Patcharin Tanya

Genome sequence of mungbean and insights into evolution within Vigna species

Mungbean (Vigna radiata) is a fast-growing, warm-season legume crop that is primarily cultivated in developing countries of Asia. Here we construct a draft genome sequence of mungbean to facilitate genome research into the subgenus Ceratotropis, which includes several important dietary legumes in Asia, and to enable a better understanding of the evolution of leguminous species. Based on the de novo assembly of additional wild mungbean species, the divergence of what was eventually domesticated and the sampled wild mungbean species appears to have predated domestication. Moreover, the de novo assembly of a tetraploid Vigna species (V. reflexo-pilosa var. glabra) provides genomic evidence of a recent allopolyploid event. The species tree is constructed using de novo RNA-seq assemblies of 22 accessions of 18 Vigna species and protein sets of Glycine max. The present assembly of V. radiata var. radiata will facilitate genome research and accelerate molecular breeding of the subgenus Ceratotropis.

Genetic Diversity Among Jatropha and Jatropha-Related Species Based on ISSR Markers

Jatropha curcas (jatropha) is a potential biodiesel crop. A major limitation in production is that jatropha remains wild with low genetic variation. Related species/genera in the Euphorbiaceae can potentially be used for its genetic improvement. In this study, we employed inter-simple sequence repeats (ISSRs) to assess genetic variation among 30 accessions of jatropha, two accessions of bellyache bush (Jatropha gossypifolia), two accessions of spicy jatropha (Jatropha integerrima), two accessions of bottleplant shrub (Jatropha podagrica), and three accessions of castor bean hybrids. Genetic relationships were evaluated using 27 of 86 ISSR markers, yielding 307 polymorphic bands with polymorphism contents ranging from 0.76 to 0.95 for IMPN 1 and UBC 807 markers, respectively. Dice’s genetic similarity coefficient ranged from 0.39 to 0.99, which clearly separated the plant samples into seven groups at the coefficient of 0.48. The first group comprised J. curcas from Mexico, the second group comprised J. curcas from China and Vietnam, the third group comprised J. curcas from Thailand, the fourth group was J. integerrima, the fifth group was J. gossypifolia, the sixth group was J. podagrica, and the last and most distinct group was Ricinus communis. Analysis of molecular variance revealed that 63% of the variability was attributable to variation among groups, while 37% was due to variation within groups. Based on Nei’s genetic distance, the population from G2 (J. curcas from China) and G4 (J. curcas from Vietnam) had the least ISSR variability (0.0668), whereas G8 (R. communis) and Jatropha spp. displayed the highest distance (0.6005–0.7211).

New microsatellite markers classifying nontoxic and toxic Jatropha curcas

Jatropha curcas (jatropha) (2n = 2x = 22) is a shrub originated from Central America and spread to Africa and Asia by Portuguese traders during 18th century. This species belongs to the family Euphorbiaceae. Jatropha seed is high in oil content and considered promising as an alternative renewable and eco-friendly energy source, especially for biodiesel. Most jatropha accessions are high in seed phorbol ester (PE), a tumour inducing substance and thus called toxic accessions. There are some low PE (nontoxic) accessions in Mexico that are locally consumed as snack foods or sweets. The toxic and nontoxic accessions cannot be distinguished morphologically (Ghosh et al. 2007; Makkar et al. 2008), thus the villagers vegetatively propagate them to avoid contamination of high PE character. Currently, the only way to identify the high and low PE seeds is by seed analysis with high performance liquid chromatography (HPLC). Jatropha breeders are looking for a cheaper and quicker way to distinguish them such as through molecular markers, provided that the location(s) of gene(s) controlling high PE content is known. To achieve this, a number of markers are required to cover all the 11 linkage groups. Basha and Sujatha (2007) reported that among 400 RAPD primers tested, 339 produced bands and 267 were polymorphic. They also tested a set of 207 ISSR primers and found that 100 of them produced bands, giving 29 polymorphic primers. They used this information to investigate genetic variation among 42 accessions of jatropha from India and a nontoxic accession from Mexico. Basha and Sujatha (2009) reported genetic variation and interspecific hybrids of Jatropha species using RAPD and ISSR as nuclear specific markers, and chloroplast microsatellite markers as organelle specific markers. Ganesh et al. (2008) studied genetic diversity of eight Jatropha species collected in India. Eighteen of 26 RAPD primers classified them into a group of J. curcas versus a group of other seven Jatropha spp. Gupta et al. (2008)

