Indian 10urnal of Experimental Biology Vol. 37, November 1999, pp. 1123-1128

Genetic similarity analysis and identification of Indian potato cultivars by random amplified polymorphic DNAs*

S K Chakrabarti , R K Birhman** & D Pattanayak

Molecular Biology Unit , Division of Genetics and Plant Breeding, Central Potato Research Institute, Shimla 171 00 I, Indi ~

Received 2 December 1998; revised 16 August 1999

Random amplified polymorphic DNAs (RAPDs) were used to fingerprint eighteen commercial Indi an potato cuitivars. A total of seventy-four distinct DNA fragments (bands), ranging from 124 to 4074 bp, were amplified by using twe lve ran­dom primers. Fifty-seven of these bands were polymorphic. Cuitivar specific DNA fin gerprints were generated by two ran­dom decamer primers (OPA-03 and OPC-04). Pair-wise genetic similarity analysis on the basis of presence or ahsence of bands reveal ed a wide range of variability among the cuitivars, thereby, suggesting a wide geneti c base of Indian potato cul ­tivars. Kinship relationshi p was not correl ated with the similarity values. Kufri swam a, which has the wil d species So/anlllll

vernei in its pedigree, showed maximum genetic divergence from other cuiti vars. Similarly, kufri badsh~h and kufri ~Ianka r

were also genetically distinct from other cu ltivars studied . Remaining fifteen cul tivars were grouped into two closely related clusters. Usefulness of RAPD an alysis in identification of cu ltivars and genetic divergence among Indian cuiti vars has becn discussed.

Emergence of agriculture as a profitab le industry and globalization o f world trade in recent years has neces­sitated formulation of lega l in struments for plant va­riety protection , regi strati on, certification and patents. For issuance of the patent the new ly bred cultivars must successfully pass the c riteria o f d isti nctness , uniformity, and stability' . Trad itionally, morpho logi­cal characters like fl ower co lour, growth habit , leaf type, sprout and tuber type, and di sease react ion have been used to define newly re leased potato culti vars. However, most of these characte rs are po lygenic and the ir express ion is a;fec ted by environment. Mor­phological characters, therefore , form poor taxonomic descriptors. Isozyme analysis has a lso been success­fu lly used to identify potato cu lti va rs2. However, the

abil ity of isozymes to di scriminate be tween c lonal variants of commerc ial cultivars is low. because of the sma ll number of loci sampl ed by this tech nique. Further, the isozyme profil es are also affected by en­vironment. At molecu la r level , res tri c tion fragment length po lymorphi sm (RFLP) technique has a lso been used to characterize potato cultivars3

,4. Unfortunate ly, detection of RFLPs is time consuming, requires de­velopment of po lymorphic DNA probes, and usually involves the use of radioi sotopes . Random ampli fied polymorphic DNA (RAPD) analysis overco mes these

* Publication No. 1584, CPR1, Sh im1 a ** Correspondent author

proble ms and provi des an a lte rnati ve to RFLPs. Polymorphism observed in RAPD anal ys is may resu lt from point mutations, inse rtio ns, del eti ons, trans loca­tions and inversions5

. These may change the sequence of primer binding si tes, a lter the size of the ampl ified fragment or prevent the successt'u l ampli f icati on of target DNA. Compared to RFLP ana lys is, RAPD pro­cedure is independent of environmental influence and ti ssue type. It is a lso less expensive, the results can be obtai ned within a short pe riod, and ex tremely small amounts of DNA is suffic ient for analysi s . These ad­vantages, together with the re lative ease o r the techni ­cal procedure, have led to use o f RAPDs in gene ti c mappin g and genotype identificat ion "·7 In this paper.

potent ial of RAPDs ha~ been described in detecting DNA polymorphism that may be used to di s tingu i~h

and identify Indi an pota to cuitiv:1rs.

Materials and Methods Eighteen potato cu lti vars (T:1bl e I ) we re used in

thi s study . Plants were grown in a g lass house and leaf sampl es for DNA ex tracti on were co ll ected whe n these were nearl y one and a half mon th o ld . A mod i­fied CTAB proced ure8 was used for ex trac ti on of ge­nomic DNA from leaves . T wo grams o f fresh lea r samples were ground to a fine powder in li quid nitro­gen using a mortar and pest le and the powder \-vas

transferred to 10 ml of pre-warmed (6:'i °C) extrac t ion bu ffer in a SO ml capacity polycarhonate centnfuge

