Recycling of superfine resolution agarose gel

Genetic markers are now routinely used in a wide range of applications, from forensic DNA analysis to marker-assisted plant and animal breeding. The usual practice in such work is to extract the DNA, prime the markers of interest, and sift them out by electrically driving them through an appropriate matrix, usually a gel. The gels, made from polyacrylamide or agarose, are of high cost, limiting their greater applications in molecular marker work, especially in developing countries where such technology has great potential. Trials using superfine resolution (SFR) agarose for SSR marker screening showed that it is capable of resolving SSR loci and can be reused up to 14 times, thus greatly reducing the cost of each gel run. Furthermore, for certain applications, low concentrations of agarose sufficed and switching to lithium borate buffer, instead of the conventional Tris-borate-ethylenediaminetetraacetic acid buffer, will further save time and cost. The 2.5% gel was prepared following the Agarose SFR™ manual by adding 2.5 g agarose powder into 100 mL 1X lithium borate buffer in a 250-mL flask with rapid stirring. Two midigels (105 x 83 mm, 17 wells) or 4 minigels (50 x 83 mm, 8 wells), 4 mm thickness can be prepared from 100 mL gel solution. A total of 1680 PCR products amplified using 140 SSR markers from oil palm DNA samples were tested in this study using SFR recycled gel. As average, the gel can be recycled 8 times with good resolution, but can be recycled up to 14 times before the resolutions get blurred

QTLs for oil yield components in an elite oil palm (Elaeis guineensis) cross

Increased modern farming of superior types of the oil palm, Elaeis guineensis Jacq., which has naturally efficient oil biosynthesis, has made it the world’s foremost edible oil crop. Breeding improvement is, however, circumscribed by time and costs associated with the tree’s long reproductive cycle, large size and 10–15 years of field testing. Marker-assisted breeding has considerable potential for improving this crop. Towards this, quantitative trait loci (QTL) linked to oil yield component traits were mapped in a high-yield population. In total, 164 QTLs associated with 21 oil yield component traits were discovered, with cumulative QTL effects increasing in tandem with the number of QTL markers and matching the QT+ alleles for each trait. The QTLs confirmed all traits to be polygenic, with many genes of individual small effects on independent loci, but epistatic interactions are not ruled out. Furthermore, several QTLs maybe pleiotropic as suggested by QTL clustering of inter-related traits on almost all linkage groups. Certain regions of the chromosomes seem richer in the genes affecting a particular yield component trait and likely encompass pleiotropic, epistatic and heterotic effects. A large proportion of the identified additive effects from QTLs may actually arise from genic interactions between loci. Comparisons with previous mapping studies show that most of the QTLs were for similar traits and shared similar marker intervals on the same linkage groups. Practical applications for such QTLs in marker-assisted breeding will require seeking them out in different genetic backgrounds and environments

Genetic Linkage Map of a High Yielding FELDA Deli×Yangambi Oil Palm Cross

Enroute to mapping QTLs for yield components in oil palm, we constructed the linkage map of a FELDA high yielding oil palm (Elaeis guineensis), hybrid cross. The parents of the mapping population are a Deli dura and a pisifera of Yangambi origin. The cross out-yielded the average by 8–21% in four trials all of which yielded comparably to the best current commercial planting materials. The higher yield derived from a higher fruit oil content. SSR markers in the public domain - from CIRAD and MPOB, as well as some developed in FELDA - were used for the mapping, augmented by locally-designed AFLP markers. The female parent linkage map comprised 317 marker loci and the male parent map 331 loci, both in 16 linkage groups each. The number of markers per group ranged from 8–47 in the former and 12–40 in the latter. The integrated map was 2,247.5 cM long and included 479 markers and 168 anchor points. The number of markers per linkage group was 15–57, the average being 29, and the average map density 4.7 cM. The linkage groups ranged in length from 77.5 cM to 223.7 cM, with an average of 137 cM. The map is currently being validated against a closely related population and also being expanded to include yield related QTLs

QTLs for oil yield components in an elite oil palm (Elaeis guineensis) cross

Increased modern farming of superior types of the oil palm, Elaeis guineensis Jacq., which has naturally efficient oil biosynthesis, has made it the world’s foremost edible oil crop. Breeding improvement is, however, circumscribed by time and costs associated with the tree’s long reproductive cycle, large size and 10–15 years of field testing. Marker-assisted breeding has considerable potential for improving this crop. Towards this, quantitative trait loci (QTL) linked to oil yield component traits were mapped in a high-yield population. In total, 164 QTLs associated with 21 oil yield component traits were discovered, with cumulative QTL effects increasing in tandem with the number of QTL markers and matching the QT+ alleles for each trait. The QTLs confirmed all traits to be polygenic, with many genes of individual small effects on independent loci, but epistatic interactions are not ruled out. Furthermore, several QTLs maybe pleiotropic as suggested by QTL clustering of inter-related traits on almost all linkage groups. Certain regions of the chromosomes seem richer in the genes affecting a particular yield component trait and likely encompass pleiotropic, epistatic and heterotic effects. A large proportion of the identified additive effects from QTLs may actually arise from genic interactions between loci. Comparisons with previous mapping studies show that most of the QTLs were for similar traits and shared similar marker intervals on the same linkage groups. Practical applications for such QTLs in marker-assisted breeding will require seeking them out in different genetic backgrounds and environments

