一门基础理论科学,它在高等农业院校的教学计划中是一门重要的专业基础课程。因此,重视《分子遗传学》课程的教学与改革创新对农林院校学生来说尤为重要。为此,笔者总结了20多年教学实践中积累的经验和体会,为农林高校《分子遗传学》教学体系的改革与创新提供资料和参考。

Cytological and Molecular Identification of Alien Chromatin in Giant Spike Wheat Germplasm

Alien chromosomes of twelve giant spike wheat germplasm lines were identified by C-banding, genomic in situ hybridization (GISH), sequence characterized amplified region (SCAR), and random amplified polymorphic DNA (RAPD). All lines showed a chromosome number of 2n = 42, five of them carried both a pair of wheat-rye (Triticum aestivum-Secale cereal) 1BL/1RS translocation chromosomes and a pair of Agropyron intermedium (Ai) chromosomes, three carried a pair of Ai chromosomes only, three others carried a pair of 1BL/1RS chromosomes only, and one carried neither 1BL/1BS nor Ai chromosome. Further identification revealed that the identical Ai chromosome in these germplasm lines substituted the chromosome 2D of common wheat (T aestivum L.), designated as 2Ai. The genetic implication and further utilization of 2Ai in wheat improvement were also discussed.

Matrix Generator (MG): a Program for Creating 0/1 Matrix from Sized DNA Fragments

A computer program called Matrix Generator (MG) was developed for transforming sized DNA fragments into a presence/absence data matrix. Dynamic computation was run to avoid errors introduced using fixed-bin-width arithmetic. MG can be used with bin sized fragments from AFLP, ISSR, RAPD, RFLP, and other molecular markers. The accuracy of MG was tested using fAFLP data of Abelia and the results show that MG results in higher resolution of taxa and is more reliable than programs of the similar usage.

Gallbladder cancer epidemiology, pathogenesis and molecular genetics: Recent update

Gallbladder cancer is a malignancy of biliary tract which is infrequent in developed countries but common in some specific geographical regions of developing countries. Late diagnosis and deprived prognosis are major problems for treatment of gallbladder carcinoma. The dramatic associations of this orphan cancer with various genetic and environmental factors are responsible for its poorly defined pathogenesis. An understanding to the relationship between epidemiology, molecular genetics and pathogenesis of gallbladder cancer can add new insights to its undetermined pathophysiology. Present review article provides a recent update regarding epidemiology, pathogenesis, and molecular genetics of gallbladder cancer. We systematically reviewed published literature on gallbladder cancer from online search engine Pub Med(http://www.ncbi.nlm.nih.gov/pubmed). Various keywords used for retrieval of articles were Gallbladder, cancer Epidemiology, molecular genetics and bullion operators like AND, OR, NOT. Cross references were manually searched from various online search engines(http://www.ncbi.nlm.nih.gov/pubmed,https://scholar.google.co.in/, http://www.medline.com/home.jsp). Most of the articles published from 1982 to 2015 in peer reviewed journals have been included in this review.

Esophageal combined carcinomas:Immunohoistochemical and molecular genetic studies

Primary esophageal combined carcinoma is very rare. The authors herein report 2 cases. Case 1 was a com- bined squamous cell carcinoma and small cell carci- noma, and case 2 was a combined squamous cell carci- noma, adenocarcinoma, and small cell carcinoma. Case 1 was a 67-year-old man with complaints of dysphagia. Endoscopic examination revealed an ulcerated tumor in the middle esophagus, and 6 biopsies were obtained. All 6 biopsies revealed a mixture of squamous cell car- cinoma and small cell carcinoma. Both elements were positive for cytokeratin, epithelial membrane antigen, and p53 protein, and had high Ki-67 labeling. The small cell carcinoma element was positive for synaptophysin, CD56, KIT, and platelet-derived growth factor-~ （PDG- FRA）, while the squamous cell carcinoma element was not. Genetically, no mutations of K/Tand PDGFRA were recognized. The patient died of systemic carcinomato- sis 15 mo after presentation. Case 2 was a 74-year-old man presenting with dysplasia. Endoscopy revealed a polypoid tumor in the distal esophagus. Seven biopsies were taken, and 6 showed a mixture of squamous cell carcinoma, small cell carcinoma, and adenocarcinoma. The 3 elements were positive for cytokeratins, epithe-lial membrane antigen, and p53 protein, and had high Ki-67 labeling. The adenocarcinoma element was posi- tive for mucins. The small cell carcinoma element was positive for CD56, synaptophysin, KIT, and PDGFRA, but the other elements were not. Mutations of KIT and PDGFRA were not recognized. The patient died of sys- temic carcinomatosis 7 mo after presentation. These combined carcinomas may arise from enterochromaf- fin cells or totipotential stem cell in the esophagus or transdifferentiation of one element to another. A review of the literature was performed.

