Scientific report: "Assessment of genetic diversity of genetic resources Lilium spp. Indigenous and imported by RAPD directive" pptx

SUMMARY In Vietnam, apart from Lilium longiflorum, Oriental lilies and its Asiatic hybrids have become very popular.. Random amplified polymorphic DNA RAPD markers were utilized for th

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Assessment of Genetic Variation in Local and Exotic

Lilium spp Germplasm Using RAPD Markers

Đánh giá đa dạng di truyền nguồn gen Lilium spp bản địa và

nhập nội bằng chỉ thị RAPD Nguyen Thi Phuong Thao, Ninh Thi Thao, Vu Quang Khanh, Nguyen Quang Thach

Faculty of Biotechnology, Hanoi University of Agriculture

TÓM TẮT

Ở Việt Nam, ngoài loài hoa loa kèn trắng Lilium longiflorum, các giống Lily thuộc nhóm Oriental

và các con lai Asiatic đã đang trở nên rất phổ biến Để thiết lập một chương trình chọn tạo giống trong nước đối với loài hoa có giá trị kinh tế này, việc thu thập nguồn gen với sự đa dạng di truyền lớn là yêu cầu tiên quyết Chỉ thị RAPD đã được sử dụng để đánh giá sự đa dạng di truyền của 32 mẫu giống đại diện của các loài/ giống Lilium bản địa và nhập nội khác nhau Trong số 20 mồi RAPD

sử dụng, chỉ có 3 mồi (chiếm 15%) là các mồi cho đa hình Ba mồi OPA10, P615 và P650 đã tạo ra được tổng số 123 băng DNA với trung bình đạt 0,8; 1,7 và 3,8 băng DNA tính trên mỗi mẫu giống nghiên cứu Mức độ đa hình khác nhau được thể hiện bởi 3 mồi này cũng đã quan sát được ở các

nhóm Lilium khác nhau Số băng DNA đa hình cao nhất với 61 băng thu được ở nhóm chứa các

giống Lily thuộc nhóm Oriental đạt 49,59%, tiếp sau đó là nhóm L longiflorum và các con lai của chúng đạt 33,3%, nhóm các loài Lily hoang dại thu thập từ Nhật Bản đạt 8,13%; nhóm các giống lily thuộc nhóm Asiatic đạt 7,32% và nhóm các loài lily bản địa của Việt Nam đạt 1,63% Phân tích RAPD cùng với việc thiết lập cây phân loại di truyền đã cho thấy sự đa dạng và mối quan hệ di truyền giữa các nguồn gen Lilium đã thu thập

Từ khoá: Chỉ thị DNA, đa dạng di truyền, đa hình DNA, Lilium, PCR, RAPD.

SUMMARY

In Vietnam, apart from Lilium longiflorum, Oriental lilies and its Asiatic hybrids have become very popular To set up a breeding program for this high economic value crop in the country, collection of germplasm with a broad genetic variation is a prerequisite for the success Random

amplified polymorphic DNA (RAPD) markers were utilized for the identification of genetic variation

of 32 representative samples of different local and exotic Lilium species and cultivars Of the 20

primers, only 3 primers (15%) showed to be informative primers that generated polymorphic patterns of PCR products A total of 123 DNA bands were obtained with primers OPA10, P615 and P650 giving an average of 0.8; 1.7 and 3.8 DNA bands per plant accession respectively Different level of genetic polymorphism as revealed by these 3 informative RAPD primers was found among

different Lilium groups The highest polymorphic DNA bands were obtained in group containing commercial Oriental Lilies with total 61 bands (49.59%), following by group of L longiflorum and

their hybrids (33,3%), group of wild species from Japan; (8.13%), group of commercial Asiatic Lilies (7.32%) and group of wild species from Vietnam (1.63%) RAPD analysis combined with the construction of phylogenetic tree revealed the genetic variation and genetic relationship between

