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GENETIC DIVERSITY ACTIVE COMPOUND ANALYSES OF A SUBSET
OF Coix lacryma-jobi L.
1Agricultural college, Guangxi University, Nanning 530004, China Hau. ThiPhucDuong2-
2National institute of medicinal materials MOH, Vietnam
The genetic diversity of 14 Coixlachryma-jobi L. cultivars were studied using simple sequence
repeats ( SSR)markers．Twelve SSR primers giving stable amplified band pattern detected 340
alleles in the collection. The average number of alleles per SSR locus were 28.33 with a range
from 15 to 45．The value of polymorphism information content(PIC) for each SSR locus varied
from 0.5764 to 0.7709 with average of 0.7001. The 14 lines were divided into 5 groups by UPGMA method based on SSR fingerprinting. Type 1: Longlin # 1, Xilin # 1, ZYYB12, Xingrenxiaobaige, qianyin # 1; Type 2: Longlin # 2, pin zhong # 1,pinzhong # 2, pinzhong # 0, pinzhong # 10, pinzhong # 11; Type 3: Yuenan huakemi; Type 4: XYYBT; Type 5: XYYH12-1.The clustered results were similar to thatbased on geographical resource and germplasmic genealogy. Using SSR markers could be an accurate and reliable method to study diversity of Coixlachryma-jobi L. germplasm.
Keywords: Coixlachryma-jobi L; Germplasm; Genetic diversity; SSR
Coix lachrymal-jobi L. is of the Poaceae. It has been used as a traditional popular medicine in ASIAN the world generally and in China, Vietnamese particularly. Coix root has been used to treat urinary tract infection inflamation, calculus of kidney, hyronephrosis….(Lợi,2006, Lee
MY,2008), Coix seed is a tonic. There is several benefitsfor the elder and childrenIt is good for
old people and childs(Lee MY,2008, Takahashi Michiko,1986). Specially, it supports to improve the secreted milk of breastfeeding women. (Lee MY,2008, Takahashi Michiko,1986).It is used as a remedy for milk supply to bearing baby women. Recently, researches which are done both domestically and abroad showing that Coix really helps to lower the level of sugar in blood and fight against cancer(Takahashi Michiko,1986).
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crops maize and oryza sativa in this research to study Coix lacryma-jobi’s genetic diversity and measure its family relationship. markers were used SSR repeating number varies significantly among individual genomes of different organisms, hence SSR can reveal higher polymorphism. SSR is a co-dominant marker featuring favorable repeatability and stability and can be analyzed with simple and convenient methods. (Kojima T, 1998, Powell W, 1996) SSR molecular marker technology has been widely applied in evaluation of plant germplasm resources, analysis of genetic diversity and genetic relations between breeding materials as well as identification, protection, development and utilization of breeding varieties, hence facilitating the direct selection of breeding materials from genetic materials (Hu Li-Qin,2015).
MATERIALS AND METHODS
Table 1 below shows fourteen Coix cultivars used in this study, in which thirteen Coix cultivars were often cultivated in China and one Coix cultivar often cultivated in Vietnam, named Yuenan huakemi (C13). This sample is selected and evaluated from six different ecological regions in Vietnam.
Table 1. Fourteen Coix cultivars used in this study
C3 Xilin #1
C4 Pinzhong #0
C5 Pinzhong #1
C6 Pinzhong #2
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C13 Yuenan huakemi
C14 Qianyin #1
The experiment was conducted in the farm yield of Guangxi University (22°51’ N, 108°17’ E, 78 m asl), Nanning, Guangxi Province, China in early Coix-growing season in 2014. The site was located in a subtropical monsoon climate zone. Daily mean temperature and solar radiation during the Coix-growing season were 25.4°C and 12.3 MJm-2d-1, respectively (Vantage Pro2
weather station, Davis Instruments Corp., Hayward, CA, USA). The soil in field Coix paddy field were taken from the research farm. The soil was an Ultisol (USDA Taxonomy) with pH 6.62, organic matter 30.59 g kg-1, NaOH hydrolysable N 129.5mg kg-1, Olsen P 10.08 mgkg-1 and NH4OAc extracted K 72.94 mg kg-1. Experiments with one experimental factorwere used. 14
cultivars, labeled from C1-C14 were set as independent variable. Each treatment was replicated three times, placed in random complex blocks. Area of each plot was set 10 m2. The total area of
experiment was 420 m2.
The amount of fertilizersis used 225kg N + 375 kg P2O5 + 300 kg KClfor each hectare. The fertilizers method included: pre plant application 40% N + 100% P2O5 + 50% KCl; seedbed application 15% N; tillering application 40% N + 50% KCl; earing application 5% N. Size of cultivating places were specified 55 cm × 40 cm.
SSR Analysis Method for Coix Lacryma-Jobi
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frequency of locus i), the genetic similarity (GS) between the materials was measured as per GS = m/ ( m + n) (m means number of common bands between genotypes and n means the number of differentially expressed bands between genotypes) and the genetic distance (GD) was measured by the equation GD = 1 － GS. (Huan Su-Hua,2004, Li Xin-Hai,2000, Yuan Li-Xing,2000).
Identification of SSR Molecular Markers
PCR Reaction System. The PCR reaction system is presented in Table 2.
