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Marker Assisted Selection - Science method

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Molecular markers are specific DNA sequences that can be used to identify and track particular genes or genetic traits of interest in plant breeding programs. These markers serve as tools to assist breeders in selecting plants with desired traits more efficiently and accurately. Molecular markers can be used to determine genetic relatedness, assess genetic diversity within populations, identify specific genes responsible for traits, and facilitate marker-assisted selection (MAS) or marker-assisted breeding (MAB) techniques.
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Molecular markers are commonly used to assess genetic variation in agronomic germplasm, analyze population structure, localize quantitative traits (QTL), or linkage mapping for gene mapping. The most interesting application of molecular markers is marker-assisted selection (MAS). Molecular marker technology enables plant breeders to select individual plants based on their marker pattern (genotype) rather than their observable traits (phenotype). This process is called marker assisted selection (MAS) or marker assisted breeding (MAB). Compared with traditional breeding programs, molecular markers can increase the efficiency and effectiveness of breeding programs. Molecular markers in the construction of linkage maps, they have numerous applications in plant breeding such as assessing the genetic variations within cultivars and germplasms. Molecular markers are one of the most powerful tools for studying genetic diversity. They are used in the study of phylogenetic relationships, selection of superior plants, and the study of similarities or differences between different specimens. Molecular markers are also used in germplasm management and marker-assisted selection (MAS) to increase the efficiency of germplasm breeding.
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Could you please recommend some molecular markers for identifying the genes for disease resistance such as blast (Pita, Ph-k, Piz), bacterial leaf blight (Xa12, Xa7, xa5, etc...), brown plant hopper (BPH17, BPH18, BPH37, etc,...). Thanks!
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Dear Sir Kalisa Alain. Thank you very much for your recommendation!
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I run my gene specific primer PCR product on 2.5% Agarose it did not show any amplification only primer dimer was seen
But the same product I used in 8% PAGE gel (with silver staining process) it shown the amplification result
What is the reason behind it...
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My only guess is that the silver stain is just extremely sensitive compared to your agarose gel. Are you using ethidium bromide or something similar for the agarose? The detection limit of this usually falls off hard below 25-50 ng while the silver stain is much better than that so it can see what the agarose gel cannot.
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Should i recalculate the concentration of the protein after adding loading buffer into the sample, so i could calculate the exact volume sample i need to add into the wells to make sure the amount of total protein is the same. And another question if i have added the same amount protein of different sample, the internal marker(eg:GADPH) is not consistent, what i should do as the mathematical calculation is not working anymore. Thanks.
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If you base your calculations on keeping the number of micrograms of protein per well the same, it will be less complicated than basing them on the protein concentration. Then the only thing you have to worry about is that the volume of the sample is not too large to fit in the well.
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Hello to everyone,
I am looking for an overexpressed marker (intra or extra cellular) in the PANC1 cell line for flow cytometry analysis. Indeed I realize coculture with cancer cells and CAF (cancer associated fibroblast), at the end of the coculture I analyze the ratio beetween cancer cell and CAF by marking the cancer cells with an overexpressed surface marker like Epcam to know the proportion of CAF and cancer cells.
I try to do this experiment with the PANC1 cell line but I have some difficulties to find the right marker because Epcam is not expressed enough to distinguish CAF and PANC1.
I already tried Epcam, EGFR, Cytokerin 7 and 8 but I can't distinguish the 2 populations .
PS1 : CAFs don't really have a specific surface marker either as far as I know ... except aSMA but it is not strong enough to distinguish the 2 populations
PS2 : I can't mark the cells with CFSE type dyes because I let my cells in culture that proliferate and it's not great with this kind of markers.
Thanking you for your help
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Markers, such as αSMA and FAP, are highly expressed in CAFs and have been widely used in order to isolate CAF populations.
I do agree that many of the CAF markers come with their own set of downsides, such as low specificity, and there is doubt whether such markers can identify all cancer-associated fibroblasts.
You may refer to the article attached. May be it could help.
Best.
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Hi,
I'm new here so sorry If I'm in the wrong place.