collected Lilium germplasm

Key words: DNA markers Lilium, DNA polymorphism, genetic variation, PCR, RAPD

1 INTRODUCTION

In Vietnam, apart from Lilium longiflorum

(so called "hoa loa kÌn tr¾ng" in Vietnamese),

Oriental and Asiatic lilies, and their hybrids,

have become very popular Remarkably, so far

Vietnam has been reported to possess three wild

species which belong to genus Lilium including

Lilium brownii F.E Brown, Lilium poilanei

Gagn and Lilium arboricola Among them, L

poilanei and Lilium arboricola have been

become very rare species in the world

To set up a breeding program for this high

economic value crop in country, collections of

germplasm with a broad genetic variation is a

prerequisite for the success Information on genetic

relationship is very important in designing crop

improvement program and management of

germplasm

Different marker techniques (Yamagishi,

1995; Fernandez et al., 1996; Niimi et al., 1996;

Roh et al 1996; Van der Meulen et al1996; Obata ,

et al., 2000; Marasek and Orlikowska, 2001;Abe et

al., 2002) have been used for studies of genetic

differentiation in Lilium populations, identification

of cultivars and varieties, introgression studies,

determination of parentage, phylogenetic analysis,

and construction of genetic maps

Among them Random amplified polymorphic

DNA (RAPD) markers is particularly well suited to

high-output systems required for plant breeding

because it is easy to perform, fast, reliable and of

relatively low cost (Williams et al., 1990) This has

been used in many studies e.g identifying Lilium

genotypes (Fern¸ndez et al., 1996; Lee et al 1996;

Choi et al., 1999; Obata et al., 2000; Yamagishi

2002); determining the hybridism of inter-specific

hybrids (Yamagishi, 1995; Obata et al., 2000;

Ploszaj et al 2005; Wiejacha et al., 2001); assisting

in breeding program for disease resistance (Van

Heusden, 2001; detecting somaclonal variation of in

vitro propagated plants (Chia-Szu Wen and Ju-Ying

Hsiao 1999; Varshney et al., 2001)

The present study was undertaken to assess the

genetic diversity of local and exotic germplasm of

Lilium using RAPD markers and the reliable

information obtained may serve as a reference for

plant breeding, gene pool diversity, and germplasm

conservation programs

2 MATERIALS AND METHODS

2.1 Plant materials

A total of 32 Lilium accessions were used for

the study (Table 2)

These were categorized into 5 major groups A:

Lilium longiflorum and their hybrids; B: wild

species from Vietnam; C: wild species from Japan; D: commercial Oriental Lilies and E: commercial Asiatic Lilies

2.2 Methods

RAPD-PCR: DNA was isolated from the leaves

of juvenile plants growing in the Greenhouse using method of Nobuo Kobayashi (1998) In PCR-RAPD reactions, 20 primers, whose sequences are given in Table 1, were used PCR was performed in a 20 ml reaction solution containing 10x PCR buffer, 40 ng

of total DNA, 3 mM MgCl2, 200 mM each dNTP, 0.2 mM primer and 0.5 unit Taq DNA polymerase (Fermentas) PCR program was set for each primer following the instruction of references (Table 1) The amplified fragments were electrophoresed in 2% agarose gels (Quiagen, USA) followed by ethidium bromide staining All amplification reactions were repeated at least three times

3 RESULTS AND DICUSSION

Of the 20 primers, only 3 primers (15%) showed to be informative primers that generated polymorphic patterns of PCR products A total of

123 DNA bands were obtained with primers OPA10, P615 and P650 giving an average of 0.8; 1.7 and 3.8 DNA bands per one plant accession respectively Figure 1 showed the example of RAPD markers generated by OPA-10

Ten-base random primers have usually been used for RAPD analysis in plant species However, Yamagishi (1995) reported that the efficiency of 10-base primers to produce polymorphic bands was

low in Lilium, i.e., 16% of the 10-base primers

produced polymorphic fragments among 13 species and 16 inter-specific hybrids In another study in

2002, Yamagishi et al indicated that long random primers have a high efficiency to produce DNA

markers in Lilium (12% of the 10-base primers

amplified polymorphic fragments between the two Asiatic hybrid lily cultivars and 54 and 67% of the 15- and 20-base random primers produced polymorphic bands, respectively) In our study using different plant clones and species, 2 out of 3 informative primers were 17-base random primers accounting for 66,7 % Long random primers might

be useful in a RAPD assay for Lilium species.

