MARKER ASSISTED SELECTION

MARKER ASSISTED SELECTION
Main features of marker aided selection
PROCEDURE FOR MAS
Marker assisted backcrossing (MABC)

1. 1 WELCOME

2. Submitted to :- Dr. K. K . Tiwari Assistant Professor, Bioscience Research Centre, SDAU, S.K. Nagar Submitted by :- Vaghela Gauravrajsinh K M.Sc. (Agri.) Reg.no:-04-AGRMA-01840-2018 SDAU, S.K. Nagar MARKER ASSISTED SELECTION Course Title : Biotechnology for Crop Improvement

3. Marker Assisted Selection (MAS) “Marker assisted selection or Marker aided selection (MAS) is an indirect selection process where a trait of interest is selected based on a marker (morphological, biochemical or DNA/RNA variation) linked to a trait of interest (e.g. productivity, disease resistance, abiotic stress tolerance, and quality), rather than on the trait itself .” This term (MAS) was first used by Beckmann and Soller (1983).. Traits that modern breeder select involve high yield, good yield and disease resistance. Traits are controlled by piece of DNA called genes. Marker are DNA segment that can be used as a flag to track genes.

4. F 2 P 2 F 1 P 1 x large populations consisting of thousands of plants PHENOTYPIC SELECTION Field trials Glasshouse trials Donor Recipient CONVENTIONAL PLANT BREEDING Salinity screening Bacterial blight screening Phosphorus deficiency plot

5. F 2 P 2 F 1 P 1 x large populations consisting of thousands of plants Resistant Susceptible MARKER-ASSISTED SELECTION (MAS) MARKER-ASSISTED BREEDING Method whereby phenotypic selection is based on DNA markers

6. Main features of marker aided selection (MAS) Application: Marker Aided Selection (MAS) is applicable to all the three groups of crop plants, viz , Self pollinated, cross pollinated and asexually propagated species. High Accuracy: The molecular markers are correlated with conventional markers. Once this work is completed molecular markers can be used with very high degree of accuracy. In other words, molecular markers have very high accuracy .

7. Rapid Method: MAS is a rapid method of crop improvement. For example, in conventional breeding when we transfer a recessive character through backcross, one selfing is required after every back cross for identification of such character. MAS permits identification of recessive alleles even in heterozygous condition and thus speeds up the progress of crop improvement work. Free from Environmental Effects: DNA markers are not affected by the environmental conditions. They are free from environmental effects and indicate simply the presence or absence of specific base sequence. The expression of conventional markers, on the other hand, is largely influenced by environmental conditions. RFLP markers maintain their identify irrespective of genetic background.

8. Permits QTL Tagging: The genes controlling polygenic or quantitative characters are scattered in different chromosomes. Hence mapping of such genes is not possible by conventional method. DNA markers offer tagging or mapping of quantitative trait loci (QTL ). Requires Sophisticated Laboratory: DNA marker techniques require well equipped laboratory facilities. It requires expensive equipment's, chemicals, glassware's and trained manpower. Thus, this is an expensive method . Health Hazards: Some of the DNA marker techniques involve radioactive labelling resulting in health hazards of people working in such laboratories. Now non-radioactive labelling techniques are also available .

9. Why Need of Marker Assisted Selection? Difficult traits (e.g. Abiotic stresses) Traits with low heritability Pyramiding of resistance genes Selection at seedling stage Distinguish homo- & heterozygote

10. PROCEDURE FOR MAS Selection of Parents Development of Breeding Population Isolation of DNA From Each Plant Scoring RFLPs Correlation with Morphological Traits

11. Selection of Parents Parents with contrasting characters or divergent origin should be chosen. The parents that are used for MAS should be pure. In self-pollinated species, plants are usually homozygous. In cross-pollinated species, inbred lines are used as parents.

12. Development of Breeding Population The selected parents are crossed to obtain F1 plants. F1 plants between two purelines or inbred lines are homogeneous. The F2 progeny is required for the study of segregation pattern of RFLPs. Generally 50-100 F2 plants are sufficient for the study of segregation of RFLP markers.