I've been working in UK Agriculture for around 10 years now and I've decided to pursue a career in R&D and plant breeding after working for a seed production company with a local breeding site. I found it all fascinating so I've started a biology degree at Open University and I'm hoping to get a job as a technician or something similar soon.
I know I'm still a fair way from my ultimate goals but in the mean time I'm looking for any good books that go into detail on agricultural plant breeding methods, plant biology, genetics and modern techniques such as Marker Assisted Selection, Doubled Haploid and tissue culture.
I'm looking for something that covers these subjects in detail but on a suitable level for a student (I like diagrams and pictures haha). One of the best looking ones I've found so far is "Plant Biotechnology and Genetics: Principles, Techniques, and Applications" by C. Neal Stewart Jr. But i could do with some advice from anyone who's read any books like this in the past.
Thanks
Stuart
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Turmeric is a polyploid and vegetatively propagated crop. Usually, F1 populations are used to identify QTLs. As this crop generally has no viable seed, what is the best approach to genarate QTLs.
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An F1 clonal propagated population should work fine for qtl mapping.
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If Mesenchymal Stromal Cells (MSCs) are used in cell therapy, what are the stromal cells and in what proportion they are recommended for cell therapy? I appreciate the guidance, reference, suggestions or recommendations.
Or if Mesenchymal stem cells are used in cell therapy, how to differentiate Mesenchymal stem cells from stromal cells? Is there any specific marker to differentiate them?
Thanks for your feedback.
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There is a new position statement paper from the International Society for Cell and Gene Therapy. They have discussed most of the queries that you have, hope it helps,
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I want to find up-to-date information on the current state of conventional (traditional) plant breeding in the world. Is the conventional breeding approach applied in the small breeding companies or crop giants like Monsanto and Syngenta now? For example in order to produce lines for subsequent marker-assisted selection (MAS) or genetic modification (GM). Or the traditional breeding approaches are currently totally substituted by the MAS and GM approaches?
If you know reviews and/or book chapters on this topic please suggest.
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i think the breeder must be create variation first and he evaluate this variability and select the good new recombination to produce a new strains or new varieties
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Suppose I have scored 6 allele for an SSR marker in 50 population with major allele frequency of 0.2448. Now how can I determine the required PIC for the marker?
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Well, I had a similar query a few months ago and finally calculated my PIC with MolKin software version 3.0 (Gutiérrez et al. 2005).
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Hi everybody. I'm wondering weather or not carboxymethylcellulose can be used and metabolized as the only carbon source in a medium for a microorganism able to produce an endocellulase. Specifically, I'm trying to use this endocellulase gene as a selection marker (no antibiotics, no auxotrophy marker). Is it possible to do that? Will the carboxymethyl glucose generated by the enzymatic reaction be metabolized by the microorganism?
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Can we use cellulose instead of CMC in Mendel' s media? Is there any other substitute in the media for CMC?
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If the experience is to find out marker assisted selection/MAS, what kind of strategy (or what kind of molecular mark) that can be best to be used?
I found several strategy lik RFLP, SSR, AFLP,, QAPD, QTL and SNP for several kind of selection for drought tolerance plant. If this research is conducted in rice plant, what kind of molecular mark is suitable for that?
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Yustina,
The methods you listed are very different in time of first application, cost, labor, and equipment demands. They are different in information value provided. If you like to test previously found markers in your plant population, you can apply RFLP, SSR, SNP methods. If you like to generate new data or study new plant - RFLP, AFLP, QAPD. But, if you plan long-term research and will use genomic data - please, concentrate your efforts on SNP.
Alex
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Would you please contribute your opinion
Upload books, bulletins,publications, Reviews, State of Art
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I agree with the opinions.  The genetic profile of indigenous chicken should be fully understood to exploit  the advantages so as to introduce into improved exotic chicken for improving their performance for both meat and egg production besides imitating the phenotypic morphology to appease the rural farmers to popularise. Suitable cross breeding tests and progeny evaluation will help, such that villager's economy and self sufficiency will definitely improve. Hence, molecular genetics has a vast scope in the preparation of vaccines against upcoming deadly diseases too.