1124 INDI AN J EXP BlOL, NOVEMBER 1999

tube. The adhering powder, if any, was washed down by adding another 5 ml pre-warmed extraction buffer. The extraction buffer contained 1.4 M, NaCI ; 20 mM, EDTA (Ethylenediaminetetraacetic acid di -sodium salt); 100 mM, Tri s-HCI (PH 8.0); 2%, CTAB (N­cetyl-N,N,N-trimethylammonium bromide); and

0.2%, ~-mercaptoethano l. The mixture was thor­

oughly stirred and incubated at 65°C for 30 min to I hr and then kept at room temperature for 10-15 min. The suspension was ex tracted with 15 ml of chl oro­form:isoamyl alcohol (24: I) to denature prote ins and facilitate phase separati on. The mixture was then centrifuged (Kubota Centrifuge) at 14,000 rpm for 20

min at 20°e. The top aqueous phase ( 15 ml) was carefully taken out in a fresh, autoclaved centrifuge tube and 10 ml ( /, vo l.) of isopropanol at room tem­perature was added. The precipitated DNA was pel­leted by centrifugation at 10,000 rpm for 10 min. The pe llet was washed in 5 ml of chilled ethanol (70%) in water, dried complete ly and then re-suspended in I ml of sterile MilliQ water. Single stranded RNA was digested with RNase A ( I 00 ~g mrl ) fo r 30 min at

37°C, extracted once with pheno l: chloroform: iso­amyl alcohol (25 :24 : I), precipitated by adding 1/ 10 th volume of ammonium acetate and 2 volume of chilled ethanol , washed with ethanol (70%) and re-suspended

Table I- List of the potato cultivars used in the present study

Cultivar

Ku fri Kumar (KK U) Ku fri Kundan (KKD) Kufri Al ankar (KAL) Kufri Chandramukhi (KCM ) Kufri Jeevan (KJE) Kufri Jyo ti (KJY ) Kufri Sheetman (KSH ) Kufri Naveen (KNA) Kufri Muthu (KM U) Ku fri Dewa (KDE) Ku fri Badshah (KB A) Kufri Sherpa (KSP) Ku fri Swarna (KSW) Ku fri Megha (KM E) Kufri Ashoka (KAS) Ku fri Jawahar (KJW)

Kufri Sutlej (KST) Kufri Kanchan (KKA)

Year of release 1958 1958 1968 1968

1968 1968 1968 1969 197 1 1973 1979 1983 1985 1989 1996 1996

1996

*

Parentage

Lumbri x Katahdin Eki shirazu x Katahdin Kennebec x ON 2090 sd 4485 x Ku fri Ku ber

M 109-3 x 698-D 3069d(4) x 28 14a( l) Craigs Defi ance x Phul wa 3070 d (4) x 692-D 3046( I) x M 109-3 Craigs Defi ance x Phul wa Kufri Jyo ti x Kufri Alankar Ultimus x Ad ina Ku fri Jyot i x (Vtn)2 62.33.3 SLB/K-37 x SLB/Z-73 EM/C- I02 1 x CP-1 468 Kufri Neelamani x Kufri Jyo ti Kufri Bahar x Kufri Alankar SLB/Z-405(a) x Pimpernel

* The line number SEII-1307 to be released as Kufri Kanchan.

in sterile MilliQ water ( I ml). Amount of DNA was quantified by gel electrophores is.

Polymerase chain reaction was pe rfo rmed in a re­

action volume of I 2 ~I containing 50 mM, KCI; 10 mM, tri s-HCI (PH 9.0); 0 . 1 %, tritonX- IOO ; 1.5 mM ,

M gCh; 100 ~M, each of dNTPs; 25 pmole, primer; 2 n g, genomic DNA; and 0.75 units, Tag DNA poly­merase (Promega). Seventeen randc m primers used in thi s study (Table 2) were purchased from Mis Operon Technology, Inc., 1000 Atl antic Ave, Al ameda, CA 9450 I , USA. Amplificati on was pe rformed in a Per­kin Elmer Thermal Cyc ler (GeneA mp PCR System 2400) . In total,35 cyc les were used, each cyc le con­

sisting of I min denaturati on at 94°C, I min anneal­

ing at 35 .5°C, and 2 min ex tension at 72°e. All PCR samples were given a 5 min pre-ampli ficat ion at 94°C and 10 min post-amplificati on at 72°e. Ampli ­fication products were separated by e lectrophores is in

agarose gel ( 1.8%) with ethidiu m bromide (0.5 ~lg

mrl ) for 6 hr at constant vo ltage (3 V cm' I). The ge l was visualized by UV transilluminator (254 n m wave length) and photographed with Po laro id 667/665 films.

