TAP CHI SINH HOC 2014, 36(3): 265-294

Cc k thut ch th DNA
DOI: 10.15625/0866-7160/v36n3.5974

CC K THUT CH TH DNA TRONG NGHIN CU

V CHN LC THC VT

Nguyn c Thnh
Vin Cng ngh sinh hc, Vin Hn lm KH & CN Vit Nam, nguyenducthanh_pcg@ibt.ac.vn

TM TT: T thp k 80 ca th k trc, cc k thut ch th DNA c bt u v pht trin

nhanh chng tr thnh lnh vc quan trng trong sinh hc phn t. Cc ch th DNA c s dng
rng ri trong nghin cu v chn lc. Cc k thut ch th DNA c xy dng pht trin cc ch
th DNA cho nghin cu a dng di truyn, pht sinh loi, phn loi, nh du v xc nh gen; cho
chn lc ngun gen v chn ging nh ch th phn t. S pht trin ca hng lot cc ch th DNA
khc nhau cng vi cc nguyn l, phng php v ng dng ca chng dn n vic cn thit xem
xt mt cch cn thn trong vic la chn k thut ph hp cho mc ch nghin cu. Khng c ch
th DNA no hin bit c th p ng y tt c cc yu cu ca nh nghin cu. Ph thuc vo
vn nghin cu, c th chn trong cc k thut ch th DNA khc nhau m mi k thut c th c
mt s c tnh cn thit. Vit Nam, mt s k thut ch DNA c bt u s dng t cui nhng
nm 90 ca th k XX. Tuy nhin, vic s dng cn hn ch v mi ch yu s dng cc k thut nh
a hnh DNA nhn bn ngu nhin, k thut cc chui lp li n gin hay tiu v tinh, k thut a
hnh di cc on nhn bn chn lc trong cc nghin cu: a dng di truyn, lp bn lin kt
phn t v chn ging nh ch th phn t thc vt. Bi tng quan ny s gii thiu tng qut v hu
ht cc k thut ch th DNA hin c v ng dng ca chng trong nghin cu v chn lc thc vt
nhm cung cp thng tin cn thit v cp nht cho cc nh nghin cu phn loi, bo tn v chn
ging trong vic la chn k thut ch th DNA ph hp.
T kha: Chn ging nh ch th phn t, chn lc ngun gen, a dng di truyn, k thut ch th
DNA, xc nh gen.

M U bn (DAF-DNA amplification fingerprinting

K t khi ch th DNA u tin c pht [28], ch th trnh t biu hin (EST-expressed
trin v ng dng cho n cui nhng nm 90 sequence tags [2], a hnh di cc chui n
ca th k XX, hng lot ch th DNA hay cn gin (SSLP-simple sequence length
gi l ch th phn t c ra i l cc ch polymorphism [38], chui a hnh nhn bn
th: ch th a hnh di cc on ct hn ch c ct hn ch (CAPS-cleaved amplified
(RFLP-restriction fragment length polymorphic sequence [6], ch th to ra do phn
polymorphism [20], chui lp ngn lin k ng PCR vi mi thoi ha-mi c v tr m
(STR-short tandem repeats [64], s lng thay c th thay th cc oligo khc nhau (DOP-
i cc chui lp li lin k (VNTR-variable PCR-degenerate oligonucleotide primed PCR
number tandem repeat [127], ch th nhn bn [170], ch th nhn bn si thay th (SDA-strand
allen c bit (AS-PCR-allele specific displacement amplification [190], chui lp li
polymerase chain reaction [97], cc oligo allen n gin (SSR-simple sequence repeat [5], vng
c bit (ASO-allele specific oligo [15], a hnh nhn bn chui DNA c m t (SCAR-
cu to si n (SSCP-single stranded sequence characterized amplified regions [137],
conformation polymorphism [136], v tr chui a hnh nucleotide n (SNP-single nucleotide
nh du (STS-sequence tagged site [134], a polymorphism [78], ch th phn ng nhn bn
hnh DNA nhn bn ngu nhin (RAPD-random bng mi n (single primer amplification
amplified polymorphic DNA [197], ch th to reactions [60], a hnh cc locus tiu v tinh
ra do phn ng PCR vi cc mi ty tin (AP- nhn bn chn lc (SAMPL-selective
PCR-arbitrarily primed PCR [195], v tr trnh amplification of microsatellite polymorphic loci
t tiu v tinh c nh du (STMS-sequence [122], tiu v tinh neo nhn bn (AMP-PCR-
tagged microsatellite site [16], du DNA nhn anchored microsatellite primed PCR [212], a

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 Nguyen Duc Thanh

hnh tiu v tinh nhn bn ngu nhin (RAMP- gen mc tiu m ch l du hiu hoc nh l c
random amplified microsatellite polymorphism nh du. Ch th di truyn bao gm c ch th
[201], chui lp li n gin gia (ISSR-inter- hnh thi v ch th phn t (isozyme, protein,
simple sequence repeat [212]), cc mi lin DNA). Tt c ch th di truyn u chim mt v
quan n allen c bit (ASAP-allele specific tr c bit trong nhim sc th (NST) v c
associated primers [57], a hnh di on gi l locus. Cc ch th di truyn thng lin
nhn chn lc (AFLP-amplified fragment length kt vi gen v c di truyn theo qui lut di
polymorphism [200], ch th PCR lng v tr truyn. Ch th phn t thng c hiu l ch
chn lc (SSI-site-selected insertion PCR [92], th DNA, cc ch th ny ch nm gn hay lin
tiu v tinh nhn bn ngu nhin (RAM-random kt vi gen v khng c hoc t nh hng n
amplified microsatellites [66], a hnh nhn bn kiu hnh.
chui c bit (S-SAP-sequence specific Ch th di truyn c th chia thnh hai loi:
amplification polymorphism [193], cc trnh t ch th truyn thng v ch th DNA. Ch th
lp li n gim neo (ASSR-anchored simple truyn thng gm ch th hnh thi (y l
sequence repeats [191] v ch th PCR o nhng tnh trng hoc c im hnh thi nh
(IPCR-inverse PCR [187]. Cc k thut ch th mu hoa, kiu hnh ht, c im sinh trng,
DNA tng ng c xy dng pht trin s bin i sc t v.v.), ch th t bo (c trng
ch th DNA cho nghin cu a dng di truyn, cu trc ca nhim sc th) v ch th sinh ha
pht sinh loi, phn loi, nh du v xc nh (cc isozyme, protein v cc cht trao i cht).
gen; cho chon lc ngun gen v chn ging nh Ch th DNA l nhng thay i trong phn t
ch th phn t. Khng c ch th DNA no hin DNA v c chia thnh nhiu loi da trn s
c c th p ng y tt c cc yu cu ca khc nhau v phng php v k thut xc nh
nh nghin cu. Ph thuc vo vn nghin s a hnh.
cu, ta c th chn trong cc k thut ch th
DNA khc nhau m mi k thut c th c mt Cc ch th DNA c s dng rng ri nht
s c tnh cn thit. Vit Nam, mt s k do s lng ch th khng hn ch. Ch th DNA
thut ch DNA c bt u s dng t cui c hnh thnh t cc loi t bin DNA khc
nhng nm 90 ca th k XX. Tuy nhin, vic nhau nh thay th (t bin im), sp xp li
s dng cn hn ch v mi ch yu s dng (thm vo hoc bt i nucleotide) hoc cc sai
cc k thut nh a hnh DNA nhn bn ngu st trong sao chp cc on DNA lp li lin k.
nhin, k thut cc chui lp li n gin hay Cc ch th DNA thng nm cc vng khng
tiu v tinh, k thut a hnh di cc on phin m. Khc vi cc ch th hnh thi v sinh
nhn bn chn lc trong cc nghin cu: a ha, ch th DNA khng gii hn v s lng,
dng di truyn v nh gi ngun gen [22, 37, khng nh hng bi yu t mi trng v giai
171, 172], lp bn lin kt phn t [98, 173] on pht trin ca cy. Ch th DNA c s
v chn ging nh ch th phn t thc vt dng nhiu trong nghin cu quan h di truyn,
[99, 104]. Bi tng quan ny s gii thiu tng pht sinh chng loi v phn loi phn t; trong
qut v hu ht cc k thut ch th DNA hin lp bn lin kt di truyn, nhn bit gen; v
c v ng dng ca chng trong nghin cu v trong chn ging bao gm nh gi a dng di
chn lc thc vt nhm cung cp thng tin truyn, nhn bit ging, chn lc cc tnh trng
cn thit v cp nht cho cc nh nghin cu v khng bnh, chng chu cc iu kin bt li
chn ging trong vic la chn k thut ch th ca mi trng, nng sut v phm cht ging.
DNA ph hp cho nghin cu v chn lc Cc k thut ch th DNA
thc vt. Mi loi ch th DNA c pht trin bng
Ch th di truyn, ch th phn t v ch th mt k thut tng ng. K thut ch th DNA l
DNA tng cn phi c cc tiu ch sau: cho a hnh
Cc ch th di truyn l cc ch th th hin cao v phn b u trong genome; cho s phn
s khc bit gia cc c th hoc cc loi khc bit r s khc nhau v di truyn, to nhiu ch
nhau. Cc ch th ny khng th hin cho cc th c lp v chnh xc; n gin, nhanh v t

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tn km; cn t mu v DNA; lin kt vi kiu v c th. Trong bng 1 l cc cc k thut ch

hnh nht nh; c th lp li trong cc nghin th DNA hin c. Trong bi ny tc gi ch gii
cu, mc sai st thp nht, ghi s liu d v thiu khi qut v c s, nguyn l, mt s bc
chnh xc, c nhiu allen (hm lng thng tin c bn, nhng u nhc im v ng dng ca
cao), khng cn bit trc thng tin v genome mt s k thut ph bin.

