rapd paper -final

8/3/2019 RAPD Paper -Final

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Variety diagnostic PCR-RAPD markers for aromatic rice varieties grown in

Eastern Part of India

Anuprita Ray, Arpita Pattanaik, K.C.Samal, S. S. Kshirsagar and G. R. Rout*Dept. of Agricultural Biotechnology,

Orissa University of Agriculture and Technology,

Bhubaneswar-751003, Odisha, India.Tele fax-0091-674-2397755

*Email- [email protected]

Running Title: Ray et al., ------- Genetic variability of aromatic rice through RAPD markers

Abstract

PCR-RAPD diagnostic marker was used to determine the genotypic identification and genetic

variation in 50 aromatic rice varieties grown in Eastern part of India. Out of thirty primers, 12

primers showed DNA amplification and polymorphism among the 50 rice varieties. A total of

104 bands appeared by using 12 decamers in 50 aromatic rice varieties. Out of which, 95 bands

are polymorphic and three are unique bands. These bands are varietal specific diagnostic markers

used for identification and also for protection of plant varieties by registration. The results

revealed that all the tested primers showed distinct polymorphism among the varieties indicating

the robust nature of RAPD markers. Most of the primers showing the highest polymorphic

information content (PIC) and resolving power (Rp). The cluster analysis indicate that the

aromatic rice genotypes are grouped into two major clusters. Among the two major

clusters, one major cluster had only two varieties and second major cluster

having 47 varieties. Based on this study, the larger range of similarity values using RAPD

markers provides greater confidence for the assessment of genetic relationships among the

varieties. The information obtained from the RAPD profile helps to identify the variety

diagnostic markers in 50 aromatic rice genotypes. Significant genetic variation at maximum

number of loci between varieties indicates rich genetic resources in rice. The intra and inter

genetically variation might be useful for breeders to improve the aromatic rice varieties through

selective breeding and cross breeding programs.

Key words: Aromatic Rice, DNA profile, Diagnostic PCR-RAPD marker, Polymorphism.

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mailto:[email protected]:[email protected]


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Introduction

Rice (Oryza sativa) belonging to the family Poaceae and subfamily Oryzoidea is the

staple food for one third of the worlds population and occupies almost one-fifth of the total land

area covered under cereals. It is grown under diverse conditions and over wide geographical

range. Most of the worlds rice is cultivated and consumed in Asia, which constitutes more than

half of the global population. Approximately 11% of the worlds arable land is planted annually

to rice, and it ranks next to wheat. The worlds rice production has doubled during last 25 years,

largely due to the use of improved technology such as high yielding varieties and better crop

management practices (Byerlee, 1996). Further scope of crop improvement depends on the

availability genetic diversity and variability and use of new biotechnological tools. There is a

rich diversity exist in rice. India is a leading country in the export of promising rice varieties

including scented rice. Aromatic rice is an important commercial commodity. It is more

preferred by the consumers all over the world because of its scent and palatability. There is a

strong need that the germplasm of this cash crop be collected preserved and characterized in

details. India has enacted a legislation as Protection of plant varieties and Farmers Right Act,

2001 for protection of plant varieties by registration. Both DUS (distinctiveness,

uniformity,stability) and molecular characteristics are essential to help the identification of

basumati rice varieties (Patra and Chawla, 2010). Indian rice varieties have been developed

traditionally by selections, hybridization and back crossing with locally adapted high yielding

lines. The number of parental lines used in the breeding programs is however quite small,

resulting in a narrow genetic base. Selection of varieties based on morphological characters are

not very reliable because major characters have low heritability and are genetically complex.

Many Indian farmers are still growing local stains under different names and they also bring

some varieties from distant places. There is a strong need to collect this germplasm and identify

the genotypes. Molecular markers based DNA sequence is found to be more reliable (Virk et al.,

1996). They represent an opportunity to provide information on the variation that exists in a

particular species within a local region. Molecular markers provide information that helps in

identifying the genotypes and their association with close relatives and phylogenetic

relationships. There are many reports that RAPD markers are unbiased and neutral markers for

genetic diversity study, genetic mapping, population genetics as well as genetic diagnostics in

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plants. The PCR-based RAPD approach using single decamer arbitrary primers requires less

amount of DNA and is technically simple as compared to other molecular markers. It can

provide the assessment of genetic distances, seed purity and resolution of uncertain parentage.

