Use of diversity array technology (DArT) to identify QTLs for physiological traits in mungbean (Vigna radiata) and soybean (Glycine max)

Vu, Thi Thuy Hang (2013) Use of diversity array technology (DArT) to identify QTLs for physiological traits in mungbean (Vigna radiata) and soybean (Glycine max). PhD thesis, James Cook University.

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This thesis reports the outcomes of research evaluating the application of Diversity Array Technology (DArT) to two important tropical legume crops, mungbean (Vigna radiata (L.) Wilczek) and soybean (Glycine max (L). Merrill.). Mungbean is comparatively under-studied, and is therefore likely to benefit from research to improve adaptation and productivity. Soybean has received more breeding attention than mungbean, but is still under-researched when compared with modern cereals like rice, wheat and maize. Drought remains an important constraint to soybean productivity in rainfed areas. DArT is a novel molecular marker technology that has been successfully applied in genetic studies in several plant species, but had not been previously applied to either of the crops of interest.

A DArT marker library/ array for mungbean was created using two mungbean cultivars, Berken and Kiloga, and two wild accessions V. radiata. ssp. sublobata, ACC 1 and ACC 87, and one or more accessions of five other Vigna species, viz. V. lanceolata, V. mungo, V. mungo var. sylvestris, V. trilobata and V. vexillata. A DArT library/ array for soybean was developed using two oilseed type cultivars, CPI 26671 and Valder, a landrace G2120, a wild soybean (G. soja) accession and one or more accessions of two wild Glycine species, G. falcata and G. tomentella. For each crop, two genomic complexity reduction methods, utilizing PstI/TaqI and PstI/BstNI restriction digests, were selected for DNA clonal library development and for the isolation in each case of 7,680 DArT clones from genomic representations of pooled DNA samples. While the PstI/BstNI method produced more polymorphic clones than PstI/TaqI for the soybean library, there was no significant difference between the two methods for the mungbean library. In the initial library evaluation, there were nearly 1,500 polymorphic clones identified for soybean. Polymorphism frequencies in mungbean were around twice those in soybean, reflecting greater diversity in the mungbean germplasm samples. The DArT marker transferability from soybean to mungbean (13.6%) was nearly five times higher than that from mungbean to soybean (3.1%). The percentage of DArT marker transferability between mungbean and several other Vigna species ranged from 3.4 to 20.2%. The genetic similarities among 11 diverse Vigna spp. samples, evaluated using the DArT mungbean library, were consistent with published information on these taxa.

These mungbean and soybean arrays were then used to evaluate the application of DArT markers for construction of genetic linkage maps and identifying putative qualitative and quantitative trait loci (QTLs). In mungbean, four F₅ recombinant inbred line (RIL) populations were derived from crosses between cultivars Berken and Kiloga and wild accessions ACC 1 and ACC 87. The F₅ RIL populations were evaluated for 54 qualitative and quantitative traits using plants grown in large pots on outdoor benches. There were large differences between the cultivated and wild parents and individual lines for all traits. Broad sense heritability estimates were moderate to high in most cases, with significant phenotypic correlations between many traits. A large number of polymorphic DArT markers were selected for the four RIL populations (1062 – 2013). The four mungbean linkage maps contained 672 to 981 DArT markers with segregation distortion levels higher in the ACC 87 (44.1 – 47.8%) than in the ACC 1 crosses (33.7 – 42.4%). Maps consisted of 15 – 19 linkage groups (LGs) and spanned lengths of 629.7 to 883.5 cM with average inter-marker distances of 0.9 – 1.2 cM. Various putative QTLs (77 – 122 QTLs) were identified for the vast majority of the 54 evaluated traits. In addition, the level of congruence across populations was reasonably strong.

