Evaluation of Mungbean Mutant Lines to Drought Stress and Their Genetic Relationships Using SSR Markers
Yuliasti1and Reflinur2
1Center for Application ISotope Radiation Jakarta Indonesia
2Indonesian Center for Agricultural Biotechnology and Genetic Resources Research and Development.JalanTentaraPelajar No. 3A, Bogor, West Java, Indonesia
A R T I C L E I N F O A B S T R A C T AIJ use only:
Received date Revised date Accepted date Keywords:
Mungbean, mutant,
drought tolerance, genetic variation, SSR markers.
Development of mungbean cultivars which is tolerance to drought stress through mutation breeding approach would enable us to anticipate climate change effect on reducing crop yield. The objective of this research was to evaluate yield performance of mungbean mutant lines that showed tolerance to drought stress, and to analyze their genetic diversity and relationship among mutant lines using SSR markers. The study was conducted in the experimental farmof Indonesian Center for Research and Development of Isotope and Radiation Technology (BATAN)in Muneng,East Java during the dry season. The experiment was laid out in a randomized block design with four replications. Five mutant lines, two parental lines as control were tested for evaluation of yield and drought tolerance under twoenvironments of two irrigation systems as treatment. The two environmental conditions consisted of optimal irrigation (at least three times: at planting, flowering and during pod filling) and Sub-optimal irrigation (two timesat planting and flowering). To evaluate genetic variation among selected mutant lines and their discrimination from parental lines in molecular level, a cluster analysis was performed using Unweighted Pair Group Method with Arithmetic Mean (UPGMA) in NTSYS software. The results showed that three mutant lines, including PsJ30, PsJ31, PsJ32 produced the highest grain yields of 1.17, 1.01, 1.04 ton/ha, respectively,compared to the other mutant lines andthe parents Gelatik (0.85 ton/ha) and Perkutut (0.87 ton/ha) as control check. Of those mutant lines,PSJ31 was the most tolerant to drought with sensitivity index value of 0.47. These mutant had been reliesed of new varieties Muri with high yield and tolerant to drought.Based on 23 SSR markers used for clustering analysis of those 3 selected mutant lines,9SSRmarkers (MB SS R033; satt137; MB SS R008; MB SS R203; MB SS R013; MB SS R021; MB SS R016 and MB SS R136; DMBSSR013 ) were successfully distinguished the three mungbean mutant lines. Taken together, the three elite mutant breeding lines obtained in present study are valuable resources for genetic variation in mungbean improvement.
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INTRODUCTION
Pulses are an integral part of Indonesian Agricultural. Among the pulse crops, Mungbean (VignaradiataL.), the third pulse crop of Indonesian has special importance in intensive crop production of the country for its short growing period, wide adaptability, low input requirements and the ability to improve the soil by fixing atmospheric nitrogen.
This crop is well suited to a large number of cropping systems and constitutes an important source of high quality protein in the cereal based diets of many people in Asia [1] (Khattaket al, 2001). Yield pulses is lesser than most of the cereals. The low productivity of the pulses may be
attributed to susceptibility to pathogenic microorganisms, asynchronous habit of pod maturity, shedding of flowers/ newly formed pods and indeterminate and long duration of growth resulting low seed yield per plant. In Indonesia, mungbean occupies about 0.7 tons per ha of dry grain, and the average potential yield of superior mungbean was only 1.20-1.75 tons per ha of dry grain. Some of the mungbean production centers are mostly arid area facing to drought
and poor in nutrientas the main
problem. Development of mungbean is a cheap solution to resolve the issue. A challenge of mungbean development in the arid area is how to
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increase the productivity and maintain the quality of land to sustainable production. However,
mungbean can be viewed as an
alternative commodity to be developed in such area, especially those with a low harvest index.