RAPD markers have been used in the analysis of rice genotypes by various researchers

(Raghunathachari et al., 2000; Rahman et al., 2007; Bhuyan et al., 2007 Ray Choudhury et al.,

2001, Patra & Chawla, 2010). However, collection of Indian aromatic rice varieties remain

unexplored as there are very scanty references available (Ray Choudhury et al., 2001). Keeping

in view, the present investigation was undertaken to estimate variety diagnostic markers for

identification and phylogenetic relationship among 50 varieties of aromatic rice grown in Eastern

part of India by using RAPD markers.

Materials & Methods

Plant Materials:

Fifty elite aromatic rice varieties were collected from the germplasm centre maintained

by the department of Plant Breeding and Genetics, Orissa University of Agriculture &

Technology, Bhubaneswar for PCR-RAPD analysis. All the varieties have been categories into

the germplasm accession number (Sl. No. 1 to 50 as per table 1). The morphological and

agronomic characteristics have also been indicated in Table 1. The seeds were shown in the

earthen pots and kept in the green house for germination. Leaf samples were collected and

subsequently stored at 20C for isolation of genomic DNA.

Genomic DNA Extraction

Genomic DNA was extracted from young leaves using N-Cetyl-N,N,N-

trimethylammonium bromide (CTAB) method described by Doyle and Doyle (1990) with

modifications. Two grams of fresh leaf material were washed in distilled water and subsequently

rinsed with 80% (v/v) ethanol and then grounded in liquid nitrogen. Ten milliliters of preheated

extraction buffer [4 % (w/v) CTAB, 0.2% -mercaptoethanol (v/v), 100 mM Tris-HCl pH 8.0,

20 mM EDTA pH 8.0, 1.4 M NaCl ] were then added per 2 g of leaf powder material and

incubated for two hours at 65 0C. The lysate was purified with chloroform : isoamyl alcohol

(24:1). The DNA pellet was resuspended in 200 l to 300 l of Tris-EDTA buffer (10 mM

Tris HCl, 1 mM EDTA, pH = 8.0). DNA was reprecipitated by adding 80% ethanol in the

presence of 0.3M sodium acetate, and palleted by centrifugation. The pelletes were lyophilized

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and resuspended in TE buffer. The RNA was removed by RNAse treatment at 37 0C for 1 hour.

For further purification, DNA solution was extracted once with equal volume of phenol and

chloroform : isoamyl alcohol (24:1) followed by two extractions with chloroform : isoamyl

alcohol (24:1). The upper aqueous phase was separated after centrifugation and mixed with

1/10th volume of 3M sodium acetate. DNA was precipitated by adding two adding two volumes

of chilled absolute alcohol, pelleted, dried in vacuum and dissolved in TE buffer. Quantification

of DNA was accomplished by analyzing the purified DNA on 0.8% (w/v) agarose gel

electrophoresis alongside diluted uncut lambda DNA as standard. DNA was further diluted with

TE to a concentration of 20ng/l for use in PCR analysis.

Primer selection

Initially, thirty decamer primers (M/S Bangalore Genei, India) were evaluated for ten

randomly chosen genotypes to test their suitability in amplifying aromatic rice. Primers were

selected on the basis of intensity of bands, repeatability of markers of genotypes. Finally, twelve

primers were selected for the analysis of 50 aromatic rice genotypes (Table 2).

PCR amplification & electrophoresis

Polymerase chain reactions (PCR) was carried out in a final volume of 25 l containing

20 ng template DNA, 100 M of each deoxyribonucleotide triphosphate, 20 ng of primer 1.5

mM MgCl2, 1x Taq buffer (10 mM Tris-HCl [pH-9.0], 50 mM KCl, 0.01% gelatin), and 0.5 U

Taq DNA polymerase (M/S Bangalore Genei, Bangalore, India). Amplification was performed in

a thermal cycler (Pelican, India) programmed for a preliminary 2 min denaturation step at 94 0C,

followed by 40 cycles of denaturation at 94 0C for 20 sec., annealing at 37 0C for 30 sec. and

extension at 72 0C for 1 min, finally at 72 0C for 10 min amplification. The details of primers

used were presented in Table 2. Amplification products were separated alongside a molecular

weight marker (1.0 Kb plus ladder, M/S Bangalore Genei, Bangalore, India) by 1.2 % agarose

gel electrophoresis in 1x TAE (Tris Acetate EDTA) buffer stained with ethidium bromide and

visualized under UV light. Gel photographs were scanned through Gel Doc System (Gel Doc.2000, BioRad, California, USA) and the amplification product sizes were evaluated using the

software Quantity one (BioRad, California, USA).

