Journal of Intellectual Property Rights Vol 6 September 2001 pp 377-388

Molecular Markers for Cultivar Identification and PBR

Syamal Krishna Ghosh, Chitore Kr Guha Sarkar

Nagarjuna Agricultural Research and Development Institute, 61, Nagarjuna Hills, Panjagutta, Hyderabad 500 082

and

Subhojit Datta

Biotechnology Centre, Indian Institute of Pulses Research, Kanpur

(Received 23 April 2001)

Identification of plant varieties is important in many areas of agriculture, marketing, research and development and for protection of Plant Breeders Right (PBR). Molecu­lar marker techniques based on DNA profiling provide novel approaches for cultivar identification. They offer advantages for comparison over morphological and bio­chemical markers, with respect to resolving power, cost effectiveness, testing at any stages of development, rapidity, environment independent expression and produce an anay of polymorphism. Markers such as RFLP, RAPD, SSR, STS, AFLP, SNP are used for DNA fingerprinting and a comparative analysis was made among these markers for cultivar identification. With the revised wro regime and privatization of agricultural research, PBR is a prerequisite for investment in plant breeding and wro has provided under UPOV the protection of plant varieties by patents or by sui generis system like PBR. Identification of plant cultiyars has become increasingly important with the requirement of PBR to demonstrate distinctness, uniformity and stability (DUS) for each new cultivar. In this context, molecular profiling helps in the finding and creation of minor variants from initial varieties and can be used to demonstrate the DUS of the variety. DNA profiling has not yet been adopted by UPOV as an essential character but this may be included as supplementary character information and it is expected to become the prefened method for characterization of cultivars in future. In this discussion an attempt was taken to know about the molecular markers for plant cultivar identification as well as their role and effectiveness.

The legal right to market a newly-bred culti­var depends on the results of statutory test­ing, which provides information regarding

distinctness, uniformity and stability (DUS). The DUS testing guarantees the quality of the new cultivar for farmers and

378 J INTELLEC PROP RIGHTS, SEPTEMBER 2001

merchants that the new cultivar is distinct from all other released cultivars as well as uniform and stable for traits evaluated in the tests. Moreover it is also used to protect PBR and encourages the continuous develop­ment of new varieties. PBR have evolved since the introduction of the concept of es­sentially derived varieties in the Interna­tional Conventions for the Protection of new Varieties of Plant (UPOV) convention of 1991. An essentially derived cultivar is one that is clearly distinguishable from the initial cultivar by phenotypic characteristic that could be a single simply inherited trait. This phenomenon has increased since the devel­opment of GE technology, which made pos­sible the introduction of a single gene into a variety.

Cultivar identification allows farmers and processors to be assured that varieties avail­able in the market are of correct genotype as specified by the breeder. Traditionally, mor­phological characters were used to identify varietal genotype and purity. The distinc­tions of cultivars are sometimes difficult on the basis of morphological characters be­cause descriptive lists from DUS tests do not provide useful morphological markers l

.

The performance in VCU (Value for Cultiva­tion and Use) trials such as yield and flower­ing are of limited value and cannot be used for cultivar identification. TIle difficulties are still more when large number of cultivars with short commercial life span have to be evaluated for discrimination. Phenology and morphological characters may not be signifi­cantly distinct and usually require growing plants to full maturity prior to classification and characterization. The phenological and morphological characters used for cultivar identification include plant growth period 2

and seed shape 3.

Biochemical methods such as isozymes have been widely used for routine testing of

parentage and monitoring genetic purity. However, these data do not provide signifi­cant discrimination to provide estimates of genetic distance 4 that can be helpful to pro­vide PBR and effective varietal identification. For most species DUS testing only relies on the comparison of morphological traits and is quite expensive and time consuming. To complement it isozyme analysis has been used in DUS testing and cultivar identifica­tion in maize, wheat, and barley. The only drawback with isozyme markers is its lim­ited availability 5 and low polymorphism level 6. Therefore, the technology, which utilizes DNA and allows complete sampling of the genome, has become useful as a dis­criminating tool. To avoid these problems DUS testing would benefit from the use of molecular markers. Molecular markers (MM) are advantageous as they are cost effective, rapid, environment independent, unlimited in number and do not require the survey of the whole crop growth period.