Development and characterization of EST-SSR markers from Jatropha curcas EST database and their transferability across jatropha-related species/genus

Jatropha curcas (jatropha) is a multipurpose plant with potential as a raw material for biofuel. In the present study, a total of 43,349 expressed sequence tags (ESTs) from J. curcas were searched for type and frequency of simple sequence repeat (SSR) markers. Five thousand one hundred and seventy-five sequences were indentified to contain 6,108 SSRs with 90.8% simple and 9.2% compound repeat motifs. One hundred and sixty-three EST-SSRs were developed and used to evaluate the transferability and genetic relatedness among 4 accessions of J. curcas from China, Mexico, Thailand and Vietnam; 5 accessions of congeneric species, viz. J. gossypiifolia, dwarf J. integerrima, normal J. integerrima, J. multifida, J. podagrica; and Ricinus communis. The polymorphic markers showed 75.56–85.19% transferability among four species of Jatropha and 26.67% transferability across genera in Ricinus communis. Investigation of genetic relatedness showed that J. curcas and J. integerrima are closely related. EST-SSRs used in this study demonstrate a high efficiency of cross species/genera amplification and are useful for identifying genetic diversity of jatropha and its close taxa and to choose the desired related species for wide crossing to improve new varieties of jatropha. The markers can also be further exploited for genetic resource management and genetic improvement of related species/genera through marker-assisted breeding programs.

A PCR-based marker for a locus conferring aroma in vegetable soybean (Glycine max L.)

Vegetable soybean (Glycine max L.) is an important economic and nutritious crop in South and Southeast Asian countries and is increasingly grown in the Western Hemisphere. Aromatic vegetable soybean is a special group of soybean varieties that produce young pods containing a sweet aroma, which is produced mainly by the volatile compound 2-acetyl-1-pyrroline (2AP). Due to the aroma, the aromatic vegetable soybean commands higher market prices and gains wider acceptance from unfamiliar consumers. We have previously reported that the GmAMADH2 gene encodes an AMADH that regulates aroma (2AP) biosynthesis in soybeans (Arikit et al. 2010). A sequence variation involving a 2-bp deletion in exon 10 was found in this gene in all investigated aromatic varieties. In this study, a codominant PCR-based marker for the aroma trait in soybeans was designed based on the 2-bp deletion in GmAMADH2. The marker was verified in five aromatic and five non-aromatic varieties as well as in F2 soybean population segregating for aroma. The aromatic genotype with the 2-bp deletion was completely associated with the five aromatic soybean varieties as well as the aromatic progeny of the F2 population with seeds containing 2AP. Similarly, the non-aromatic genotype was associated with the five non-aromatic varieties and non-aromatic progeny. The perfect co-segregation of the marker genotypes and aroma phenotypes confirmed that the marker could be efficiently used for molecular breeding of soybeans for aroma.

Mapping of the genomic regions controlling seed storability in soybean (Glycine max L.)

Seed storability is especially important in the tropics due to high temperature and relative humidity of storage environment that cause rapid deterioration of seeds in storage. The objective of this study was to use SSR markers to identify genomic regions associated with quantitative trait loci (QTLs) controlling seed storability based on relative germination rate in the F2:3 population derived from a cross between vegetable soybean line (MJ0004-6) with poor longevity and landrace cultivar from Myanmar (R18500) with good longevity. The F2:4 seeds harvested in 2011 and 2012 were used to investigate seed storability. The F2 population was genotyped with 148 markers and the genetic map consisted of 128 SSR loci which converged into 38 linkage groups covering 1664.3 cM of soybean genome. Single marker analysis revealed that 13 markers from six linkage groups (C1, D2, E, F, J and L) were associated with seed storability. Composite interval mapping identified a total of three QTLs on linkage groups C1, F and L with phenotypic variance explained ranging from 8.79 to 13.43%. The R18500 alleles increased seed storability at all of the detected QTLs. No common QTLs were found for storability of seeds harvested in 2011 and 2012. This study agreed with previous reports in other crops that genotype by environment interaction plays an important role in expression of seed storability.

Development of interspecific and intergeneric hybrids among jatropha-related species and verification of the hybrids using EST-SSR markers

Jatropha curcas (jatropha) is an important non-edible oilseed crop with potential as a raw material for biofuel production. Although J. curcas has 30-35% oil content in its seeds, it has low seed yield (< 2 ton/ha) and thus cannot become an economically viable crop. However, jatropha has many related species and genera such as J. integerrima, J. multifida, J. podagrica and Ricinus communis that are suitable for interspecific and intergeneric hybridization. The desirable features that can be obtained from these species are high number of inflorescences from J. integerrima, large fruit size from J. multifida, high oil content from J. podagrica and raceme-type inflorescence from R. communis. We were initially successful in producing hybrids between J. curcas and these related species. Hybridity was confirmed using expressed sequence tag (EST)-simple sequence repeat markers developed from the J. curcas EST database.