Amelanotic malignant melanoma of the esophagus:Report of two cases with immunohistochemical and molecular genetic study of KIT and PDGFRA

The author reports herein two cases of amelanotic malignant melanoma of the esophagus. Case 1 is an 87-year-old woman who was admitted to our hospital because of nausea and vomiting. Endoscopic examination revealed an ulcerated tumor of the distal esophagus, and a biopsy was taken. The biopsy showed malignant polygonal and spindle cells. No melanin pigment was recognized. Immunohistochemically, the tumor cells were positive for melanosome （HMB45）, S100 protein, KIT and Platelet derived growth factor receptor-α （PDG- FRA）. The patient was treated by chemotherapy and radiation, but died of systemic metastasis 12 mo after the presentation. Case 2 is a 56-year-old man presenting with dysphagia. Endoscopic examination revealed a polypoid tumor in the middle esophagus, and a biopsy was obtained. The biopsy showed malignant spindle cells without melanin pigment. Immunohistochemically, the tumor cells were positively labeled for melanosome,S100 protein, KIT and PDGFRA. The patient refused operation, and was treated by palliative chemotherapy and radiation. He died of metastasis 7 mo aEer the admission. In both cases, molecular genetic analyses of gene （exons 9, 11, 13 and 17） and PDGFRA gene （exons 12 and 18） were performed by the PCR direct sequencing method, which showed no mutations of KIT and PDGFRA genes. This is the first report of esophageal malignant melanoma with an examination of the expression of KIT and PDGFRA and the mutational status of K/T and PDGFRA genes.

Construction of a cDNA library for sea cucumber Acaudina leucoprocta and differential expression of ferritin peptide

Acaudina leucoprocta is an edible sea cucumber of economic interest that is widely distributed in China. Little information is available concerning the molecular genetics of this species although such knowledge would contribute to a better understanding of the optimal conditions for its aquaculture and its mechanisms of defense against disease. Therefore, we constructed a c DNA library and, based on bioinformatics analysis of the sequences, the functions of 75% of the c DNAs were identifi ed, including those involved in cell structure, energy metabolism, mitochondrial function, and signal transduction pathways. Approximately 25% of genes in the library were unmatched. The gene for A. leucoprocta ferritin was also cloned. The predicted amino-acid sequence of ferritin displayed signifi cant homology with other sea-cucumber counterparts but indicated that it was a new member of the ferritin family. Semiquantitative real-time RT-PCR indicated the highest levels of ferritin m RNA expression in the intestine. A polyclonal antibody of ferritin was also produced. These data provide a set of molecular tools essential for further studies of the functions of ferritin protein in A. leucoprocta.

Factors affecting genomic selection revealed by empirical evidence in maize

Genomic selection(GS) as a promising molecular breeding strategy has been widely implemented and evaluated for plant breeding, because it has remarkable superiority in enhancing genetic gain, reducing breeding time and expenditure, and accelerating the breeding process. In this study the factors affecting prediction accuracy(rMG) in GS were evaluated systematically, using six agronomic traits(plant height, ear height, ear length, ear diameter,grain yield per plant and hundred-kernel weight) evaluated in one natural and two biparental populations. The factors examined included marker density, population size, heritability,statistical model, population relationships and the ratio of population size between the training and testing sets, the last being revealed by resampling individuals in different proportions from a population. Prediction accuracy continuously increased as marker density and population size increased and was positively correlated with heritability; rMGshowed a slight gain when the training set increased to three times as large as the testing set. Low predictive performance between unrelated populations could be attributed to different allele frequencies, and predictive ability and prediction accuracy could be improved by including more related lines in the training population. Among the seven statistical models examined, including ridge regression best linear unbiased prediction(RR-BLUP), genomic BLUP(GBLUP), Bayes A, Bayes B, Bayes C, Bayesian least absolute shrinkage and selection operator(Bayesian LASSO), and reproducing kernel Hilbert space(RKHS), the RKHS and additive-dominance model(Add + Dom model) showed credible ability for capturing non-additive effects, particularly for complex traits with low heritability. Empirical evidence generated in this study for GS-relevant factors will help plant breeders to develop GS-assisted breeding strategies for more efficient development of varieties.

