15강_분자표지이용 육종

Ⅰ. 식물유전체의 구조

Ⅱ. 분자표지

III. 분자표지 이용 육종

1900 1920 1940 1960 1980 20001800

IntroductionSeparation method

HybridizationUse of hybrid vigorChromosomal variationGene mutationTissue cultureCell fusionTransformationGenome editing

Pedigree/bulk method, etcBackcrossingRecurrent selectionAnther cultureMarker-assisted selection Genomic selection

Cre

atio

n o

f varia

tion

Sele

ction

meth

ods

시대별 육종기술의 발전 개요

분자육종이란= 전통육종학 + 분자유전학 + 분자생물학 + 유전체정보= 전통육종기술 + 분자표지(유전체) 육종기술 + 형질전환육종기술

Marker-assistedselection

Molecular markers

Goal: Introduction of new allelesGenetic recombination

New variety

GeneGenomics

Molecularmap

QTL X

Geneticfixation

Marker-assisted breeding

Transgenic plants

Vector

Selection system

Promotor

GM crops

Foreigngene

Hybridization breeding

Goal: Creation of new variationIntroduction of new genes

Transgenic breeding

Geneticfixation

Genome-assisted breeding

phylogenetic tree of all species with sequenced genomes

Published genomes in black. Species marked in lighter shades of gray are less complete or less available

1. 식물유전체의 구조

(1) Polyploidy is common in plants species.

Wood T E et al. PNAS 2009;106:13875-13879

Polyploid incidence and speciation frequencies across major groups of vascular plants.

국화아강류

장미아강류

진정쌍떡잎

Examples of polyploid crops

Triploid crops: apple, banana, citrus, ginger, watermelon

Tetraploid crops: apple, durum or macaroni wheat, cotton, potato, cabbage,

leek, tobacco, peanut, kinnow, grapes, Pelargonium

Hexaploid crops: chrysanthemum, bread wheat, triticale, oat, kiwifruit

Octaploid crops: strawberry, dahlia, pansies, sugar cane,

oca (Oxalis tuberosa)

* Some crops are found in a variety of ploidies: tulips and lilies are

commonly found as both diploid and as triploid; daylilies (Hemerocallis

cultivars) are available as either diploid or tetraploid; apples and kinnows

can be diploid, triploid, or tetraploid.

(2) Genome organization

Characteristics of plant genomes

• Nuclear genome

– Linear, with multiple chromosomes

– Highly variable in size (within & between species)

– Highly variable chromosome number (between species)

• Chloroplast genome:

– Circular and highly conserved (gene order & seq)

– Size: 100-300 kb (av = 140 kb, 110 genes)

Arabidopsis = 154 kb, 87 genes (DNA Res 6(5):283)

rice = 135 kb, 110 genes (MGG 217:185)

• Mitochondrial genome:

– Circular and less conserved,

Intra-molecular recombination to give the two smaller molecules: (turnip) 218 kb

-------> 135 kb + 83 kb

– Size: 200-2500 kb

Arabidopsis = 367 kb, 57 genes (Nat Gen 15:57)

rice = 491 kb, 35 genes+10 ORFs

variation in estimated genome sizes in base pairs

(Mol. Pl. Breed. P.69)

Genomic organization, content: sizes and corresponding composition of six major model organisms (Kosi Gramatikoff , 2007)

Genome =

coding sequences

+ non-coding sequences

Only 36.3% of the 25,426 predicted genes for A. thaliana are classified. For rice, only 20.4% of the 53,398 complete predictions, with both initial and terminal exons, could be classified. (Jun et al, 2002)

(NCBI, Gramene 2015.12)

Gene identification in Arabidopsis and rice

(In rice)

Character groupNumber of genes

isolated

2011 2015

Growth and development 40 410

Quality components 13 83

Tolerance to environmental stresses 24 154

Reproduction 18 167

Disease resistance 17 118

Insect resistance 1 7

Others 57 102

Total 170 1,041

~ 3% of whole genes of rice

Tandem repeats : - Telomeric and subtelomeric repeats

- Tandem repeats in centromere

• Satellite DNA: main component of functional centromeres, and form the main structural constituent of heterochromatin

Minisatellite/VNTR(Variable number tandem repeat): a short series of bases 10-60 bp, 20~50 repeats

Microsatellite/SSR(simple sequence repeat)/STR(short temdem repeat): repeating sequences of 2-6 bp, 3~100 repeats