Drought is a worldwide problem, constraining global crop production and quality seriously. Moreover, recent global climate change issue has made this situation becomes more seriously. Therefore, it is important to develop stress tolerant crops to cope with the increasing food requirements and as drought is a major stress which adversely affects plant growth and productivity[2]
(Mahajan and Tuteja 2005).. Drought is often accompanied by relatively high temperatures, which promote evapotranspiration and affects photosynthetic kinetics, thus intensifying the effects of drought and further reducing crop yields. It is anticipated that the occurrence of drought in many food-producing regions will increase significantly in response to climate change [3] (Collins et al. 2008;
[4] Reynolds and Ortiz 2010).Drought stress eventually results in a wide gamut of various biochemical and physiological modifications (Garg 2010) . Induced mutation has played a very vital role in altering the genetic make up of genotipes not at chromosomal but even at molecular level. [7]
( Alan, 2007). Mutation breeding may be an effective tool for generating variability in the existing varieties and selecting desirable characters lines which would be proved to be an ideal crop for abiotic stress. This technique has been widely used in efforts to breed abiotic stress tolerance with some success [8] (Gaiet al. 2006,[9] Spreeth and De Ronde 2006). An intensive mungbean breeding effort in1Center for Application ISotope Radiation ( CAIR BATAN ) began in 1975 by mutation breeding, resulting in the release of Camar 1991 and Muri varieties 2013 which gave higher yield and tolerant to drought. Genetic relationships of four mungbean mutant lines have been evaluated.
Genetic relationships among accessions are helpful for designing future breeding efforts for yield, quality, and pest resistance improvement [10]
(Wang et al., 2006a). Evaluation of genetic diversity is very important for crop plant genetic resources. DNA-based molecular markers provide powerful tools in studying genetic diversity and
population structure techniques for analyzing molecular markers such as simple sequence repeats (SSRs) [11] (Chapuis & Estoup, 2007, [12]
Lung’aho et al., 2011), and single nucleotide polymorphisms (SNPs) [13] (Ganal, Altmann, &
Röder, 2009)are now available. SSRs or microsatellite markers, due to their codominance and high polymorphism, are particularly attractive for studying genetic structure and the relationships between species[14] Saxena, Saxena, Kumar, Hoisington, & Varshney, 2010; [15] Zhang, Blair, &
Wang, 2008). Microsatellites or simple sequence repeat (SSR) markers are short tandem repetitive DNA sequences with a repeat length of a few (one to five) base pairs. This marker have become a good choice for a wide range of applications such as genetic mapping and genome analysis, genotype identification and variety protection, seed purity evaluation and germplasm conservation, diversity studies, paternity determination and pedigree analysis, gene and quantitative trait locus analysis and marker-assisted breeding [16] (Archana and Jawali, 2007).
SSR have higher resolving power for detecting population structure and estimating genetic diversity than SNP in a sample of 303 accessions of domesticated soybean and its wild progenitor G. Soja ,this research was found by [17]
Li et al. (2010)., SSRs have been used successfully in estimation of genetic diversity and relationships among soybean genotypes in different populations [18] (Narvel et al., 2000a [19] Sutakom, 2004; The geneticrelationships among mutant lines are helpful for designingfuture breeding efforts for yield, quality and pest resistanceimprovement [10] (Wang et al., 2006a). New molecular breeding strategies such as marker-assisted recurrent selection (MARS) and genomic selection (GS) or genome-wide selection (GWS) are discussed as options to be integrated in crop improvement programmes for developing the next generation of drought-tolerant crops [20] (Reyazul Rouf Mir • Mainassara Zaman- Allah et l Nese Sreenivasulu • Richard Trethowan
•Rajeev K. Varshney. 2012}
Paucity of polymorphic molecular markers in mungbean has been a limiting factor in application of molecular tools for its genetic improvement. In the research, 23 SSR markers used
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for clustering analysis of those 3 selected mutant lines,9SSRmarkers (MB SS R033; satt137; MB SS R008; MB SS R203; MB SS R013; MB SS R021;
MB SS R016 and MB SS R136; DMBSSR013 ) were successfully distinguished the three mungbean mutant lines
The present study was aimed to evaluate yieldperformance of mungbean mutant lines toler- ance to drought stress, and to analyze their genetic diversity and relationship among mutant lines using SSR markers.