Bng 1. Cc k thut ch th DNA

K hiu Tn ting Anh Tn ting Vit
K thut khng s dng PCR
RFLP Restriction Fragment Length Polymorphism a hnh di on ct hn ch
REF Restriction Endonuclease Fingerprinting Ly du ct hn ch
K thut s dng PCR
AFLP Amplified Fragment Length Polymorphism a hnh di nhn bn chn lc
RAPD Randomly Amplified Polymorphic DNA DNA a hnh c nhn bn ngu nhin
AP-PCR Arbitrarily primed PCR PCR vi mi ngu nhin
ARMS Amplification Refractory Mutation System H thng t bin chu nhit nhn bn
ASAP Arbitrary Signatures from Amplification Cc du hiu ngu nhin t nhn bn
SPAR Single Primer Amplification Reaction Phn ng nhn bn vi mi n
SPLAT Single Polymorphic Amplification Test Php th nhn bn a hnh n
S-SAP Sequence-Specific Amplification Polymorphisms a hnh nhn bn chui c trng
ASLP Amplified Sequence Length Polymorphism a hnh di chui nhn bn
CAPS Cleaved Amplification Polymorphic Sequence Chui a hnh nhn bn c ct hn ch
CAS Coupled Amplification and Sequencing Gii trnh t v nhn bn kt hp
DAF DNA Amplification Fingerprint Du nhn bn DNA
SAMPL Selective Amplification of Polymorphic Loci Nhn bn chn lc cc locus a hnh
K thut ch th da trn cc tiu v tinh
ISSR Inter-Simple Sequence Repeats Chui lp li n gin gia
ISTR Inverse Sequence-Tagged Repeats Chui lp li ngc c nh du
MP-PCR Microsatellite-Primed PCR PCR vi mi tiu v tinh
RAHM Randomly Amplified Hybridizing Microsatellites Tiu v tinh lai c nhn bn ngu
nhin
RAMPs Randomly Amplified Microsatellite a hnh tiu v tinh c nhn bn ngu
Polymorphisms nhin
VNTR Variable Number Tandem Repeats S lng thay i cc chui lp li lin k
Cc k thut ch th PCR cc chui c trng
STS Sequence-Tagged-Site V tr chui nh du
SCAR Sequence Characterised Amplification Regions Vng nhn bn chui c m t
SSLP Single Sequence Length Polymorphism a hnh di chui n gin
SSR Simple Sequence Repeats Cc chui lp li n gin
STMS Sequence-Tagged Microsatellite Site V tr chui tiu v tinh nh du
Cc k thut ch th gen nhy
REMAP Retrotrasposon-Microsatellite Amplified a hnh tiu v tinh gen nhy ngc
Polymorphism c nhn bn
RBIP Retrotrasposon-Based Insertion Polymorphism a hnh s gn da trn gen nhy ngc
IRAP Inter- Retrotrasposon Amplified Polymorphism a hnh gen nhy ngc gia c nhn bn
TD Transposable Display Biu l gen nhy
Cc k thut ch th nhn khc
ITS Internal Transcribed Spacer Vng m trong c sao m
SNP Single Nucleotide Polymorphism a hnh nucleotide n
OLA Oligonucleotide Ligation Assay Phn tch gn Oligonucleotide
SSCP Single Stranded Conformation Polymorphism a hnh cu to si n
ASO Allele Specific Oligonucleotide Oligonucleotide c trng allen
ASH Allele-Specific Hybridization Lai allen c trng

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 Nguyen Duc Thanh

Cc k thut ch th lc lp
cpSSR Chloroplast Simple Sequence Repeats Chui lp li n gin lc lp
RFLPA Restriction Fragment Length Polymorphism Phn tch a hnh di on ct gii
cpDNA Analysis cpDNA hn DNA lc lp
tRNAs Chloroplast Transfer RNAs Cc RNA vn chuyn lc lp
Cng ngh h tr hin i
DarT Diversity array Technology Cng ngh sp xp a dng
NGS Next-geneation sequencing Gii trnh t th h th hai

Cc k thut ch th DNA c chia thnh nh du vi DNA sau khi ct hn ch bng

cc nhm chnh l: cc k thut khng s dng enzyme ct hn ch v c thm truyn ln
phn ng PCR v cc k thut s dng PCR. mng lai bng phng php Southern. Kt qu l
Ngoi ra, cn phi k n mt s cng ngh h to ra hnh nh cc phn on DNA khc nhau.
tr ht sc hiu qu cho pht trin ch th v Cc hnh nh phn on DNA khc nhau ny
nhn bit a hnh ch th DNA nh cng ngh c to nn do s thay th, thm vo hay bt i
sp xp a dng (Diversity array Technology) ca cc nucleotide hoc do a hnh nucleotide
v cng ngh gii trnh t th h th hai (Next- n. K thut RFLP c tin hnh theo cc
generation sequencing). bc: ct DNA bng mt hoc vi enzyme ct
Cc k thut ch th DNA khng s dng PCR hn ch; cc phn on DNA sau c phn
tch trn gel agarose v thm truyn ln mng
K thut a hnh di on ct hn ch lai. Vic xc nh cc phn on DNA c tin
(RFLP) hnh bng lai cc phn on DNA ny vi on
Trong k thut a hnh di on ct hn d c nh du hunh quang hoc phng x
ch (Restriction Fragment Length c th pht hin bng phn ng hunh quang
Polymorphism-RFLP), a hnh DNA c xc hoc phim chp phng x (hnh 1).
nh bng cch lai on d DNA (DNA probe)

Hnh 1. Cc bc tin hnh k thut RFLP: Sau khi tch chit, DNA genome c ct bng enzyme
ct hn ch, cc phn on DNA sau c phn tch bng in di trn gel agarose, ri c
thm truyn bng phng php Southern ln mng nylon v c lai vi cc on d c nh
du bng phng x hoc hunh quang, cui cng l hin trn phim hoc nhum bng cht hin mu

268

Cc ch th RFLP c mc a hnh cao, ng
tri (nn c th phn bit c cc c th d hp
t v ng hp t) v kh nng lp li cao. K
thut RFLP cng cho php phn tch ng thi
nhiu mu. Tuy nhin k thut ny khng c
dng rng ri v mt s hn ch nh: cn nhiu
DNA cht lng cao, phi pht trin th vin
on d cho tng loi, cn thng tin v trnh t
to on d, khng thun tin cho vic t
ng ha, mc a hnh v s lng locus
trn ln phn tch thp, i hi nhiu thi gian,
tn km. Trong nhng thp nin 80 v 90 ca
th k trc, k thut RFLP c s dng trong
lp bn genome [54, 96], xc nh ging
[82]. RFLP c dng trong nghin cu quan
h gia cc nhm phn loi gn [119], a dng
di truyn [42], trong lai to v chuyn gen vo
c th khc (introgression) bao gm c tri gen
gia cy trng v c di [36].
Cc k thut da trn phn ng PCR
Vi s pht trin k thut phn ng chui
trng hp (polymerase chain reaction-PCR)
dn n nhng tin b vt bc trong vic ci
tin cc k thut xy dng cc ch th DNA da
trn phn ng PCR [123]. Cc k thut ch th
DNA da trn phn ng PCR, ngoi hai k
thut s dng rng ri l RAPD v AFLP, da
vo s nhn bn cc chui hoc vng DNA
khc nhau c th chia ra mt s nhm nh: k
thut ch th cc chui c trng, k thut ch th
tiu v tinh, k thut ch th gen nhy.
K thut a hnh DNA nhn ngu nhin (RAPD)
C s ca k thut RAPD l s nhn bn
DNA genome bng phn ng PCR vi cc mi
ngu nhin to ra s a hnh DNA do s ti
sp xp hoc mt nucleotide v tr bt mi
[197].
Mi s dng cho k thut RAPD l cc mi
ngu nhin, thng l 10 nucleotide v c nhit
ko di mi thp (34-37oC). Mc d trnh t
mi RAPD l ngu nhin nhng phi t c
hai tiu ch l: t l GC ti thiu phi l 40%
(thng l 50-80%) v khng c trnh t baz
u xui v ngc ging nhau. Sn phm PCR-
RAPD thng c phn tch trn gel agarose
1,5-2,0%.
K thut RAPD khng cn thng tin v
genome ca i tng nghin cu v c th ng
Cc k thut ch th DNA
dng cho cc loi khc nhau vi cc mi chung.
Hn na, k thut RAPD n gin v d thc
hin. Nhng k thut RADP c hn ch l sn
phm PCR khng n nh do mi ngn, nhit
bt mi thp; ngoi ra, k thut ny to ra cc
ch th tri do khng phn bit c cc c
th d hp t vi cc c th ng hp t. RADP
s dng nhiu trong nghin cu a dng di
truyn gia cc loi thc vt [84, 85, 176, 177,
179, 180, 185], trong nghin cu c im ca
ging v nh gi bin i di truyn [114],
trong xc nh loi [52, 153] v xc nh con lai
[10, 152].
K thut PCR vi cc mi ngu nhin
(Arbitarily Primed PCR-AP-PCR) v ly du
bng nhn bn DNA (DNA amplification
fingerprinting-DAF) l hai k thut c pht
trin c lp v l hai dng bin th ca k thut
RAPD. i vi AP-PCR ch dng mt mi n
c di 10 n 15 nucleotide. K thut c
thc hin khc vi PCR bnh thng l hai chu
k u c tin hnh iu kin khng cht
ch (nhit bt mi thp) sau cc chu k
sau PCR c tin hnh trong iu kin cht
ch bng vic tng nhit bt mi. Trong
trng hp DAF th ch s dng mt mi ngu
nhin ngn hn 10 nucleotide v sn phm PCR
c phn tch bng gel polyacrylamide. Vi
DAF, mi s dng ngn nhng nng mi cn
cao, phn ng PCR gm hai chu k nhit ch
khng phi ba chu k nh RAPD. K thut AP-
PCR khc vi RAPD v DAF l: phn ng PCR
chia thnh ba bc, mi bc c cht ch v
nng ca cc thnh phn phn ng khc
nhau. Nng mi nhng chu k u cao,
mi di 20 nucleotide hoc hn.
K thut a hnh di on nhn chn lc
(Amplified Fragment Length Polymorphism-
AFLP)
K thut AFLP c s dng pht hin
a hnh DNA. Phn tch AFLP c kt hp c
RFLP v PCR bng vic gn cc chui nhn
bit vo mi hay cn gi l chui tip hp
(adapter) nhn chn lc cc phn on DNA
c ct hn ch. Cc cp mi thng to c
t 50 n 100 bng trong mt phn tch. S
lng bng ph thuc vo s nucleotide chn
lc trong t hp mi. K thut AFLP cho php
ly du DNA t bt k ngun gc no.
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 Nguyen Duc Thanh

