Development of Gene-Based InDel Markers on Putative Drought Stress-Responsive Genes and Genetic Diversity of Durian (Durio zibethinus).

Insertion-deletion (InDel) markers are co-dominant, relatively abundant and practical for agarose gel genotyping. InDel polymorphism usually affects gene functions. Nucleotide sequences of durian (Durio zibethinus) are available, but InDel makers have not been well established. This study aimed to develop drought-related gene-based InDel markers for durian through bioinformatic analysis of RNA-Seq datasets. The polymorphism of the markers was verified in 24 durian genotypes local to Thailand. Bioinformatic analysis indicated 496 InDel loci having lengths more than 9 bp. To evaluate these InDel markers, 15 InDel loci were selected. Nine markers were successfully amplified a clear polymorphic band pattern on 2% agarose gel. The polymorphic information content (PIC) of these nine markers ranged from 0.1103 to 0.5808. The genetic distance between the 24 genotypes ranged from 0.222 to 0.889. The phylogeny based on the nine InDel loci distinguished the 24 genotypes and divided samples into four groups. This set of gene-based InDel markers on putative drought-responsive genes will be useful for genetic studies.

Potential receptors in Fenneropenaeus merguiensis ovary and role of saxophone, the bone morphogenetic protein receptor, in ovarian development.

Receptors, which play an initial role in signaling pathways in several physiological processes, including reproduction, are among the several molecular factors that control ovarian development in organisms. This study aimed to identify and study receptors potentially involved in controlling the reproductive process of female banana shrimp, Fenneropenaeus merguiensis. Ovarian transcriptomes derived from 4 developmental stages were generated by RNA sequencing. A total of 53,763 transcripts were obtained from the de novo assembled transcriptome, and 663 genes were identified as receptors. Among them, 185 receptors were differentially expressed during ovarian development. Fifteen of these differentially expressed receptors showed distinct expression patterns that were validated by RT-qPCR. Bone morphogenetic protein receptors (BMPR) and their signaling genes were investigated for their roles in shrimp vitellogenesis. The expressions of F. merguiensis saxophone (FmSax), a BMP type I receptor, and BMP type II receptor (FmBMPRII) as well as FmMad, FmMed, and FmSMAD3 were significantly altered during ovarian development. RNA interference was used to investigate the role of FmSax in vitellogenesis. The result indicated that the expression of vitellogenin (Vg) was significantly reduced in both ovary and hepatopancreas of FmSax-knockdown shrimp compared to control shrimp. Furthermore, in FmSax-silencing shrimp, FmBMPRII, FmMad, and FmMed expressions were decreased as well as Vg expression. These findings suggest that FmSax positively regulates Vg synthesis via the BMP signaling pathway.

Long non-coding RNA profile in banana shrimp, Fenneropenaeus merguiensis and the potential role of lncPV13 in vitellogenesis.

The long non-coding RNAs (lncRNAs) have been known to play important roles in several biological processes as well as in reproduction. This study aimed to identify lncRNA in ovary female banana shrimp, Fenneropenaeus merguiensis, and investigate the potential role of lncPV13 in the vitellogenesis. After the in silico identification of the ovarian transcriptome, a total of 24,733 putative lncRNAs were obtained, and only 147 putative lncRNAs were significantly differentially expressed among the ovarian development stages. To validate the in silico identification of lncRNAs, the 16 lncRNAs with the highest differential expression in the transcriptome analysis were evaluated by RT-qPCR. The 6 lncRNAs showed higher expression levels in the mature stage than in the previtellogenic stage and were found in several tissues such as in eyestalks, brains, thoracic ganglia, gills, and muscle. Furthermore, most candidate lncRNAs were amplifiable in Litopenaeus vannamei's and Penaeus monodon's DNA but not in Macrobrachium rosenbergii's DNA, suggesting some lncRNAs are expressed in a species-specific manner among penaeid shrimp. In this study, the lncPV13 was investigated for its vitellogenin regulating function by RNA interference. The result indicates that the lncPV13 expression was suppressed in the ovary on day 7 after the injection of double-stranded RNA specific to lncPV13 (dslncPV13), while vitellogenin (Vg) expression was significantly decreased. In contrast, the gonad inhibiting hormone (GIH) expression was significantly increased in the lncPV13 knockdown shrimp. However, the oocyte proliferation was not significantly different between control and lncPV13 knockdown shrimp. This suggests that lncPV13 regulate Vg synthesis through GIH inhibition. Finally, our findings provide lncRNA information and potential lncRNAs involved in the vitellogenesis of female banana shrimp.

Indel marker analysis of putative stress-related genes reveals genetic diversity and differentiation of rice landraces in peninsular Thailand.