Understanding biological functions through molecular networks

The completion of genome sequences and subsequent high-throughput mapping of molecular networks have allowed us to study biology from the network perspective. Experimental, statistical and mathematical modeling approaches have been employed to study the structure, function and dynamics of molecular networks, and begin to reveal important links of various network properties to the functions of the biological systems. In agreement with these functional links, evolutionary selection of a network is apparently based on the function, rather than directly on the structure of the network. Dynamic modularity is one of the prominent features of molecular networks. Taking advantage of such a feature may simplify network-based biological studies through construction of process-specific modular networks and provide functional and mechanistic insights linking genotypic variations to complex traits or diseases, which is likely to be a key approach in the next wave of understanding complex human diseases. With the development of ready-to-use network analysis and modeling tools the networks approaches will be infused into everyday biological research in the near future.

Clinical cytogenetics and molecular cytogenetics

The short report will be focused on helping our students to understand commonly used conventional and cutting edge cytogenetic techniques and their clinical applications, the advances and drawbacks of each technique, and how to pick the right test(s) for a specific patient in order to achieve a proper diagnosis efficiently and economically.

AN EPIDEMIOLOGY AND MOLECULAR GENETIC STUDY ON BREAST CANCER SUSCEPTIBILITY

Objectives. To investigate the genetic susceptibility for breast cancer of Chinese, a hospital-based case-control study, pedigree survey and molecular genetic study were conducted. Methods. Logistic regression model and stratification methods were used in the risk factors analysis. Li-Mantel-Gart and Falconer methods were used to analyze the segregation ratio and heritability. Polymerase chain reaction(PCR) and polyacrylamide gel electrophoresis were used to detect AI, G-banding technique was used to detect the chromosome aberration of peripheral blood lymphocyte. Results. Family history of breast cancer is related to enhanced breast cancer risk significantly, OR is 3905(95%CI=1079～1413), and it widely interacts with other risk factors. Accumulative incidence of breast cancer in first degree relatives is 999%, which is larger than that in second, third degree and non-blood relatives. Segregation ratio is 0021, heritability among first degree relatives is 356±58%. Frequencies of LOH at BRCA1 and BRCA2 loci in sporadic breast cancer are 612% and 577% respectively. In the sibs, both of them show LOH at D13S173 locus, and high frequencies of chromosome aberrations were observed. Conclusions. Genetic susceptibility contributes to breast cancer occurrence of Chinese, and its racial variation may be one of the important reasons for the large difference of incidence between western and eastern countries.

Charaterization of Citrus Hybrid ＂Huangguogan＂ through the Combination of Morphological and Molecular Markers

Huangguogan, an obvious Citrus hybrid, is suitable for transportation and export and ripens in March or April. Because of late season, it may playa significant role in fruit market. However, its origin is still unconfirmed. The aim of this study was to clarify the possible parentage of Huangguogan via the combination of morphological and molecular markers including simple sequence repeat （SSR） and chloroplast simple sequence repeat （cpSSR）. Analysis of morphological traits including leaf stalk length, phylliform index and fruit shape index indicated that Huangguogan had similarities in morphology with Sweet orange. The SSR Cluster Analysis showed that Huangguogan was clustered together with Hongju tangerine and revealed -80% genetic similarity. They illustrated a close genetic distance between Huangguogan and Hongju tangerine. In addition, the bands of2 polymorphic cpSSR were identical in Huangguoggan and Sweet orange. Consequently, it is likely that its female parentage was the sweet orange （Citrus sinensis （L.） Osbeck） and its male parentage was the tangerine （Citrus reticulata Blanco）.

The Variety Selection and Testing for Effect on HepG-2 Cell Line of Dioscorea membranacea Pierre （Hua Khao Yen Tai） Extract Using Molecular Genetics

