Randomly amplified polymorphic DNA (RAPD) technique was applied to assess the genetic variations and phylogenetic relationships in genetic differentiation within 4 Chromium-treatment Leersia hexandra. The fresh leaves of Leersia hexandra cultivated on the condition of chrome pollution and exogenous organic acids were used as experimental material. The genomic DNA of Leersia hexandra was extracted by using CTAB method. The results showed that different samples of Leersia hexandra exhibited DNA polymorphism when using the random primer S43, S51and S55 as the primers in the RAPD reaction. One specific DNA band about 1000 bp was found in the sample which treated with 10 mmol/L concentration EDTA when used the S43 primer to RAPD. The obvious differences between different EDTA-treatment levels suggest that EDTA has certain effects on enrichment to heavy metals of Leersia hexandra, it will be more favored to Leersia hexandra accumulation of chromium when EDTA concentration increased.
Leersia hexandra Swartz, a perennial marshy plant, has been reported to be a Cr-accumulating plant with high tolerance to Cr. Under nutrient solution culture, it did not show any obvious symptoms of Cr toxicity when Cr concentrations in the leaves reached 5608 mg∙kg−1 dry weight [
Previous and continuous researches show that environmental factors have a significant impact on the plants [
Random Amplified Polymorphic DNA (RAPD) is a kind of molecular marker based on PCR. It uses l0 bp random primers amplifying the different DNA fragments in genome to show the polymorphism [
At present, there are not molecular biology reports on heavy metal chromium enrichment of L. hexandra, and little molecular ecology study on plants to heavy metal pollutants under the long -term effect. Reports on using RAPD to analyze heavy metal pollution on plant population genetic diversity are increasing recently, Li et al. (2007) used RAPD to analyze genetic diversity and genetic differentiation of Dicranopteris dichotoma popula- tions and lead-zinc mine tailings in population which grow in nature potential [
Plant material handling
L. hexandra collected from Guilin city, China, and artificial cultivated in sunlight greenhouse. 400 mg chrome metal Cr3+ (CrCl3) to per kg were added to soil when L. hexandra planted. EDTA solution of 0 mmol/L, 2.5 mmol/L, 5 mmol/L and 10 mmol/L concentration were prepared to handle L. hexandra plant, 3 repeats for every kind of concentration gradient, joined the appropriate EDTA solutions to L. hexandra plant every other week, and added a total of 3 times.
Reagents and solutions
2 × CTAB extracting buffer (2% CTAB, 1.4 mol/L NaCl, and 20 mmol/L EDTA, and 100 mmol/L Tris-Cl, pH 8); phenol/chloroform/isoamyl alcohol (25:24:1), chloroform/isoamyl alcohol (24:1), β-mercaptoethanol; 3 mol/L NaAc, (pH 5.2) isopropyl alcohol; TE solution (10 mmol/L Tris-Cl, 1 mmol/LEDTA, pH 8); 75% alcohol; alcohol; 1 × TAE buffer (40 mmol/L Tris, and 20 mmol/L HAc, 1 mmol/L EDTA, pH 8.0), Taq DNA polyme- rase; agarose; Lambda DNA/EcoR I + Hind III Marker.
Instruments and equipments
General refrigerators; HVE-50 high pressure sterilization pot (Japan Hirayama), LEGEND MICRO17 high speed centrifuge (United States saimofei Fisher); trace moving liquid (Japan Nichipet EX); T1Thermocyler am- plification apparatus (Germany BIOMETRA); DYY-12 Sanheng Multiple use Electrophoresis apparatus (Bei- jing Liuyi); DYCP -34A Electrophoresis tanks (Beijing Liuyi); MRS-1200048U scanner (Shanghai, Zhongjing technology); Furi FR-980 gel imager (Shanghai, Furi technology).