Genetic assessment of rice landraces is important for germplasm evaluation and genetic resource utilization. Rice landraces in peninsular Thailand have adapted to unique environmental stresses over time and have great significance as a genetic resource for crop improvement. In this study, rice landraces derived from rice research centers and farmers from different areas of peninsular Thailand were genetically assessed using 16 polymorphic InDel markers from putative stress-related genes. A total of 36 alleles were obtained. The average PIC value was 0.27/marker. The FST varied from 0.46 to 1.00. Genetic diversity was observed both within and between populations. AMOVA indicated that genetic variations occurred mainly between populations (70%) rather than within populations (30%). The dendrogram, population structure, and PCoA scatter plot clearly demonstrated the differentiation of the two major groups, i.e., landraces from upland and lowland rice ecosystems. The unique alleles of Indel1922, -2543, -6746, -7447 and -8538, which lie in genes encoding putative WAX2, heavy metal-associated domain-containing protein, GA20ox2, PTF1, and PLETHORA2, respectively, were only found in rice from upland ecosystems. Putative WAX2, GA20ox2, and PLETHORA2 are likely related to drought and salt stress. Our findings demonstrate the diversity of landraces in peninsular Thailand. The preservation of these landraces should be facilitated with effective markers to maintain all variant alleles and to protect the genetic diversity. Indel1922, -2543, -6746, -7447 and -8538 have the potential to differentiate upland rice from lowland rice. Furthermore, Indel1922, -6746 and -8538 might be effective markers for drought and salt tolerance.

Cassava (Manihot esculenta) transcriptome analysis in response to infection by the fungus Colletotrichum gloeosporioides using an oligonucleotide-DNA microarray.

Cassava anthracnose disease (CAD), caused by the fungus Colletotrichum gloeosporioides f. sp. Manihotis, is a serious disease of cassava (Manihot esculenta) worldwide. In this study, we established a cassava oligonucleotide-DNA microarray representing 59,079 probes corresponding to approximately 30,000 genes based on original expressed sequence tags and RNA-seq information from cassava, and applied it to investigate the molecular mechanisms of resistance to fungal infection using two cassava cultivars, Huay Bong 60 (HB60, resistant to CAD) and Hanatee (HN, sensitive to CAD). Based on quantitative real-time reverse transcription PCR and expression profiling by the microarray, we showed that the expressions of various plant defense-related genes, such as pathogenesis-related (PR) genes, cell wall-related genes, detoxification enzyme, genes related to the response to bacterium, mitogen-activated protein kinase (MAPK), genes related to salicylic acid, jasmonic acid and ethylene pathways were higher in HB60 compared with HN. Our results indicated that the induction of PR genes in HB60 by fungal infection and the higher expressions of defense response-related genes in HB60 compared with HN are likely responsible for the fungal resistance in HB60. We also showed that the use of our cassava oligo microarray could improve our understanding of cassava molecular mechanisms related to environmental responses and development, and advance the molecular breeding of useful cassava plants.

A genome scan for quantitative trait loci affecting cyanogenic potential of cassava root in an outbred population.

BACKGROUND: Cassava (Manihot esculenta Crantz) can produce cyanide, a toxic compound, without self-injury. That ability was called the cyanogenic potential (CN). This project aimed to identify quantitative trait loci (QTL) associated with the CN in an outbred population derived from 'Hanatee' × 'Huay Bong 60', two contrasting cultivars. CN was evaluated in 2008 and in 2009 at Rayong province, and in 2009 at Lop Buri province, Thailand. CN was measured using a picrate paper kit. QTL analysis affecting CN was performed with 303 SSR markers. RESULTS: The phenotypic values showed continuous variation with transgressive segregation events with more (115 ppm) and less CN (15 ppm) than either parent ('Hanatee' had 33 ppm and 'Huay Bong 60' had 95 ppm). The linkage map consisted of 303 SSR markers, on 27 linkage groups with a map that encompassed 1,328 cM. The average marker interval was 5.8 cM. Five QTL underlying CN were detected. CN08R1from 2008 at Rayong, CN09R1and CN09R2 from 2009 at Rayong, and CN09L1 and CN09L2 from 2009 at Lop Buri were mapped on linkage group 2, 5, 10 and 11, respectively. Among all the identified QTL, CN09R1 was the most significantly associated with the CN trait with LOD score 5.75 and explained the greatest percentage of phenotypic variation (%Expl.) of 26%. CONCLUSIONS: Five new QTL affecting CN were successfully identified from 4 linkage groups. Discovery of these QTL can provide useful markers to assist in cassava breeding and studying genes affecting the trait.

Development of simple sequence repeat markers from expressed sequence tags of the black tiger shrimp (Penaeus monodon).

In this study, we describe the development of expressed sequence tag-simple sequence repeat markers from expressed sequence tags of the black tiger shrimp (Penaeus monodon) deposited in public sequence databases. A total of 46 primer pairs were designed and screened on 26 individuals of P. monodon from a natural population. Of these, 16 primer pairs showed polymorphic profiles with between two and five alleles per locus. The average unbiased and direct count heterozygosities were 0.4662 and 0.3516, respectively. Cross-amplification was tested with five individuals of Penaeus vannamei and polymorphic products were detected at five loci.