Interspersed repeats: from a few copies to as many as 100,000 copies per genome

• Transposon(transposable elements) families- Class I: retrotransposons

- Class II: DNA-mediated elements

non-coding sequences

Classification of transposable elementsClass I (retrotransposons)

․transpose via an RNA intermediate ․use of reverse transcriptase

․autonomous and non-autonomous elements

․replicative transposition

․Many retrotransposons have long terminal repeats (LTRs) at their ends

that may contain over ~100 bp to over 5 kb in size

․large portion of plant genome (up to 80%)

․likely to have evolved from retrovirus

* Non-LTRs:

LINEs (Long interspersed nuclear elements), SINEs (Short interspersed nuclear elements)

Class II (DNA-mediated elements)

․no RNA intermediate

․non-replicative transposition (cut and paste)

․normally a moderate portion of plant genome (<10%)

* MITE (miniature inverted transposable elements) (Zheng et al (2000) PNAS 97: 1160)

․plants, animals, nematodes, insects, fish

․short length(125-500bp), terminal inverted repeats (10-15bp),

high copy number (>10,000)

․associated with 5' regulatory regions of genes

․mechanisms of transposition unknown

․may be one of the major sources of novel variation

․over 100,000 MITEs in the rice genome (representing some 6% of the total genome)

Nat 436:793(2005)

The human genome might contain as few as 20,000 genes, comprising less than 1.5% of the total genome sequence(Nat Reviews, Genet 6:699 (2005)

Crop Year publ. Mocot/Dicot Chr(#) Size(Mb) Gene(#) Repeat(%)

Oryza sativa rice 2005 monocot 12 389 37,544 26Populus trichocarpa black cottonwood 2006 dicot 19 485 45,555 NAVitis vinifera grape 2007 dicot 19 505 29,585 27Carica papaya papaya 2008 dicot 9 372 28,629 43Cucumis sativus cucumber 2009 dicot 7 367 26,682 24Sorghum bicolor sorghum 2009 monocot 10 818 34,496 62Zea mayes maize 2009 monocot 10 2,300 32,540 85

Glycine max soybean 2010 dicot 20 1,115 46,430 57Ricinus communis castor bean 2010 dicot 10 320 31,237 50 Malus x domestica apple 2010 dicot 17 742 57,386 67Jatropha curcas jatropha 2010 dicot NA 380 40,929 37Theobroma cacao cocoa 2011 dicot 10 430 28,798 24Fragaria vesca strawberry 2011 dicot 7 240 34,809 23Solanum tuberosum potato 2011 dicot 12 844 39,031 62Cucumis sativus cucumber 2011 dicot 7 367 26,587 NABrassica rapa chinese cabbage 2011 dicot 10 485 41,174 40Cannabis sativa hemp 2011 dicot ? 820 30,074 NACajanus cajan pigeon pea 2011 dicot 11 833 48,680 52Phoenix dactylifera date palm 2011 monocot 18 658 28,890 40Solanum lycopersicum tomato 2012 dicot 12 900 34,727 63Cucumis melo melon 2012 dicot 12 450 27,427 NALinum usitatissimum flax 2012 dicot 15 373 43,484 24Gossypium raimondii cotton D 2012 dicot 13 880 40,976 60Azadirachta indica neem 2012 dicot NA 364 20,169 13Citrullus lanatus watermelon 2012 dicot 11 425 23,440 45Gossypium raimondii cotton D 2012 dicot 13 880 37,505 61Prunus mume chinese plum 2012 dicot 8 280 31,390 45Musa acuminata malaccensis banana 2012 monocot 11 523 36,542 44Hordeum vulgare barely 2012 monocot 7 5,100 30,400 84Triticum aestivum wheat 2012 monocot 21 17,000 94,000 80Pyrus bretschneideri pear 2013 dicot 17 527 42,812 53Cicer arietinum chickpea 2013 dicot 8 738 28,269 49Hevea brasiliensis rubber tree 2013 dicot 18 2,150 68,955 72Prunus persica peach 2013 dicot 8 265 27,852 37Aegilops tauschii wheat DD 2013 monocot 7 4,360 43,150 66Triticum urartu wheat AA 2013 monocot 7 4,940 34,879 67Capsicum annum Hot pepper 2014 dicot 12 3,480 34,903 NA

Michael and Jackson(2013) The Plant Genome 6(2):1-7

작물 유전체 염기서열 분석 현황

유전자와 비유전자(non-coding sequence) 부위에품종간/생물개체간/집단간에 왜 다양한 변이가 존재하는가?