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I fed in my diploid data (29 samples, 18 primers (loci) and average of 8 bands/locus) for dominat analysis for Shanon Index, total allele diversity, percentage polymorphism etc sucessful. But at the last comment of analysis, the dialog box showing OUT MEMORY was displayed. How do i overcome this problem?
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"OUT OF MEMORY" always shows up when the data input file does not meet the requirements, in all the cases it has happened to me - it was a space/gap left at the end of the row
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microsatellite markers used as marker assisted selection (MAS) in wheat, rice or barley for commercial breeding program
which is the perfect allele size
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In these times of smokescreens and race for money, maybe I'll tell a heresy, but the only way to create a variety (e.g. wheat) is to walk the experimental field from morning to evening and that’s the only "marker" that really works. We are living times of terms invention - "phenotyping", "genotyping", “genomics” and so on. Molecular geneticists get so amazed when they “discover” something that geneticists in Agronomy have known for decades that they must to inventing the whole name and the “new” scientific field! Paradigm is “epigenetic”. For “centuries” agricultural geneticists have known something we called “prolonged modifications”. Few decades ago, molecular geneticists have come to the same knowledge, and they got so excited by the “invention” that they invented the whole knew field - Epigenetic! The similar case is the story of “Genomics”. This is due to the entry of molecular biologists in breeding and agronomy. They have no idea about the real genetics and the interaction of genotype and the environment, in particular. They are still viewing genes, almost as isolated units of heredity, and they are late in the comprehension of how the genome works about 30 years, on average, comparing to agricultural geneticists and breeders. They are even proud with the statement that “the advantage of molecular markers is that they exclude the effect of the environment” (!?) and in breeding programs we are relying on GE interaction! If we followed the major genes effects, than we do not need molecular markers, or to be more accurate the costs usually do not correspond to the result. If we follow the effect of minor genes, or QTL correlation to the phenotypic variation for a specific trait, results are commonly totally unconvincing. In other words, a lot of water gonna pass under the bridge, while molecular biologists come up with something that can replace boots and walking the experimental field!
So my dear and respectable colleague, use the SSR in studying genome variability, eventually, in early generations, when you still have no seed enough to establish a field trials. However, the variability in F2, for example, is so huge, that you gotta be a Rockefeller to follow it by SSR. We've had about 3000 crosses per year just in wheat. Can you imagine, using SSR to follow genetic variation in F2! You can use them in fingerprinting, as well, but sometimes and for the internal use, a simple and cheaper approaches could do the work. However, if you liked to get the job done, and to create a real commercial “beauty”, get on your boots, put your hat on, and do some “phenotyping” straight in the nature. No man has ever made a variety in the lab with artificial light instead the Sun above and with no windows to smell the nature around, yet.  :)
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Do you know this one? So, Please let me know the reason.
Recently I tried to do test the possibility of using the geneticin in A. fumigatus in the 400 ug/ml concentration. But it is not working. I am trying to figure out the reasons. Help me please
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Geneticin should be toxic to fungi and yeasts at 400 ug/ml concentration, have you tried reducing the geneticin concentration to 250 ug/ml? It might work.
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Fusarium head blight, powdery mildew, wheat yellow mosaic bymovirus, and sharp eyespot are important diseases of wheat in the middle and lower reaches of Yangzi River. Pyramiding resistances to these diseases is not an easy work. 
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I would suggest Genomic selection - which can pickup contribution from different yield limiting factors. 
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Replies from respected fellows will highly be appreciated
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As stated before, the right answer is "it depends". The complexity of the trait under investigation will determine the number of QTLs that are important for selection. You also need to remember that a single QTL might contain multiple genes. Therefore; depending on the trait, a single QTL can explain most of the variation observed. By definition, the trait will still be considered a complex trait as multiple genes are controlling its expression. For the selection process per se, I would say that you should focus on the QTLs with minor effects which are really hard to be selected phenotypically. But everything depends on your final goal! Trying to use marked assisted selection for all QTLs controlling a trait might come with a really high cost and complexity. I hope that can help.  
