Alternative selectable markers for potato transformation using minimal T-DNA vectors

Alternative selection systems for plant transformation are especially valuable in clonal crops, such as potato (Solanum tuberosum L.), to pyramid transgenes into the same cultivar by successive transformation events. We have modified the pGPTV series of binary vectors to construct pMOA1 to pMOA5, resulting in a series of essentially identical binary vectors except for the presence of different selectable marker genes. These selectable marker genes are tightly inserted between the left and right T-DNA borders and confer resistance to kanamycin (nptII), hygromycin (hpt), methotrexate (dhfr), phosphinothricin (bar), or phleomycin (ble). The T-DNA of all the vectors is based on the minimal features necessary for plant transformation, with no extraneous DNA segments that may be unacceptable to regulatory authorities for general release of transgenic plants. A series of unique restriction sites exists between the right border and each selectable marker gene for subsequent insertion of useful genes. We have also developed improved culture procedures for potato transformation and used the pMOA1 to pMOA5 binary vectors to define stringent selection conditions for each marker gene. Combining these advances improved the frequency of recovering transformed potato plants while maintaining a low frequency of escapes. The relative efficiency of recovering transgenic potato lines with each selectable marker gene can be summarised as: kanamycin resistance>hygromycin resistance>phosphinothricin resistance>phleomycin resistance>methotrexate resistance.     

分子标记及其在鸟类分子生态学研究中的应用

分子生态学是一门相当新的分支学科,从前人的研究工作来看,分子标记在这个学科中应用非常广泛,本文描述了RFLP、RAPD、MinisatelliteDNA,MicrosatelliteDNA和AFLP这5个分子标记的优缺点及其在鸟类分子生态学中的应用和研究概况。     

Multiple Paternity in the Nurse Shark, Ginglymostoma Cirratum

For over a decade, we have been studying the reproductive behavior of the nurse shark, Ginglymostoma cirratum, in the Dry Torugas off the Florida Keys, an important mating and nursery ground for this species. In the course of these studies, we have used a variety of tags and tagging protocols to monitor individual animals. Here we report the use of molecular methods for the genetic analysis of nurse sharks. Specifically we have analyzed genetic variation at the MHC II alpha locus using the polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) analysis of the amplified products. We found this technique to be a relatively rapid and reliable method for identifying genetic differences between individual sharks. Applying this method to a family of sharks consisting of a mother and 32 pups, we demonstrate that at least four fathers must have fathered this brood. Multiple paternity in the nurse shark suggests a mechanism by which populations of this species may maximize genetic variability. This seems especially valuable for philopatric species whose migratory movement, and thus potential for genetic diversity, is limited.     

Molecular markers reveal a strong genetic differentiation between two European relic tree species: Zelkova abelicea (Lam.) Boissier and Z. sicula Di Pasquale, Garfì & Quézel (Ulmaceae)

Chloroplast (trnL) and ribosomal (ITS2)sequences and chloroplast DNA (PCR-RFLP andSSR) markers were analysed in two relicUlmaceae tree species: Zelkova abelicea,from Crete, and Z. sicula, from Sicily.The analysis of the plastidial trnLintron and of ITS2 ribosomal sequences revealedtheir divergence from the related speciesZ. carpinifolia, widespread in the Caucasianregion; one base substitution in the trnLintron was detected between the twoMediterranean species, thus suggesting theirrecent separation. Molecular markers(plastidial PCR-RFLP and SSR) showed an evidentgenetic differentiation between Z. siculaand Z. abelicea, the two species beingcharacterised by different haplotypes. Nowithin population variation was detected usingdifferent chloroplast markers inZ. abelicea and Z. sicula. Paleobotanicaldata proved that the genus Zelkova wasabundant and widespread in central Italy untilit became extinct in the continental part ofEurope during last glaciation events andsurvived only in two Mediterranean islands. Thesegregation of the two Mediterranean relicspecies might have occurred as a consequence ofthe strong reduction of their distribution andthe following geographic isolation. Geneticdrift may have determined the drastic reductionof within stand diversity as observed in othersmall, peripheral and geographically isolatedplant populations. The priorities forconservation programs are discussed in thelight of the different genetic resourcesrepresented by the two taxa.     

Nonanchored Inter Simple Sequence Repeat (ISSR) markers: Reproducible and specific tools for genome fingerprinting

Many molecular marker techniques are available today. PCR-based approaches are in demand because of their simplicity and requirement for only small quantities of sample DNA. Nonanchored inter simple sequence repeats (ISSRs) are arbitrary multiloci markers produced by PCR amplification with a microsatellite primer. They are advantageous because no prior genomic information is required for their use. We found the technique stable across a wide range of PCR parameters. Polymorphisms were abundant among 7 dicot species tested with 2 tri-nucleotide and 2 tetra-nucleotide primers. Thus, nonanchored ISSR markers are a good choice for DNA fingerprinting.     

Genetic Modification of Short Rotation Popular Wood: Properties for Ethanol Fuel and Fiber Productions

Opportunities for matching wood chemical and physical properties to manufacturing and product requirements via genetic modification have long been recognized. Exploitation is now feasible due to advances in trait measurement, breeding, genetic mapping and marker, and genetic transformation technologies. With respect to classic selection and breeding of short-rotation poplars, genetic parameters are favorable for decreasing lignin content and increasing specific gravity, but less so for increasing cellulose content. Knowledge of functional genomics is expanding, as is that needed for eventual application of marker-aided breeding, trait dissection, candidate gene identification, and gene isolation. Research on gene transfer has yielded transgenic poplars with decreased lignin and increased cellulose contents, but otherwise normal growth and development. Until effective marker-aided breeding technologies become available, the most promising approach for enhancing ethanol fuel and fiber production and processing efficiencies centers on selecting and breeding poplars for high wood substance yields and genetically transforming them for decreased lignin and increased cellulose contents.     

沙田柚系列遗传关系的RAPD标记研究

采用 RAPD标记技术分析了沙田柚系列 1 2个样品的遗传关系。利用经筛选具多态性的 1 4个 1 0碱基随机引物对 1 2个样品进行 DNA随机扩增 ,获得清晰可重复的位点 99个 ,其中多态性位点 5 8个 ,占 5 8.89% ;在部分样品中检出了特异性 RAPD标记 1 9个 ,占 1 9.1 9% ;通过样品间遗传相似性系数比较与 UPGMA聚类分析 ,并根据历史事实可知 ,广西沙田柚、梅州金柚为沙田柚的无性繁殖系 ,软枝系沙田柚、沙田柚早熟单株为沙田柚的芽变后代 ,梅花早柚、菊花心沙田柚、冬瓜圈沙田柚、段氏柚、垫江沙田柚、古老钱沙田柚为沙田柚的实生变异品系。     

大肠杆菌编码区碱基片段的分析研究

对大肠杆菌1231个编码开始区域和1307个编码终止区域内的6碱基片段、4碱基片段和3碱基片段进行了统计,发现绝大多数4碱基和3碱基模式出现在盯对频率小于2的范围之内;在这两个区域中,出现最多的碱基片段是多聚A;编码开始区域和编码终止区域的碱基构成模式有明显区别;编码开始区域里GGA类的稀有片段恰恰是SD区域最偏好的碱基片段;以TAG或CTA构造的3碱基模式为编码开始区和编码终止区的禁用模式。