K thut AFLP c tin hnh theo mt s kiu gen c mi quan h gn [69, 159], nghin
bc nh trong hnh 2: u tin DNA genome cu cu trc di truyn ngun gen [8, 68] v
c ct bi enzyme ct hn ch ct khng nh gi phn ha di truyn trong qun th
thng xuyn (EcoRI hoc PstI) v ct thng [186, 196].
xuyn (MseI hoc TaqI). Cc phn on to ra
sau khi ct hn ch c gn vi cc on kt
ni c hai u. Cc on kt hp ny c
thit k sao cho v tr nhn bit ban u ca
enzyme ct hn ch khng khi phc li sau khi
gn. Phn ng PCR ch xy ra khi cc mi c
th gn vi cc phn on c trnh t kt ni
cng vi cc cp baz b sung vi cc
nucleotide chn lc. Vic nhn bn c thc
hin hai ln trong iu kin cht ch vi cc
mi b tr vi cc on tip hp v 1 n 3
nucleotide chn lc u 3. Phn ng PCR ln
u (tin nhn) c tin hnh vi t hp mi
cha mt nucleotide chn lc. Phn ng PCR
ln hai (nhn chn lc) c tin hnh vi cc
cp mi c 1 n 3 nucleotide chn lc. Do mc
chn lc cao, cc mi khc nhau ch mt
nucleotide s cho cc b phn on nhn bn
khc nhau. Cc mi vi 1, 2 v 3 nucleotide
chn lc s gim s phn on c nhn bn
Hnh 2. Cc bc tin hnh k thut AFLP:
tng ng l 4, 16 v 64. S nucleotide chn
DNA genome c ct bng enzyme EcoRI v
lc ph hp thay i theo kch thc genome
MseI to ra cc on ct hn ch, cc on
ca loi. Cc phn on AFLP c phn tch
ct c gn vi on kt ni EcoRI v MseI,
bng gel polyacrylamide kt hp nhum bc
tip theo cc on ct gii hn c gn on kt
(AgNO3) hoc bng my gii trnh t t ng.
ni c nhn bn bng PCR vi cc mi
Phn tch AFLP khng d nh RAPD EcoRI v MseI tng ng c thm mt
nhng hiu qu hn RFLP. K thut ny c mt nucleotide (tin nhn), sn phm PCR nhn
s u im nh: c tin cy v lp li cao; c c nhn vi t hp cc mi EcoRI v
khng cn thng tin v trnh t DNA ca c th MseI c thm 1 n 3 nucleotide (nhn chn
nghin cu; cho nhiu thng tin do c kh nng lc), sn phm PCR chn lc c phn tch
phn tch s lng ln locus a hnh vi mt t trn gel polyacrylamide v nhum bc quan
hp mi trn mt gel v c th cho thy cc st cc bng.
locus c th; cc s liu c th lu gi trong c
K thut ch th da trn cc tiu v tinh
s d liu so snh.
(microsatellite) hay chui lp li n gin
Bn cnh cc u im, AFLP c mt s (simple sequence reapeats).
nhc im nh: phi qua nhiu bc mi c
y l nhm k thut s dng cc cp mi
kt qu; DNA cn phi sch, khng c cc cht
c trng nhn cc vng genome c cc
c ch hot ng ca enzyme ct hn ch; i
nucleotides lp li cc v tr khc nhau bao
hi cng sc v gi thnh cao. K thut to ra
gm: k thut chui lp li n gin hay cn gi
ch th tri, khng phn bit c ng hp t
l k thut a hnh di cc chui n gin
v d hp t.
(Single Sequence Length Polymorphism-SSLP),
AFLP c s dng trong nghin cu lp k thut v tr chui tiu v tinh nh du
bn genome [14, 173], a dng di truyn [53, (Sequence-Tagged Microsatellite Site-STMS),
103, 176, 178] v quan h chng loi gia cc k thut chui lp li gia (Inter-Simple

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 Cc k thut ch th DNA

Sequence Repeat-ISSR) v k thut a hnh cc hnh, c th phn tch bn t ng, khng s

tiu v tinh c nhn bn ngu nhin dng phng x, c th s dng cc DNA c
(Randomly Amplified Microsatellite (ancient DNA-aDNA). SSR c th phn bit cc
Polymorphisms-RAMP). Chng ta s xem xt c th c mi quan h gn. im hn ch quan
ln lt cc k thut ny trong cc phn sau. trng ca k thut ch th SSR l cn phi c
K thut chui lp li n gin (Simple trnh t genome da vo c th thit k
Sequence Repeats-SSR) cc cp mi c th v ti u ha iu kin cc
mi cho tng loi trc khi s dng. Hin nay,
Chui lp li n gin (Simple Sequence SSR l ch th c chn cho cc nghin cu h
Repeats-SSR) [169], tiu v tinh (microsatellite) s php l, di truyn qun th v nghin cu
[107], chui lp li trc sau ngn (Short ng vt hoang d. thc vt SSR c s
Tandem Repeats-STRs) hay a hnh di chi dng trong nghin cu a dng di truyn [53,
n gin (simple sequence length 71,109,117, 121, 171, 172, 175, 183, 210,],
polymorphisms-SSLPs) [115] l s lp li cc trong chn cp lai (43, 71], trong xc nh con
chui nucleotide ngn t 2-6 nucleotide [30]. lai [1] v trong lp bn lin kt phn t [39,
SSR (SSLP, STMS) tr thnh k thut ch 110, 173].
th phn t quan trng trong c ng vt v thc K thut a hnh di cc chui n gin
vt. SSR rt a hnh do t bin tc ng ln s (Single Sequence Length Polymorphism-SSLP)
n v lp li. S thay i hay s a hnh ca l k thut ch th to cc chui lp li vi
SSR l kt qu ca s khc nhau v di cc di khc nhau nm gia hai gen. Cc chui c
on lp li trong genome do qu trnh trao i di khc nhau l do s lng trnh t lp li
cho khng cn hoc do s gim nucleotide trong chui khc nhau do qu trnh trao i
trong qu trnh sao chp. SSR khng nhng ph cho khng cn hoc do s gim nucleotide
bin m cn bin ng mnh v s lng kiu trong qu trnh sao chp. C hai qu trnh ny
lp li trong genome sinh vt nhn thc. S u c th dn n gim hoc tng s lng n
khc nhau allele ca SSR l kt qu ca s thay v lp li trong genome. S khc nhau v di
i s lng n v lp li trong cu trc tiu v ca SSLP c s dng nh gi s thay i
tinh. Cc chui lp li thng n gin v cu di truyn gia cc c th trong loi.
to bi 2, 3 hoc 4 nucleotide.
K thut SSLP ging nh k thut SSR,
K thut SSR c thc hin bng phn cng c tin hnh bng cc cp mi c hiu
ng PCR vi mi SSR xui v ngc. Sn nhn bn cc chui n gin nm gia hai gen,
phm PCR c phn tch trn gel sn phm PCR c phn tch trn gel
polyacrylamide kt hp nhum bc (AgNO3) polyacrylamide.
hoc bng my gii trnh t t ng.
SSLP c s dng trong nghin cu a
Vic pht trin ch th SSR c tin hnh dng di truyn [12], xc nh ging [67], xc
theo mt s bc nh: xy dng th vin SSR, nh d hp t [135] v lp bn di truyn [5,
xc nh locus SSR, xc nh vng ph hp 202].
thit k mi, PCR vi cc mi c thit k,
K thut v tr chui tiu v tinh nh du
nh gi v phn tch mu bng, nh gi a
(Sequence-Tagged Microsatellite Site-STMS)
hnh ca sn phm PCR.
[21, 181, 182] l k thut ch th nhn bn cc
K thut SSR c mt s u vit hn cc ch chui lp li v tr c nh du bng cc
th khc nh: i. Cho nhiu allen trong mt locus; cp mi c hiu. K thut ny cng c s
ii. Phn b u trong genome; iii. SSR cho dng nhiu trong nghin cu a dng di truyn,
thng tin c th hn so vi di truyn ty th theo phn tch genome [86], lp bn di truyn [51,
ng m (v c mc t bin cao) v di truyn 198] v nhn bit ging [47].
theo c b v m; iv. L ch th ng tri; v. C
u im ni bt ca SSR, SSLP v STMS
tnh a hnh v c th cao; vi. C th lp li
l cc ch th ny c th s dng chung cho mt
cc th nghim, s dng t DNA, r v d tin
s loi cy. Chng hn STMS ca cy u ng