13. Isolation of DNA The main advantage of MAS is that DNA can be isolated even from the seedlings and we need not to wait for flowering or seed development stage. The DNA is isolated from each plant of F2 population. Standard procedures are available for DNA isolation. The isolated DNA is digested with specific restriction enzyme to obtain fragments of DNA. The DNA fragments of different sizes are separated by agarose gel electrophoresis. The gel is stained with ethidium bromide and the variation in DNA fragments can be viewed in the ultraviolet light.

14. Scoring RFLPs The polymorphism in RFLPs between the parents and their involvement in the recombinants in F2 population is determined by using DNA probes. The labelled probes are used to find out the fragments having similarity. The probe will hybridize only with those segments which are complementary in nature. Generally 32P is used for radioactive labelling of DNA probe. Now non-radioactive probe labelling techniques are also available. In this way RFLPs are determined.

15. Correlation with Morphological Traits The DNA marker (say RFLPs) are correlated with morphological markers and the indirect selection through molecular markers is confirmed. Once the correlation of molecular markers is established with morphological markers, MAS can be effectively used for genetic improvement of various economic traits.

16. Marker assisted backcrossing (MABC ) A backcross program based on markers is known as marker-assisted backcrossing (MABC ). Molecular markers have been generally used for foreground selection often for background profiling, sometimes for background selection, and only occasionally for recombinant selection. When markers are used for foreground as well as background selections, the backcross scheme is often called complete line conversion, full MAS, or simply MABC.

17. Molecular assisted backcrossing based on three types of selection and those types of selection achieve three objective of backcross Foreground selection Foreground selection refers to using marker that are tightly linked to the gene of interest in order to select for the target allele or gene . Background selection Codominant markers distributed throughout the genome enable selection of plants having the highest proportion of the recurrent parent genome. Recombinant selection Codominant markers located on either side of the target gene can be used to select for rare recombinants that do not have the donor genome beyond these markers.

18. Foreground selection Term foreground selection given by- Hospital and Charcosset. Marker Assisted Selection is indirect selection for the target gene/QTL based on molecular markers closely linked to the target gene/QTL. This is most common application of MAS and is called foreground selection. The closer is the marker to the gene/QTL, the greater will be the efficiency of foreground selection . For example:- when a marker and a gene/QTL are separated by 5 cM, the association between their alleles would change in about 5 % of the progeny. As a result, the marker genotype will incorrectly predict the gene/QTL allele in these 5 % of plants. Therefore, the distance between the marker and the target gene/QTL should be less than 5 cM; ideally, the marker should be allele specific or, at least, gene-based. Whenever the markers are more than 5 cM away from the gene/QTL, a pair of flanking markers, i.e., one marker located on either side of the gene/QTL , should be used. This would minimize the chances of incorrect prediction of the allelic state of the gene/QTL by the marker genotype .

19. Background Selection Term background selection given by - Hospital and Charcosset (1997 ). Molecular markers distributed throughout the genome can be used to monitor and aid the recovery of genomic regions, except for the target region, of one of the parents. This strategy is called background selection.

20. Most commonly used markers in MAS Restriction fragment length polymorphisms ( RFLPs ) were the first molecular markers used to diagnose genetic variability in organisms . RFLP uses restriction enzymes to digest (cut) the DNA molecule and identify regions linked to a trait. The most widely used markers in major cereals are called simple sequence repeats ( SSRs ) or microsatellites . They are highly reliable (i.e. reproducible ), co-dominant in inheritance, relatively simple and cheap to use and generally highly polymorphic . There are two pre-requisites for marker assisted selection. These are: a tight linkage between molecular marker and gene of interest, and H igh heritability of the gene of interest

21. MABC for Gene Pyramiding The concept of gene pyramiding was proposed by Nelson (1978) Nelson (1978) to develop crop varieties with durable resistance to diseases by bringing together few to several different oligogenes for resistance to the given disease. Introgression of two or more genes from a single DP is relatively simple: the DP is crossed with the RP, and the F1 and the subsequent progeny are repeatedly backcrossed to the RP. But when the genes to be pyramided are present in different DPs, each DP is used in a separate backcross program with the RP to recover the target gene from each DP in the genetic background of RP either in heterozygous or homozygous state. Finally, the pyramided version of RP having all the target genes is recovered from this hybrid by selfing coupled with selection.