Amplified DNA fragments (bands) were scored for each variety as I (band present) and 0 (band absent) with band number I be ing the sma ll est fragment. The data was analyzed with the software (NTSYS-PC) 9.

The data matrix obtained fo r presence or absence of fifty -seven RAPD bands was analyzed according to Ne i and Li lo definition of the geneti c s imil arity- Sij = 2a/(2a + b + c), where Sij is the s imil arity between two indi viduals (i and j ), a is the number of bands present in both i and j , b is the number of bands pres­ent in i and absent in j , and c is the number of bands present in j and absent in i. Matri x o r s imil arity was analyzed by UPGMA method II . Dendogram was c re­ated with TREE program. Dendogram was graphi­cally represented as a pheneti c tree.

Results Five of the seventeen random primers (-29%)

tried in thi s experiment d id not produce any amplifi ed product. Amplificati on of tota l genomi c DNA fro m the eighteen tetraploid culti vars with the remaining twelve decamer primers produced a tota l of seventy­four fragments (bands) ranging in s ize from 124 to 3074 bp ; out of which 57 (-77%) were polymorphic (Table 2). The number of fragments produced by a primer ranged from 2 (OPB-06) to 12 (OPC-04). Pat­tern of RAPD fragments produced by the random

CHAKRABARTI et al. : GENETIC SIMILARITY ANALYSIS AND IDENTIFICATION OF INDI AN POTATO 11 25

primer OPA-03 is shown in Fig. I. Similarity matrix for all the pair-wise combinations of cultivars is pre­sented in Table 3. Similarity values ranged from 0.24 to 0.82 (ca . 0.60) with a standard deviation of 0 .12. This indicated a fair range of variability in similarity values suggesting a wide genetic base of eighteen cultivars tested in the present experiment. Cultivar kufri swama, represented by a solitary branch in the phenogram (Fig. 2) , had maximum genetic divergence

from the remaining cultivars. Average similarity of kufri swama with other cultivars was 0 .36 (range

from 0.24 to 0.65 with standard deviation ±0.09). In case of kufri badshah and kufri alankar, pair-wi se similarity values with other culti vars studied ranged from 0.41 to 0.64 and 0 .34 to 0 .70, respecti vely, and they formed distinct branches in the phenogram. Re­maining fifteen cultivars could be grouped into two major clusters . Group I consisted of kufri jyoti , kufri

Table 2-Total number of amplified fragments and number of polymorphic fragments generated by PCR using 17 random decamer primers.

Primer Sequence No. of amplified No. of polymor- Size (bp) (5'-3') fragments phic fragments

OPAOI CAGGCCCITC 6 5 4 38-20 12 OPA02 TGCCGAGCTG 6 4 265-438 OPA03 AGTCAGCCAC 12 8 124- 1245 OPA04 AATCGGGCTG Nil Nil Nil OPA05 AGGGGTCTTG 6 4 210-151 9 OPA 17 GACCGCITGT 3 3 199-923 OPA 19 CAAACGTCGG 4 2 93 1-2 105 OPB 06 TGCTCTGCCC 2 2 515-639 OPBI4 TCCGCTCTGG Nil Nil Nil OPC04 CCGCATCTAC 12 II 490-4074 OPC 13 AAGCCTCGTC Nil Nil Nil OPC 20 ACTTCGCCAC 6 6 264-1 756 OPDOI ACCGCGAAGG Nil Nil Nil OPD08 GTGTGCCCCA 5 5 282- 1650 OPE 09 CTTCACCCGA Nil Nil Nil OPE 20 AACGGTGACC 4 4 48 1-1 442 OPH 07 CTGCATCGTG 8 3 306-3900

Total 74 57

" 3 :·~l 6 7 H 9 1(; ~ 1 12 13 ' , 15 13 ! '7 . ,.

:1 r'..:l L. ,+ j!-t' it)

Fig. I-RAPD pattern of 18 Indi an commercial potato cultivars generated by primer OPA-03 . M-I Kb molecular weight ladder; I-kll fri jyoti ; 2-kufri chandramllkhi ; 3-kll fri muthll ; 4-kll fri badshah; 5-kllfri sherpa; 6-kufri swarna; 7-kllfri slltlej ; 8- kll fri jawahar; 9-kll fri jeevan; 10-kufri kanchan; II-ku fri alankar; 12-kllfri kllndan; 13-kufri kllmar; 14-kufri megha; 15-kllfri 'dewa; 16-kufri ashok a; 17- kll fri sheetman; 18-ku fri navecn.