MATERIALS AND METHODS Plant Materials
A total of nine mungbean mutant lines were used in this study. These mutant lines were derived from induced mutations through gamma rays irradi- ation at the Indonesian Center for Research and De- velopment of Isotope and Radiation Technology (BATAN). The yield of these mutant lines under both normal and drought stress conditions were tested to select a promisingly mungbean mutant lines tolerant to drought stress with high yield. The two control varieties, including Gelatik and Perkutu were included in this study. The selected mutant lines which showed tolerant to drought and perform high yield were then subjected to molecular analysis using SSR markers in order to discriminate the se- lected mutant lines from the parental lines and to de- tect variation among selected mutant lines at molec- ular level
Field experiment
Direct drought tolerance test for those mu- tant lines was conducted in the experimental station of BATAN atMuneng,East Java province during the dry season of 2012.The experiment was laid out in a randomized block design RCBD).
Nine mutant lines with two varieties as control with four Replication and twoenvironments consisted oftwo irrigation system namely: optimal irrigation at least three times: at planting, flowering and during pod filling) and Sub-optimal irrigation, that is two timesat planting and flowering. The growth and yield of the selected drought tolerant plants were evaluated in the field condition.
Molecular analysis
DNA extraction and SSR analysis
Five mutant lines showing high yield under drought condition were selected and subjected to molecular analysis. DNA was extracted from young leaves using a standard cetyltrimethylammonium bromide (CTAB) method [21] (Gelvin and Schilper- oort, 1995). The concentration of the DNA samples was determined by a spectrophotometer and DNA samples were diluted to 50 ng/μl for analysis using SSR markers. A total 23 SSR primers derived from mungbean [22] (Chen et al 2004; [23] Somta et al 2008; [24] Somta et al 2009) and soybean se- quences[25] (Hisano et al 2007; [26] Xin et al 2012) were used in this study. PCR analysis was conducted in a volume of 15 μl and contained ge- nomic DNA, 0.25 μM of each primer (forward and reverse), 0.125 mM of each dNTP, 0.15 units of TaqDNA polymerase, 1 × reaction buffer. The PCR amplification was performed in a thermal cycler and consisted of an initial denaturation step of 3 min at 94°C, followed by 35 cycles at 94°C for 45 s, 47 to 57°C (depending upon the primer pair) for 30 sec, 72°C for 1 min, and 1 cycle of 72°C for 10 min.
PCR products were separated on 6% (w/v) non-de- naturation polyacrylamide gel and visualized by ethidium bromide. The ethidium bromide stained gels were documented using chemidoc (Biorad, USA).
Data analysis
Combined analysis of variance based on Random Complete Block Design (RCBD) and comparison of quantitative traits means based on Duncan’s new multiple range test (DNMRT) were performed in SAS (2001).
Drought tolerance was measured using Drought Sensitivity Index (S) as described by [27]
Fischer and Maurer (1978). In summary, S = (1- Y/Yp) / (1-X/Xp), where (Y) = average of pod num- ber, pod dry weight, root length, root dry weight of a drought-treated genotype; (Yp) = average of vari- ables of optimally grown genotype; (X) = variable value of all drought-treated genotypes; and (Xp) = variable values of all optimally grown genotype.
Plants were classified as tolerant if having sensitiv- ity Index of < 0.5, mildly tolerant if 0.5 <S < 1, and
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sensitive if S > 1.
PCR products amplified by SSR markers were scored visually for their presence (1) or ab- sence (0). To measure the informativeness of the markers, the polymorphism information content (PIC) for each SSR locus was calculated according to the formula [30] (Weir, 1996): PIC = 1- (Σpi2), where, i is the total number of alleles detected for a SSR marker, and pi is the frequency of the ith allele in the set of the 20 genotypes investigated. A den- drogram was constructed based on Jaccard’s simi- larity coefficient (with unweighted pair group method and arithmetic average (UPGMA) using the NTSYS-pc version 2.02 [31] (Rohlf, 2000).
RESULT AND DISCUSSION
The data on Mungbean yield was subjected to statistical analysis and significant differences were found among the mutant lines and control plant under drought condition (Table 1). Grain yield of mungbean crop is a function of cumulative effect of various yield components, which are influenced by genetic make-up of variety, various agronomic practices and environmental conditions. Table 1 also showed that mungbean could grow and yield better under drought condition.PSj S31 produced grain yield 1.17 ton ha-1. These yields were significantly higher compared to their original variety Gelatik (0.85 ton ha-1) and the national check varieties, Perkutut (0.87 ton ha-1) under drouhgt condition(Table 1).