The primer sequences
The primer sequences used in this study are shown in
Extraction and purification for L. hexandra genomic DNA
NO | (5′→3′) | NO | (5′→3′) | NO | (5′→3′) | NO | (5′→3′) |
---|---|---|---|---|---|---|---|
S41 | ACCGCGAAGG | S51 | AGCGCCATTG | S82 | GGCACTGAGG | S94 | GGATGAGACC |
S42 | GGACCCAACC | S52 | CACCGTATCC | S83 | GAGCCCTCCA | S95 | ACTGGGACTC |
S43 | GTCGCCGTCA | S53 | GGGGTGACGA | S84 | AGCGTGTCTG | S96 | AGCGTCCTCC |
S44 | TCTGGTGAGG | S54 | CTTCCCCAAG | S85 | CTGAGACGGA | S97 | ACGACCGACA |
S45 | TGAGCGGACA | S55 | CATCCGTGCT | S86 | GTGCCTAACC | S98 | GGCTCATGTG |
S46 | ACCTGAACGG | S56 | AGGGCGTAAG | S87 | GAACCTGCGG | S99 | GTCAGGGCAA |
S47 | TTGGCACGGG | S57 | TTTCCCACGG | S88 | TCACGTCCAC | S60 | ACCCGGTCAC |
S48 | GTGTGCCCCA | S58 | GAGAGCCAAC | S89 | CTGACGTCAC | S81 | CTACGGAGGA |
S49 | CTCTGGAGAC | S59 | CTGGGGACTT | S92 | CAGCTCACGA | S93 | CTCTCCGCCA |
Applied an improved ctab method potential [
Primers screening
Used L. hexandra genomic DNA extracted as DNA templates for RAPD random primers screening. Primers are used in this research process all synthesized by Shanghai bio-engineering technology services company li- mited. Random primers with clear RAPD amplification and stability response were screened for final primers of L. hexandra RAPD molecular markers.
RAPD amplification reaction of L. hexandra
We used L. hexandra genomic DNA extracted as DNA templates for RAPD amplification reaction. Opti- mized reaction system: 25 μl total volume, 18.5 μl double distilled water, 2.5 μl 10 uffer liquid, 2 μl Mg2+ con- centration for 20 mmol/L, 0.5 μl dNTP concentration for 200 μmol/L, 0.5 μl primer concentration for 0.4 μmol/L, 1 μl template DNA originated from DNA extraction diluted 4 times with TE buffer liquid, 2U/25 μl Taq enzyme. Circulation system after optimized: for 5 min 94˚C predegeneration, then for 40 cycles, followed by 1 min 94˚C predegeneration, 1 min 36˚C annealing, 1 min 72˚C stretch. Last, 7 min 72˚C complete the extension potential [
Agarose gel electrophoresis results of L. hexandra leaves genomic DNA is shown in
Used L. hexandra genomic DNA as a template, for screening of the 46 UBC random primers, results showed that 29 UBC hadn’t amplified products or obscured and couldn’t tell, other 17 UBC random primers amplified a strip of clear and stable, that account for 36.9% of total primers. They varied approximately 200 bp or 3000 bp size, each primer amplification of fragments between changes in 1 - 7. Part L. hexandra leaves DNA RAPD primers screened results are as shown in
Screened 46 random primers (see
mmol/L and the third sample appeared DNA polymorphism at about 564 bp (as shown by the arrow in the 3rd sample). EDTA concentration of the fourth and fifth sample was 5 mmol/L, and the fifth sample appeared DNA polymorphism at about 700 bp (as shown by the arrow in the 5th sample). EDTA concentration of the sixth and seventh sample was 10 mmol/L, and the 7th appeared DNA polymorphism at about 1000 bp (as shown by the arrow in the 7th sample).