• 자연 돌연변이 유전자 변형• 교잡변이 유전자 재조합• 염색체 변이

- 구조적 변이 유전자 그룹 변형- 수적 변이 이수체, 배수체

(DNA) 변이의 원인유전자변화

비유전자 부위 변화1) No 표현형 다양성

(단지 DNA만 다양) 2) ??

다양성 유발

• 자연 돌연변이• 교잡변이• 염색체 변이

- 구조적 변이- 수적 변이

변이의 결과

DNA의 다양한 차이를 어떻게 감별하는가?

(1) 유전자 분리 비교 --- 분리한 유전자의 염기서열, m-RNA, 단백질(2) 염기서열 비교 --- 전체 또는 일부분(유전자 또는 비유전자 부위)의 염

기서열을 분석 비교 : resequencing, YAC/BAC/PAC/cosmid contigsequencing

(3)DNA마커 비교 --- DNA마커가 위치하는 특정부위(유전자 또는 비유전자 부위 모두) 비교 : 염기서열, 전기영동 결과

차세대염기서열분석방법

20G genome sequencing~3,000 US$<1 week

염기서열 분석방법 발달

$1,245(3000Mb)

II. Molecular marker (분자표지; 분자 마커)

1. 마커(= Genetic marker )의 개념과 종류2. Protein marker

3. DNA marker

마커 (=표지)?

1. 마커(= Genetic marker )의 개념과 종류

Genetic marker (유전표지인자): A gene with (a known location on a chromosome) and a clear-cut phenotype used as a point of reference--- Morphological marker, molecular marker

벼에서 형태적유전표지인자들)

(1) Morphological marker: 염색체위치가동정된형태적돌연변이유전자들

Partial map of morphological markers in rice

(2) Molecular marker (분자표지) : protein (enzyme) 또는 DNA marker

: (염색체위치가동정된) protein (enzyme) 이나 DNA 염기서열을전기영동(electrophoresis)으로감별하여이용하는마커

(3) Marker 종류별비교

Morphological marker

(형태표지)

Molecular marker (분자표지)

Protein /enzyme DNA

Deleterious

Epistatic

Limited number

Recessive/dominant

Environmently sensitive

Coding regions only

Low natural variation

Cheap

Neutral

Non-epistatic

Limited number

Codominant

Less sensitive

Coding regions only

More natural variation

More expensive

Neutral

Non-epistatic

Unlimited number

Codominant

Insensitive

Coding/non-coding

Much natural variation

Most sensitive

2. Protein marker(1) Seed storage protein

- seeds are a rich source of stable and abundant proteins

- seeds represent a well-defined developmental stage

- seeds are easily stored and transported

- each protein band in an electrophoretic profile usually represents

a direct gene product

Total 단백질추출

전기영동 품종간차이감별

밀 품종의종자단백질 변이

(2) Isozyme markers

Isozyme: Also called allozymes. These are multiple forms of an enzyme

that differ from each other by either the substrate acted upon; their

maximum activity; or their regulatory properties.

Uses include the identification of genotypes and the assessment of the

amount of genetic variation in plants.

Leucine aminopeptidase polymorphism in Dreissena polymorpha

(Molluscs 조개류). Sixteen different individuals were analyzed.

참고: Gel Electrophoresis of DNA

Agarose (한천) gel electrophoresis

3. DNA marker

Agarose gel in a dry condition

Amount of agarose in

gel(%[w/v])

Efficient range of

separation oflinear DNA

molecules (kb)

0.3 5~60

0.6 1~20

0.7 0.8~10

0.9 0.5~7

1.2 0.4~6

1.5 0.2~3

2.0 0.1~2

Agarose 농도별로 분명하게 구분(separation) 할 수 있는DNA 단편의 크기

준비물: agarose, TAE/TBE buffergel casting tray, comb 등

Agarose gel 제작

12 3

4

56 7

Agarose gel 제작 과정

12 3

4

5

6

Electrophoresis (전기영동) 과정

7

준비물: agarose gel, TAE/TBE buffer, DNA, electrophoresis kit, dye(blue juice), UV illuminator, pipette 등

