We have 13 soybean cultivars and 13 microssatilite loci. I need to know how many seeds I have to use to extract DNA from each cultivar to adequately represent the cultivar in these analysis.
De nada!
That's a tough one. Personally, I'd probably grow out a set of each variety (10s to 100s of individuals, depending on available space) and assess their variability phenotypically to give myself a rough idea of how many to sample from each. If they look pretty uniform, you might just take a few samples at random from each variety. If a variety appears to be a mix of two types, you could collect a couple of samples from each type. If a variety is highly variable, you should probably stop and consider whether your analysis would be meaningful on such a mixture. If you decide to proceed with the analysis of a highly variable variety, you should take a representative, random sample. "Representative" is subjective, but you should hope to see multiple instances of at least one genotype within such a sample (the "true" variety).
Be sure to analyze each sample separately (don't mix samples from the same variety).
Oh, and if you are not familiar with the crop you should find a good clean, uniform variety that you can trust to be your control, so you can differentiate the genetic variability of your varieties from the environmental variability.
Boa sorte!
If your cultivars are genetically uniform (such as homozygous inbreds or F1 hybrids), then each seed is identical - you only need to sample a single individual per cultivar.
Thank you very much Dr. Haggard. It should be homozygous inbreds, but I am not sure about the purity of the seeds. If we consider a variety, for example, is there a minimum sample size in this case?
De nada!
That's a tough one. Personally, I'd probably grow out a set of each variety (10s to 100s of individuals, depending on available space) and assess their variability phenotypically to give myself a rough idea of how many to sample from each. If they look pretty uniform, you might just take a few samples at random from each variety. If a variety appears to be a mix of two types, you could collect a couple of samples from each type. If a variety is highly variable, you should probably stop and consider whether your analysis would be meaningful on such a mixture. If you decide to proceed with the analysis of a highly variable variety, you should take a representative, random sample. "Representative" is subjective, but you should hope to see multiple instances of at least one genotype within such a sample (the "true" variety).
Be sure to analyze each sample separately (don't mix samples from the same variety).
Oh, and if you are not familiar with the crop you should find a good clean, uniform variety that you can trust to be your control, so you can differentiate the genetic variability of your varieties from the environmental variability.
Boa sorte!
Olá Maria da Glória!
Acho que a Francismar, da Embrapa Soja, é uma boa referência para você tirar essa dúvida. Os contatos são: [email protected] ou 43-3371-6265.
Porém, como penso que você irá extrair DNA das sementes, então a amostra que você tem já é bem representativa da cultivar e é só moer os grãos e retirar as subamostras que você precisa (2 ou 3 no máximo). Acho que só duplicatas já bastam.
Um abraço.
Oi Vanoli, saudade de você. Me esqueci que agora você está na Embrapa Soja. Poderia ter feito a pergunta diretamente para vc. Nós estamos com uma demanda para implementar no laboratório a técnica de diferenciação de cultivares utilizando marcadores microssatélites. E iremos receber as amostras das cultivares que estão registradas no SNPC. Daí eles querem saber a quantidade de sementes para enviar. Li trabalhos que utilizam uma única planta por cultivar, outros que utilizam 50 sementes e até 100 sementes. Como imagino que tenha restrição na quantidade de semente que pode ser encaminhada, pensei em pelo menos 200 sementes, que daria pra processar em processador e extrair o DNA e certamente, seria bem representativa da cultivar, como vc mencionou. No caso da soja, acho que é mais simples, o problema são as outras espécies, como braquiária e etc.
Grande abraço.
Hi Maria,
1. I wish that English can be used for discussion, so other members can benefit from these discussions.
2. I attach one paper (2013) for you (see attachment). They were genotyping 30 soybean cultivars with 2 methods ( conventional PCR evaluated in polyacrylamide gel and PCR with UTSP evaluated by capillary electrophoresis ). In the paper, they used 50 seeds for each cultivar (p.271, yellow highlight). You can take a look at their results from using 50 seeds, and use it as a reference for preparing your study. The group is also from Brazil.
3. Brazil is the second largest soybean-producing country in the world. In 2013, Brazil produced 82.72 million metric tons of soybeans ( U.S. produced 89.48 million metric tons). [ http://www.statista.com/statistics/267270/production-of-soybeans-by-countries-since-2008/ ]. Thank to soybean scientists like you, we now have better soy milk to drink and tofu (etc.) to eat.
Hi Dr. Yuan-Yeu Yau!
Thank you very much for your interesting answer. The paper you sent me will be very hepfull in my analysis. Forgive me for answering in Portuguese in the previous post. I used the same language to answer my colleague from Brazil, but you are right I should have used English for the benefit of the group.
Have a nice day.
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