1126 INDIAN 1 EXP BIOL, NOVEMBER 1999

jawahar, kufri sutlej, kufri ashoka, kufri jeevan, kufri kanchan, kufri chandramukhi and kufri kundan and group II encompassed kufri muthu , kufri megha, kufri sherpa, kufri kumar, kufri naveen, kufri dewa and kufri sheetman. Maximum simi larity (0.82) was ob­served between kufri sutlej and kufri ashoka. Kinship relationship among different cultivars could not be predicted from the similarity data. For example, similarity value between kufri dewa and kufri sheet­man (full-sibs) was only 0 .70, which was lower than 0 .82 as recorded between two unrelated cultivars ku­fri ashoka and kufri sutlej. Similarly , a wide range of variability was observed among six half-sib combina­tions (0.37 to 0.66) and four progenitor x progeny combinations (0.33 to 0.78; Table 3).

Binary data matrix generated by recording pres­ence and absence of fragments amplified by individ­ual primer and their combination were analyzed by the software (NTSYS-PC) to find out minimum num­ber of primers required to distinguish all the eighteen potato cultivars. It was observed that data matrix gen­erated by two primers (OPA-03 and OPC-04) were sufficient to clearly distinguish all the eighteen culti­vars.

Discussion Authentic identification of cultivars IS necessary

both for breeders to ensure protec tion of inte llectual property right and fo r end users like farmers , proces­sors and consumers. The traditional method of identi­fying cultivars by morphological charac ters is now gradually being replaced by more re li able protein or DNA profiling largely because of several limitations of morphological data l 2

. In recent years DNA based fingerprinting techniques are bei ng favoured because protein profiles are often influenced by environment and tissue type. DNA based identification of cu lti var exploits the inherent polymorphi sm in the base se­quences of genomic DNA between two indi vidual which arises due to base subst itution, de leti on or in­sertion, transposi tion, gene in version, unequal ex­change during meiotic recombinati on and polymerase slippage at the replication fork . Initi al ly, restric ti on fragment length polymorphi sm (RFLP) was the usual method to generate individual spec ific DNA fin ger­print' .4·I J

. Recently a variety of DNA fin gerprinting techniques like random amplified polymorphic DNA), DNA amplification finge rprinting (DAF)6, arbit raril y primed PCR (AP-PCR)7, and amplified fragment length polymorphism (AFLP) 14 have been developed which do not require prior investmen t in terms of se­quence analysis, primer synthesis or characterization of DNA probes . RAPDs are the most favoured method largely because of relat ive ease with which

Nei's Genetic Similarity

0-36 0-60 0 ·72 O·S4 ~--------~------------------~--------~!KJY

~

-

'---

-

r-

L....-..-.i

~

rC I

KJW KST KAS KJE KKA KeN! KKD KMU KME KSP KKU KNA KDE KSH KAL KBA KSW

Fig. 2-Phenogram of 18 Indian commercial potato cultivars based on RAPD analysis. [K1Y-kufri jyoti ; KM U-ku fri mu thu : KCM-k ll fri chandramukhi; KSP-kufri sherpa; KAS-kufri ashoka; KKU-kufri kumar; KNA-kufri naveen; KME-kufri megha; KDE-kllfri dewa; KB A­kufri badshah; KSH-ku fri sheetman; KST-kufri slitlej; K1W-kufri jawahar; KKD-kufri kundan; K1 E-kufri jeevan; KKA-kllfri kanchan ; KAL-kufri alankar; KSW-kufri swama).

n Table 3-Nei 's genetic similarity matrix of 18 rndian potato cultivars ::I:

:» A

KJY KSP KSW KST KJW KJE KKA KAL KKD KKU KME KDE KAS KSH KNA ;;0 Cultivar KCM KMU KKA :» c:l :»

KJY 1.00 ;;0

::l ~ KCM 0.66 1.00 ~

KMU 0.69 0.62 1.00 Cl l"I1 Z

KBA 0.56 0.64 0.55 1.00 l"I1 -l n Vl

KSP 0.67 0.67 0.76 0.56 1.00 3::: t=

KSW 0.33P 0.27 0.39 0.41H 0.40 1.00 :» ;;0

=i KST 0.81 0.74 0.61 0.59H 0.69 0.30 1.00 -<

;Z KJW 0.78P 0.68 0.57 0.63H 0.54 0.37H 0.79 1.00 F=

-< KJE 0.64 0.68 0.57H 0.55 0.62 0.33 0.64 0.67 1.00

Vl

Vi

KKA 0.75 0.63 0.62 0.43 0.63 0.33 0.74 0.68 0.74 1.00 ;Z 0

KAL 0.67 0.70 0.63 0.49P 0.64 0.34 0

o.66P 0.52 0.52 0.60 1.00 l"I1 Z ::l

KKD 0.61 0.71 0.57 0.59 0.62 0.33 0.70 0.67 0.73 0.59 0.52 1.00 :!l n :»