Electrophoresis (전기영동)

gel

dye

Genotype 또는 품종 간 DNA 구성의 차이 존재

이유: 품종간 또는 종간 유전자 및 염색체의 변이

가. 제한효소처리에의한차이감별 : RFLP 마커

나. 특정부위의증폭에의한차이감별 : PCR-based 마커

RAPD 마커

Microsatellites (SSR) 마커

AFLP 마커

STS 마커

SNP 마커 등

DNA markers (DNA 표지)

품종간 염기서열 차이의예)

Rice 품종간 DNA 변이1번 염색체 1S end

Japonica: NipponbareIndica 93-11

(1) DNA 마커의 종류

가. RFLPs - restriction fragment length polymorphisms

Genomic DNA에 제한효소를 처리하고 절단된 단편들의 크기를비교하여 그 차이를 마커로 활용하는 방법

과정

1. Genomic DNA 추출2. 적절한 제한효소(restriction enzymes)를 처리(37C)하여 로 DNA를 절단3. Agarose gel 전기영동으로 절단된 단편들을 퍼트림4. Gel에 퍼트려진 DNA단편들을 membrane으로 옮김 : southern blotting

5. 작은 용기에서 Hybridize with radioactively labeled DNA probe(s)

6. Autoradiography

7. Scoring

제한효소 (restriction enzymes)

RFLP 과정

Probe :A small piece of nucleic acid that has been labelled with a radioactive

isotope, dye, or enzyme and is used to locate a complementary nucleotide

sequence or gene on a DNA molecule.

--- Genomic clone, cDNA(A duplex DNA, representing an mRNA, carried

in a cloning vector.) 등

0.4N NaOH

Marker

NameType

CDO102RFL

P

[ View Detail

s ]CDO105

RFL

P

[ View Detail

s ]CDO1053

RFL

P

[ View Detail

s ]CDO1056

RFL

P

[ View Detail

s ]CDO1081

RFL

P

[ View Detail

s ]CDO1083

RFL

P

[ View Detail

s ]CDO109

RFL

P

[ View Detail

s ]CDO1091

RFL

P

[ View Detail

s ]CDO116

RFL

P

[ View Detail

s ]CDO1160

RFL

P

[ View Detail

s ]CDO12

2

RFL

P

[ View Detail

s ]

RFLP Probes in oat

Interpretation of results

벼 12품종 genomic DNA를 probe RZ5로 hybridization 한 후5개의 제한효소로 처리한 RFLP 변이

1 2 3 4 5 6 7 8 9 10 11 12

1 2 3 4 5 6 7 8 9 10 11 12

1 2 3 4 5 6 7 8 9 10 11 12

bp

23,130

9,4166,557

4,361

2,3222,027

Size marker : 매우 다양한 종류--분리하고자 하는 DNA 단편의 크기에 따라 적절한 것 사용

예)20000

10000

7000

5000

4000

3000

2500

2000

1500

1200

1000

850

700

500

400

300

200

100 bp

1% TopVisionHindIII /EcoR1

Molecular wt marker

나. PCR based 마커

PCR(: polymerase chain reaction)

: 소량의 DNA를 단시간에 대량으로 증식하는 방법.DNA 감정을 하는 경우 그 분석에는 다량의 DNA가 필요하다. 그러나 범죄수사에서 범인이나 피해자의 신원을 확인하려면 현장에 남겨진 머리털 1개나 약간의 혈흔이라는 극히 소량의 DNA를 포함하고 있는 샘플에서 DNA를추출한 것만으로는 충분치 않다. 이것을 해결한 것이 PCR법(폴리메라아제연쇄 반응법)이다. PCR법은 DNA 감정이나 유전자 진단에서 빠질 수 없는기술이다.

PCR법에서는 우선 DNA에 열을 가하여 2개의 사슬로 나눈다. 이것에 ‘프라이머’라고하는 짧은 DNA를 추가하여 냉각하면 프라이머가 DNA에 결합한다. 이것에 DNA 폴리메라아제라는 효소를 더하면 프라이머 부분이 출발점이 되어 DNA가 복제된다. 이 ‘가열 및 냉각’이라고 하는 1사이클로 DNA는2배가 된다. 이것을 수십 회 반복하면 약 1시간에 DNA는 수십억 배로 불어난다. 이 가열ㆍ냉각을 자율적으로 하는 장치가 개발되어 있기 때문에 매우간단히 짧은 시간에 DNA를 증폭할 수 있다.