EDTA concentration of the first was 0 mmol/L (contrast). Comparison between the second and third and the first samples, the third appeared DNA polymorphism at about 564 bp (as shown by the arrow in the 3rd sample). Comparison between the fourth and fifth sample and the first sample, both appeared strip absence at about 1375 bp (absence strip as shown by the arrow in the first sample), in addition, the fourth sample appeared strip ab- sence at about 700 bp (absence strip as shown by the arrow in the first sample). Comparison between the sixth and seventh and the first sample, the sixth sample appeared strip absence at about 700 bp (as shown by the arrow in the first sample), and the seventh sample appeared DNA polymorphism at about 1000 bp (as shown by the arrow in the 7th sample).
RAPD amplification results of primer S51
EDTA concentration of the first was 0 mmol/L (contrast). Comparison between the second and third and the first sample, both appeared DNA polymorphism at about 947 bp (as shown by the arrow in the 2nd and 3rd sample). Comparison between the fourth and fifth sample and the first sample, both appeared DNA polymor- phism at about 947 bp (as shown by the arrow in the 4th and 5th sample), in addition, the 5th sample appeared DNA polymorphism at about 1904 bp and 3530 bp ( as shown by the arrow in the 5th sample), and both the samples appeared strip absence at about 700 bp (strip absence as shown by the arrow in the first sample). Compared to the sixth and seventh sample, the contrast showed no difference.
RAPD amplification results of primer S55
EDTA concentration of the first was 0 mmol/L (contrast). Comparison between the second, the third and the first sample, the second sample appeared DNA polymorphism at about 1548 bp (as shown by the arrow in the 2nd sample). Comparison between the fourth, the fifth sample and the first sample, the fifth sample appeared DNA polymorphism at about 947 bp (as shown by the arrow in the 5th sample). Comparison between the sixth and seventh and the first sample, both samples appeared DNA polymorphism at about 1548 bp (as shown by the arrow in the 6th and 7th sample).
Study on the evolution of pollution can help to understand how plants adapt to the environment and evolve, to understand biological adaptation mechanism under pollution. Knowledge on the genetic diversity can be used in future breeding programs potential [
Heavy metal pollution is an important form of soil pollution, the past researches focused on heavy metal toxic effect and mechanism of plants, and study on migration, accumulation, distribution and other aspects on heavy metals in the plant’s tissues, organs and ecological systems potential [
This study first screen primers of L. hexandra RAPD polymorphicing, and screen random primers S43 (GTCGCCGTCA), S51 (AGCGCCATTG), S55 (CATCCGTGCT) from the 46 random primers to be random primers of L. hexandra RAPD molecular markers preliminary. And then used S43, S51, S55 as primers on the polymorphism of different samples for research. The combined use of both RAPD and ISSR markers for the genetic analysis of the cultivar varieties have been performed earlier potential [
Compared EDTA concentration of 5 mM /kg, 10 mM /kg, 2.5 mM /kg with the control, the former expansion is larger than that of the latter. Different degrees of difference in comparison between different EDTA concentrations and control of treated RAPD results of L. hexandra, this shows that EDTA has certain effects during enrichment of L. hexandra to heavy metals. But perhaps, in a certain scope, it will be more favourable to L. hexandra accumulation of chromium metal when EDTA concentration increased. L. hexandra in the accumula- tion of chromium metal is also the process of chromium metal tolerance, and cause changes in gene eventually, to make it more resistant chromium metal against.
Different L. hexandra samples exhibited DNA polymorphism when using the random primer S43, S51 and S55 as the primers in the RAPD reaction. One specific DNA band about 1000 bp was found in the sample which was treated with 10 mmol/L concentration EDTA when used the S43 primer to RAPD. The obvious differences be- tween different EDTA-treatment levels suggest that EDTA has not much effects on enrichment to heavy metals of L. hexandra, so we should do more work to research the L. hexandra accumulation of chromium mechanism when EDTA concentration increased.
This research was funded by Guangxi Nature Science of Foundation (2011GXNSFA018012), Key Discipline Physical Geography Established in Guilin University of Technology and Guangxi Key Laboratory of Hidden Metallic Ore Deposits Exploration Guilin University of Technology, Guilin, China.