MMs have been successfully applied in reg­istration activities like cultivar identifica­tion7

, controlling seed purity of hybrids 8,

etc. MMs could also be used for checking the genetic relatedness 5 between cultivars, for reducing the number of reference varieties for comparison and would improve the com­parison of morphological traits.

Biochemical Markers

Several biochemical methods such as fatty acid profile by GLC, glucosinalates by HPLC and isozymes are available but such tech­niques detect only a limited degree of poly­morphism l , are sensitive to environmental and developmental variation 9 and the dis­crimination between different genotypes is not always possible 10. Electrophoresis of seed proteins has been a prominent test for cultivar identification in crops such as

SYAMAL KRISHNA GHOSH et at.: MOLECULAR MARKERS .. .. . 379

wheatll, cotton l2, etc. HPLC has replaced I .;: . 13 . 14 t electrop 10resls lor maize , nce , e c.

DNA Markers

1. RFLP

DNA hybridization as a tool for detecting specific DNA sequences after electro­phoratic separation was first described by Southern 15' and since then it has became an indispensable tool for molecular analysis. This technique allows probes to hybridize with filters containing DNA which has been digested with restriction enzymes (RE). The RE generates an array of DNA fragments and the length differences between homolo­gous fragments of DNA from different culti­val'S are caused by changes in primary sequence of DNA as a result of point muta­tion / insertion or deletion of DNA/ DNA rearrangement. Such differences when pre­sent, can be detected by DNA hybridization

M · fi . ti' 16 and used as a M m ngerpnn ng .

Among the various DNA markers, RFLPs were the first to be developed and used in human genome mapping 17 and later

. 18 t adopted for plant genome mappmg 00. RFLPs are co-dominant and can identify unique locus with very reliable detection of polymorphism, and can be used for popula­tion studies and diversity classification. RFLP, the first molecular technique to be used in plant breeding has witnessed a revo­lution due its application in crop improve­ment programmes and subsequent use in cultivar identification and registration. Dis­advantages of RFLP technology include rela­tively slow process, less cost effectiveness, producing fewer numbers of discriminating loci 19 and requiring large quantities of DNA samples.

Intra cultivar polymorphism of DNA mark-h b 'd tifi d' . 20 B P 21 ers as een I en e m nce , . na us

22 and sunflower using RFLPs. RFLPs have been also used for the pUrPose of varietal . d 'fi . . t t 23 . 24 d I entt Icatton 111 po a 0 , maize , an whea~5 .

2. PCR- based Multilocus-Profiling Techniques

The amplification of genomic DNA using short oligonucleotide primers results in mul­tiple amplification products from the loci dis-

26 Th' tributed through out the genome . IS developm~nt le~ to the use o~ oliionu­c1eotide pnmers m genome mappmg and fingerprinting 27. Depending on the amplifi­cation conditions or product separation and detection, the arbiratory primer amplifica­tion methods were tenned as randomly am­plified polymorphic DNA CRAPD) 26, arbitrarily primed PCR CAP-PCR) 27. These methods are comparatively simple and appli­cable to any genome and their added advan­tage is that they can provide infonnation on numerous loci that give strength in distin­guishing individuals for cultivar identifica­tion, detennining parentage, fingerprinting and differentiating genotypes.

A. RAPD: In this technique the amplifica­tion products are separated on agarose gels and then stained with ethidium bromide and visualized under UV light. RAPD is a simple, quick and convenient procedure requiring much smaller quantities of template DNA and requires no previous sequence infonna­tion for the fingerprinting of cultivar genomes. It is an effective, cost-efficient method for genotype identification and pedi­gree analysis. RAPDs have allowed cultivar and genotvne identification in pearl mille~8,

'(. 29 30 d B. napus , soybeans , mango an ap-ple31. Using RAPD sunflower lines and Fl hybrids32 were characterized and relation­ships33 between sunflower in bred lines were detennined.