Mutant lines /Variety
Yield (t/ha)
Optimal Condition Drought Condition
PsJ S-30 1.25 d 1.17 e
PsJ S- 31 1.53 b 1.01 f
PsJ S-32 1.46 bc 1.04 f
PsJ-6 1.31 d 0.95 g
PsJ-19 1.49 bc 1.00 fg
PsJ-21 1.58 b 0.95 g
PsJ-BII-17 1.71 a 0.96 g
PsJ-BII-5 1.37 c 0.95 g
PsJ-BII-15 1.35 c 1.01 f
Gelatik 1.31 d 0.85 h
Perkutut 1.35 c 0.87 h
Rerata (t/ha) 1.42 0.97
CV 5% 15. 25
The highest yielding was related to PSj S31 mungbean mutant lines. Under drought conditions mutant line PSj S31 had the best grain yield com- pared to the parent (Gelatik ) (Table 1). Drought is deleterious for plant growth, yield and mineral nutri- tion [28] (Garg et al. 2004; [ 29] Samarah et al.
2004) and is one of the largest limiting factors in agriculture [30] (Reddy et al. 2004). Considering Seed weight and pods number plant-1 is one of the major components of grain yield, the superiority of seed weight and pods number plant-1 is important in plant breeding to improve grain yield [31]
(Thrikawela and Bandara 1992). From these data it could be concluded that the mutant line PSj S31 was a promising line to be developed as dual purpose mungbean, as grain especially when grown in dry land. Mutant are candidates for new varieties high yield tolerant to drought.
Mutant lines/
Varietas
Sensitivitas Indeks
Berat 100
Ket .
Berat kerin
Ket .
Berat kering akar
Ket.
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butir g Biji
PsJ S-30 -0.395 T 1.100 TL 1.032 TL
PsJ S- 31 0.214 T 0.468 T -0.003 T
PsJ S-32 -3.253 T 0.839 MTL 0.337 TL
PsJ-6 0.627 MTL 0.793 MTL 0.527 TL
PsJ-19 -1.970 TL 1.027 TL 1.229 TL
PsJ-21 1.559 TL 1.371 TL 0.926 TL
PsJ-B11-17 2.166 TL 1.605 TL 1.568 TL
PsJ-BII-5 4.048 TL 0.916 MTL 2.158 TL
PsJ-BII-15 2.085 TL 0.678 MTL 1.335 TL
Gelatik 2.314 TL 1.111 TL 0.872 MTL
Perkutut 2.996 TL 0.996 MTL 0.737 MTL
Keterangan
= Toleran (T
= MidleToleran (MTL
= Supcetible (S)
The sensitivityindex data of mutants under drought condition are presented in Table 2. Based on calculation on S value on seed weight 100g -1;
seed dryand dry root weightand dry root weight PsJ S 31 mutant line had from drought condition were classified as tolerant with S value0.214; 0.468 and – 0.03, respectively, “Gelatik were sensitive had S value 2.314; 1.111; 0.872 and “perkutut” were sensitive with S value of 2.996; 0.996;
0773.Relatively high drought index sensitive value
was found for the mutant lines PsJ S31. This low sensitve index < 0.5 indicates that these mutant lines were more tolerant to the condition of drought with regard to the ability of producing grain yield.
In other words, their grain yield production was reduced only less than 30% under drought condition. PsJ S 31 mutant line described by BATAN as highly tolerant to drought stress compared to the parental lines Glatik and Perkutut national control variety.Stress, especially in the growing stage, reduces the capacity of the source plants for the source and sink is forced to balance the number of flowers and pod production to reduce the stress that can handle grain filling period and also reduced the final yield. The results of this study are in agreement with Asaduzzaman et al. [32]
(2008) investigations believe that moisture stress reduces grain yield of Mungbean and maximum negative effects of drought obtained with once irrigation during growth season. [33] Reddy et al.
(2004) also obtainedthe similarAsaduzzaman et al., [ 32] (2008). Rafiei Shirvan and Asgharipur [33]
(2009) also obtained.
