Application of molecular markers in rice

bài giảng chọn giống lúa sử dụng chỉ thị phân tử khái niệm và ứng dụng thực tế, Application of molecular markers in rice, Application of molecular markers in rice ,Application of molecular markers in rice

Application of Molecular

Markers In Rice

Classification of main molecular markers

Specific primer

Based on DNA hybridization

Based on PCR amplification

Single nucleotide polymorphism

RAPD 、 AFLP

AP － PCR 、 ISSR

SSR 、 STS 、 SCAR 、 CA

PSSSCP 、 TGGE 、 DGGESNP

RFLP

Technologic Comparison of Main Molecular Markers

ISSR Yes High dominance High Medium/high Low STS Yes High Co-dominance /

dominance High Medium/high LowSRAP/

EST Yes Medium Co-dominance High Medium Low

Demands of ideal molecular markers

Ideal molecular markers must meet the following demands:

(1) Have high polymorphism;

(2) Codominance heredity, meaning that they can distinguish heterozygous and homozygous genotype in diploid;

(3) Can distinguish allele;

(4) Spread all over the genome;

(5) well-distributed in the genome except special sites;

(6) Have no pleiotropism;

(7) Have a simple and fast detection method （ e.g experiment procedures are easily

automatized ） ;

(8) Have the development cost and use-cost as low as possible;

(9) Have excellent repeatability between laboratories （ available for data exchange ）

RFLP (Restriction Fragment Length Polymorphism)

RFLP has been widely used in genetic map construction of genomes, gene mapping

， as well as biological evolution and classification study RFLP is based on

different species (individuals) whose genomes have endonuclease restriction sites of the base mutation, or restriction sites between the bases of the insertion or deletion which results in restriction fragment size changes Such a change can be carried out through specific detection probe hybridization, which can discern differences of varieties (individual) in the level of DNA (ie, polymorphism) and multiple probes can be established by comparison of the evolution of biological relations and

classification The used probes come from the same species or different species of cloned genomic DNA and are located in different chromosomal loci, which can be used as a molecular marker to build molecular map

When a trait (gene) coseparates with the molecular markers, it

indicates that the trait (gene) links to the molecular markers The

exchange value between the molecular markers and traits means the distance of the target gene and molecular markers Then genes can be located in the molecular genetic map After different restriction

endonucleases cut the genomic DNA, there will be different types of cutting fragments Therefore, restriction enzymes and molecular

markers can be combined to study Common restriction endonucleases are Hind , BamH , EcoR , EcoRV and Xba The more molecular Ⅲ, BamH Ⅰ, EcoR Ⅰ, EcoRV and Xba Ⅰ The more molecular Ⅰ, EcoR Ⅰ, EcoRV and Xba Ⅰ The more molecular Ⅰ, EcoR Ⅰ, EcoRV and Xba Ⅰ The more molecular Ⅰ, EcoR Ⅰ, EcoRV and Xba Ⅰ The more molecular markers can build a more saturated map Construction of saturated RFLP map is one of the main objectives of the study

 Characteristics of RFLP

1 RFLP markers are stable and reliable and the detection is never

influence by environmental conditions and the impact of developmental stages

2 RFLP markers are codominant among alleles

3 There is no epistatic effect between the non-alleles

4 RFLP markers derive from genomic DNA of its own variation and are

virtually unlimited in the number

The research of identification would not be desirable for a large number

of individuals(>200) because the current RFLP technology is still expensive and laborious with the complicated operation and requires a large quantity of DNA samples

RAPD ( Random amplified polymorphim )

• RAPD technology is built on the basis of PCR technology It is using a series of (usually hundreds of ) different base sequences

These sequences use random single-stranded oligonucleotide (usually

10 polymer) as primers for the study of genomic DNA for PCR

amplification Through polyacrylamide or agarose gel electrophoresis separation and EB staining or autoradiography to detect radioactive PCR products of polymorphic DNA fragments, the PCR products reflect the polymorphism of DNA fragments corresponding to

genomic region of DNA polymorphism

 Characteristics of RAPD ：

 RAPD technique is simple and easy to grasp compared to RFLP It

not onlyovercomes the shortcomings of dificient isozyme loci but also avoids the disadvantages of RFLP and the complicated operation What’s more, it doesn’t need the isotopic molecular hybridization, so the general laboratory can use this technique

 Most RAPD molecular marker are dominant markers, only a small number can be developed into codominant markerd Therefore, information provided is limited and covers up the dominant homozygote and heterozygote distinction Meanwhile, markers themself are completely dominant

 RAPD technique has a high false positive rate, if the experimental condition is not very stringent, there will be a higher false positive rate;

 RAPD technique demands a high purity and is sensitive to DNA reaction condition with a bad repeatability;

 For the markers attained by experiments, they must be transformed into other kinds of markers to suceed to map genes and enrich the genetic map

AFLP (Amplified restriction fragment polymorphism)

• Netherlandish scientists Zabeau and Vos developed the detection technology on DNA polymorphism of molecular markers in 1993 AFLP technology is based on the PCR

reaction of a selective restriction fragment amplification

method Due to the different size of genomes of different

species, the genome DNA will lead to different sizes of

restriction fragment after restriction endonuclease digestion.