In this research, Dioscorea membranacea Pierre was studied by using 30 samples from Khao Ruak Sub-district, Chai Badan District, Lop Buri Province, in Thailand. In this research, some morphology was studied including shapes, leaves, stem colors, epidermal cells, stomata sizes and stipules. To study the genetic relationships, the AFLP technique and computer program were used. The Dioscorea membranacea Pierre was classified into 2 groups according to its phylogenetic type： the first group was ＂Hua Khao Yen Tai-Nuea＂ （Smilax corbularia Kunth）, and the second group included 30 further samples of Hua Khan Yen Tai （Dioscorea membranacea Pierre）. The ethanolic crude extract was also applied to test the anti-proliferative activity in the liver hepatocellular carcinoma （HepG2） cell lines which illustrates the characteristics of apoptosis： cell shrinkage, membrane blebbing and nuclear condensation. The expression ofBax gene is increased more than that of the control group while Bcl-2 gene which is anti-apoptotic is decreased. Furthermore, the result of western blot analysis reveals the up-regulation of Bax protein and down-regulation of Bcl-2 protein when compared with untreated cells. This might indicate that ethanolic crude extracts of Hua Khao Yen Tai could induce apoptosis and anti-proliferative on HepG2 cell lines, The results also revealed that some morphology cannot be used to predict which Dioscorea membranacea Pierre plants would be most effective.

Development of Three Multiplex PCR Primer Sets for Ark Shell(Scapharca broughtonii)and Their Validation in Parentage Assignment

Scapharca broughtonii is a commercially important and over-exploited species.In order to investigate its genetic diversity and population structure,43 novel polymorphic microsatellites were isolated and characterized.The number of alleles per locus ranged from 3 to 22 with an average of 6.93,and the observed and expected heterozygosities varied between 0.233 and 1.000,and 0.250 and 0.953,with an average of 0.614 and 0.707,respectively.Three highly informative multiplex PCRs were developed from nine of those microsatellites for S.broughtonii.We evaluated and validated these multiplex PCRs in 8 full-sib families.The average polymorphism information content(PIC) was 0.539.The frequency of null alleles was estimated as 3.13% of all the alleles segregation based on a within-family analysis of Mendelian segregation patterns.Parentage analysis of real offspring demonstrated that 100% of all offspring were unambiguously allocated to a pair of parents based on 3 multiplex sets.Those 43 microsatellite loci with high variability will be helpful for the analysis of population genetics and conservation of wild stock of S.broughtonii.The 3 sets of multiplex PCRs could be an important tool of pedigree reconstruction,population genetic analysis and brood stock management.

Application of Molecular Genetic Methods in Anthropological and Paleodemographic Studies of Fragmentary and Damaged Skeletal Material from Rescue Excavations

The purpose of this study was to demonstrate the application of molecular genetic methods in anthropology and paleodemography in case where the examined bone material is damaged and fragmented, and where the skeletal remains of children are investigated. The application of traditional anthropological methods is limited, especially if sex determination of children and fragmentary skeletal remains is detected. Sex typing genetic markers （SRY, amelogenin） were used for sex determination of children and undetermined fragmentary skeletal remains from the burial site Pohansko, south outer precincts （Czech Republic）. This is an approximately 1,200 year-old burial site （the Great Moravian period）, which was excavated during rescue excavations. After the genetic analysis, sex was determined in more than half of the investigated samples. The results of the genetic analysis were used for completion of demographic data of this archaeological site. The results of sex determination of several samples were independently verified by the Institute of Criminology in Prague （Czech Republic）, using the PowerPlex ESX 17 System （Promega）. This study showed the suitability of modem molecular genetic methods to skeletal anthropology and paleodemographic analyses.

Discriminating ability of molecular markers and morphological characterization in the establishment of genetic relationships in cultivated genotypes of almond and related wild species

A total 23 morphological traits, 19 AFLP-primer combinations, 80 RAPD primers and 32 SSR primer pair were used to compare the informativeness and efficiency of random amplified polymorphic DNA （RAPD）, amplified fragment length polymorphism （AFLP） and simple sequence repeat （SSR） markers in establishing genetic relationships among 29 almond cultivars and three related wild species. SSRs presented a high level of polymorphism and greater information content, as assessed by the expected hetrozygosity, compared to AFLPs and RAPDs. The lowest values of expected hetrozygosity were obtained for AFLPs; however AFLPs showed the highest efficiency, owing to their capacity to reveal large numbers of bands per reaction, which led to high values for various types of indices of diversity. All the three techniques discriminated almond genotypes very effectively, except that SSRs failed to discriminate between ＇Monagha＇ and ＇Sefied＇ almond genotypes. The correlation coefficients of similarity were statistically significant for all the three marker systems, but were lower for the SSR data than for RAPDs and AFLPs. For all the markers, high similarity in dendrogram topologies was obtained, although some differences were observed. All the dendrograms, including that obtained by the combined use of all the marker data, reflect relationships for most of cultivars according to their geographic diffusion. AMOVA detected more variation among cultivated and related wild species of almond within each geographic group. Bootstrap analysis revealed that the number of markers used was sufficient for reliable estimation of genetic similarity and for meaningful comparisons of marker types.