Data analysis

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RAPD reactions were performed twice for each primer with DNA sample of each genotype and

only reproducible bands were included in the study. Furthermore, gel images of the genotypes

were carefully and independently scored by two persons. Amplifications were scored as discrete

variables, using 1 to indicate presence and 0 for absence of band. A binary matrix was obtained

by visual scoring of the bands. Efficiency of discrimination was assessed in terms of the number

of polymorphic markers generated and the ability to generate unique band. Pairwise-similarity

matrices based on RAPD data were determined using Jaccards similarity coefficient (Sneath and

Sokal, 1973).The average of similarity matrices was used to generate a tree by

UPGMA (Unweighted Pair-Group Method Arithmetic Average) using NTSYS-

PC, version 2.0 (Rohlf 1995).

Results and Discussion

The present investigation offers an optimization of primer screening for

evaluation of genetic relationship of 50 varieties of aromatic rice through

RAPD markers. Ten genotypes were used for screening primers obtained

from different series for amplification by using polymerase chain reactions.

The results showed that A-, B- and C-series primers produced relatively

more amplification fragments compared to N- and D-series decamer primers.

The amplification generated by primers OPB-12, OPC-08 and OPA-08

produced maximum number of DNA fragments; the size of the DNAfragments ranged from 200 to 2500 base pairs. Primer OPA- 08 amplified 14

fragments whereas; OPB-12 produced 11 bands (Table 2). It was also noted

that some of the primers did not show any amplification by using the ten rice

genotypes. The twelve decamers produced good amplification of RAPD

fragments. Among the thirty primers, twelve primers were selected to

analyze the genetic relationship among the 50 aromatic rice genotypes

through RAPD markers. The reproducibility of the amplification product was

tested with two independent extractions. Most of the amplification reactions

were duplicated. Only bands that were consistently reproduced across

amplifications were considered for the analysis. Bands with the same

mobility were considered as identical fragments, receiving equal values,

regardless of their staining intensity. When multiple bands in a region were

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difficult to resolve, data for that region of the gel was not included in the

analysis. As a result, twelve informative primers were selected and used to

evaluate the degree of polymorphism within 50 varieties of aromatic indica

rice under family Poaceae and subfamily Oryzoidea. The maximum and minimum

number of bands were produced by the primers OPA-08 and OPC-07

respectively (Table 2). A total of 109 amplified fragments was scored across

the 50 varieties of aromatic rice for the selected primers, and was used to

estimate genetic relationships among themselves. The patterns of RAPD

produced by the primers OPC-08, OPB-12 and OPA-08 are shown in the

Figures 1A-C. The genetic variation through molecular markers has been

highlighted in a number of rice genotypes (Raghunathachari et al., 2000;

Ray Choudhury et al., 2001; Rahman et al., 2007). The genetic similarity as

determined by RAPD fingerprinting also corresponded considerably with the

known pedigrees. The similarity matrix was obtained after multivariate

analysis using Nei and Lis coefficient (data not shown). The similarity matrix

was then used to construct a dendrogram with the UPGMA method (Figure

2). The dendrogram shows two major clusters within 50 varieties of aromatic

indica rice. Among the two major clusters, one major cluster had only two

varieties (Kukudajata and Manasi) and other major cluster divided into twominor clusters. First minor cluster had only one variety i.e. ``Kalajeera`` and

second minor cluster having 47 variety. Second minor cluster again divided

into two sub-minor clusters; first sub-minor cluster having twenty two

varieties. Among the twenty two varieties, ``Jalaka``, ``Dangarbasumati``

and ``Gangabali`` having 100 % similarity among themselves in particular

with panicle length. The variety ``Basumati`` and ``Basumati-1`` making

same cluster with 65% similarity in days to flowering and panicle length. The

variety ``Gopalabhog`` and ``Kalikati`` having 84% similarity with respect

to days to flowering, panicle length and panicle number. The variety

``Basnasapuri`` ``Sagadadhuli`` having 80% similarity among themselves

and also 52% similarity with variety ``Kalajera` . The second sub minor

cluster having twenty five varieties and divided into two clusters. First cluster

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having four varieties with 30 to 50% similarity among themselves (``Nanu``,

``Sirimula``, ``Thakurabhoga`` and ``Jalaka``). Second cluster having 21

varieties with two sub clusters. First sub cluster having two varieties i.e.