 Characteristics of AFLP

 It has a very high level of polymorphism which

is stable and reliable But it goes with a large

quantity of DNA template and complex steps At the same time the cost is too high and it is a

patented technology.

ISSR (inter simple sequence repeat)

• ISSR molecular marker is a new technology on the basis of SSR markers Its basic principle is that in the SSR of the 5 'or 3' end 1 to 4 base are anchored in the form of purine or pyrimidine Then amplify DNA sequences Repeat sequence and anchoring base is randomly selected PCR products separated by polyacrylamide or agarose gel electrophoresis, can produce more amplified fragment than RAPD Therefore, ISSR technique is a fast, reliable and information-rich

genomic DNA fingerprinting

Micro-satellite or SSR (simple sequence repeat)

DNA consisting of 1 to 6 nucleotides repeats of basic units, is widely distributed in the genome of different locations, and the length is generally

200bp below.

 Abide to Mendelian genetics, is codominant ；

 Each site is determined by primer sequences to facilitate mutual exchanges and cooperation in different laboratories and to

develop primers to obtain the information which can be repeated

in different laboratories and shared

 Advantages of SSR

1) SSR does not require the use of isotopes, reducing

harm to staff ；

2) SSR experiment has little amount of DNA, reducing the work of a

large number of extraction; the experiment does not require Southern transfer, hybridization etc

3) SSR materials , used in the experiment without having sexual

generation, can be used for any single parent or any part of the material,for example DNA in the mitochondria and chloroplast studies may be used

STS (sequence-tagged site)

• Sequence-tagged site is a class of tags defined

by specific sequence primers The biggest

advantage of technology-specific PCR is that it has a very reliable information, rather than

RAPD, AFLP and random RFLP probe

generating the existence of ambiguity.

EST (expressed sequence tags)

• EST (expressed sequence tags) provides a valuable resource for the development of molecular markers Compared to the traditional markers from genomic DNA, EST-based molecular markers are a new

molecular marker and have its obvious advantages, such as the simplicity and high information ,resulting

in a lot of important value.

SCAR (Sequence-characterized Amplified Region)

• Sequence-characterized Amplified Region marker is usually

transformed from RAPD In order to improve the applied stability of

found RAPD markers, we can design a pair of primers (18-24 base

around) according to its base sequence We can also sequence the end

fragments of the RAPD markers and add 14 bases to its end of sequence into the original 10 bases, becoming the complementary primer of the

original RAPD fragment Then carry out PCR amplification using the

primers specific to genomic DNA and we can get the same specific size of cloned fragments This molecular marker-specific DNA markers after

transformation is called SCAR

• SCAR marker is a kind of dominant marker in the form

of existence of amplified fragments, but sometimes in the form of the length Compared with the RAPD markers, SCAR markers can be amplified under stringent

conditions, resulting in good stability, repeatability With the development of research work, there will be more and more SCAR markers to be developed, which will play a huge role in the molecular marker-assisted breeding

 Application of Molecular Markers

in Molecular Biology

 1 For species-specific identification and protection of new varieties

 2 Determine the evolutionary relationship

 3 Track the exogenous gene

 4 Identify the specific chromosome DNA fragment to find the target genes linked to molecular markers for gene mapping and gene isolation

 5 Genetic mapping

 6 Hybrid rice seed purity detection

Here is the brief introduction

about gene location, molecular

breeding and hybrid rice seed purity detection using SSR in our lab:

1.Gene Location Using SSR

Locate semi-dwarf gene in rice

Use Xiaoxiang’ai and Xiangzao143(earlly indica) for

parents and their 116 plants of F2 to construct a tall gene pool and a dwarf gene pool.Then utilize 359

microsatellite primers distributed in chromosomes 1-12 to analyze polymorphism of the two gene pools.

• The rapid extraction method is used for each rice leaf to extract DNA Then analyze PCR amplification and we find that RM249 on chromosome 5 has

polymorphism for 116 dwarf individuals to verify the chain relationship between the dwarf gene We

conclude that the genetic distance between marker RM249 and target gene is about 1cM So it is a new dwarf gene.

