Molecular Analysis of Synedrela Nodiflora ( L . ) Gaertn . Resistance Against Fomesafen using Rapd Markers

Both morphological and protein studies reveal that differences are observed between resistant Synedrella nodiflora against fomesafen and its susceptible wildtype. These are, however, more or less influenced by environmental factors, so that molecular analysis employing DNA markers is necessarily required. The methods involved total genomic DNA extraction using modified CTAB protocol following Doyle & Doyle (1990), RAPD marker amplification and visualization of RAPD markers prior to data analysis. Ten RAPD markers were used, but only seven of them showed polymorphism. Calculation of genetic distance and variation was carried out employing PopGen software. Based on the RAPD markers used in this study, it can be concluded that genetic distance between susceptible and resistant S. nodiflora is higher than that within susceptible samples supporting our previous morphological and protein data, although genetic variation among susceptible individuals seems to be significantly high.


Introduction
Synedrella nodiflora is a weed species commonly found to grow very rapidly in legume crops potentially causing serious agronomic problems.As many as 3,000 seeds ready to grow can be produced by a single plant, since they are not subject to dormancy.This eventually results in high variabilty.
When these diverse plant individuals are controlled with fomesafen at a lethal dose, considerably various resistance against the herbicide will arise, and recurrent application in a long period of time may increase the resistance Susceptible and resistant S. nodiflora against fomesafen show morphological differences, one of which is in terms of plant vigor.Resistant plants are larger in size in compare to that of its susceptible wildtype.
Moreover, the resistant type has also relatively better phylotaxis, which is represented by higher leaf area index.The plant is vigorous with wider and more compact leaves, indicating higher competitive capacity for survival than its susceptible wildtype, resulting in higher capacity to compete with legume crops, such as ground nut.Consequently, this reduces the yield of ground nut.Therefore, attention should be paid any time to prevent the development of resistant S. nodiflora in certain area, so that it is easier to manage.
The vigor of susceptible S. nodiflora varies morphologically, so that genetic differentiator among susceptible individuals is necessarily required to prevent potentially susceptible plants changing to resistant.Study on plastidal PPOase, an enzyme responsible for chlorophyll and heme biosynthesis, reveals that a protein band of 22.906 kDa observed in resistant S. nodiflora is absent in the susceptible wildtype.So, it is assumed that this protein band can be resistance marker of S. nodiflora against Reflex application (Dwiati & Susanto, 2015).
Both morphological and protein characters are, however, more or less influenced by environmental factors since they are phenotypes resulting from the interaction between genotype and environment.To provide more constant and accurate markers, molecular analysis by the use of DNA markers is necessarily required.RAPD is one of DNA markers widely used in the studies of genetic diversity in various crop species, including those in terms of plant resistance.Runtunuwu et al. (1999) reported the use of RAPD markers for coconut resistance against Phytophthora palmivora which is successfully identified by DNA polymorphism among five extremely resistant cultivars and another five extremely susceptible cultivars.Tartarini (1995) has also shown RAPD polymorphism for scab resistance among apple cultivars.Since no RAPD charac-terization for S. nodiflora resistance against fomesafen has been reported so far, here we are presenting RAPD profile to verify our previous morphology and protein studies.

Materials and Methods
Susceptible S. nodiflora wildtype seeds were obtained from Karang-wangkal Purwokerto, while the resistant types were generated by gradually increasing sublethal applications fomesa-fen on the wildtype plants for seven generations until individuals resistant to lethal dose were obtained.Fomesafen used in this study was formulated by Syngenta and known commercially as Reflex.The chemicals used included those for DNA extraction with modified CTAB protocol, PCR kit (Promega), RAPD primers (OPA7 5'-GAAACGGGTG-3', OPA8 5'-GTGACGTAGG-3', OPA11 5'- Technologies (Almeda, USA) and agarose electrophoresis compo-nents.The main equipments used were minibead beater (Biospec), genequant (General Electric), 1 4 , 0 0 0 r p m c e n t r i f u g e ( T h e r m o ) , thermocycler (Boeco), electrophoresis set (Biorad), UV transiluminator and digital camera.

Total genomic DNA extraction
Both fresh resistant and susceptible S. nodiflora leaves were weighed to 0.1 g and cut into small pieces before put into microtube with a bead.
Then 800 µl o preheated CTAB in 65 C for 30 minutes was added, after which the leaf pieces were crushed in minibead beater for four minutes.
o This sample was heated in 65 C for 60 minutes while gently mixing in every 10 minutes was conducted by inverting the tube before putting it in room tempera-ture for two minutes before 500 µl chloroform isoamyl alcohol was addded.This was then vortexed for five minutes and centrifugated at 12,000 rpm for 15 minutes.Supernatant was moved cautiously into new microtube and then mixed gently with 1/10 volume of 3M sodium acetate, after which 2/3 volume of cold isopropanol was added.The mixture was kept in freezer for 24 hours before centrifugated at 12,000 rpm for 10 minutes.Supernatant was discarded, while pellet was washed with 500 µl 70% ethanol.The suspension was centrifu-gated at 12,000 rpm for five minutes.Supernatant was removed, while pellet was air dried before dissolved in 50 µl TE buffer.The DNA solution was measured for concentration and purity using genequant and then stored o at 4 C. Once the DNA concentration was measured, alliquots of 2.5 ng/µl were made by diluting the solution stock (Doyle and Doyle, 1990 with modification).