``Basumatidhan`` and ``Dulhabhog``. Second sub cluster having 19

varieties and divided into two clusters. One cluster having one variety

``Kaminibhog-2`` with 30% similarity with other 18 varieties. Second cluster

again subdivided into two clusters i.e. one having 8 varieties and others

making two clusters. One having 6 varieties

(``Sujata``,``Heerakani``,Sheetabhog``,``Ganjam Local-2``,

``Tulasiphulla`` and ``Kalikati``) and other having 4 varieties (``Nalidhan``,

``Nuadhusura``, ``Thakurasuna`` and Dhoiabankoi) with more than 50%

similarity among themselves. Molecular markers show better resemblance

with the pedigree as compared to morphological markers. Patra and Chawla

(2010) reported that the biochemical and RAPD molecular markers helps to

establish the distinctiveness of basumati rice varieties. Genetic variation is

important in maintaining the developmental stability and biological potential

of the genotype. The results indicate that there was very close variation

among the varieties. These results suggest that the use of different RAPD primers would

enable to asses the genetic diversity of aromatic rice variety as reported earlier in other variety of

rice (Blair et al., 1999). Joshi et al (2000) studied the genetic diversity and phylogenetic

relatedness in Oryza by ISSRmarkers. The present study showed that the higher percentage of

polymorphism as compared with other molecular marker as reported earlier (Galvan et al., 2003,

Mohapatra et al., 2005). Bhuyan et al (2007) illustrated the genetic diversity in traditional

lowland rice grown in Assam using both RAPD and ISSR markers. Further, Youssefet al(2010)

used both RAPD and ISSR markers to identify the new promising drought tolerant lines of rice

under drought stresses. These traits with molecular differences commented upon in this

investigation suggest that these rice varieties were belongs to indica rice with introgressions from

wild rice land races. Significant genetic variation at maximum number of loci between varieties

indicates rich genetic resources in rice. The intra and inter genetically variation might be useful

for breeders to improve the aromatic rice through selective breeding and cross breeding

programs.

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Acknowledgement

The authors wish to acknowledge to Department of Biotechnology, Government of India

for providing funding for student research under PG HRD program.

References

Bhuyan N., Borah BK., Sarma RN. (2007) Genetic diversity analysis in traditional lowland rice

(Oryza sativa L.) of Assam using RAPD and ISSR markers. Current Sci., 93:967-972.

Blair MW, Panaud O, McCouch SR (1999). Inter-simple sequence repeat (ISSR) amplification

for analysis of microsatellite motif frequency and fingerprinting in rice (Oryza sativa L.).

Theor. Appl .Genet. 98: 780-792.

Byerlee D (1996). Knowledge-Intensive Crop Management Technologies: Concepts, Impacts,

and Prospects in Asian Agriculture. International Rice Research Conference, Bangkok,

Thailand, 3-5 June, 1996.

Doyle JJ, Doyle JL (1990) Isolation of plant DNA from fresh tissue.Focus 12: 1315

Joshi SP, Gupta VS, Aggarwal RK, Ranjekar PK, Brar DS (2000) Genetic diversity and

phylogenetic relationship as revealed by Inter simple sequence repeat polymorphism in

the genus Oryza. Theor. Appl. Genet. 100: 1311-1320.

Mohapatra A, Rout GR (2005) Identification and analysis of genetic variation among rose

cultivars using random amplified polymorphic DNA.Z Naturforschung60C: 611617

Patra N. , Chawla HS (2010) Biochemical and RAPD molecular markers for establishing

distinctiveness of basumati rice (Oryza sativa L.) varieties as additional descriptors for

plant variety protection. Indian Journal of Biotechnology, 9:371-377.

Rahman SN., Islam MS., Alam MS., Nasirudin KM (2007) Genetic polymorphism in rice

(Oryza sativa L.) through RAPD analysis. Indian Jour Biotechnology, 6: 224-229.