5’-AGCCTGTCAA-3’

dNTP (A,T,C,G)

Taq polymerase

Buffer

A. RAPD 마커 : Randomly Amplified Polymorphic DNA 마커

: random primer를이용하여 DNA를 PCR로증폭하여품종간차이를감별하는

마커 * agarose gel, polyacrylamide gel

과정

1. DNA 추출

2. PCR reaction soln 조제하여 PCR

Templete DNA (Genomic DNA)

dNTP(A,T,C,G)

Primers (Random(arbitrary) primers) * Operon 회사: 10-mer

Buffer

Taq polymerase

* PCR temp. profile: 94C 7분,

94C 1분, 55C 1분, 72C 2분 : 35 cycles

72C 7분

3. PCR product (=amplicon)를전기영동 (보통 agarose gel)

4. Ethidium bromide로염색하여 uv-lamp에서관찰 band(;amplicon) 조사

OPA16 프라이머 (Random 10-mer)에의해PCR 증폭한벼 15개품종간 RAPD 변이

Description Sequence

Tube A-13 5'-CAGCACCCAC-3'

Tube A-15 5'-TTCCGAACCC-3'

Tube A-01 5'-CAGGCCCTTC-3'

Tube A-03 5'-AGTCAGCCAC-3'

Tube A-06 5'-GGTCCCTGAC-3'

Tube A-08 5'-GTGACGTAGG-3'

Tube A-02 5'-TGCCGAGCTG-3'

Tube A-04 5'-AATCGGGCTG-3'Tube A-05 5'-AGGGGTCTTG-3'

Tube A-07 5'-GAAACGGGTG-3'

Tube A-09 5'-GGGTAACGCC-3'Tube A-10 5'-GTGATCGCAG-3'Tube A-11 5'-CAATCGCCGT-3'

Tube A-12 5'-TCGGCGATAG-3'

Tube A-14 5'-TCTGTGCTGG-3'

Operon Tech.의 random 10-mer Set A 01-15

B. Microsatellite (SSR) 마커: 특정부위에서반복염기서열의품종간차이를감별하는마커로서해당부위의염기서열을분석후 primer를제작이용한다.

* agarose gel 대신에 polyacrylamide gel 사용

(-)

(+)

Microsatellite typing can be multiplexed and automated

• Flourescence-labelled primers 사용• sequencing gel Genotyper 이용

Microsatellite typing can be multiplexed and automated

P1 P2 F1

F1

P1

P2

Co-dominance (공우성) 마커

P1 P2 F1

F1

P1

P2

Dominance (presence/absence) (우성) 마커

Herozygote 감별가능

C. AFLP 마커 : amplified fragment length polymorphism

: 제한효소로절단한단편을 random primer를이용 PCR로 증폭하여감별하는마커 * agarose gel 대신에 polyacrylamide gel 사용

AFLP gel for rice

Direction of electrophoresis

AFLP autoradiographs of 14 rice

cultivars using two primer

combinations. The First 14 lanes

are from primer combination

92S04/92G12 and the second 14

are from primers 93B11/92F06.

Cultivars are 1=M-103, 2=M-201,

3=M-202, 4=M-203, 5=M-204,

6=M-401, 7=ltalica Livorno, 8=L-

202, 9=L-203, 10= Labelle.

11=Black Gora, 12=IR40931-26,

13=87-Y-550, 14=WC1403.

Entries 11 and 12 are indica types

and the others are japonica types.

Both primers are +3 bases.

Amplified bands were labelled

with 33 P and run on sequencing

D. STS (Sequence-tagged Sites) 마커

: 목표부위의 염기서열을 분석하여 primer를 제작 PCR하고 품종간 증폭된 부위의 차이를 감별하는 마커

• SCARs (sequence-characterized amplified regions)

- single-locus marker derived from RAPD

fragment

- More reproducible

Cloning

Sequencing

Design of PCR primer

PCR

• CAPS (: cleaved amplified polymorphic sequence)

- locus-specific marker

- Amplicon digested with a restriction enzyme

( PCR amplified product analyzed by RFLP)

- Polymorphisms detected as differences in fragment sizes

A 품종 B 품종

PCR product

Restriction enzyme 처리

A B

• Sequence DB-based STS marker

- Use of sequence differences caused by indels

- Design primers PCR of amplicons

NCBI http://www.ncbi.nlm.nih.gov/TIGR http://www.tigr.org/RGP (rice) http://rgp.dna.affrc.go.jp

. . . . .