380 J INTELLEC PROP RIGHTS, SEPTEMBER 2001

B. AP-PCR: It is a versatile method that generates fi ngerprints of genomes using ar­bitrarily selected primers 27 under condi­tions where the primers initiate synthesis on DNA, even when the match with the tem­plate is impeliect. AP-PCR has been used is genetic mapping, phylogenetics, taxonomy and molecular genetics of animals and plants34

. DNA profiles based on AP-PCR are both time and cost effective35 and are used for cParentage determination of maize hybrids3

.

C. AFLP: Itisa powerful DNA marker tech­nique and helps in the construction of very high-density molecular maps for application in genome research, positional cloning and genotyping. AFLP is based on the detection of DNA restriction fragments by PCR ampli­fication37

. The restJiction fragments are am­plified by liga ting the double stranded adapter seq uences to the ends of restriction sites which serves as 'u niversal' b inding sites for primer annealing in PCR In this way, restriction fragments of a particular segmentofDNAcan be amplified withAFLP primers corresponding to the restriction site and adapter sequence. The AFLP primers result in selective amplification of those frag­ments in which the primer extension match the nucleotides flanking the restriction site and the number of fragments to be amplified can be selected. Restriction fragment pat­terns generated by the AFLP technique are called AFLP fingerprints and gives polymor­phism. It provides a large number of mark­ers in a single analysis without requiring sequence information for their development 37

It is used for genetic diversity analysis and relationships between inbred lines in sun­flower38

, wheat39 and for genetic linkage mapping in lice 40. AFLP have very attractive properties for DUS testing in rapeseed5 and

for estimating the level of relatedness be­tween cultivars.

Do Microsatellite Markers: Microsatel­li tes are tandemly repeated nucleotide units of 1-6 base pairs and alleles usually differ in the number of repeated units. Microsatel­lites are generally co-dominant and highly polymorphic, wide spread in the genome, detectable by simple PCR based assays and thei r multiplexing potential makes them highly attractive markers for genotyping, identification and population studies. Mi­crosatellite markers are also known as sim­ple sequence repeats (SSRs) 4 1 and are rapidly becoming popular for identiiication of individuals. The SSRs are small repetitive DNA sequences present through out the eukaryotic genome and provide the basis of PCR based co-dominant marker system. Length polymorphism is created when PCR products from different individuals vary in length as a result of variation in the number of repeat units in the SSR.

Microsatellitc markers are used for geno­type identiiication in soybean43

, rice44, suo­

flower45• pea rl millet28

, e tc. In r ice , micro satellites have demonstrated ~olymor­ph ism between46,47 and within20, 4 rice va­rieties and Charters et at. 1 used SSR marker for identifying the variability at the inter and intra-cultivar levels of oilseed rape cultivars. In addition, inter simple sequence repeat­PCR aSSR-PCR), DNA sequences between two have also been applied for DNA finger­printing of maize48

, wheat49 ,etc.

E . Single Nucleotide Polymorphisms (SNPs): SNPs are the new generation MMs for individual genotyping needed for MAS and identification of cultivars. SNPs are bial­leHc and very stable in inheritance than other marker systems. However, the disad­vantage of being biallelic nature is overcome by the abundance of SNPs. According to

SY. CAL KRISHNA GHOSH et at.: MOLECULAR MAJU<ERS .....

estimate, one SNP occurs in every 100-300 bp in any genome, thus making them the most abundant MM known so far50. SNPs seem to be more abundant in plant systems, occurring at one in 20 bp in wheat 51 and one in 70 bp in maize52, as recorded in certain regions of their respective genomes. RFLP, RAPD and SSRs are MMs where gel based assays are required , therefore being time consuming and expensive. Whereas, SNPs can be detected by non-gel based assays, and are represented by sites and DNA se­quences that differ by a single base50. Sev­eral methods50 are available for detection of S Ps, which are fo und within a gene or in its close proximity. SNPs have already been used for cul tivar identification in barJey53.