Raghunathachari P., Khanna VK., Singh US, Singh NK (2000) RAPD analysis of genetic

variability in Indian scented rice germplasm (Oryza sativa L.). Current Sci., 79 (7): 994-

998.Ray Choudhury, P., Kohli S., Srinivasan K., Mohapatra T., Sharma RP (2001) Identification and

classification of aromatic rices based on DNA fingerprinting. Euphytica, 118:243-251.

Rohlf FJ (1995) NTSYS-PC Numerical taxonomy and multivariate analysis system. Version

1.80, Exeter Software, Setauket, New York

Sneath, P.H.A., and R.R. Sokal. (1973) Numerical taxonomy: The principles and practice of

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numerical classification. W.H. Freeman, San Francisco, CA.

Virk PS., Ford-Lloyd BV., Jackson MT., Pooni HS., Clemeno TP., Newbury HJ (1996)

Predicting quantitative variation within rice germplasm using molecular markers.

Heredity, 76: 296-304.

Youssef MA., Mansour A., Solliman SS (2010) Molecular markers for new promising drought

tolerant lines of rice under drought stress via. RAPD-PCR and ISSR markers. Jour. of

American Sci., 6: 355-363.

Table 1. Morphological characteristics of aromatic rice varieties used for molecular analysis.

Accessio

n no.

Name of GenotypesDays

To

flowering

Plant

Height

(cm)

Panicle

Length

(cm)

Panicle

Number

No of

Fertile

grains

Fertility

(%)

1000

grain

weight(g)

Harves

t

index

Potential

Yield(q/ha)

1 Nanu 103 133.9 26.6 9 165 76.6 11.0 0.36 17.482 Basmatidhan 91 122.4 22.3 6 116 88.2 12.8 0.37 18.623 Basmati 100 120.0 26.7 8 109 74.1 11.8 0.43 23.954 Basumati-1 100 103.3 24.1 9 62 65.9 16.9 0.21 23.955 Dholabankoi 102 127.1 27.1 9 169 86.5 12.4 0.37 29.426 Jalaka 104 107.4 24.7 6 112 89.6 14.2 0.31 22.817 Kalikati 101 133.1 24.9 7 164 92.9 10.9 0.34 21.39

8 Dulhabhoga 100 125.2 23.0 7 141 85.3 18.9 0.41 17.659 Kanakachampa 98 135.6 24.5 7 146 76.2 12.1 0.38 18.6210 Gopalabhoga 102 121.5 24.6 7 125 78.0 11.5 0.39 18.5711 Jala 103 115.4 24.5 10 136 75.7 11.6 0.33 18.7112 Dangarabasamati 101 126.0 22.8 7 135 82.3 15.6 0.39 19.2013 Kaminibhoga-2 103 129.0 25.2 7 145 82.3 11.9 0.43 19.7714 Kalajeera 104 129.9 25.6 8 123 89.9 14.4 0.38 25.55

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Accessio

n no.

Name of GenotypesDays

To

flowering

Plant

Height

(cm)

Panicle

Length

(cm)

Panicle

Number

No of

Fertile

grains

Fertility

(%)

1000

grain

weight

(g)

Harves

t

index

Potential

Yield(q/ha)