AGGCAATGCCGGTAATTTGGCGCCAATGATATCCCAGGCAATGCCGGTAATTTGGCGCCAATGATATCCC

GGATTATCCTGCTGCAACTTGTGCCAATATACTACCGGATTATCCTGCTGCAA-----------TATACTACC

GCTCTCAATTCCGTAAT--TTAAGGCAACCTCCATCGCTCTCAATTCCGTAATGTTTAAGGCAACCTCCATC

TTTGGCCAGGAGATTCCAAGATCTCGAATCCTGTACTTTGGCCAGGAGATTCCAAGATCTCGAATCCTGTAC

A품종B품종

PCR

136bp127bp

A품종B품종Sequence blast 결과

E. SNP 마커: Single Nucleotide Polymorphisms

• Polymorphism corresponding to

differences at a single nucleotide position

• Codominant marker

• No PCR-based and Gel-based

• Highly technology – DNAchips

Fertile plants MS plants

M H H Ho H Ho H

◄undigested◄digested

dCAPS marker genotype of F2 plants from Hwacheong ms-h(t) Hwacheong,

classified by the phenotype. ( M: 100bp ladder, H: hetero- zygous, Ho: homozygous)

○ dCAPS 방법으로 SNP 마커 분석

exon 14intron14

exon 15 exon 16intron15

AGGAAG (single nucleotide substitution)

Wild

Mutant

3' 5'

74-bp

100

200

300

예) UGPase 유전자 돌연변이 확인

: SNP 확인을 위한 primer-제한효소 조합은 Web 디자인 order

• 전기영동을 하지 않고도 SNP 차이를 감별할 수 있다.• PCR 산물을 전기영동하지 않고, 기기안에서 온도를 서서히 올리면서 이중나선이풀리는 시점의 curve를 가지고 감별한다. (염기쌍별로 수소결합 강도가 다른 성질을 이용)

• dsDNA-binding dye의 농도를 실시간 측정하여 분석 그래프 작성* 형광염료는 이중나선시에만 발현함.

○ HRM (high resolution melting) 분석기의 이용한 SNP 검출

신속 정확하고비용 저렴하다

384 well

G/G

A/G

A/ASNP의 homo/hetero 상태 감별

SNP의 감별

벼 indica(93-11)와 japonica(Nipponbare)의 sequence 비교

Feltus et al. 2004. Genome Res. 14:1812

DNA표지 genotyping 방법의 발전

RFLP마커

PCR-기반마커

SNP마커

Fluidigm: BioMark™ HD System

Illumina: BeadExpressR System

Labtech: HRM Capillary electrophoresisFragment Analyzer

~1,000점/일/인

>30,000점/일/인

○ Highthroughput SNP genotyping 방법

대용량 자동화 분자표지 분석시스템 : Array Tape과 TaqMan chemistry 방식

- 230,400 Data Point/6 hour 분석 가능

※ Array Tape 방식 : 1~2 작물의 대량 분석이 용이

대용량 자동화 샘플 전처리시스템 (동영상)

대용량 자동화 분자표지분석시스템(Douglas Scientific)

전자동 샘플 추출시스템

로봇의 팔 전자동 샘플

종자산업진흥센터의 분자표지 분석 장비

(조영일, 2016)13/41

DNA마커 간의 유용성 비교

RFLPPCR 기반 마커

RAPD AFLP SSR STS SNP

분석 원리 제한효소임의

프라이머선택적PCR

SSR 반복정도

InDel 길이DNA 칩/HRM/제한효소 등

소요 DNA양 (㎍) 10 0.02 0.5~1.0 0.02 0.02 0.02

Gel 사용 Yes Yes Yes Yes Yes Yes/No

Genome내분석 가능 마커 수

많음 매우 많음 매우 많음 매우 많음 매우 많음 매우 많음

마커 형태 공우성 우성 우성 공우성 공우성 우성/공우성

재현성 매우높음 Fair 매우높음 매우높음 매우높음 매우높음

염기서열정보 필요 No No No Yes Yes Yes

분석법 쉬운 정도매우복잡

쉬움 복잡 쉬움 쉬움 쉬움