22. Transgene Pyramiding Pusa basmati 1 , one of the first product of molecular breeding . Genetic transformation technology can be used for transgene pyramiding by sequential transformation , co-transformation or transformation with linked transgenes. Datta et al . (2002) pyramided the BB resistance gene Xa21 from transgenic line TT-103 with the Bt gene for insect (yellow, striped, and pink stem borers and leaf folder) resistance and the RC7 chitinase gene for sheath blight ( Rhizoctonia solani ) resistance from the transgenic line TT-9. Lines TT-103 and TT-9 were crossed, and the F2 plants were genotyped for the presence of Xa21, Bt, and RC7 chitinase transgenes using PCR and Southern hybridization. The F2 plants were also assayed for their reaction to BB and sheath blight pathogens and to yellow stem borer. Plants having the three transgenes and showing the best bioassay performance were advanced to F3. The F3 plants were analyzed as above and plants homozygous for the three transgenes and with the best bioassay performance were selected and advanced to F4. The pyramided lines were resistant to BB but showed variable levels of resistance to sheath blight .

23. Advantages of MAS 1. Foreground selection greatly facilitate selection for such traits whose phenotypic evaluation is cumbersome , tedious, time consuming , destructive, and/or dependent on specific threshold conditions. 2. MAS permits backcrosses to be made in succession during introgression of recessive genes. 3. MAS allows selection for the target traits in off-season nurseries/greenhouses ; this allows two to four generations to be taken each year. 4. MAS can be done in the seedling stage . This permits the use of selected plants for hybridization in the same generation even when the target traits relate to fruit and seed

24. 5. In the case of MABC, MAS can accelerate recovery of the recurrent parent genotype (background selection ). 6 . In case of gene introgression from unadapted germplasm, recombinant selection helps minimize/eliminate linkage drag . 7. MAS allows selection for horizontal resistance and greatly facilitates gene pyramiding as well as trait stacking . 8. MAS enables stacking of oligogenic and polygenic resistance to obtain more durable and effective resistance to diseases. This objective cannot be achieved without MAS .

25. Limitations of MAS Tightly linked, widely applicable, and reliably diagnostic markers are available for only a limited number of target traits. MAS is a costly method and requires well trained manpower for handling of sophisticated equipment's, isolation of DNA molecule and study of DNA markers. The detection of various linked DNA markers (AFLP, RFLP, RAPD, SSR, SNP etc .) is a difficult, laborious and time consuming task. MAS sometimes involves use of radioactive isotopes in labelling of DNA, which may lead to serious health hazards. It has been reported that MAS may become less efficient than phenotypic selection in the long term. The use of MAS is more difficult for QTL because they have minor cumulative effects and are greatly influenced by environmental conditions and genetic background .

26. Achievements The first report on application of MAS concerned resistance to soybean cyst nematode (Concibido et al . 1996). The first variety developed by MAS was a maize hybrid offered for commercial cultivation in the USA in 2006 by Monsanto, USA. Two rice varieties, Cadet and Jacinto, developed through MAS have been released in the USA; they have unique cooking and processing qualities, including reduced amylose content. In Indonesia, rice varieties Angke and Conde were developed by transfer of BB resistance genes; they were BB resistant and gave 20 % higher yields than IR64. In Australia, barley varieties Sloop SA and Sloop Vic were developed by MABC to transfer multiple disease resistance into the popular variety Sloop In India – Rice improved PB-1, S ambha Mahsuri - 2

27. Improved Samba Mahsuri (RP Bio-226) Improved Samba Mahsuri  (RP Bio-226) is a high yielding fine grain  rice variety . In a collaborative project, scientists from CSIR-CCMB and the ICAR-Indian Institute of Rice Research   worked together to develop it. The variety was developed using  Marker assisted selection  and has three major bacterial  blight  resistance genes Xa21, xa13 and xa5 . All India Coordinated Rice Improved Project (AICRIP) conducted trails in multiple locations across India, and found positive results for resistance to bacterial blight.