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As seen in table 2, plant accessions were

categorized into 5 major groups A, B, C, D and E

Different level of genetic polymorphism as revealed

by these 3 informative RADP primers was found

among different groups The highest polymorphic

DNA bands were obtained in Group D with total 61

bands (49.59 %), following by group A (33, 3 %), C

(8.13 %), E (7.32 %) and B (1.63 %) However,

three primers failed to produce any scorable band in

individual plant of Lilium longiflorum from Gialam,

Lilium poilanei Gagn from Sapa, Lilium oriental

“Crystal Star” and Lilium asiatic hybrid “Quinta”

from the Netherlands and Lilium asiatic from Japan

This might suggest the distinct DNA sequences of

these species

Within group A of L longiflorum, different

DNA bands were recorded indicating the genetic

variation have occured and they seemed to differ

from those lately imported such as Lilium

longiflorum “Deliana” or Lilium longiflorum

“Snow Queen” In 2007, Trinh Thi Viet Chung also detected the morphological variations among

population of L longiflorum collected in different

locals in Vietnam This might be the result of selection and adaptation process of different clones

since L longiflorum was the earliest cultivar that

has been introduced into Vietnam since 18 century Figure 2 showed the phylogenetic dendrogram

of 32 individual accessions with 3 informative primers using the program NTSYSpc 2.02h The result showed that they can be divided in two major groups which in turn can be divided into smaller subgroups.The results of genetic analyses showed

that the local and exotic Lilium using in this study

is composed of genetically different clones This would offer possibilities for identifying the germplasm collections, including material for a breeding program in the country

Table 1 Primers used in RAPD analysis, their sequence and references

Table 2 List of plant materials and number of amplified products obtained

with primer OPA10, P615 and P650

Number of amplified products

A Lilium longiflorum and their hybrids

B Wild species from Vietnam

C Wild species from Japan

D Commercial Oriental Lilies

E Commercial Asiatic Lilies

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Fig 1 RAPD patterns generated by primer OPA-10 Lanes M: 1 kb size marker; 1-32: L oriental

“Nostalgia”; L longiflorum 1;L formolongo; L poilanei Gagn.; L longiflorum 2; L longiflorum 3; L oriental hybrid “Cassandra”; L oriental “Sorbonne”; L oriental - trumpet “Gradisca”; L oriental

“Acapulco”; L hybrid “Leslie”; L oriental “Tiber”; L oriental hybrid “Siberia”; L oriental

“Stargazer”; L longiflorum “Deliana”; L oriental “Crystal Star”; L asiatic hybrid “Royal Show” ; L longiflorum 4; L oriental “Giacondo”; L oriental “Mondriann”; L oriental “Valadores”; L oriental hybrid “Salinas”; L oriental hybrid “Shalloon”; L asiatic; L brownii; L.asiatic hybrid “Freya”; L asiatic hybrid “Quinta”; L longiflorum 5; L longiflorum “Georgia”; L longiflorum “Snow Queen”; L

formosanum;L speciosum

Fig.2 Dendrogram obtained from RAPD pattern of 27 Lilium accessions

4 CONCLUSION

Obviously with the ever-increasing

production of new cultivars, the importance of

accurate tools for assessing the genetic background and species or cultivar genuineness has become more important

M 16 15 14 13 12 11 10 9

8 7 6 5 4 3 2 1

M 32 31 30 29 28 27 26 25

24 23 22 21 20 19 18 17

The RAPD technique provides a useful

approach for evaluating genetic differentiation,

particularly in Lilium species, which is still poorly

known genetically In this work, RAPD analysis

combined with the construction of phylogenetic

tree revealed the genetic variation and genetic

relationship between the local and exotic lily

cultivars and certain wild lily species from

Vietnam However, more efficient primers will be

required to bring more reliable information which

will asset the breeding program for Vietnam its self

using valuable indigenous germplasm such as L

poilanei and L aboricola

Acknowledgements

This research was supported by fundamental

scientific research in life science program code

number 6 006 08 (2006-2008) from the Ministry of

Science and Technology

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