RAPD marker amplification and electrophoresis
All of the seven RAPD primers used in this study result commonly in polymorphic bands in various plant species.PCR amplification of the RAPD markers was performed in a total volume of 10µl consisting of 5 µl Gotaq green, 2.25 µl NFW, 2.5 µl genomic DNA as PCR template and 0.25 µl primers.This PCR mixture was preheated at The PCR products were run in 1.5% gel electrophoresis gel at 75 V, 400 A for 40 minutes.After soaked in ethidium bromide, the gel was put on UV transiluminator and documented using digital camera.The RAPD profile was analyzed statistically using GenAlex.The visualized pattern of RAPD markers was converted into binary data (0 means fragment is absent and 1 means fragment is present).The calculation of genetic distance and variation was carried out employing PopGen software.

Results and Discussion
Resistant S. nodiflora leaves are found significantly larger than those of susceptible individuals.The resistant leaves are 8.76 ± 0.69 cm in length and 5.33 ± 0.44 cm in width, while the susceptible ones are 7.76 ± 0.34 cm in length and 5.05 ± 0.30 cm in width.
In addition to size, the positioning of resistant leaves seems likely to be better than that of susceptible ones, which is represented by relatively larger leaf area index.More vigorous plant with more ideal leaf positioning is also observed (Table 1), indicating that resistant S. nodiflora compete better for survival in compare to the susceptible wild type (Fig 1A and 1B).Nevertheless, leaf thickness of resistant individuals is less than that of susceptible ones.Different number of fragments are to some extent due to different allele frequencies in each locus.When the frequency of most common allele reaches 0.95 or more, the locus is said to be monomorphic, since the presence of the other rare allele can be neglected.Oppositely, when the frequency of most common allele is less than 0.95, it means that the other allele can also be found in the population and the locus is said to be polymorphic.The presence of certain fragments may increase heterozygosity indicating higher genetic diversity (Harris, 1994.Yuwono (2006) noted that a template DNA may not be successfully amplified by a primer because of no homologous site available between both.As well, the random primer used may anneal at two distant sites in the template causing the failure of DNA polymerase to amplify.
The amplified RAPD markers using seven selected primers were shown in Table 2.The DNA band intensity of individual primer is highly affected by primer binding site distribution in template DNA and the concentration and purity of template DNA containing polysaccharide and phenolic com-pounds.Primer competition for annealing sites in template DNA may lead to different number of amplified fragments (Roslim, 2001).
Polymorphism results from different positions of primer annealing at genomic DNA strand or different sequences among individuals.In addition, polymorphism can also be caused by substitution, deletion or insertion resulting in the loss of primer recognition sites (Salam, 1994).
It can be seen from Table 2 that polymorphism among individuals based on the amplified markers using seven selected primers exists.Polymorphism indicates genetic diversity of a species and its response to evolution process and predicts the existence of a species in the future (Solikhin, 2006).
The percentage of polymorphic bands obtained is sufficiently high ranging from 85.7 to 90.9%.Two primers (OPB-7 and OPB-10), however, show low polymorphism, i.e. 66.7 and 40.0%respectively (Table 2).Poerba and Martanti (2008) noted that primer selection for RAPD analysis influences the band polymorphism produced d u e t o s p e c i f i c a n n e a l i n g s i t e s .Consequently, the polymorphic DNA bands resulting from individual primer show differences both in number and size.Polymorphism exists both within susceptible S. nodiflora samples from Bogor and between these samples and that from Purwokerto, which is also shown by scatter plot diagram (Figure 4).Two samples of susceptible S. nodiflora from Purwokerto are present in quadrant II, while the other one lays on quadrant III.On the other hands, resistant S. nodiflora samples cluster in quadrant II, but they lay apart from the two sus ceptible samples, meaning that resistant and susceptible samples are genetically different from each other.Imron et al .(2000)noted that the level of genetic diversity determines genetic quality of a population.Lower genetic diversity may result in some deleterious traits, such as lower environmental adaptability including that against herbicide stress.Higher variation is, however, observed within the three susceptible samples from Purwokerto, since one of them is present in the same cluster as that for the resistant samples.

Conclusion
Based on the RAPD markers used in this study, it can be concluded that genetic distance between susceptible and resistant S. nodiflora against fomesafen is higher than that within susceptible samples supporting our previous morphological and protein data, a l t h o u g h g e n e t i c v a r i a t i o n a m o n g susceptible individuals seems to be significantly high.
5 min, followed by 30 PCR cycles o comprising denaturation at 95 C for 45 sec, o annealing at 36 C for 1 min and elongation at o 72 C for 1 min 30 sec respectively.Final o elongation was conducted at 72 C for 7 min o before stored at 4 C.

Figure 1 .
Figure 1.Performance of 7 week-old weed S. Nodiflora.A. susceptible B. resistant A B

Table 1 .
Comparison between susceptible and resistant S. nodiflora vigor

Table 3 .
Indeks similaritas antara sampel S. nodiflora resisten dan S. nodiflora rentan Similarity index between sample 3.1 and 3.2 is only 0.545 and that between sample 3.2 and 3.3 is only 0.688, meaning that the three susceptible S. nodiflora samples from Karangwangkal are relatively diverse.The scatter plot data corresponds to dendogram analysis illustrated in Figure5.It is shown that resistant and susceptible S. nodiflora is separated in two different clusters with a similarity coefficient of 0.68 and ground nut from Balikabi, Malang as outgroup sample.