１． Xiangzao143(tall parent)

２． Xiaoxiang’ai(dwarf parent)

３． F 2 tall gene pool

４． F 2 dwarf gene pool

１２３４

Locate cold-tolerance gene in rice

By using 89 microsatellite markers (SSR) , we constucted a linkage map for a RIL

population developed from a cross between japonica variety Yukihikari and indica variety Erjiuqing ,and identified the quantitative

trait loci (QTLs) for cold tolerance related

characters.

 We mapped two QTLs , controlling

seedling growth at low temperature They were RM104 on chromosome 1 and

RM160 on chromosome 9 , and the

phenotypic variation explained by each QTL was 23.5% and 15.3% respectively.

likelihood intervals for QTLs of seedling cold

seedling cold tolerance (seedling growth)

I：： Erjiuqing geno type improve the type

seedling height at low temperature

RM247 RM277

RM235 RM17

RM257 RM160

RM205

RM311

RM271 RM258 RM171 RM294A RM333

RM286 RM332 RM167 RM202 RM287 RM21 RM206 RM224 RM114 RM44

RM145 RM324 RM341

RM263 RM318 RM250 RM213 RM138

RM22

RM218 RM251 RM282 RM16 RM135

RM293 RM130 RM85

RM335

RM252 RM241 RM303

RM127

RM159 RM13 RM289 RM164 RM173 RM274 RM334

RM276 RM136 RM3

RM11

12

15

15 15

18

17

17

17 17

17 17

2cM

RM20A

12

 By using 79 microsatellite markers (SSR) , we constucted a linkage map for an F 2 population of

118 individuals developed from a cross between seedling cold tolerance variety Hokkai 289(a

japanese Japonica variety ) and cold susceptible variety Dular (an Indian indica variety ) ,and

found locus RM160 on chromosome 9 affected seedling growgth, chlorosis wither and dying related characters The phenotypic variations explained by this QTL were 17.19%,33.27% and

7.47%, respectively.

 We located a QTL RM244 on chromosome 11 , controlling chilling injury and could explain 49.3% of the total phenotypic variation.

likelihood intervals for QTLs of seedling cold tolerance

cold tolerance

（

（ seedling

growgth, chlorosis, wither and dying ）

improve resistance

RM247

RM277

RM235 RM17

RM294A

RM333

RM286

RM332 RM167

RM202

RM21

RM224 RM114 RM44

RM145 RM324 RM341

RM263 RM318 RM250 RM213 RM138

RM22

RM218 RM251

RM282 RM16 RM135

RM293 RM130

RM85

RM335

RM252 RM241 RM303

RM127

RM159

RM13

RM289 RM164 RM173

RM274 RM334

RM276 RM136 RM3

3 Molecular marker-assisted selection breeding

DNA marker-assisted selection technology: use DNA markers closely linked to the target traits to pick out individuals with related genes indirectly By the means of the technology, we can not only select ideal individuals accurately and steadily at the early breeding stage, but also resolve the problem of recessive obscurity, so as to accelerate the breeding process and improve the breeding efficiency.

QTL analysis and gene mapping

cold resistance indetiification

cold tolerance gene mapping

(cold resistance indetiification )

(CAPs 、 STS marker selection)

 we are now carrying on the molecular polymerization breeding by SCAR markers Y2668L and R251 , which are closely linked to the mapped cold tolerance gene at the booting stage.

Figure 1 physical map near recombination

G181 on rice chromosome 11

Figure 2 high-resolution physical and molecular map

of the cool tolerance gene on rice chromosome 11

S13316A G389A CAPs pattern of Xiangwan 31/

Normal detection technology for seed purity

Modern detection technology SSR markers

Technic flow Equipments

Meaning of seed purity and

authenticity detection

 National Hybrid Rice acreage is increasing year

by year

 Every year the use of hybrid rice seed is about 250 million

kilograms so the quality of seeds (mainly the authenticity and purity) inspection and supervision is particularly important

 The quality of hybrid rice seeds is more common, with the key

being the quality of authenticity and purity

Methods of authenticity and seed purity detection

Physical and chemical methods

Modern technology Protein fingerprint

technology （ isozyme 、 seed storage protein and monoclone antibody ）

DNA fingerprint technology Form analysis by computer simulation

 Seed form detection

 Distinguish this species and other species according to seed form qualities such as grain type,top shape etc

 This method is time-saving, simple, fast and intuive.However, its accuracy is affected by environmental factors

 The morphological differences commercial species becomes smaller and smaller because of the narrow germplasm, therefore, relying on the seed morphology to identify species is becoming increasingly difficult 。