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 Nguyen Duc Thanh

(Pisum sativum L.) c th dng cho cy u ca ISSR l to ra cc ch th tri [60, 191] v

chu (Cicer arietinum L.) v STMS ca u s d ng nht ca cc sn phn nhn bn do
chu c th dng cho u lng (Lens culinaris s di chuyn ng thi.
L.) v u tm thuc chi Vicia [7], STMS ca
cy h u c th dng cho lc [22].
K thut chui lp li n gin gia (Inter-
Simple Sequence Repeat-ISSR)
ISSR l nhng ch th c nhn bn bng
PCR vi mt mi b tr vi tiu v tinh
(microsatellite) ch. Mi bng tng ng vi
chui DNA c gii hn bi cc tiu v tinh
o ngc. Kt qu dn n a locus, c s a
hnh cao v to ra cc ch th tri. ISSR-PCR l
k thut nh gi kiu gen nhanh, khng t; s
a hnh da trn s thay i trong cc vng nm
gia cc tiu v tinh. K thut ny khng cn
thng tin v trnh t, to c nhiu locus, c
tnh a hnh cao v to ra ch th tri [120]. K
thut ISSR l k thut nhn bn on DNA nm
gia hai vng lp li ging ht v ngc chiu Hnh 3. Nhn bn chui lp li nm gia hai
nhau (hnh 3). K thut ny s dng cc tiu v chui (CT)n ngc chiu nhau bng mi n
tinh nh cc mi trong phn ng PCR vi mt (GA)n: A. Mi n (GA)n khng neo c th bt
mi cho nhiu locus ch nhn bn ch yu cp bt k v tr no trong vng (CT)n ca
cc chui lp li n gin vi di khc nhau. DNA khun nn vic nhn bn khng c
Cc tiu v tinh s dng nh mi trong k thut chun, B. Mi n (GA)n c hai nucleotide
ISSR c th l 2, 3, 4, hoc 5 nucleotide. Cc (NN) neo u 3 bt cp vo vng c th trn
mi s dng c th khng phi mi neo hoc l DNA khun v to bng r rng, C. (GA)n c
mi neo u 3 hoc 5 vi 1 n 4 nucleotide hai nucleotide (NN) neo u 5 bt cp vo
thoi ha ko n cc chui bn cnh. K thut vng c th trn DNA khun nhn bn c
ISSR s dng mi di (15 n 30 nucleotide) v mt phn vng lp li to ra cc bng ln.
vy nhit bt mi cao dn n n nh cao
ca phn ng. Sn phm c di 200-2000 bp ISSR c dng trong nghin cu a dng
nn c th phn tch trn c gel agarose v di truyn nhiu loi cy trng khc nhau nh la
polyacrylamide. [79, 149], la m [124], k [156], nho [3], khoai
lang [70], m [199], to ta [131] v.v.
K thut ISSR c s dng rt rng ri
trong nghin cu a dng di truyn [58, 149], K thut a hnh tiu v tinh c nhn bn
nghin cu c im di truyn trong qun th ngu nhin (Randomly Amplified Microsatellite
[145], ly du di truyn [18, 29], nh du gen Polymorphisms-RAMP)
[147], xc nh cy trng [47, 108], phn tch Cc k thut ch th da vo tiu v tinh cho
ngun gc [48], xc nh s thay i genome mc a hnh allele cao nhng li tn nhiu
[101] v nh gi con lai [105]. K thut ISSR cng sc. K thut RAPD khng tn km
c mt s li th so vi cc k thut khc l c nhng mc a hnh thp. dung hon yu
th phn bit c cc kiu gen gn v khng im ca hai k thut ny, k thut RAMP c
cn thng tin v trnh t gen ca cy nghin xy dng [201]. K thut ny s dng mi c
cu. Ging nh RAPD, ISSR l k thut nhanh, nh du cha u 5 neo v u 3 lp li
d tin hnh. N u vit hn RAPD l cho nhn DNA genome vi s c mt hoc vng
nhiu thng tin v c th lp li cc th mt cc mi RAPD. Sn phm nhn c phn
nghim do mi di hn. Nhc im ch yu tch trn gel polyacrylamide bin tnh. Do mi

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 Cc k thut ch th DNA

lp li c nh du nn ch c sn phm ca c u lp li di (Long Terminal Repeat (LTR)

mi neo c xc nh. Nhit bt cp ca retrotransposon) v gen nhy khng c u lp
mi neo thng cao hn mi RAPD 10-15oC. li di (non-LTR retrotransposon) bao gm yu
V vy, nhit bt mi cao ch c mi neo t LINE (Long INterspersed repetitive Element)
c ko di hiu qu. cc chu k PCR vi v SINE (Short INterspersed repetitive
nhit bt mi thp, c hai mi neo v mi Element). Hai nhm ph ny c phn bit
RAPD u ko di. Do chng trnh PCR bng s c mt v vng mt u lp li di
c thit lp sao cho c s chuyn gia nhit (LTR) u. Tt c cc nhm u c b
bt mi cao xung thp trong qu trnh phn sung cc dng khng ph thuc tng ng thiu
ng. Hu ht cc phn on nhn c ch vi mt hoc nhiu gen quan trng cho s chuyn
cc mi RAMP s bin mt khi c cc mi v nh: cc yu t c u lp li ngc nh
RAPD v cc mu bng khc nhau nhn c (Miniature Inverted repeat Terminal Elements-
bi cng mt mi RAMP v cc mi RAPD MITEs) cho nhm II, SINEs cho non-LTR
khc nhau cho thy cc mi RAPD cnh tranh retrotransposons v gen nhy ngc c u lp
vi mi RAMP trong chu k vi nhit bt li nh (Terminal-Repeat retrotransposons In
mi thp. RAMP c s dng trong nghin Miniature-TRIMs) v phin bn gen nhy li
cu a dng di truyn mt s loi cy nh la ln (large retrotransposon derivatives-LARDs)
mch [157, 201] v o [33]. cho LTR retrotransposons.
Cc k thut ch th phn t da vo gen nhy Gen nhy ngc (retrotransposons) cho
(Transposable element-based molecular php to ra h ch th phn t tuyt vi bi
markers) chng c chui di v bo th, a hnh thm
Gen nhy l cc yu t di truyn c kh nucleotide c to ra bi hot ng sao chp.
nng thay i v tr ca chng trong h gen. Gen Vic b sung nucleotide mi gip cho vic t
nhy c pht hin u tin ng [111]. C chc cc hin tng b sung tm thi trong
hai loi gen nhy. Loi I l gen nhy ngc dng ging v nh vy c th s dng xc
(retrotransposon), y l yu t ngn v di nm nh ph h v pht sinh loi [63]. Phn tch
ri rc trong nhn, l yu t m ha mRNA v phn t da vo gen nhy ngc c thc hin
c mc thay i v tr trung bnh. Hin tng vi vic nhn bn bng mi tng ng vi gen
gen nhy loi I to ra cc bn sao mi trong khi nhy v mi ph hp vi vng gen bn cnh.
bn gc vn gi nguyn v tr ban u. Cc a hnh gen nhy c mt s k thut sau: K
bn sao c to ra theo hai giai on: u tin thut a hnh chui c th c nhn bn
l phin m t DNA thnh RNA v tip theo l (Sequence-Specific Amplified Polymorphism-
RNA to thnh c phin m ngc thnh S-SAP) l nhn bn DNA nm gia v tr gn
DNA. Bn sao mi c gn vo v tr mi ca gen nhy v v tr ct hn ch cng vi
trong genome. Gen nhy loi II (gen nhy chui tip hp [193]; k thut a hnh gen nhy
DNA) cu to bi gen nhy DNA, loi ny thay ngc gia c nhn bn (Inter-Retransposon
i v tr bng c ch ct v gn [56]. Chng Amplified Polymorphysm-IRAP) l nhn bn
t ri khi v tr ban u gn vo v tr tip chui DNA nm gia hai gen nhy gn nhau
nhn. Da vo c im cu trc, gen nhy cn hoc chui lp li cui trc tip di (Long
phn chia thnh di nhm, trn h, h v di Terminal Direct Repeats-LTR) c nhn bn;
h da trn s nh hng ca cc khung c k thut a hnh tiu v tinh gen nhy ngc
m; vo s c mt, nh hng, di v trnh c nhn bn (Retrotransposon-microsatellite
t lp li bn trong v vo di v trnh t Amplified Polymorphism-REMAP) c tin
nhn i v tr ch to nn do s thm hnh bng vic nhn bn phn on nm gia v
nucleotide [56]. tr gn ca gen nhy v v tr tiu v tinh; k
thut a hnh v v tr gn (insersion) da vo
Gen nhy ngc (retrotransposons) hay gen gen nhy ngc (Retrotransposon-based
nhy loi I chia thnh hai nhm ph khc nhau Insersion Polymorphism-RBIP) c s dng
cu trc v chu k chuyn v l gen nhy xc nh cc locus b gen nhy chim hoc