Choice of Markers for Cultivar Identification

All DNA markers have their own advantages and disadvantages but fo r diversity analysis multilocus profiling techniques have their strengths in fingerprinting, identification and characterization, and are preferable for genotyping and population studies. Classical phenotypic methods of identification are not always sufficient to solve these problems because of instability of morphological char­acters as well as an inability to use such information for identification at juvenile stages or from isolated plant parts. The choice of appropriate DNA profiling tech­nique is dependent on the aim of the testing. Under PVR the formal obligation is the dem­onstration of the DUS criteria. Practical fea­sibility of the tests ask for it to b e straightforward, inexpensive, reliable, re­producible and capable of unambiguous analysis.

The PCR based DNA fingerprinting tech­niques offer a number of advantages over RFLPs54, as they require small amount of

DNA, involve fewer steps and are therefore faster, technically straightforward, do not involve radioactivity, readily automated and the vast range of potential primer sequence available gives the technique the great diag­nostic power. It is generally concluded that DNA profiling technique via PCR will also be much cheaper than RFLP analysis. Ragot and Hoisington:i5 uggested that RAPD analyses is cheaper for small sample sizes and RFLP analysis becomes cost effective fo r larger sample sizes. Any comparison of cost between PCR and RFLP must take into account the type of PCR based marker to be used, costs of developing suitable primers. costs of developing the technique and the sample size to be screened51 . RFLP is pref­erable over isozyme pattern since RFLP can distinguish between several Cll Itivars be­longing to the same isozyme group56. The frequency of polymorphism of RFLP mark­ers limits its application in distinguishing closely related cultivars.

As described earlier that in RAPD prior DNA sequence information is not required to study the anonymous genomes but the inevi­table trade offs with this technique is that the amplification is perfOlmed under conditions of low stringency54. Other probiem with RAPD is a low incidence of non-inherited bands as found in maize57, reproducibility amongst laboratories58 and band domi­nance. RFLP and RAPDs detect low level of polymorphism in wheat59. Many of the limi­tations can be overcome with careful study and can provide valuable supplementary evi­dence54.

The main advantages of AP-PCR over RFLP are increased speed of analysis and the amount of DNA required36. Detection of polymorphism by using AP-PCR or RAPD technology is faster and less laborious than by using RFLP technology, as long as prim-

382 J INTELLEC PROP RIGHTS, SEPTEMBER 2001

ers of approximately the same length and GC content are used in a given set of experi­ments, so that other reaction parameters can be kept constant36.

SSRs are abundant, ubiquitous in presence, hypervariable in nature and have high poly­morphic infonnation content60

. It has also been shown that the use of a limited number of microsatellites is adequate to discriminate even the most closely related wheat61 and barely62 genotypes. DNA fingerprinting by SSR is most infonnative as compared to isozyme and RAPD in case of sunflower lines and hybrids32. SSR markers overcome many of the inherent problems of RAPD analysis and provide highly reproducible, infonna­tive, co-dominant markers. Microsatellite markers may prove to be the DNA profiliny method of choice for primary DUS criteria5 .

The ISSR- PCR produces reliable and highly polymorphic band profiles63 than either RFLP & RAPD analyses. ISSR-PCR is tech­nically simple and quick, where PCR prod­ucts can be generated, fractionated and detected within 9 hours. Microsatellites have been shown to be ten times more poly­morphic than RFLPs in plants 64 and is being increasingly explored for their application in plants. Among the available molecular mark­ers, AFLP is a powerful technique for culti­var identification 65.

Different kinds of markers have been devel­oped and used for identification of markers for agronomically important genes and the identification of cu1tivars, lines and hybrids but such studies do not focus on the impor­tant problem which markers have to be used optimally for variety identification purposes. The greatest challenges faced are to reduce the cost of analysis as well as the risk of confusing one of these elite genotypes with a randomly chosen genotype taken from a large sample66. The recent development of

PCR to amplify DNA and the use of RAPD and AP-PCR has resulted in a potentially useful tool for cultivar identification. With prudient selection of primers, it appears to offer a reliable method of cultivar identifica­tion and have advantages over many of the chemotaxonomic and biochemical methods used for the purpose.