KKU 0.63 0.67 0.67 0.50 0.72 0.32 0.75 0.59 0.62 0.70 0.64 0.66H 1.00 -l <5 z

KME 0.55 0.66 0.77 0.52 0.71 0.52 0.58 0.44 0.6 1 0.59 0.67 0.64 0.73 1.00 0 "T1

KDE 0.49 0.57 0.68 0.53 0.65 0.65 0.56 0.56 0.67 0.54 0.50 0.63 0.62 0.72 1.00 z 0 ;;

KAS 0.70 0.73 0.67 0.53 0.76 0.34 0.82 0.66 0.72 0.73 0.68 0.66 0.78 0.70 0.62 1.00 Z -0 0

KSH 0.54 0.5 1 0.67 0.45 0.59 0.38 0.58 0.49 0.53 0.63 0.5 1 0.45 0.64 0.63 0.70F 0.68 1.00 -l :» -l

KNA 0.53 0.62 0.74 0.49 0.67 0.24 0.64 0.52 0.60 0

0.55 0.46 0.60 0.78 0.69 0.64 0.75 0.62 1.00

H-Half Sib; F-Full Sib: P-Progen~ x Progenitor; For fu ll names of the culti vars refer to Table I .

N -.J

1128 INDIAN J EXP BIOL, NOVEMBER 1999

the technique can be practiced in any molecular biol­ogy laborator/ 5

.

For PCR amplification a reaction volume of 25 III has been used by most of the workers ' 6

-' 8

. In our ex­periment, however, no difference in banding pattern was observed whether a reaction volume of 50, 25, or 12 III was used. Therefore, we used a reaction volume

of 12 III throughout our experiment to economize on PCR reaction. Unlike North American '6. ' 8, Japanese l9

and Korean20 potato cultivars, the genetic base of In­dian cultivars was found to be reasonably wide. This was expected since 30 different parental lines are rep­resented in the pedigree of the 18 cultivars tested in the present study (Table 1). The distinctness of kufri swarna from all other cultivars can also be explained by the fact that it has the diploid wild species Sola­num vernei (resistance source for cyst nematode) in its pedigree. Regarding relationship between similar­ity and kinship relation, conflicting results are avail­able in the literature. Hosaka et al. 19 have studied the genetic relationship of 73 Japanese potato cultivars and reported that RAPD banding patterns of closely related cultivars are clustered together and concluded that banding patterns are reflections of the pedigree relationship. Demeke et al. '? while studying genetic diversity of 28 North American potato cultivars, how­ever, have observed glaring exceptions to this obser­vation and recorded that varieties with close kinship can often be genetically as diverse as varieties with no immediate relationship. Our ·observations also demonstrated that kinship relationship could not be reflected in the similarity of the banding pattern. This is probably due to highly heterozygous tetraploid na­ture of the potato genome.

In our study five primers failed to produce any am­plified fragments. The reasons for failure of these primers to amplify could not be explained . Similar observation has been reported by Hosaka et al. 19, Ci s­neros & Quiros2 1

, and Sosinski & Douches ' 8. We

could distinguish all the 18 cultivars using only 2 primers . The primer OPA 03 alone could resolve the 18 potato cultivars in to 12 groups, while OPC 04 alone could distinguish 16 groups . The data matrix generated by these two primers could distinguish all the 18 cultivars. In similar experiments, Demeke et al. l

? have used two primers (131 & 184) to distin­guish 36 potato cultivars and clonal variants . Sosinski and Douches l8 have used 10 decamer primers to dis­tinguish 36 North American potato cultivars. Organi­syan et al. 22 have used three primers to d istinguish

eight Russian cultivars and 6 species. It is conc luded from the present experiment that commercia l potato cultivars can be identified by RAPD fingerprinting. RAPD analysis can also determine genetic divergence among the cultivars and within the breed ing stock. This is the first report of usi ng RAPD to fingerprint commercial potato cultivars in India .

Acknowledgement We are thankful to Drs G S Shekhawat, Director

and P C Gaur, Head of the Division, for providing necessary facilities. Technical he lp provided by Mr C M S Bist and Ms Manju Bala is gratefully ac­knowledged.

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