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 Nguyen Duc Thanh

locus rng; k thut biu l gen nhy v cc s kin b sung nucleotide [189].
(Transposable Display-TD). Cc k thut ch th PCR cc chui c trng
K thut S-SAP c s dng u tin K thut vng nhn bn chui c m t
trong nghin cu v tr ca gen nhy BARE1 (Sequence Characterized Amplified Regions-
(barley retrotransposable element 1) trong la SCAR)
min [93]. V c bn, y l dng n gin ca
k tht AFLP. Vic nhn bn c thc hin c th s dng cc ch th xc nh c
vi mi c hiu cho gen nhy v mi c hiu bng cc mi ngu nhin (nh RAPD, AFLP
cho on tip hp MseI (MseI-adaptor). S-SAP v.v.) v khc phc nhc im mn cm vi
c s dng trong nghin cu a dng di iu kin phn ng ca cc phn ng vi mi
truyn v lp bn [138, 144]. ngu nhin, k thut ch th SCAR c pht
trin v s dng. K thut SCAR l k thut ch
Trong k thut IRAP v REMAP a hnh th da vo PCR to ra cc phn on DNA ti
c xc nh bng s c mt hoc vng mt cc locus xc nh bng s dng cc mi
ca gen nhy ngc ti locus ring bit. Mt nucleotide c th [137]. SCAR c tin hnh
phn ng PCR c th to c khong 30 bng. bng cch tch dng sn phm PCR vi cc mi
Cc ch th ny c a hnh cao v c th s ngu nhin sau c trnh t hai u on tch
dng trong nh gi quan h trong loi. IRAP dng. Da vo trnh t hai u ny thit k
c s dng nghin cu c im h gen cp mi vi di 15 n 30 bp nhn bng
[126]. IRAP v REMAP c s dng pht n vi kch thc tng t nh phn on
hin s ging nhau gia cc ging la [22]. c nhn dng. S a hnh c xc nh bng
K thut RBIP c pht trin nh s dng s c mt hay vng mt ca bng c nhn
gen nhy PDR1 ca Pisium sativum [50]. Trong bn hoc s sut hin a hnh v chiu di ng
k thut ny, cn phi c thng tin v trnh t thi chuyn ch th tri thnh ch th SCAR
u 5 v 3 ca cc vng k bn gen nhy. Khi ng tri. K thut SCAR c dng phn
s dng mi c th cho gen nhy cng vi mi tch th vin genome [31], xc nh cc locus
thit k ko di ti vng k bn s cho sn c th [137], chn ging nh ch th phn t
phm t DNA khun c cha nucleotide b [95].
sung. Mt khc, cc mi c th cho c hai K thut v tr chui nh du (Sequence-
vng k bn s to sn phm nu khng c b tagged Sites-STS)
sung nucleotide. S a hnh c xc nh trn
gel agarose hoc lai vi phn on PCR tham Chui v tr nh du (STS) l vng ngn
kho (tham chiu). y l k thut kh tn km trong genome (c di 200-300 cp baz) m
v phc tp so vi cc k thut gen nhy khc. trnh t ca n khng th c bt c ni no
RBIP c s dng trong nghin cu a dng di khc trong genome. Chui trnh t duy nht ny
truyn v tin ha u ng (Pisum) [77]. c th nhn bn c bng PCR. Trnh t DNA
ca STS c th c cc yu t lp li, v cc trnh
K thut TD l dng ci tin ca k thut t xut hin bt c u trong genome, nhng
AFLP, k thut ny cho php xc nh ng trnh t hai u ca STS l duy nht. Chng ta
thi nhiu yu t phin m (TEs) t cc dng c c th tng hp cc mi duy nht b sung vi
s lng bn sao cao. Trong k thut ny, sn trnh t hai u STS v nhn on STS bng
phm PCR c to ra nh cc mi neo v tr PCR. Vi ngha rng, STS bao gm cc ch th
ct hn ch ca hai enzyme BfaI v MseI v yu nh microsatellites (SSRs, STMS or SSRPs),
t gen nhy. Cc gen nhy c xc nh bng SCARs, CAPs v ISSRs.
PCR gn (ligation-mediated PCR) bt u trong
phm vi gen nhy v nhn bn t phn chui k K thut STS l s dng PCR nhn chn
bn n v tr ct hn ch c th. Sn phm lc v tr nh du (STS) bng cp mi c hiu
PCR c th phn tch trn gel polyacrylamide. gn vi hai u ca chui STS vi iu kin ca
TD c s dng u tin khm ph s bn phn ng sao cho ch nhn c chui STS ch
sao ca nhm gen nhy dTph1 (TIRs) Petunia m khng phi chui no khc trong genome.

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 Cc k thut ch th DNA

Sn phm PCR c phn tch trn gel agorose no. K thut ny rt hu ch cho vic theo di
hoc polyacrylamide. u im ca k thut cc t bin bit trong qun th phn ly
STS l to ra cc ch th ng tri, c th phn [128] v pht trin ch th, c bit l ch th
bit c d hp t v c tnh lp li cao v cc SNP lin kt vi gen/QTL phc v cho chn
mi s dng di. Nhc im ca k thut ny ging [40, 100].
l cn bit v trnh t vng STS v cn u t K thut a hnh cc chui lin quan c nhn
cho vic pht trin mi c hiu. bn (Sequence-Related Amplified
STS c s dng cho nghin cu a dng Polymorphism-SRAP)
di truyn [139], nhn bit gen [89], so snh bn K thut SRAP [102] l k thut nhn bn
[25], xc nh gen lin kt [125], xc nh cc khung c m (ORFs). Trong k thut ny
thun ht lai [206] v nh gi tnh khng bnh dng hai mi ngu nhin c di 17 n 21
[13]. nucleotide v giu AT hoc GC nhn phn
Cc k thut ch th da trn phn ng PCR on bn trong gen cho a hnh c xc
khc nh. Cc mi cu to bi cc thnh phn: cc
K thut cc chui a hnh nhn bn c ct chui li (core primers) vi 13-14 nucleotide,
(Cleaved Amplified Polymorphic Sequences- trong 10 -11 nucleotide u bt u t u 5
CAPS) l trnh t khng c th (hay cn gi l chui
ri) tip theo l cc trnh t CCGG cho mi
K thut ch th CAPS l gii php s dng xui v AATT cho mi ngc. Cc mi li
cc chui DNA ca ch th RFLP c lp c gn vi ba nucleotide chn lc u 3.
bn pht trin ch th PCR nhm trnh Chui ri ca mi ngc v xui phi khc
bc lai Southern phc tp. V th, k thut nhau v c th di 10-11 nucleotide. Nm chu
CAPS cn c gi l k thut PCR-RFLP k u vi nhit ko di mi l 35oC, 35 chu
[93]. K thut CAPS c tin hnh bng ct k thip theo nhit ko di mi l 50oC. Cc
hn ch sn phm PCR ca cc locus c th phn on DNA nhn bn c phn tch bng
vi mt hoc nhiu enzyme ct hn ch v phn gel acrylamide bin tnh. K thut SRAP kt
tch cc on DNA c ct trn gel agarose hp s n gin, tin cy v d dng c trnh
hoc polyacrylamide. Cc cp mi c thit k t cc bng chn lc. SRAP hng ti chui m
da trn thng tin v trnh t ca DNA hoc ha trong h gen to ra cc ch th ng tri. a
cDNA trn ngn hng gen hoc trnh t cc hnh cc chui nhn bn to nn do hai s kin
bng RAPD c tch dng. Ch thi CAPS l l s thay i di phn on DNA do b
ch th ng tri v l cc locus c th c sung hoc mt nucleotide to ra ch th ng tri
dng phn bit ng hp t v d hp t v s thay i nucleotide to ra ch th tri.
cc loi thc vt [93]. K thut CAPS c hn SRAP c dng lp bn [34, 106, 166]
ch v s t bin c th lm mt hoc to thm v nghin cu a dng di truyn [59, 163].
cc v tr nhn bit ca cc enzyme ct hn ch.
khc phc hn ch ny, Michae & Amasino K thut a hnh cu to si n (Single Strand
(1998) [118] a ra k thut bin th khc gi l Conformation Polymorphism-SSCP)
dCAPS. Trong phn tch dCAPS, v tr nhn a hnh cu to si n l k thut ci tin
bit ca enzyme ct hn ch c SNP c a phn tch s thay i v tr ca cc chui DNA
vo sn phm PCR bng mi cha mt hoc n trn in di gel polyacrylamide trung tnh
nhiu im bt hp (mis-match) vi khun xc nh a hnh to nn bi s cun khc
DNA. Sn phm PCR ci tin ny sau c nhau ca si n DNA do cc thay i kh thy
ct bng enzyme ct hn ch v s c mt hay trong chui nucleotide [136]. Phn tch SSCP l
vng mt ca SNP c xc nh bng mu ct cng c mnh cho vic nh gi sn phm PCR
hn ch to ra. CAPS v dCAPS l phng phc tp khi hai si DNA t cng sn phm
php xc nh SNP ct sn phm PCR c bit PCR thng chy tch ri trn SSCP gel, v th,
bng cc enzyme ct hn ch. y l k thut r c hai kh nng ghi nhn a hnh v gii quyt
thc hin, t tn km so vi bt k phng php vn a hnh bn trong chui ca sn phm