Table 2 SSR-PCRreaction system
Reaction components 20ul Usage amount
Ultra-pure water 14.4 µL
10×Buffer 2.00 µL
10mmol/LdNTPs 0.40 µL
10µmol/L Upper primer 1.00 µL
10µmol/L Lower primer 1.00 µL
DNA(40g/µL) Template 1.00 µL
TaqDNA Polymerase (5U/ul) 0.20 µL
The PCR procedure was used the NPTII gene consisted of an initial denaturant at 94℃ for 5 minute, follwed by 35 cycles of 94℃for 30 s, 53℃ for 30 s, 72℃for 5minute and a final 10 – minute elongation step at 10℃.
Agarose Gel Electrophoresis as PCR Production: The final production of PCR reaction was treated in the 2.5% to 3.5% agarose gel electrophoresis and the Gel Rednucleic acid dye, and through 140V voltage for 2 hours and finally imaged on the gel ultraviolet imager and then the imaging result was stored on the computer.
www.ijsernet.org Page 64 RESULTS AND DISCUSSION
Analysis of SSR Marking Result
This study implemented comparative selection among 3000 pairs of random primers for oryza sativa and 40 pairs of random primers for maize and took 12 pairs (8 for oryza sativa and 4 for maize) with favorable polymorphism for SSR analysis. SSR detected 340 allele variations on 14 subject coix lacryma-jobi germplasms and 28.33 alleles on average (ranging from 15 to 45) at each SSR locus. The PIC values of SSR loci ranged from 0.5764 to 0.7709 and averaged at 0.7001. Primer C10291 had the largest PIC while Primer ccl2 had the smallest PIC (see Table 3). Figure 1 is the electrophoretogram of Primer RM1151 with the most allele variations detected by SSR analysis and shows that 1kb marker produced 9 fairly clear and separated bands. In fact, the bands of all varieties were fairly clear and separated, but XYYBT (Variety 9) and XYYH12-1 (Variety 11) showed no bands in their lanes. Analysis said the weak mismatching between the selected primers and Variety 9 and 11could be the cause. Overall, the PIC values of all primers were not high probably due to the fact that the primers were not extracted from coix lacryma-jobi.
Fig.1 SSR analysis of primer named RM1151
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Table 3 SSR primers,the PIC value amplified in 14 tears aermplasm
No. Primer Alleles PIC
1 ccl2 24 0.5764
2 RM482 22 0.5785
3 umc1999y3 33 0.6648
4 umc1705wl 15 0.6933
5 C0319 20 0.6950
6 umc1506k12 31 0.7055
7 umc2015k3 31 0.7222
8 S1803 27 0.7298
9 YW1BC9 42 0.7404
10 RM1151 45 0.7575
11 C0315 23 0.7675
12 C10291 27 0.7709
Analysis on Inter-Germplasm Genetic Variation
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Table 4 Genetic similarity coefficient of 14 tears aermplasm
C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13 C14
C2 0.45 1.00
C3 0.63 0.41 1.00
C4 0.53 0.75 0.44 1.00
C5 0.42 0.88 0.37 0.79 1.00
C6 0.38 0.73 0.33 0.73 0.86 1.00
C7 0.49 0.74 0.47 0.81 0.76 0.70 1.00
C8 0.51 0.69 0.37 0.72 0.76 0.66 0.77 1.00
C9 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.00
C10 0.58 0.33 0.43 0.64 0.33 0.27 0.37 0.43 0.00 1.00
C11 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.00
C12 0.55 0.27 0.50 0.64 0.36 0.28 0.38 0.39 0.00 0.67 0.00 1.00
C13 0.33 0.23 0.33 0.29 0.26 0.28 0.28 0.28 0.00 0.35 0.00 0.47 1.00
C14 0.54 0.39 0.50 0.61 0.37 0.29 0.46 0.37 0.00 0.52 0.00 0.63 0.43 1.00
Cluster Analysis on the 14 Subject Germplasms
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Fig.2 Clustering map of SSR markers
Table 5 Results of clustering analysis with SSR markers
Group No. GermplasmName
Type 1 Longlin # 1, Xilin # 1, ZYYB12, xingrenxiaobaige, qianyin # 1
Type 2 Longlin # 2, pinzhong # 1, pinzhong # 2, pinzhong # 0,pinzhong # 10, pinzhong # 11
Type 3 Yuenan huakemi
Type 4 XYYBT
Type 5 XYYH12-1
CONCLUSION AND SUGGESTION
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In this study, 4 pairs of maize primers and 8 pairs of oryza sativa primers with favorable polymorphism were used to classify 14 subject Coix lacryma-jobiLvarieties into 5 types based on the SSR analysis on the DNA of Coix lacryma-jobiL: Type 1：Longlin # 1, Xilin # 1,
ZYYB12, Xingren xiaobaige, qianyin # 1；Type 2：Longlin # 2, pinzhong # 1, pinzhong # 2,
pinzhong # 0, pinzhong # 10, pinzhong # 11；Type 3：Yuenan huakemi；Type 4:
XYYBT；Type 5 : XYYH12-1.
In this study, the glyceryl trioleate contented in all 14 varieties of Coixlachryma-jobi L. oil were all above 0.69%. Qianyin #1 (C14) and Pinzhong #11 (C8) had the highest glyceryl trioleate content. Since the growth time of Pinzhong #11 (C8) was longer, Qianyin #1 (C14) was chosen for the nitrogen management experiment.
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