Molecular biology of pancreatic cancer

In spite of continuous research efforts directed at early detection and treatment of pancreatic cancer, the outlook for patients affected by the disease remains dismal. With most cases still being diagnosed at advanced stages, no improvement in survival prognosis is achieved with current diagnostic imaging approaches. In the absence of a dominant precancerous condition, several risk factors have been identified including family history, chronic pancreatitis, smoking, diabetes mellitus, as well as certain genetic disorders such as hereditary pancreatitis, cystic fibrosis, familial atypical multiple mole melanoma, and Peutz-Jeghers and Lynch syn- dromes. Most pancreatic carcinomas, however, remain sporadic. Current progress in experimental molecular techniques has enabled detailed understanding of the molecular processes of pancreatic cancer development. According to the latest information, malignant pancre- atic transformation involves multiple oncogenes and tumor-suppressor genes that are involved in a variety of signaling pathways. The most characteristic aberrations （somatic point mutations and allelic losses） affect onco- genes and tumor-suppressor genes within RAS, AK-I- and Wnt signaling, and have a key role in transcription and proliferation, as well as systems that regulate the cell cycle （SMAD/DPC, CDKN2A/p16） and apoptosis （TP53）. Understanding of the underlying molecular mechanisms should promote development of new methodology for early diagnosis and facilitate improvement in current approaches for pancreatic cancer treatment.

A preliminary phylogeny of Tipula and taxonomic placement of the subgenus Tipula (Sivatipula) (Diptera: Tipuloidea) based on mitochondrial COI gene

The large genus Tipula Linnaeus,1758 contributes heavily to the biodiversity of the family Tipulidae.However,the monophyly of Tipula has not yet been verified.The subgenus Sivatipula Alexander,1964 is possibly the most confusing subgeneric taxon in the genus Tipula because of its members’particularly long antenna and one-armed posterior immovable apodeme on semen pump,which makes its subgeneric position uncertain.In this research,the sequences of cytochrome oxidase I(COI)for 19 Tipula species and five taxa from other genera are analyzed.Considering the molecular evidence on genetic distance as well as phylogenetic analysis and morphological information,our results indicate that(1)the genus Tipula is not resolved as monophyletic in phylogeny based on neighbor joining(NJ)and maximum likelihood(ML)trees because the subgenus Sivatipula doesn’t form a monophyletic clade with the remaining subgenera of Tipula;and(2)Sivatipula may deserve a generic status since it forms an independent phylogenetic line.

A Review of Molecular Makers Applied in Cowpea （Vigna unguiculata L. Walp.） Breeding

In recent years, with the rapid development of molecular biology, molecular markers have been widely used in genetic breeding of various crops including cowpea. However, molecular researches in cowpea are lack of systematic summary. This review presents an overview of accomplishments on different aspects of molecular markers in cowpea genetic breeding, such as genetic diversity analysis, genetic linkage map construction, QTL mapping, etc. Furthermore, it provides the discussion of some existing problems about molecular markers applied in cowpea breeding and the prospect of the future development. The authors find that SSR is the most frequently used molecular marker, while SNP has not been used in the genetic diversity analysis of cowpea. And the authors also conclude that more QTL of cowpea should be located and more molecular markers linked to resistance gene should be found. This will be useful for scientists and breeders to research cowpea with the aid of molecular markers, thus accelerating improvement of cowpea varieties.

Genetic Variation Analyses of nsp2 Gene of PRRSV in Ningxia Hui Autonomous Region of China

To gain a better understanding of the genetic diversity and evolution of PRRSV in the Ningxia Hui Nationality Autonomous Region(Ningxia) of China,the nsp2 genes from a series of PRRSV strains collected from the region in 2007 were partially sequenced. These sequences were then analyzed along with the classical strain(ch-1a) and two other epidemic strains SD(3) and SD2006. Comparison of the nucleotide sequence with ch-1a indicated that nsp2 genes of seventeen Ningxia isolates(NX strain) have deletions of 87 nucleotides. Sequence analysis indicated that homology between the Ningxia strain and ch-1a was 60.3%-79.9% in the nucleotide sequence,and homology between the NX strains and SD strains was 80.3%-98.8% in the nucleotide sequence. The nsp2 genes of the seventeen isolates had 74.9%-100% nucleotide sequence identities with each other. This study was undertaken to assess the regional variation of prevalent PRRSV and to establish a sequence database for PRRSV molecular epidemiological studies.