275
 Nguyen Duc Thanh

PCR t vng ging ht nhau trong h gen ca nht cho ti nay. ng c 60 n 100 bp c
cc c th nghin cu. Nguyn l ca phng mt SNP [35], ngi 90% thay i trnh t l
php phn tch SSCP l da trn thc t: si thay i nucleotide n v c 1000 bp c mt
n DNA c cu to nht nh. Thay i cu SNP [155].
to bng s thay i mt nucleotide s lm cho thc vt, SNP ang c thay th cho
si n DNA chuyn ng khc i trong iu SSR nh ch th cho cc ng dng trong di
kin in di trn gel khng bin tnh. V th, truyn v chn ging. SNP c s lng ln, n
mu nguyn thy v mu t bin s c mu nh, hiu qu, cho php t ng ha, v ngy
in di khc nhau. Phn tch SSCP c tin cng kinh t hn [44, 46]. Hn na, SNP xy ra
hnh theo cc bc: i. nhn bn PCR chui c cc vng m v khng m ca DNA nhn
DNA quan tm, ii. bin tnh sn phm PCR, iii. v lc lp. Ngun SNP hin ang c pht
lm lnh chui DNA bin tnh to si n, trin v thng tin rng ri cho nhiu ng dng
iiii. xc nh s di chuyn khc nhau gia cc la. Cc ngun ny bao gm c s d liu SNP,
si n trong iu kin in di trn gel khng phng tin xc nh cc SNP mang nhiu
bin tnh. C th quan st s khc nhau bng thng tin cho cc mc ch ng dng v b SNP
nh du phng x, nhum bc, bng cc mi cho nh gi c thit k theo t hng phc
PCR c nh du bng mu hunh quang v v mc ch chn lc nh ch th phn t.
in di mao qun. PCR-SSCP l k thut nhanh,
n gin, nhy dng cho xc nh cc t bin Mc d SNP c cc u vit hn cc k thut
b sung, thm vo v bt i nucleotide trong khc, n vn c mt s nhc im nh: s hn
phn on DNA c nhn bn. y l cng c ch khm ph cc SNP trong cc c th khng
quan trng cho phn tch gen c bit l xc phi hnh mu do s tn km v kh khn trong
nh t bin im. K thut ny ging k thut cc cng ngh ang c s dng pht hin
RFLP v n c th gii on nhng thay i SNP.
allen ca cc tnh trng di truyn. Khc vi C rt nhiu phng php pht hin SNP
RFLP l SSCP c th xc nh c a hnh bao gm cc phng php lai (lai allele c
DNA v t bin nhiu ch trong phn on bit, SNP microarray), cc phng php s
DNA. PCR-SSCP hunh quang l dng k thut dng cc emzyme (RFLP, PCR, phng php
thch ng ca SSCP trong s nhn bn chui ko di mi, s dng 5-nuclease v.v.), cc
DNA ch c tin hnh vi mi nh du phng php da trn tnh cht vt l ca DNA
hunh quang. S hn ch ca k thut SSCP l i vi cc sn phm PCR (SSCP, in di
vic xy dng ch th SSCP mt nhiu cng sc, gradient nhit , sc k lng cao p bin tnh
t v khng t ng ha c. K thut SSCP v.v.) v phng php gii trnh t. c gi c
c xy dng xc nh t bin [136] sau th tm hiu su hn v cc phng php pht
c s dng xc nh bin i ca chui hin SNP trong cng b ca Kwork & Chen
DNA [162] v nhn dng gen [204]. (2003) [94] v tng quan ca Jehan &
K thut a hnh nucleotide n (Single Lakhanpaul (2006) [76].
Nucleotide Polymorphism-SNP) Chin lc trc tip in hnh cho khm
Nhng thay i mt nucleotide trong trnh ph cc SNP l gii trnh t cc sn phm nhn
t genome ca cc c th trong qun th c bn cc locus c th (locus specific
gi l a hnh nucleotide n (SNP). Cc v tr amplification-LSA) t nhiu c th hoc xc
th hin SNP trong genome l ni m nh trnh t cc chui th hin c nh du
chui DNA c phn bit bi mt baz duy (gii trnh t cc EST) [165, 188]. Cc chin
nht khi hai hoc nhiu c th c so snh. S lc trc tip khc c th k n nh: gii trnh
khc nhau v nucleotide ny c th dn n thay t ton b h gen hoc gii trnh t cc vng i
i tnh trng c bit hay kiu hnh, hoc c din. Nu c cc d liu v cc chui cn so
th l s thay i trung tnh c th c s dng snh trn mng thng tin i chng hoc cc c
nh gi s a dng trong tin ha. SNP l s d liu khc th c th tin hnh cc so snh
dng thay i trnh t trong genome ph bin pht hin ra SNP [61]. Phn tch trc tip s

276
 Cc k thut ch th DNA

khc nhau trong trnh t gia nhiu c th vi khc nhau.

s lng ln cc locus c th t c bi cng Nghin cu a dng di truyn s dng ITS
ngh gii trnh t th h th hai (next- c th tin hnh bng vic gii trnh t trc tip
generation sequencing). Gii trnh t li hay ti vng quan tm t cc c th khc nhau sau
gii trnh t (re-sequencing) c s dng tin hnh xy dng cy phn loi da vo s
xc nh s thay i cc c th c th cho cc liu so snh cc trnh t. Cng c th xc nh
ch th di truyn phn t v thy c vai tr s thay i ca cc chui bng ct hn ch vng
ca gen. Qu trnh ti gii gii trnh ton b ITS bng cc enzyme ct hn ch v phn tch
genome (whole-genome re-sequencing) s dng cc phn on bng gel agarose hoc
cng ngh c ngn (short-read technology) bao polyacrylamide. Nhng thay i v cc chui
gm s so snh nhng b hng triu chui c to ra bng k thut ct hn ch vng ITS hay
ln lt tng nucleotide vi chui genome so cn gi l a hnh di on ct gii hn
snh. Bng k thut ny c th xc nh c s (PCR-RFLP) c th dng trong phn loi.
bin i trong chui nucleotide ca mu nghin
Do s bo th trong cu to bc hai, ITS
cu v i chng.
c xem nh cng c sc bn cho vic so snh
SNP c s dng trong lp bn di tin ha ca c th nhn thc [184]. S d vy l
truyn [88, 146, 203], trong nghin cu a dng do vng ITS bo th cao trong loi nhng li
di truyn [53, 83], trong nhn bit ging [74] v thay i cc loi khc nhau. i vi nhiu c
trong chn ging [62]. th, ITS2 trong rRNA c t chc xung quanh
K thut phn tch vng m trong trong c trung tm bo th chung ca cu trc bc hai t
sao m (Internal Transcribed Spacer-ITS) bn vng xon c to ra. C s d liu v
cu trc ca ITS2 cha hn 288.000 cu trc
Cc gen RNA ribosome (hay ribosome
tin on cho cc chui ITS2 bit hin nay.
DNA-rDNA) l cc phn ca cc n v lp li
Cc trnh t ny ca ITS2 m ra mt kh nng
c sp xp theo th t. Chng c tm thy
mi l kt hp cc thng tin v cu trc trong
cc v tr nhim sc th c gi l cc vng
nghin cu tin ha.
t chc nhn (nucleolar organizing regions-
NORs). DNA ribosome nhn c hai vng sao ITS c s dng trong nghin cu quan h
m: ITS-1 nm gia tiu phn nh (16S-18S) v di truyn v phn loi [151, [211], trong xc
5,8S ca gen v ITS-2 nm gia 5,8S v tiu nh con lai [148, 192], trong nghin cu ngun
phn ln (23S-28S) ca gen. Hai vng ny v gc, pht sinh loi [90] v trong nghin cu m
tiu phn 5,8S c gi chung l vng sao m vch thc vt [207].
bn trong (ITS). Cc k thut ch th DNA lc lp
Cc vng ITS c di 600 n 700 bp l DNA t bo cht gm DNA lc lp v DNA
cc vng tin ha nhanh nn c th thay i v ty th. Cc ch th DNA lc lp cng nh ty th
trnh t cng nh di. Cc vng bn cnh tr nn rt hiu qu trong nghin cu a dng di
ITS li rt bo th nn c s dng thit k truyn v phn loi. Phn tch DNA lc lp
cc mi chung cho nhn bn vng ITS. cung cp nhng thng tin v a dng di truyn
S bn sao cc on lp li ca rDNA ln thc vt tng ng vi cc DNA nhn. H gen
ti 30 000 trong mt t bo. iu ny lm cho lc lp c tnh bo th cao v mc t bin thp
ITS tr thnh i tng l th cho nghin cu hn so vi h gen nhn. V vy, ch th lc lp
tin ha, pht sinh loi [11] v a dng di truyn c s dng rng ri trong nghin cu a dng
[140]. ITS l k thut quan trng cho phn loi di truyn. Cc k thut ch th DNA lc lp
phn t cc nhm phn loi (taxon) c mi lin c s dng bao gm: phn tch v tr ct hn
kt gn, bi v ITS c tnh bo th cao trong ch ca DNA lc lp (cpDNA), phn tch cc
loi nhng li thay i cc loi khc nhau RNA vn chuyn (tRNA) lc lp, phn tch SSR
[26]. Cc nghin cu v pht sinh loi da vo lc lp (cpSSR) v phn tch trnh t cpDNA.
nrDNA hay cc chui ITS cho php hiu bit K thut phn tch v tr ct hn ch DNA lc
su v tin ha v s lai to cc loi thc vt lp (RFLP cpDNA)