Identification of Amplified Fragment Length Polymorphism Fragments Linked to Fruit Skin Colour of Oil Palm (Elaeis Guineensis Jacq.)

Oil palm (Elaeis guineensis Jacq.) is an important commercial oil crop. It can be classified by its fruit colour into two types: a) nigrescens (Nig) type which is dark violet (unripe) and turning to reddish violet (ripe), and b) virescens (Vir) type which changes from green colour (unripe) to orange (ripe). Vir and Nig are monofactorially inherited, with Vir dominant over Nig. The different degrees of ripeness during harvesting of the fruits would influence the quantity and quality of oil in the mesocarp. The oil palm harvesting system is based on a minimum ripeness standard whereby the workers use the number of fruits detached from a bunch as a measure of its ripeness. Most of the oil palms grown commercially are the Nig type, being the more common type in the wild. However, Vir is an economically important trait as it is much easier to determine the degree of ripeness in Vir fruits. Molecular markers are powerful tools with the potential to influence plant breeding. Segregating populations for fruit colour CBP line which is dura x tenera cross and NPC1 line, tenera x tenera cross were obtained from Pamol Plantation. Obtaining high quality DNA from mature leaves was difficult. Therefore, a protocol of DNA isolation was developed in this study after 12 different extraction methods were attempted. The aim of this project was to identify Amplified Fragment Length Polymorphism (AFLP) primer combinations and markers that have the potential to distinguish the fruit skin colour trait of oil palm by using the AFLP-based Bulked Segregant Analysis (BSA) technique. Of the 64 primer combinations, 10 primer combinations for CBP line and four primer combinations for NPC1 line were selected. In study 1, only three bands that showed 100% specificity to fruit colour differentiation which are regarded as fruit skin colour-specific markers were obtained for both lines, respectively. In study 2, there were four and eight specific bands which showed 80% probability of significant association to the fruit skin colour trait in CBP and NPC1, respectively. Primer B12 (E-ACT/M-CAT) generated fragments 142.13 bp and 355.76 bp as Vir-specific markers for CBP line and NPC1 line, respectively. It gave 83.30% (CBP) and 83.70% (NPC1) accuracy to distinguish Vir in study 2. Primer combination B13 (E-ACT/M-CTA) targeted 253.79 bp as the Nig–specific marker for both lines. It gave 76.50% (CBP) and 75.70% (NPC1) of confidence in differentiating the trait in study 2. The sequences of the AFLP markers were considered to be too short for reliability as specific markers. For an efficient MAS, it is therefore highly desirable to convert the linked markers into sequence-specific primers, such as STS, SCAR, STMS or CAP. Dice similarity coefficient (Nei and Li 1979) chosen to estimate the genetic similarity of the progenies studied, which gave average similarities of the CBP progenies of 0.790±0.057 and the NPC1 progenies of 0.761±0.089. The values derived from this study were almost similar showing that the progenies are variable only at their segregating trait. The dendrograms generated by cluster analysis using NJ based on similarity coefficients indicated the applicability and reliability of AFLP polymorphism for distinguishing the two varieties in both lines

Dna markers and mapping of quantitative trait loci for yield and bunch quality in Deli dura X Yangambi pisifera oil palm (Elaeis guineensis) population