Advantages of Molecular Markers

-DNA sequences of an organism are independent of environmental influences.

·-Simple Mendelian inheritance. -Infinite number of polymorphic

markers can be identified ---DNA allows tests on any tissue at

any stage of the plant development.

- -Simultaneous evaluation of multiple loci is possible.

Molecular Markers and PBR

PBR67 is a legal mechanism to maintain trade secrets for a limited period to enjoy the benefits by the breeder or company. wro agreement also envisages protection of IPR of member countries through a special agreement, viz. TRIPS which covers protec­tion of wide ranging IPRs such as patents, trademarks, copyrights and PBR The WTO provides scope for the plant varieties to be protected by patents or by sui generis system such as PBR provided under the UPOV. Agreement on TRIPS stipulated that 'mem­bers shall provide for the protection of plant varieties either by patents or an effective sui generis system or by any combination thereof" and therefore PBR system is evoked for the effective sui generis system of plant varieties.

The purpose of UPOV to ensure PBR is to have an exclusive property right on new

SYAMAL KRISHNA GHOSH et at.: MOLECUU\R lv1ARKERS .. ... 383

plant varieties in order to provide incentive to the development of agriculture and to safeguard the interests of plant breeders and seed industries. upav prescribes guide­lines that member states are to set PBR laws as sui generis system. A set of characters with detailed parameters is established for th e de novo description of new varieties. New varieties must be distinct, uniform and sta­ble 4. Distinctness is determined by compari­sons to descriptions of previously released varietie s. Revised upav (March 1991) treaty has made provisions to manage and restrict commercialization of close copies of initial varieties throu~h the concept of essen­tially derived variety . Identification of plant cultivars has become increasingly important with the requirement of PBR to demonstrate DUS for each new cultivar. In this context molecular profiling makes the monitoring of minor variants from initial varieties a quick and relatively simple method68.

With the revised wra scenalio and privati­zation of agriculture the IPR and PBR are a prerequisite for private investment into plant breeding. Private investment will not occur without effective measures to prevent misap­propriation. Similarly, the use of proprietary variety will not be allowed if there is no sharing of revenues to cover R&D of the initial variety4. However, molecular markers are more quick and accurate in revealing genetic identification and provide significant differences among the genotypes.

DNA profiling has not yet been adopted by upav as an essential character but after some time this may be included as supple­mentary character information. The evalu­ation of DNA profiling technique as a tool for describing DUS characters under the upav system was under consideration for particu-

69 lar crops ' . In the revised upav (1991) con-vention the enactment of PBR may be

facilitated by DNA profiling technique which will help in extending the PBR in essentially derived varieties from a pro­tected variety and harvested material. It may playa key role in the resolution of disputes over the derivation of varieties or the identity of harvested material.

Conclusion

Mankind stands at a technological threshold that has no parallel in history. We are enter­ing a world in which humanity will start to manage evolution in more direct ways. It is an unknown world that promises great bene­fits and poses numerous policy dilemmas. ll1e creation of the WTa in 1995 is of con­siderable importance to the future of agricul­ture and presents s ome e thical and socio-economic issues in India. In India, TRIPS has received much special attention and it has been seen as an opportunity. It has also been viewed with apprehension by some, particularly in the contest of plant variety registration (PVR).

The need for cultivar discrimination is re­flected in the number of alternative methods developed in the recent years. According to International Seed Testing Associations (ISTA) 70 the common rapid tests that were done by the laboratories were biochemical and electrophoratic analysis, which can be now accomplished by genetic fingerprinting using DNA markers. However, DNA finger­printing is expected to become the preferred method for characterization of cultivars in future. The recent developments in marker technology can be used to demonstrate the DUS of the variety. The data generated through DNA profiling can be tendered as supplementary information in PBR registra­tion. The DNA marker data in addition to morphological characters will be the most powerful tool as the basis for plant variety

384 J INTELLEC PROP RIGHTS, SEPTEMBER 2001

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