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Hi everybody
I have some specific markers for drought tolerance in wheat. Can I use the same marker for drought tolerance in barley? In other words, if I run some experiment to test drought tolerance in some barley genotypes and then I use the specify maker for drought tolerance (in wheat) to genotype the barley lines. If I found a significant association between marker and drought tolerant trait in barley, is it possible to say like a qtl?
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Hi
it depends on what the markers are for, some traits are generic for drought tolerance, others not , there are species differences as pointed above; many traits also work together or in a hierarchy, so it is hard to say. Try running the markers and see what you get... 
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Hi everyone. can you help me with a procedure  for marker assisted selection. A website link or PDFs where l can find the answer will be greatly appreciated. THANK YOU IN ADVANCE
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hope these enclosed publication will help you, in addition to  above said papers
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I have found following summary last week, but unfortunately I have not saved the link:
2. Main objectives of the project – Project summary
The 21st century is a time of continuing population growth. If we are to shape and profit from plant breeding, for the mutual benefit of the consumer and of industry, we must integrate innovative methods for generating variability and fixing genotypes. The use of doubled haploids, particularly in combination with marker-assisted selection, has sped up transgenic and non- ransgenic breeding programmes and broadened the gene pool from which attractive traits are selected. Despite the well-established advantages of doubled haploids and their enormous benefit for breeding, they are still difficult to obtain in many important crop species. As a result, an enormous and costly effort needs to be invested by industry if they are to be universally employed. Here we identify crucial bottlenecks to the development of doubled-haploid technology and propose a work-plan for their removal. Based on our preliminary results, we postulate that epigenomic regulation is a crucial regulator of plant cell
Reprogramming and hence totipotency. Epigenetics defines a set of mechanisms mostly consisting of chromatin alterations that modulate DNA transcription, dictate tissue and cellular complexity and bring the phenotype into being. Chemical probes targeting key enzymes of epigenetic regulation are likely to be crucial to manipulate the plant cell epigenome and shift differentiation  processes from the default pathway towards embryo formation.
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Sorry, I am not answering your question directly. Just a suggestion, if you use FIREFOX as your browser (or any other browser should have similar function), you can probably click the "OPEN MENU" link (on the top-righ most of the screen), then click the "History" icon.
In the newly open side-window, you can scan which of the recently opened/closed website containing the information you are looking for. Probably you have to scroll further down if it was allready a couple of days/weeks/months, depending on how active you are opening/closing websites. Click on that link, and you should go back to the website where you read your info.
Good luck
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I intend to study the characterization of indigenous chickens in Zimbabwe using molecular markers. Nothing of that sort has been attempted and I need collaborators. Please contact me if you are interested
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Dear Never
The huge achievements in poultry industry ( in all world ) done with classical approaches of poultry breeding , selection and mating systems , I did not work against success of molecular genetics but this technique promise the world to do some things have economically worth value , when the molecular approaches become part of large scale poultry production , the breeding programs will be dramatically change .
Good Luck
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The primers we are using are also the ones validated in several studies and PCR condition is very specific (touchdown). Thanks for your time.
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What is the template you try to amplify? Is it of the same type as in the original study you refer to?
And is the PCR mixture also exactly the same? The [MgCl2] for example, has a huge influence on the Tm of the primers.
I know from experience with qPCR that samples with less template have more primer dimers. When primer dimers are formed, and you make serial dilutions of this template, the more diluted the template is, the higher the primer dimer peak in the dissociation curve analysis.
I agree that primer design algorithms cannot fully predict the behaviour of the primers, also because the starting conditions are not always entered/known exactly.
A lot of primer design algorithms have an option for a 3'GC-clamp which of course give primers with a high 3'end Tm. Although I sometimes doubt if a few complementary bases could really anneal when the annealing temperature is >50 degrees higher then the Tm of the complementary part.
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Hi, I have always been using synaptophysin as a presynaptic marker, unfortunately as other presynaptic markers its penetration in the tissue is very low, usually less than 3 or 4 microns when using with 0.2% triton. Does anyone know of a synaptic vesicle marker with better penetration?
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Dear Jan,
We tried several things to solve this issue, and indeed we saw an increase in penetration of synaptic markers when using higher concentrations of detergents, however it increases the myelin signal as well. 