Increased modern farming of the oil biosynthesis efficient oil palm, Elaeis guineensis Jacq., has propelled it to be the world's largest source of edible oil today. However, further oil yield improvement by conventional breeding is increasingly limited by lengthy time and costs due to long reproductive cycles, large plant size and an evaluation period of 10-15 years. Molecular tools which allow rapid, large scale evaluation over a short time, independent of plant age, will be particularly valuable in the face of such constraints. Towards such a goal, the aim of this particular study was to construct a genetic linkage map of a high yield oil palm population using DNA markers and to identify Quantitative Trait Loci (QTLs) related to oil yield components. This was followed by configuration of Quantitative Trait Alleles (QTA) with favourable and unfavourable effects on their respective oil yield components. The mapping population was a high-yielding Felda breeding cross, coded DA41, represented by 118 progeny palms. Besides the genotypic data generated in this study, phenotypic data of 21 yield components were available from ongoing field trials. The DNA markers employed for genotype data were microsatellites (SSR), Amplified Fragment Length Polymorphism (AFLP) and Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) markers. A total 804 segregating marker loci (489 SSRs, 307 AFLPs and 8 PCR-RFLP) were used for final linkage analysis and map construction. The map of DA41 (ARK86D X ML161P) was 2398.8 cM long with 512 marker loci (368 SSRs, 135 AFLPs and 9 PCR-RFLPs), at an average 32 markers and a range of 15-59 markers per linkage group, and an average map density of 5cM. The linkage group length was 77.5 cM to 223 cM, with an average of 150 cM. Taking the yield components phenotype data on board resulted in the detection of 164 QTLs associated with oil yield components. The QTLs had an average confidence region of 15.4 cM and no marker interval exceeded 50 cM. In the DA41 population, cumulative QTL effects increased in tandem with the number of QTL markers, matching the QT+ allele for each of the traits tested. The many QTLs detected per trait suggested that the traits studied are polygenic with many genes of individual small effects on independent loci. However, the scope of the study did not rule out or rule in epistasis between different QTLs affecting a particular trait. Furthermore, several QTLs probably also show pleiotropic effects as seen by QTL clustering of inter-related traits on almost all the linkage groups, confirming the complexity of the genetic architecture of not only oil yield but also its components in the oil palm. The overall picture suggests that certain regions of the chromosomes are richer in the genes that affect the expression of a particular yield component trait and encompass pleiotropic, epistatic and heterotic effects. Hence, it will not be surprising if a large proportion of the identified additive effects from QTLs actually arise from digenic interactions between loci. For practical applications from this work, it will be necessary to test these yield component QTLs in a broader array of genetic backgrounds and in different environments. Also, more closely linked markers or flanking markers to the QTLs should be sought because recombinations between the markers and QTLs can occur when transferring the results from one population to another. Clearly, while this study has generated results that can be used in initial marker-assisted selection (MAS) for oil palm breeding, such as in population selection and enrichment, more detailed knowledge of marker-trait association will further contribute to more precise applications

Therapeutic insects

To some, insects invoke an uncomfortable feeling; phrases like creepy crawlies come to mind. But as the saying goes, don't judge a book by its cover; and so it is with insects. The ubiquitous cockroach (Blatta orientalis Linnaeus, Periplaneta americana Linnaeus, Periplaneta P. australasiae Fabricius) can be used to detoxify, soothe down blemishes, carbuncles and swellings, and reduce the poison if bitten by snake or insects. The cockroach is dried and the wings and feet discarded before use

Putative regulatory candidate genes for QTL linked to fruit traits in oil palm (Elaeis guineensis Jacq.)

Palm oil is among the most important vegetable oils, contributing to a quarter of the world’s oils and fats market. The oil palm (Elaeis guineensis Jacq.) fruitlets, which are the source of palm oil, vary from 8eight to 20 g in weight. Palm oil content in the fruitlets is approximately 45 – 50 % by weight and an increase in the percentage of mesocarp-to-fruit is likely to have a positive effect on oil yield. In this study, we report a quantitative trait loci (QTL) associated with two yield related components, namely fruit and mesocarp content in a commercial breeding population (Deli dura x Yangambi pisifera). The QTL confidence interval of about 12 cM (~6.7 Mbp) was fine-mapped with 31 markers (17 SNPs and 14 SSRs) consisting of 20 nuclear markers derived from the maternal parent, six paternal and five co-segregating markers. Interestingly, inheritance of the paternal alleles leads to a larger difference in both fruit and mesocarp weight, when comparing genotypes in the progeny palms. Candidate genes and transcription factors were mined from the QTL region by positioning markers on the oil palm EG5 genome build. Putative genes and transcription factors involved in various biological processes including flower organ development, flowering, photosynthesis, microtubule formation, nitrogen and lipid metabolism were identified within this QTL interval on pseudo-chromosome 3. This genome-based approach allowed us to identify a number of potential candidate gene markers associated with oil palm fruit and mesocarp weight which can be further evaluated for potential use in marker-assisted breeding

Integrated linkage map of FELDA's oil palm cross DA41 (ARK 86×ML 161) using MAPRF7 programme with Kosambi mapping function (Linkage Group I–IV).

<p>Marker names are shown to the right of each LG, with map distances (in cM) to the left. The map consists of 479 marker loci (331 SSRs, 142 AFLPs and 6 PCR–RFLPs) with 168 anchor points. Markers indicated in normal front are from map ARK86 while markers in italics are from map ML161, and markers representing an anchor point are underlined. Marker types and designations are as follows: SSRs (CNI, DHP, mEgCIR, PJ, sEg, sMg and sMo); RFLPs (CA, CB, MET and SFB); AFLPs (EAAC, EAAT, EACA, EACC, EACT, EAGA and EAGG).</p