SSR Analysis
SSRs evaluation carried out in present study could be useful to determine variation among mu- tant lines and to discriminate mutants from the wild type in molecular level.Of the total of 23 c SSR primers used in discriminatingof 3 selected mung- bean mutants and the parental lines, 12 primers ex- hibited polymorphism (Table 3). A total of 28alle- les were detected with an average alleles per locus was 0.85, ranging from 2 to 4. The PIC value, re- flecting allele diversity for particular marker, ranged from 0.19 (DMBSSR013, DMBSSR016, and DMB- SSR021) to 0.63 (MBSSR033) with an average of 0.35. Diversity index for the 12-marker set was in range of 0.21–0.69 with an average of 0.42. The mean PIC value and diversity index were indicative of low levels of genetic diversity in the tested mung- bean mutant lines.
Table 3.Summary statistics of 12 polymorphic SSR primers genotyped on mungbean mutant lines.
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SSR primer
Major.Allele.Frquenc
y AlleleNo GeneDiversityHeterozygosity PIC
MBSSR033 0.3750 4.0000 0.6875 1.0000 0.6299
MBSSR086 0.5000 3.0000 0.6250 1.0000 0.5547
Satt137 0.7500 2.0000 0.3750 0.5000 0.3047
MBSSR008 0.7500 2.0000 0.3750 0.0000 0.3047
MBSSR063 0.5000 2.0000 0.5000 1.0000 0.3750
MBSSR203 0.7500 2.0000 0.3750 0.5000 0.3047
Gaat47 0.5000 2.0000 0.5000 1.0000 0.3750
Gat216 0.5000 2.0000 0.5000 1.0000 0.3750
DMBSSR013 0.8750 2.0000 0.2188 0.2500 0.1948
DMBSSR021 0.8750 2.0000 0.2188 0.2500 0.1948
DMBSSR016 0.8750 2.0000 0.2188 0.2500 0.1948
DMBSSR136 0.7500 3.0000 0.4063 0.5000 0.3706
Mean 0.6667 2.3333 0.4167 0.6042 0.3482
Based on UPGMA cluster analysis, those mungbean mutant lines were clearly differed with the wild type in which they were grouped into two clusters, and two outliers (Figure 2). The major clus- ter I was of the wild type. While, the cluster II con- tained mutant lines which were separately grouped into cluster I. 2 out liers..Of the 23 SSR markers used in clustering 8 SSRmarkers (MB SS R033;
satt137; MB SS R008; MB SS R203; MB SS R013;
MB SS R021; MB SS R016 and MB SS R136) ) were successfully distinguished the three mungbean mutant lines.
M MB1 MB2 MB3 MB10 M
Figure 1. SSR profile of mungbeanmutant lines amplified by MBSSR086 { MB1, MB2, MB3, M10 MB1= Mungbean Mutant 1, MB2= Mungbean Mutant 2; MB3= Mungbean Mutan 3…., MB10=
wild type Lanes M, 100 bp
Coefficient
0.71 0.78 0.84 0.90 0.96
MB10MW
MB1
MB2
MB3
MB10
Figure 2. Clustering of mungbean and related Vigna species derived from an unweighted pair group mean average (UPGMA) cluster analysis using the Jaccard’s similarity coefficient based on adzuki bean.
SSR primers were chosen as DNA markers in present study because they produced multipl bands for one or more accession. These multiple bands may be due to annealing at more than one lo- cus or duplication of primer binding sites in the cross-species legumes. This indicates a high level of sequence conservation among the flanking regions of microsatellites. This sequence conservation formed the basis of cross-species utilization of SSR primers in assessing phylogenetic relationships across species and genera.
Conclution
Taken together, we conclude that the three elite mutant breeding lines obtained in present study are valuable resources for genetic variation to im- prove mungbean breeding program in Indonesia.
Through selection processes and direct screening for drought tolerance, the number of puta- tive mutant lines has been obtained. In dry season, the mutant PSj S 31 had significant higher grain yield compared to the original variety (Gelatik).
These mutant lines were released new variety tolerance to drought in 2013.
ACKNOWLEDGEMENT
Financial supports from PATIR BATAN are greatly appreciate. Thank the Ministry of re- search and Technology for including mungbean in the grant program 2009- 2011
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