The fact that is specific for synaptic molecules is probably related to the saturation of antibodies on the surface of the tissue, especially glutamatergic synaptic markers such as synaptophysin or vglut1. Typically you see very high intensity of antibodies on the surface, which might create some esteric impediment, blocking antibodies to get deeper in the tissue. Since detergents can improve (limitedly) penetration, you might be able to increase penetration if you remove completely lipids using clarity-processed tissue.
Hope it helps
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My understanding is you plant many varieties in the field or greenhouse. You observe and measure for your desired traits. You genotype your entire population. This is where I am stuck.
How does phenotype observations + genotype sequences = molecular markers that we can one day use for marker assisted selection (MAS)?
How do I find molecular markers for desirable traits such as rhizomes, non shattering, and large seed size in wheat and sunflower?
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Jacob,
Identification of marker for a desired trait requires two things, genotype data and phenotype data. Genotype data is converted into a genetic linkage map depending upon recombination frequencies between markers. This map can be obtained  using MAPMAKER, MAP-MANAGER, JOINT-MAP etc.
Once the map is generated, we can identify marker-trait association using map data and phenotype data. This can be done using QTL-cartographer software. The closest marker can be used in MAS.
I would suggest you to read following article-
Collard B. C. Y. et al. (2005) Euphytica 142, pp 169-196
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I want to search markers for MAS screening of Xa-10 gene of BLB resistant population, does anybody have marker for the same?
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Kindly read attached research paper.
All the details about CAPS primer for Xa13 gene in rice and its position describe in this paper..
The details about primer, sequence and for restriction digestion RE are written below: 
M582 F: CTTGATTTCTAACTCTTCTGCTC
R: TTTGTCTTTCTCTCTATGCAGG
582 HindIII or DraI
M491 F: AGTAATGGATGCAGTGTGGGGC
R: TTGTTTGCTCACTCCACCCTTC
491 StyI
M604 F: CAACGCCTATCTTCTGCATTTC
R: GTGACCCTAGTTTCTGGTTATG
604 XbaI
M419 F: CATCAGCAACCCCGTGAAAACG
R: GATGGCAATGTACCGCGAATAC
419 PvuII
M593 F: TCAGGGAGATGTTCTGGCCTTC
R: CTTATTCGAGGATAGTCGTCAC
593 AvaII
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I want to generate a marker for the RF gene.
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Following articles should help you. Good Luck
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Can anyone succinctly explain how one differs from the otherand why a breeder would use one over the other?
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Very good illustrations in the form of diagrams differentiating among conventional breeding, marker assisted selection, marker assisted recurrent selection, and marker assisted back crossing are posted in the Integrated Breeding Platform (IBP). Here are the links for those:
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Javascript.
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Dear Awais Rasheed ·Thanks for the reply. But I have checked it 3 times and getting same results. can you tell me the other possibilities
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In genomic selection, the model estimates the substitution effect of each SNP, which is the difference between the average effect of its two alleles. GEBV is then obtained as the sum of the substitution effects over all SNPs.
Is it really the same thing as the breeding value, which is the sum of the average effect of all the alleles?
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There is a clear answer in Vitezica et al 2013 genetics. They explained that when the matrice of molecular data (ie the Z matrix) is computed according to Van Raden (ie taking into account allele frequencies), then the product "Z * SNP effects estimated by GS" gives the breeding value of each individual.
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In many crops the genomes have sequenced and plenty of markers are available for most of the important traits. Is it possible to develop a high yielding variety having all desirable quality traits coupled with resistance/tolerance to important biotic and abiotic stresses?
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A genotype is more or less adapted to a given environment. An intensive cultivar usually is depressed under drought conditions. A stable-yielding cultivar will not compensate for large inputs, etc. Except predictable environmental factors (soil, latitude), there are unpredictable. So I think that there are no perfect varieties but growers need to have a system of complementary varieties. Actually, gene interactions, genotype - environment interaction, and coenotic nature of many important traits (they arise at coenotic level only and cannot be reduced to lower levels of material organization) make MAS for complex traits much more complicated then it is sometimes advertised.