In soybean, one F₇ and two F₆ RIL populations derived from crosses between CPI 26671, Valder and G2120 were used in phenotypic evaluation and QTL mapping with DArT markers for physiological drought stress response traits. The RILs were grown in deep cylindrical pots in the glasshouse, and exposed to severe water deficit followed by re-watering. Traits recorded included relative water content (RWC), epidermal conductance (gₑ), and recovery in growth following re-watering. The drought stress responses in the parental plants and RIL populations were broadly consistent with prior studies: As plant available water (PAW) in the soil declined, both RWC and gₑ declined, with the relation between RWC and gₑ exponential rather than linear as in previous studies. Analysis of variance showed significant differences at both population and genotypic levels for all key traits. However, there were large environmental effects on most traits, which resulted in high coefficients of variation and low estimates of broad sense heritability. The three individual linkage maps contained 196 – 409 DArT markers and 15 – 22 LGs with the aggregate length ranging from 409.4 to 516.7 cM. An integrated soybean map was constructed consisting of 759 DArT markers, 27 LGs and an expanded length of 762.2 cM. Total numbers of putative QTLs identified in the CPI 26671 x G2120 (CG) and VG (Valder x G2120) populations were 106 and 34 respectively. In each of the population, 10 LGs harboured QTLs associated with RWC, gₑ and recovery ability, of which five similar LGs contributed to drought tolerance. A BLAST (Basic Local Alignment Search Tool) search for sequences of 19 selected DArT markers linked to QTLs conditioning drought response traits indicated that 18 DArT markers were unique and aligned to 12 soybean chromosomes. Comparison of these DArT markers with other markers associated with drought-related QTLs in previous studies confirmed that five of them overlapped whereas the remaining 13 had not been previously identified. However, except for chromosome 15, the chromosomes with which the DArT QTLs in the CG and VG populations were associated were ones that had been shown to harbour drought-related QTLs in previous studies.

This study is the first showing DArT development in mungbean and soybean and its application for manipulating QTLs associated with wild physiological traits in mungbean and drought tolerance in soybean. DArT was successfully developed for both species, with more polymorphisms evident in the mungbean than in the soybean arrays. The study demonstrated that DArT provides high quality markers which can be used for diversity analyses, the construction of high-density genetic linkage maps and for QTL analysis. Meanwhile the marker transferability between arrays enabled plausible discrimination of genetic relationships between related taxa.

In both mungbean and soybean, the large numbers of DArT markers that were generated contributed to relatively tight resolution in the genetic maps, enhancing the power for QTL detection. Potentially useful QTLs/ markers were identified for many traits in mungbean, including some potentially useful ones such as resistance to powdery mildew and thrips, late flowering, hardseededness and perenniality, and in soybean, for RWC, gₑ and recovery after drought stress. In mungbean, further research is needed to identify an appropriate approach for the construction of an integrated map from the four RIL populations used in this study.In both species, follow-up research is required to verify the QTLs detected in this study before they can be used for marker-assisted selection in mungbean and soybean breeding programs. Nonetheless, the QTL analyses based on DArT markers in this study have been shown to be useful in the genetic dissection of both qualitative and quantitative traits in both species, and it is apparent that DArT markers will offer advantages for a range of molecular breeding and genomics applications.

Item ID: 40601
Item Type: Thesis (PhD)
Keywords: array processors; clonal library; crop biochemistry; crop breeding; crop physiology; crop selection; DArT; diversity array technology; drought stress response; drought; effect of drought on; epidermal conductance; genomic complexity reduction; genotyping; Glycine max; leaf area maintenance; morphological traits; morphology; mung bean; mungbean; physiological traits; plant DNA; plant genetics; plant phenology; polymorphism; QTL; quantitative trait loci; recovery; relative water content; soybean; transferability; Vigna radiata
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Publications arising from this thesis are available from the Related URLs field. The publications are:

Chapter 3: Vu, T.T. Hang, Lawn, R.J., Bielig, L.M., Molnar, S.J., Xia, L., and Kilian, A. (2012) Development and initial evaluation of diversity array technology for soybean and mungbean. Euphytica, 186 (3). pp. 741-754.

Other publications:

Vu, Hang T.T., James, A.T., Lawn, R.J., Bielig, L.M., and Kilian, A. (2015) Use of DArT molecular markers for QTL analysis of drought-stress responses in soybean. I. Phenotypic evaluation of traits. Crop & Pasture Science, 66 (8). pp. 802-816.

Vu, Hang T.T., Kilian, A., James, A.T., Bielig, L.M., and Lawn, R.J. (2015) Use of DArT molecular markers for QTL analysis of drought-stress responses in soybean. II. Marker identification and QTL analyses. Crop & Pasture Science, 66 (8). pp. 817-830.

Date Deposited: 01 Oct 2015 03:01
FoR Codes: 07 AGRICULTURAL AND VETERINARY SCIENCES > 0703 Crop and Pasture Production > 070303 Crop and Pasture Biochemistry and Physiology @ 50%
07 AGRICULTURAL AND VETERINARY SCIENCES > 0703 Crop and Pasture Production > 070305 Crop and Pasture Improvement (Selection and Breeding) @ 50%
SEO Codes: 82 PLANT PRODUCTION AND PLANT PRIMARY PRODUCTS > 8204 Summer Grains and Oilseeds > 820405 Soybeans @ 100%
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