Development of RAPD Markers and SCAR Markers Linked to Bentazon Susceptible Lethality Gene in Rice

Rice cultivar Norin 8 and its mutant Norin 8m harbour bentazon resistance trait and bentazon susceptibility trait respectively. A total of 360 arbitrary 10-mer oligonucleotide primers were screened on the genomic DNA of Norin 8 and Norin 8m with RAPD technique. Among which, five primers produced seven polymorphic RAPD bands between Norin 8 and Norin 8m. Amplified RAPD polymorphic products were cloned and sequenced. The sequences were used to design primers for PCR. Five SCAR markers, SCAR/G18/883, SCAR/G18/890, SCAR/G18/919/948, SCAR/D10/1237 and SCAR/F03/1186, were developed from OPG18/943, OPG18/972, OPD10/1248 and OPF03/1198. F-2 progeny of 320 individuals was analyzed to map SCAR markers in relationship to ben or Ben genes. SCAR markers of SCAR/G18/883, SCAR/G18/890, SCAR/G18/919/948 were shown to cosegregate with ben or Ben genes, and SCAR/D10/1237 to be linked of Ben gene with a distance of (14.8 +/- 2.1) cM. The genetic linkage to ben gene and SCAR markers was identified by a pair of near isogenic lines H121 and Hben121. Southern blotting analysis and segregation ratio of F-2 progeny revealed that OPG18/943 and OPG18/972 were single-copy in genome, and locus of OPG18/943 and OPG18/972 were allelic and sequence tagged sites. It is the first report on molecular markers linked to ben or Ben genes. The markers are useful to marker-assisted selection for the breeding and tag ben gene with map-based cloning.

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.

RAPD-SCAR Markers: An Interface Tool for Authentication of Traits

The versatility of the PCR technique is that several kinds of primers can be explored for genome analysis depending on the purpose of study. The easy to access and low cost PCR-based markers include Random Amplified Polymorphic DNA (RAPD). The RAPD markers are easy to develop but lack of reproducibility makes it less reliable and obstacles to their further use in authentication of traits. In addition, other PCR and non PCR based markers like Amplified Fragment Length Polymorphism (AFLP), Simple Sequence Repeat (SSR) and Restriction Fragment Length Polymorphism (RFLP) are also employed in authentication of traits with certain restrictions vis-à-vis use of radioactive materials, high cost and requirement of sequence information etc. Therefore, this problem can be overcome by converting RAPD markers to more robust sequence characterized amplified regions i.e. SCAR markers. SCARs are locus specific, co-dominant in nature and amplified by PCR using specific 15 - 30 bp DNA fragments. For developing SCAR markers, primers are designed from the nucleotide sequences of a cloned RAPD fragments linked to a trait of interest. SCAR markers are easy to develop and reliable tools for DNA fingerprinting. This mini review is an attempt to summarize efficacy of RAPD-SCAR interface in authentication of traits.

Development of a stable SCAR marker for rapid identification of Ganoderma lucidum Hunong 5 cultivar using DNA pooling method and inter-simple sequence repeat markers

The cultivar Ganoderma lucidum Hunong 5 was obtained using cross-breeding. Hunong 5 has high commercial value due to its high polysaccharide and triterpene content, This is the first report of using a DNA pooling method to develop a stable sequence characterized amplified region （SCAR） marker for rapid identification of the G. lucidum Hunong 5 cultivar. The SCAR marker was developed by first generating and sequencing a distinctive inter simple sequence repeat （ISSR） fragment （882 bp） from G. lucidum Hunong 5 cultivar. A stable SCAR primer pair GLH5F/GLH5R were obtained to identify the cultivar and the SCAR marker is a DNA fragment of 773 bp.

Development of a SCAR marker for male gametophyte of Gracilariopsis lemaneiformis based on AFLP technique

The red alga Gracilariopsis lemaneiformis(Bory) is an economically valuable macroalgae. As a means to identify the sex of immature Gracilariopsis lemaneiformis, the amplifi ed fragment length polymorphism(AFLP) technique was used to search for possible sex- or phase-related markers in male gametophytes, female gametophytes, and tetrasporophytes, respectively. Seven AFLP selective amplifi cation primers were used in this study. The primer combination E-TG/M-CCA detected a specifi c band linked to male gametophytes. The DNA fragment was recovered and a 402-bp fragment was sequenced. However, no DNA sequence match was found in public databases. Sequence characterized amplifi ed region(SCAR) primers were designed from the sequence to test the repeatability of the relationship to the sex, using 69 male gametophytes, 139 female gametophytes, and 47 tetrasporophytes. The test results demonstrate a good linkage and repeatability of the SCAR marker to sex. The SCAR primers developed in this study could reduce the time required for sex identifi cation of Gracilariopsis lemaneiformis by four to six months. This can reduce both the time investment and number of specimens required in breeding experiments.