Mapping quantitative trait loci for yield-related traits in soybean ( Glycine max L.)

Development of soybean cultivars with high seed yield is a major focus in soybean breeding programs. This study was conducted to identify genetic loci associated with seed yield-related traits in soybean and also to clarify consistency of the detected QTLs with QTLs found by previous researchers. A population of 135 F2:3 lines was developed from a cross between a vegetable soybean line (MJ0004-6) and a landrace cultivar from Myanmar (R18500). They were evaluated in the experimental field of Kasetsart University, Kamphaeng Saen, Nakhon Pathom, Thailand in a randomized complete block design with two replications each in 2011 and 2012 growing seasons. The two parents exhibited contrasting characteristics for most of the traits that were mapped. Analysis of variance showed that the main effects of genotype and environment (year) were significant for all studied traits. Genotype by environment interaction was also highly significant for all the traits. The population was genotyped by 149 polymorphic SSR markers and the genetic map consisted of 129 SSR loci which converged into 38 linkage groups covering 1156 cM of soybean genome. There were 10 QTLs significantly associated with seed yield-related traits across two seasons with single QTLs explaining between 5.0% to 21.9% of the phenotypic variation. Three of these QTLs were detected in both years for days to flowering, days to maturity and 100 seed weight. Most of the detected QTLs in our research were consistent with earlier QTLs reported by previous researchers. However, four novel QTLs including SF1, SF2 and SF3 on linkage groups L and N for seed filling period and PN1 on linkage group D1b for pod number were identified in the present study.

Interspecific jatropha hybrid as a new promising source of woody biomass

An interspecific hybrid Jatropha curcas (Jc) x Jatropha integerrima (Ji) was developed between jatropha (Jc) from Mexico and Ji from Thailand. Jc is a large canopy plant with soft wood, while Ji is a tall plant with semi-hard wood. The F-1 hybrid and their parents were grown under field conditions at a spacing of 1m x 1.5 m. One-year-old plants were harvested and determined for biomass yield, calorific value and chemical composition of wood, as well as for heterosis (hybrid vigour) of these characters. The F-1 plants gave an average fresh wood weight of 18.07 kg/plant, a moisture content of 46.56%, a dry wood weight of 9.56 kg/plant and a wood density of 0.62 g/cm(3). The F-1 hybrid had less ash (2.60%) than Jc (6.93%), but a higher heat value of wood (18.73 MJ/kg) than Jc (17.77 MJ/kg). Heterosis over mid-parent was very high and positive in dry wood weight, fresh wood weight and number of secondary branches at 542, 310 and 450%, respectively, while negative heterosis was found in moisture content (-24.86%). The desirable traits found in the F-1 hybrid can be fixed by cutting propagation of the selected plants.

Genome sequence of Jatropha curcas L., a non‐edible biodiesel plant, provides a resource to improve seed‐related traits

Summary Jatropha curcas (physic nut), a non‐edible oilseed crop, represents one of the most promising alternative energy sources due to its high seed oil content, rapid growth and adaptability to various environments. We report ~339 Mbp draft whole genome sequence of J. curcas var. Chai Nat using both the PacBio and Illumina sequencing platforms. We identified and categorized differentially expressed genes related to biosynthesis of lipid and toxic compound among four stages of seed development. Triacylglycerol (TAG), the major component of seed storage oil, is mainly synthesized by phospholipid:diacylglycerol acyltransferase in Jatropha, and continuous high expression of homologs of oleosin over seed development contributes to accumulation of high level of oil in kernels by preventing the breakdown of TAG. A physical cluster of genes for diterpenoid biosynthetic enzymes, including casbene synthases highly responsible for a toxic compound, phorbol ester, in seed cake, was syntenically highly conserved between Jatropha and castor bean. Transcriptomic analysis of female and male flowers revealed the up‐regulation of a dozen family of TFs in female flower. Additionally, we constructed a robust species tree enabling estimation of divergence times among nine Jatropha species and five commercial crops in Malpighiales order. Our results will help researchers and breeders increase energy efficiency of this important oil seed crop by improving yield and oil content, and eliminating toxic compound in seed cake for animal feed.