277

Phn tch ct hn ch DNA lc lp (cpDNA)
c s dng rng ri trong nghin cu cc loi
v cc bin i trong loi. Phn tch s thay i
di cc on ct hn ch cpDNA l phng
php u tin trong phn tch cpDNA cho mc
ch nghin cu pht sinh loi. Phng php
phn tch ct hn ch cpDNA bao gm phng
php n gin nh so snh ton b cc on ct
hn ch genome lc lp trn gel agarose n lp
bn so snh trc tip cc v tr ct hn ch
bng lai Southern. Phn tch im ct hn ch
cpDNA c nhiu u vit trong nghin cu pht
sinh loi: i- k thut n gin so vi nhiu k
thut phn t khc; ii- h gen lc lp ln
c th c c nhiu v tr ct cho mt enzyme;
iii- cc v tr ct hn ch th hin gn nh mu
ngu nhin ca cpDNA; iv- cc thay i mang
thng tin ph h xy ra cc v tr nm trn
vng khng m; v- phn tch im ct hn ch
khng b nh hng bi s nhim nh i vi
gii m sn phm PCR. Tuy nhin, do tnh bo
th ca cpDNA nn cc loi gn nhau thng
kh c a hnh. Phn tch v tr ct gii hn
cpDNA cng c thc hin trn cc chui gen
(matK, atpB, ndhF, rbcL), cc vng khng m
(ndhF v trnL intron) v cc vng m gia cc
gen lc lp (trnH-trnK, psbC-trnS, trnD-trnS,
trnT-trnF, trnL-trnF v.v.) bng vic ct sn
phm PCR nhn bn cc gen v cc vng ny
bng cc enzyme ct hn ch. Phng php ny
cn c bit n l phng php PCR-RFLP
cpDNA. Phn tch v tr ct hn ch l phng
php so snh gin tip s thay i trong
genome. so snh trc tip cn tin hnh c
trnh t gen sau so snh.
Phn tch ct hn ch cpDNA c s dung
trong nghin cu tin ha, phn loi, a sinh
hc [133], nghin cu a dng di truyn v bin
i cpDNA [141, 174, 176, 177, 180].
K thut phn tch cc RNA vn chuyn lc lp
Cc RNA vn chuyn (tRNAs) lc lp l
cc i phn t c tin ha di cc p lc phm c di khc nhau tng ng vi vng
mi trng nh cc yu t thch ng trong s
chuyn i tt c cc dng sng, nhng cng
dn ti cc thay i cu trc v chc nng. Ni
cch khc tRNAs rt a dng v vai tr (tng
hp thnh t bo, tng hp porphyrin cho
chlorophyll, bin i u N ca protein, khi
278
Nguyen Duc Thanh
ng phin m ngc, ti m hnh ha lipid
trong s b sung vo tng hp protein ph
thuc ribosome) v cu trc. Mt c im ca
cc vng tRNA l rt bo th v v th cho php
nhng thay i cu trc di dng thay th,
thm bt nucleotide (indel-insertion and
deletion) v chui lp li. Nhng thay i ny
cung cp nhng thng tin quan trng v s tin
ha.
Trong phn tch cc RNA vn chuyn, cc
cng trnh nghin cu thng s dng cc vng
m gia cc gen, c bit l gia hai gen trnL-
trnF. Vng m gia hai gen trnL-trnF c
di nh hn 500 bp. T vng bo th, hai mi
c thit k nhn vng m ny cc
loi [167]. Vi cc mi chung ny v mc a
hnh cao trong vng m trnL-trnF lm cho
vng ny tr thnh ch th tt cho nghin cu
phn tch nhiu loi thc vt [32]. Cc trnh t
khc nhau gia cc loi trong vng m ny c
th xc nh bng in di trn gel
polyacrylamide, phn ng PCR-SSCP hoc
thm tr in di trn gel agarose. Ngoi vng
m gia gen trnL-trnF, mt s vng m khc
nh cc vng m gia cc gen trnH-trnK,
psbC-trnS, trnD-trnS, trnT-trnF cng c s
dng [129]. Cc vng khng m (intron) ca
gen trnL (UAA) cng c s dng nhiu cho
phn tch tin ha thc vt v cho pht trin ch
th xc nh thc vt cho gen l m c th
a bi cng vi kh nng khm ph quan h tin
ha ca cc loi lin quan [205].
K thut Simple sequence repeats lc lp
(cpSSR)
SSR lc lp (cpSSR) hay tiu v tinh lc lp
(chloroplast microsatelitte) c tm thy trong
tt c cc h gen lc lp c gii trnh t
hon ton v hng trm trnh t lc lp cha
c gii trnh t ht. Vic nhn bn bng PCR
cc cpSSR ny bng cc mi c th vi cc
vng nm bn u 5 v u 3 to ra cc sn
cpSSR. SSR lc lp (cpSSR) khc vi SSR
nhn (nSSR) ch cpSSR cu to bi kiu
nucleotide n lp li t 8 n 15 ln. cpSSR
tin ha nhanh hn cc vng gen khc trong h
gen lc lp [75] v c xc nh nhiu loi
cy. Cho n nay, cc nghin cu cho thy
 Cc k thut ch th DNA

cpSSR cho mc a hnh cao hn phn tch
ct hn ch cpDNA (RFLP-cpDNA) [142].
H gen lc lp c di truyn vi nhiu bn
sao trong phn chia mitos v meios, v vy, n
tr thnh i tng nghin cu phn ha do tri
gen trong cu trc di truyn v tri gen ngoi t
nhin [65] v xc nh s suy gim a dng
di truyn [116]. Di truyn theo m, n bi, v
bn cht khng ti t hp ca h gen lc lp lm
n tr thnh phng tin hu hiu trong nghin
cu tin ha. cpSSR c s dng trong
nghin cu nhiu loi cy nh la, la mch,
thng, g v.v. [45, 142,143, 180].
K thut phn tch trnh t cpDNA
Trnh t DNA cung cp cng c cho phn
tch so snh trc tip. Tiu ch chn trnh t
DNA cho phn tch bao gm: i. trnh t phi
di c thng tin v tin ha di truyn cc
v tr nucleotide, ii. mc a dng ph hp vi
vn tin ha di truyn c t ra, theo
Ritland v Clegg [151] mc a dng t 5 n
15% l ph hp, mc ny cung cp s
lng c im cn thit. Phn tch trnh t
cpDNA c u im l cn rt t cpDNA khun.
phn tch trnh t cpDNA, cng ging nh
phn tch trnh t DNA ni chung, trc ht l
nhn cc gen, vng gen hay vng m cc gen
lc lp bng cc mi c hiu, sau c th
nhn dng trc khi c trnh t hoc c trnh
t trc tip sn phm PCR. Cui cng l so
snh trnh t bng cc phn mm chuyn dng.
Nhiu nghin cu s dng trnh t gen
rbcL cho nghin cu phn loi v tin ha loi
[132] mt s khc s dng trnh t gen ndhF
[55] v gen matK [91]. Trnh t cc chui vng
m gia cc gen hay trnh t cc chui khng
m ca cc gen hoc gia hai gen (trnL (UAA),
rbcL-atpB cng c s dng [130, 208]. Trnh
t cc vng gen rbcL, matK, atpF-atpH IGS,
psbK-psbI IGS v trnH-psbA IGS cn c
dng cho m vch (barcode) cc loi lan [87].
Cng ngh th hin a dng (Diversity Arrays
Technology-DArT)

Hnh 4. S cc bc tin
hnh DArT

A. Pa-nen a dng. Genomic

DNA ca cc mu nghin cu
c gp vi nhau. Sau
DNA c ct bng cc
enzyme ct hn ch c chn
v gn vi cc tip hp. Mc
phc tp ca genome c
gim thiu bng PCR vi cc
mi c nucleotide chn lc.
Cc phn on i din c
nhn bn bng cc mi c
hiu cho vector v nhn dng,
sn phm PCR c tinh sch
v gn ln slide.
B. S khc nhau gia hai mu th hin bng DArT. Hai mu genome c chun b theo phng
php m t (A). Mt mu c nh du hunh quang mu xanh, mt mu c nh du mu
, trn vi nhau v lai vi pa-nen a dng. T l mc tn hiu xanh/ c o mi ln v s
khc nhau v t l tn hiu cho thy s th hin khc nhau ca cc phn on DNA [73].

Cng ngh th hin a dng (DArT) l cng trin khc phc mt s nhc im ca cc
ngh phn tch kiu gen c tnh ph hp rng v cng ngh ch th phn t khc nh RFLP,
hiu qu v kinh t. Cng ngh ny c pht AFLP v SSR [4]. y l cng ngh khc phc

