Cross-specific amplification of microsatellite DNA markers in Shorea platyclados

In the megadiverse forests of Southeast Asia, hundreds of timber species are economically important but the population genetics of only a few taxa are known. Cross-specific amplification of microsatellite loci among closely related taxa could enhance our ability to study and manage previously unstudied species. We successfully utilized STMS markers in Shorea platyclados, originally developed for Shorea curtisii. The six primer pairs we tried successfully produced PCR products of expected sizes. The number of alleles observed ranged from 10 to 14 and an average of 12 alleles were detected per locus. A high expected and observed heterozygosity was observed and it ranges from 0.718 to 0.827 among all populations across all six loci tested. Microsatellite DNA markers are highly polymorphic, co-dominant, reproducible, and amenable to high throughput genetic analyses. Overall, the cross- specific amplification of microsatellite loci appears to be complicated by numerous factors. While the approach may be effective for local management and conservation ofpoorly known species, the results must be carefully interpreted.

Detecting mislabeling and identifying unique progeny in Acacia mapping population using SNP markers

Acacia hybrids offer a great potential for paper industry in Southeast Asia due to their fast growth and ability to grow on abandoned or marginal lands. Breeding Acacia hybrids with desirable traits can be achieved through marker assisted selection(MAS) breeding. To develop a MAS program requires development of linkage maps and QTL analysis. Two mapping populations were developed through interspecific hybridization for linkage mapping and QTL analysis. All seeds per pod were cultured initially to improve hybrid yield as quality and density of linkage mapping is affected by the size of the mapping population. Progenies from two mapping populations were field planted for phenotypic and genotypic evaluation at three locations in Malaysia,(1) Forest Research Institute Malaysia field station at Segamat, Johor,(2) Borneo Tree Seeds and Seedlings Supplies Sdn, Bhd.(BTS) field trial site at Bintulu, Sarawak, and(3) Asiaprima RCF field trial site at Lancang, Pahang. During field planting, mislabeling was reported at Segamat, Johor, and a similar problem was suspected for Bintulu, Sarawak. Early screening with two isozymes effectively selected hybrid progenies, and these hybrids were subsequently further confirmed by using species-specific SNPs. During field planting, clonal mislabeling was reported and later confirmed by using a small set of STMS markers. A large set of SNPs were also used to screen all ramets in both populations. A total of 65.36% mislabeled ramets were encountered in the wood density population and 60.34% in the fibre length mapping population. No interpopulation pollen contamination was detected because all ramets found their match within the same population in question.However, mislabeling was detected among ramets of the same population. Mislabeled individuals were identified and grouped as they originated from 93 pods for wood density and 53 pods for fibre length mapping populations.On average 2 meiotically unique seeds per pod(179 seeds/93 pods) for wood density and 3 meiotically unique seeds per pod(174 seeds/53 pods) for fibre length mapping population were found. A single step statistical method was used to evaluate the most informative set of SNPs that could subsequently be used for routine checks for mislabeling in multi-location field trials and for labelling superior clones to protect breeder’s rights. A preliminary set of SNPs with a high degree of informativeness was selected for the mislabeling analysis in conjunction with an assignment test. Two subsets were successfully identified,i.e., 51 SNPs for wood density and 64 SNPs for fibre length mapping populations to identify all mislabeled ramets which had been previously identified. Mislabeling seems to be a common problem due to the complexity involved in the production of mapping populations. Therefore, checking for mislabeling is imperative for breeding activities and for analyses such as linkage mapping in which a correlation between genotypic and phenotypic data is determined.

Evaluation and Bulked Segregant Analysis of Major Yield QTL qtl12.1 Introgressed into Indigenous Elite Line for Low Water Availability under Water Stress

Near isogenic lines carrying large-effect QTL （qtl2.1）, which has a consistent influence on grain yield under upland drought stress conditions in a wide range of environments, were evaluated under water stress in the fields. The line which gave higher yield under drought was crossed with a local elite line, PMK3, and forwarded to F2：3 generation. Significant variation was found among the F2：3 lines for agronomic traits under water stress in the fields. Low to high broad sense heritability （H） for investigated traits was also found. Water stress indicators such as leaf rolling and leaf drying were negatively correlated with plant height, biomass and grain yield under stress. Bulked segregant analysis （BSA） was performed with the markers in the vicinity of qUl2.1, and RM27933 was found to be segregated perfectly well in individual components of drought resistant and drought susceptible bulks which were bulked based on yield under water stress among F2：3 lines. Hence, this simple and breeder friendly marker, RM27933, may be useful as a potentially valuable candidate marker for the transfer of the QTL qtl12.1 in the regional breeding program. Bioinformatic analysis of the DNA sequence of the qtl12.1 region was also done to identify and analyze positional candidate genes associated with this QTL and to ascertain the putative molecular basis of qUl2.1.

Intraspecific genetic variability,differentiation and evolutionary relationships revealed through microsatellite loci in seven economically important Calamus species

Population density,species richness and critical population parameters are crucial in determining the levels of gene diversity in dioecious species of the genus Calamus.The extent of intraspecific and intrageneric genetic variability in Calamus from the southern Western Ghats of India was studied using 26 microsatellite markers by sampling 227 individuals belonging to seven economically important species.The heterozygosity of microsatellite loci ranged from zero to 0.78.Average gene diversity within species was 0.13;in all species it was 0.18 and amongst species was 0.06.The Shannon Information Index was the lowest for Calamus metzianus(0.11),whereas it ranged from 0.16 to 0.26 for other species.The expected heterozygosity varied from 0.08 to 0.18.Calamus hookerianus and Calamu travancoricus showed the highest genetic differentiation(44%)revealed through Fst values,whereas the lowest(22%)was observed between Calamus gamblei and Calamu thwaitesii.Population structuring and phylogenetic analysis differentiated the seven species.Due to overexploitation and loss of rare alleles,small populations could lead to fertilization between closely related individuals,resulting in inbreeding and increasing the risk of extinction.This could be important for species such as C.metzianus where allelic polymorphism was 23%,whereas for all other species it was 38%to 46%.Genetic diversity“micro-hotspots”were identified from the protected area network of the southern and central Western Ghats with highest observed heterozygosity.Four microhotspots from the Agasthyamalai Biosphere Reserve and the Pushpagiri Wildlife Sanctuary may be possible for long-term conservation programs.The findings of this study lay a strong foundation for strengthening protected area networks,especially areas with intermediate levels of disturbance.

Characterization of mean stem density,fibre length and lignin from two Acacia species and their hybrid

The objective of this study was to compare the wood properties related to wood pulp quality of two widely planted Acacia species viz.Acacia mangium Willd.and Acacia auriculiformis A.Cunn.Ex Benth.and their hybrid.Acid insoluble lignin content(Klason),mean stem density and fibre length differed considerably among the species and hybrids.A.mangium possessed a high percent of lignin content compared to A.auriculiformis and the Acacia hybrid.However,mean stem density of A.auriculiformis was higher than A.mangium and the hybrid.Fibre length of heartwood tissues was generally shorter than that of sapwood tissues.The hybrid had longer fibres than the parent species.Lignin was negatively correlated with mean stem density.Generally,the wood properties of the hybrid were superior to its parent species.The significant intraspecific variation observed for wood properties of Acacia species could be used in breeding superior hybrids combining desirable traits of the two species.Considering thedifficulty involved in accurately measuring the lignin content compared to mean stem density,selection for plants with low lignin content can be achieved by indirect selection of high mean stem density.