15 Kalikati-1 102 148.2 27.5 6 153 69.0 11.2 0.36 21.5416 Badsahbhog 104 135.9 27.4 9 159 90.6 11.5 0.33 24.48

17 Ganjam local-1 98 123.0 23.9 8 137 86.5 11.8 0.39 19.2018 Karpurakranti 102 130.9 24.5 7 88 74.6 13.6 0.39 17.6519 Basnaparijat 104 117.6 24.3 10 147 91.5 11.1 0.34 22.2220 Basnasapuri 104 145.2 25.5 11 173 79.4 11.0 0.38 25.0721 Saragadhulli 103 129.5 25.6 10 132 82.0 11.8 0.42 19.2322 Magura 108 111.7 21.90 7 112 83.0 17.6 0.30 17.0623 Kalajeera 100 125.6 25.0 7 125 85.6 13.9 0.40 18.4724 Lajakulibadan 103 136.0 26.7 9 150 80.4 12.5 0.37 28.4225 Kalajauvan 101 140.3 29.8 4 143 90.9 13.5 0.31 18.6026 Kukudajata 103 136.2 26.2 9 140 79.6 12.4 0.43 19.5927 Gatia 107 136.2 23.2 8 135 82.3 18.0 0.39 24.3728 Jaiphulla 102 105.0 20.6 6 107 83.7 19.2 0.41 25.6829 Gangaballi 97 125.4 25.0 9 139 93.9 11.6 0.41 24.4830 Manasi 101 115.4 24.6 9 117 94.4 13.2 0.40 23.4831 Chatianaki-1 99 140.7 29.0 7 123 72.0 12.5 0.38 19.5932 Thakursuna 103 108.1 26.8 6 185 84.2 11.5 0.37 28.9933 Bishnubhog 100 129.8 22.8 7 195 95.9 13.3 0.39 27.3634 Dhobaluchi 91 103.9 18.8 7 123 83.6 23.4 0.57 27.4635 Srimula 105 125.9 25.2 8 131 76.4 11.1 0.31 21.5936 Ganjam local-2 105 130.0 24.5 7 128 87.0 13.9 0.40 27.2937 Thakurbhog 106 127.4 28.8 10 145 89.9 12.9 0.33 28.12

38 Nuakalajeera 105 136.1 27.2 7 159 97.8 11.0 0.32 20.7839 Pimpudibasa 100 124.5 26.4 6 160 85.9 13.3 0.37 18.5740 Heerakani 98 117.6 25.2 10 175 88.0 11.0 0.40 23.1641 Chatianaki 98 147.2 29.5 6 126 71.9 12.8 0.41 18.8442 Baranamgomati 104 147.9 25.4 8 97 88.9 19.8 0.41 22.6143 Nalidhan 105 141.1 27.7 13 174 93.1 12.3 0.37 24.0744 Sheetabhog 103 120.6 23.6 8 145 84.4 10.7 0.40 19.1345 Nuadhusura 104 125.3 25.5 8 110 75.7 15.9 0.41 20.7246 Tulasiphulla 98 110.8 23.2 9 108 79.4 13.7 0.49 10.4947 Sujata 96 115.3 24.7 9 140 90.0 14.1 0.51 26.3248 Neelabati 108 110.6 26.9 7 160 86.2 14.0 0.34 23.45

49 Chinikamini 100 126.2 28.0 8 119 81.0 12.3 0.39 18.0450 Khosakani 104 127.8 27.4 7 174 87.3 13.9 0.37 28.41

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Table 2. DNA profile of 50 genotypes of aromatic rice by using RAPD primers.

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Prime

r

CodeSequence

Total

no. of

of

bands

Polymo

rphic

band

Uniq

ue

band

Rare

band

(< 5%)

Low

Freque

ncy

band

(5-

30%)

High

Frequ

ency

band

>30%

%age

Polymorp

hism

Size

Range

(bp)

Resolvi

ng

Power

(Rp)

Avera

ge

PIC

Value

OPC-8

5

TGGACCGG

TG 3

11 11 1 1 6 3 100300-1700

4.8 0.91

OPB-12

5CCTTGACG

CA 311 11 1 1 8 1 100

215-2500

2.68 0.98

OPA-85

GTGACGPAGG 3

14 14 0 1 10 3 100200-

25005.64 0.88

OPA-15

CAGGCCCTTC 3

10 9 0 0 7 2 90.00230 1500

3.120.512

OPA-25

TGCCGAGC

TG 3

9 7 0 1 6 0 77.78320

2500

2.980.318

OPA-35

AGTCAGCCAC 3

7 5 0 0 3 2 71.43375 2200

3.900.412

OPA-45

AATCGGGCTG 3

9 6 1 0 5 0 66.67320

1900

3.120.448

OPA-55

AGGGGTCTTG 3

7 7 0 0 4 3 100.00260 1350

3.870.437

OPC-07

5GTCCCGAC

GA 36 4 0 0 1 3 66.67

320-2850

3.76 0.384

OPC-15

5

GACGGATCAG 3

7 5 0 1 3 1 71.43 390-3450

3.98 0.436

OPD-08

5GTGTGCCC

CA 38 7 0 0 5 2 87.50

340-3150

2.95 0.378

OPD-10

5

GGTCTACACC 3

10 9 0 1 7 1 90.005002700

4.40 0.570

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