28. Orange-Fleshed Sweet Potato Orange Fleshed Sweet Potato is highly rich in beta-carotene . Beta-carotene is an excellent source of Vitamin A . Orange-Fleshed Sweet Potato is high in fiber and has a good taste. There is also the presence of magnesium in the OFSP. These properties combined improves digestion a great deal . Orange-Fleshed Sweet Potato has anti-inflammatory properties. This is basically due to the presence of beta-carotene, vitamin C and magnesium. They are very effective in curing internal and external inflammations . Beta-carotene is a major antioxidant and together with phosphorus, iron, Vitamin C and Vitamin B complex and iron. They are a very good immune booster.

29. Vivek QPM 9 - an Early Maturing QPM Maize Hybrid for India Maize one of the major sources of calorie and protein. However , it is deficient in essential amino acids  viz ., lysine and tryptophan . Quality protein maize (QPM) with  opaque -2 gene along with associated modifiers contains twice as much lysine and tryptophan and 30% less leucine than the normal maize. The reduced level of zein further improves the nutritional quality of the QPM.

30. HHB 67- Improved HHB 67 released in India in 1990 by ICAR. It is extra-early, requiring less than 65 days from sowing to grain maturity. Recent surveys have, however, indicated that HHB 67 has started to suceptible to Downy Mildew (DM), showing up to 30% disease incidence in farmers’ fields. The parental lines of HHB 67 were subjected to DM resistance maintenance breeding. Restriction fragment length polymorphism-based marker assisted backcrossing with elite donor parent ICMP 451 was used to add DM resistance to male parent H 77/833-2. The result was several improved resistant versions of H 77/833-2. Additional genes for DM resistance were backcrossed into female parent 843A/B from donor ICML 22 using conventional progeny-based greenhouse disease screening of pot-grown seedlings. Conventional backcross transfer of DM resistance to improve 843A/B took nearly nine years (1991-1999), while marker-assisted backcross transfer of DM resistance to improve H 77/833-2 was completed in just over three years (1997-2000).

31. Improved PUSA RH10 Pusa RH10, the widely cultivated superfine grain aromatic rice hybrid, and its parental lines Pusa6B and PRR78 are susceptible to bacterial blight (BB) disease caused by  Xanthomonas oryzae  pv.  oryzae . Pusa1460 , a Basmati rice variety, was utilized as the donor for introgressing BB resistance genes  xa13  and  Xa21  into Pusa6B and PRR78 using a marker-assisted backcross breeding program. The markers RG136 and pTA248 linked to BB resistance genes  xa13  and  Xa21 , respectively, were used for foreground selection . Improved lines of Pusa6B and PRR78 showed yield advantages of up to 8.24 and 5.23%, respectively. The performance of the BB-resistant version of Pusa RH10 produced by intercrossing the improved parental lines was superior to the original Pusa RH10.

32. References Marker-Assisted Plant Breeding: Principles and Practices by B.D . Singh , A.K. Singh. Plant Breeding Principles and Methods by B.D . Singh. Gupta , Hari & Mahajan, Vinay & Agarwal, P & Bisht, G & Pant, M. (2010). Vivek QPM 9 maize: A Hybrid for Himalayan Hills and Peninsular Region. Indian Farming. 59. 10-12. Basavaraj, S.H., Singh, V.K., Singh, A. et al . Mol Breeding (2010) 26: 293. https://doi.org/10.1007/s11032-010-9407-3