279

nhc im tn km v thi gian ca cc k
thut da trn vic lai DNA v c th phn tch
hng ngn locus trong mt ln th nghim.
DArT c bit ph hp cho phn tch kiu gen
cc loi a bi vi h gen ln. Cng ngh ny
c th ly du (fingerprint) ton b h gen bng
vic ghi nhn s c mt hoc vng mt cc phn
on DNA trong h gen i din c hnh
thnh t cc mu DNA genome. Cng ngh
DArT gm nhiu bc nh: gim thiu s phc
tp ca mu DNA (bng gp DNA ca cc mu,
ct DNA bng enzyme gii hn v PCR vi cc
mi c nucleotide chn lc), to th vin DNA,
a cc th vin ln bn knh, lai vi cc DNA
c nh du hunh quang trn bn knh, qut
bn knh xc nh tn hiu lai, ghi chp s
liu v phn tch (hnh 4).
DArT gim thiu s phc tp ca mu DNA
nhn i din ca mu. S gim thiu ny
da trn vic kt hp ct hn ch v gn chui
tip hp v km theo s nhn bn bng PCR.
Ch th DArT cho loi mi c khm ph bng
vic sng lc th vin hng ngn phn on
DNA t genome i din c chun b t vic
gp cc mu DNA cha ng s a dng ca
loi. H thng phn tch chip vi im
(microarray platform) lm cho qu trnh khm
ph hiu qu v tt c cc ch th trn mt phn
tch DArT c th c ghi nhn ng thi. i
vi mi phng php gim thiu phc tp c
mt b ch th DArT ring ph hp cho phn
tch DArT ring r. Do , s lng cc ch th
cho mi loi nht nh ch ph thuc vo mc
bin i trong loi v s phng php gim
thiu phc tp c sng lc.
Cng ngh gii trnh t th h th hai (Next-
Generation of Sequencing Technology)
S pht trin cc cng ngh gii trnh t s
lng ln (high throughput sequencing) lm
cho vic gii trnh t DNA tr nn c bit quan
trng cho cc nghin cu v a dng di truyn,
tin ha, bo tn sinh hc v chn ging. Cc
cng ngh ny c tim nng loi b mt trong
cc tr ngi i vi thc hin tip cn h gen
trong cc c th khng phi l m hnh bao gm
cc c th thiu thng tin v trnh t genome.
Cc cng ngh ny trnh c s tn km, phc
tp v sai lch phng php gii trnh t da
280
Nguyen Duc Thanh
vo nhn dng bng nhn bn trc tip t DNA
khun [17, 113].
Cng ngh gii trnh t th h th hai (hay
cn c coi l cng ngh gii trnh t s lng
ln) gm ba nhm chnh l gii trnh t bng
phng php tng hp, gii trnh t bng
phng php gn v gii trnh t phn t n.
Gii trnh t bng phng php tng hp
ging nh k thut gii trnh t ca Sanger tc
l xc nh thnh phn nucleotide bng pht
hin tn hiu quang ha trong qu trnh gn cc
nucleotide vo si DNA b tr bng enzyme
DNA polymerase. Trong k thut Sanger, s kt
thc chui dideoxynucleotide c dng xc
nh trnh t, cn trong gii trnh t bng
phng php tng hp th DNA c ct thnh
nhiu phn on ri gn vi chui tip hp sau
nhn dng tng tn hiu hunh quang
hoc tn hiu ha hc. C ba h thng gii trnh
t bng phng php tng hp. Ba h thng ny
khc nhau v di c, v phng php nhn
dng v c nh, bao gm: h thng 454 ca
Roche, Solexa ca Illumia v SOLiD ca
Applied Biosystem.
H thng Roche 454 gii trnh t bng
phng php da trn nguyn tc tng hp cao
nhit (pyrosequencing), si n DNA khun
c gn vo ht siu nh (microbead) v nhn
bn bng PCR nh tng (emulsion PCR)
[112]. PCR nh tng l phn ng PCR s
dng hn hp phn ng PCR thng thng v
hn hp phn ng PCR dng nh tng nc
trong du [158]. H thng Roche 454 u tin
ch c c on 100 bp, hin nay do ci tin
c th c on n 800 bp.
H thng Illumina ca Solexa gii trnh t
da vo vic n gin ha phng php xy
dng th vin v o u bng phng php
hunh quang dn n vic to ra cc on c
c di 35 bp [17]. H thng ny s dng vic
nhn bn cu pha cng (solid-phase bridge
amplification), trong cc on tip hp u
5 v 3 c gn vi mi u ca khun DNA.
Mt u ca khun sau c nh vi c
cht. Cc on tip hp c lai vi cc mi
xui hoc ngc c nh to thnh cu ni, to
thun li cho vic nhn bn to ra cc sn
phm nhn bn c nh vi c cht v to
 Cc k thut ch th DNA

thnh cc nhm khun ging nhau do lm t n c nhiu li th so vi gii trnh t th h

tng cng s pht hin bng quang ha. Vi h th hai. c bit l vic n gin ha s chun
thng HiSeq 2000 c th to khong 6 t on b mu v c th s dng DNA km cht lng
c cho 540 n 600 Gb trong 11 ngy hoc c nng thp, ng thi trnh c cc
(http://www.illumina.com). li trong qu trnh nhn bn khun bng PCR.
H thng Ion Torrent Phng php ny cng s dng vic gii trnh
(http://www.iontorrent.com) l h thng gii t trc tip RNA nn loi b c cc sai lch
trnh t th h th hai duy nht xc nh trnh t trong nhn bn cDNA. Hin nay, c hai thit
khng da vo cc cht hunh quang m da b gii trnh t theo phng php gii trnh t
vo vic o s thay i pH do vic gii phng phn t n l Helicos - Helicos Genetic
H+ khi gn nucleotide bng cng ngh bn dn Analysis System (http://www.helicosbio.com)
(semiconductor) [154]. Bng vic b sung lin v PacBio RS SMS ca Pacific BioSciences
tc cc nucleotide, my c th nhn bit (http://www.pacificbiosciences.com).
nucleotide no c gn vo si ang ko di. Do s khc nhau v di on c, mc
Gii trnh t bng phng php gn l gii ch ca tng cng ngh gii trnh t l khc
trnh t bng tng hp c s dng DNA nhau. Vi on c ngn v gi thnh thp ca
polymerase lm phng tin ko di trong qu Solexa v SOliD th hai cng ngh ny ph hp
trnh xc nh trnh t DNA. Gii t bng cho gii trnh t ton b h gen, trong trnh
phng php gn khai thc s mn cm ca t genome mi c th lp rp v so snh vi
DNA ligase vi s bt cp sai (mismatch) trnh t tham chiu (trnh t genome ca loi
xc nh trnh t DNA. Phng php ny s ang tn ti). Cng ngh gii trnh t Roche
dng cc oligonucleotide d c kch thc khc 454 vi chui c di (c th ti 800 bp) cng
nhau v c nh du bng cht hunh quang c th s dng nhn c tng th bc u
nucleotide cn xc nh, cc phn on DNA v h gen v h sao chp ca loi.
khun c mi vi cc chui neo ngn bit Cng ngh gii trnh t th h mi c s
to iu kin cho s lai vi cc on d. dng trong nghin cu m vch DNA th h
DNA ligase c b sung vo phn ng ni mi-next-geneation DNA bacoding [81, 161],
cc on d c nh du hunh quang vi trong xc nh t bin [19], trong nghin cu
mi v khun. Hnh nh hunh quang c thit phn loi v pht sinh loi [41, 164], trong
lp xc nh on d no c gn. Qu nghin cu bin i h gen v phin m c
trnh ny c lp li vi vic s dng cc b th a bi [72] v trong pht trin ch th DNA
d khc nhau cho cc DNA khun nghin cu nh SNP v SSR [9, 209].
xc nh trnh t nucleotide. H thng gii
trnh t h tr gn v pht hin oligonucleotide KT LUN V KHUYN CO
(Supported Oligonucleotide Ligation and
Detection-SOLiD) ca Life Cho n nay, c rt nhiu k thut c
Technologies/Applied Biosystems (http:// s dng pht trin ch th DNA phc v cho
www.appliedbiosystems.com) gii trnh t bng nghin cu v chn lc thc vt. Mi k thut
phng php gn c th to ra chui DNA 0,1 u c nhng im mnh v im yu. Vic la
n 4 Gb trong mt n by ngy vi gi thnh chn k thut ch th DNA ph hp v hiu qu
trong khong 3400 n 8500 -la M. cn da vo cc c im: v mc cho a
hnh ca cc ch th s dng cao hay thp, s
Gii trnh t phn t n cn gi l gii lng locus c th nhn c trn ln phn tch
trnh t th h th ba (third-generation nhiu hay t, kiu di truyn ca ch th tri hay
sequencing). Phng php ny to ra cc tn ng tri, mc n nh ca k thut xy
hiu nhn bit s gn nucleotide bng quang dng ch th c th lp li c, v mc
ha trong qu trnh gii trnh t t phn t n gin hay phc tp ca k thut, s lng v
nucleic acid n. V th c th loi b vic nhn cht lng DNA cn thit cho phn tch, mc
bn khun. iu ny lm cho gii trnh t phn kh d trong s dng ch th, mc t ng

281
 Nguyen Duc Thanh

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DNA MARKER TECHNIQUES IN STUDY AND SELECTION OF PLANT

Nguyen Duc Thanh

Institute of Biotechnology, VAST

SUMMARY

Since the 1980s, DNA marker techniques have been invented and developed quickly to become the most
significant development in the field of molecular biology. The DNA markers have been widely used in study
and selection of plants. The DNA techniques have been developed for DNA markers used in studies of
genetic diversity, phylogeny, classification, gene tagging and identification; in germplasm and marker-
assisted selection. The presence of various types of DNA markers, and differences in their principles,
methodologies, and applications require careful consideration in choosing one or more of such methods. No
DNA markers are available that fulfill all requirements needed by researchers. Depending on the nature of
each study, one can choose among the variety of DNA marker techniques, each of which combines at least
some desirable properties. In Vietnam, the use of DNA marker techniques began at the end of 1990s.
However, the application was limited with only few techniques such as random amplified DNA
polymorphism, microsatellites or simple sequence repeats and amplified fragment length polymorphism.
Those techniques were used in the studies of plant genetic diversity, molecular mapping and marker-assisted
selection. This review provides an overview on most of the available DNA marker techniques and their
utilities in the study and selection of plants with the aim to provide researchers and breeders necessary
information for choosing appropriate DNA marker techniques.
Keywords: DNA marker techniques, gene identification, genetic diversity, germplasm selection, marker-
assisted selection.

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