Development of RAPD-based SCAR Marker for Rapid Identification of Trichoderma harzianum

Based on previous morphological classification and RAPD analysis, we recovered and sequenced genomic DNA of a specific fragment obtained from No. 45 Trichoderma harzianum strain in RAPD amplification. A pair of specific primers was synthesized and the RAPD markers were successfully converted into SCAR markers. The SCAR molecular marker exhibited the specificity that could differ T. harzianum from other Trichoderma strains. This pair of molecular primers was used for specific PCR amplification of 45 Trichoderma strains, and T. harzianum strains could amplify a 779 bp DNA band while other strains had no amplification products.

Heavy metal induced DNA changes in aquatic macrophytes: Random amplified polymorphic DNA analysis and identification of sequence characterized amplified region marker

Plants have been used as good bio-indicators and genetic toxicity of environmental pollution in recent years. In this study, aquatic plants Hydrilla verticillata and Ceratophyllum demersum treated with 10umol/L Cd, 5 umol/L Hg, and 20 umol/L Cu for 96 h, showed changes in chlorophyll, protein content, and in DNA profiles. The changes in DNA profiles included variation in band intensity, presence or absence of certain bands and even appearance of new bands. Genomic template stability test performed for the qualitative measurement of changes in randomly amplified polymorphic DNA （RAPD） profiles, showed significant effect at the given concentration of metals. Cloning and sequencing of bands suggested that these markers although may not be homologous to any known gene but its conversion as a sequence characterized amplified region （SCAR） marker is useful in detecting the effects of genotoxin agents.

Phenotypic and genetic variation between two populations of the Chinese yellow pond turtle, Mauremys mutica (Cantor, 1842)

Mauremys mutica(Cantor,1842)is an endangered species in China.Main phenotypic variations inbody color,body weight,body shape,clutch size,egg size,and hatchling size were revealed betweenthe southern and northern populations.Both populations have the phenomenon of'larger male'sexualsize dimorphism(SSD),especially in the southern population.Furthermore,genetic variations betweenthe two populations were analyzed by RAPD band patterns of 30 random individuals in each population.The average genetic distance was 0.299±0.108 among the samples of two populations.The average ge-netic distance between southern and northern populations was 0.305±0.046.Cluster analysis indicatedthat all the individuals from the southern and northern populations were clustered among themselves toform two distinct clades.A total of 20 population-specific RAPD fragments were scored from 16 primers,and could be used as RAPD markers for distinguishing the southern and the northern population.Basedon the nucleotide sequences of two RAPD markers,two pairs of SCAR primers(SC1-S and SC2-S)weredesigned,which could be used as SCAR markers for the southern population.According to the significantphenotypic and genetic variations,we suggested that the northern population and southern populationmight be considered as two separate taxa,the'northern taxon'and the'southern taxon',and the con-servation should be respectively conducted on the two taxa.

Development and utilization of new sequenced characterized amplified region markers specific for E genome of Thinopyrum

Species containing E genome of Thinopyrum offered potential to increase the genetic variability and desirable characters for wheat improvement. However, E genome specific marker was rare. The objective of the present report was to develop and identify sequenced characterized amplified region （SCAR） markers that can be used in detecting E chromosome in wheat background for breeding purpose. Total 280 random amplified polymorphic DNA （RAPD） primers were amplified for seeking of E genome specific fragments by using the genomic DNA of Thinopyrum elongatum and wheat controls as templates. As a result, six RAPD fragments specific for E genome were found and cloned, and then were converted to SCAR markers. The usability of these markers was validated using a number of E- genome-containing species and wheat as controls. These markers were subsequently located on E chromosomes using specific PCR and fluorescence in situ hybridization （FISH）. SCAR markers developed in this research could be used in molecular marker assisted selection of wheat breeding with Thinopyrum ehromatin introgressions.