Rice Genomics - Science topic

Rajadurai Gothandaraman

Thank you very much. I will try once as per your suggestions.

Hello Alice. I experimented with gene gun transformation in prickly pear cactus. Stable transformation rate is very low (less than 1 percent in some species). Try experimenting with different bead sizes and pressures to get the right spread and density to increase your chances (use cross sections to see how far and dense the bead penetration is). Also consider using an RFP instead of a GFP as your reviewers will inevitably ask how you know it's not auto-fluorescence. General rule is if you still have fluorescence after two weeks it's stable transformation. This protocol is like playing cards at the casino, the more hands you play the better chances you have at winning! Good luck!

hello,

Primarily we should get to know that for what purpose we are carrying out molecular studies and based on that analysis can be done. like,

Stat tools:- ArleQin, GenAlEx, Molkiv, Power marker, NTSYS, STRUCTURE, MEGA and Darwin.

*If you are using genic SSRs-

Stat Tools: - Windows QTL Cartographer, TASEL.

all the best

Also check the results of this study https://www.researchgate.net/publication/336747205_Rice_of_Northeast_India_harbor_rich_genetic_diversity_as_measured_by_SSR_markers_and_ZnFe_content

I do this work with JoinMap and MapQTL but the module rQTL is the go to for people who like working in R. SNP filtering varies depending on the methods used but generally you want markers to cover at least 90% of the individuals within the population. Once your QTL location is mapped to a physical location within the rice genome, you can consider that location +/- 1 mB the QTL.

Indica and japonica rice are the tow sup species of O. sative. ofcourse there are distinct morphological differencesbetween them. But they are have difernecs at genome level as well as genome size, number of ggenes etc, its better to read the genome sequencing articles for both, it will make you clear Asif Ali . I have compiled this data as assignment for Plant Functional Genomics, but I cant find that file to send you.

I think the reason for the failure of fertilization

You can immerse rice anthers in the Alexander staining solution and let it stain overnight. Then put the anther on a microscopic slide and gently dissect the anther to release pollen with a forcep or syringe needles. Add more stainning solution if needed and inspect under a stereomicroscope.

I am also getting a faint band in non transgenic rice while doing PCR with hygromycin primers. Is this is a common problem for hpt in rice?

That's so kind of you Oreci, thank you very much!

Hi Katrina,

It is recommended that if your data is skewed you have to do data transformation depending upon the type of data you have (percentage data etc). You may use angular transformation methods for the transformation. Once after you finish data transformation you may use the data for the QTL analysis.

Many researchers proposed similar theories for manipulating C3 genomes to be C4, and confer nitrogen fixation property to Poaceae members. As Yuan-Yeu said, its not easy to confer more than one gene!

I can direct you to this link

Dr. Wen Yao,

Thank you for your suggestion. The link you provided is not accessible, can you make available updated link.

Thanks,

Dear Syed Adeel Zafar if you still have trouble getting it amplified at the new TM you could do two consecutive PCRs. First a PCR of 25 uL for about 20 cycles. Then use one microliter of this PCR as template in a new PCR with fresh primers/ Taq etc. Run it about 30-35 cycles. This method has often helped me to amplify a difficult template. If you get too much product from the second PCR you may want to run less cycles or dilute the 1st PCR product 1:10 or 1:100 and then use as template for the 2nd PCR. You can use the same primers, they don't have to be nested primers.

In rice reference genome is well characterized,

You may use following approaches

1) Find loci probe seq based on DEGs from affimatrix chip

2) Blast search hits with high match can be seach on KEG pathway

3) Also may try GO term search of blast hits

I totally agree with DR Biswas, but I am simply extending it a little because I am not certain what you really expect. Maybe your question relates to what you decide to set as your goal. If you use yield / duration as.a selection factor you will get a different kind of outcome than if you go for one maximum duration and select for yield. Likewise would you be better off selecting for days duration or heat units? (Some climate considerations might me involved in that case.)

I am actually not sure how the GTF file is build but it could work something like this in a Linux terminal. Let's assume the gene id field is in column 12 and the gene description is in column 10

cat japonica.gtf | awk '{print $10"\t"$12}' > output.definition

However, I am not sure about the GTF format, where the important columns are stored etc.

Also I have to mention (but I assume you know that): not having replicates in differential expression is considered bad experimental design. While I do not know the reasons for your choice, you will probably not be able to discern technical from biological noise...

plz refer

www.ncbi.nlm.nih.gov › NCBI › Literature › PubMed Central (PMC)

@Andrew Thank you for you reply. i needed the genome data for some particular varieties, to be used as reference. My varieties are not their in the project.

Attached paper describes current progress and future challenges of C4 rice development. You can also find some helpful information through accessing The C4 Rice Consortium (http://c4rice.irri.org/)

Crossing msms male serile mutant with the male fertile line (MsMs) will give 100% of fertile plants (Msms). Pollen from the heterozygous must be used to cross with msms sterile plants and the progeny will segregate 50% msms: 50% Msms. It would be useful to have some marker  associated to the sterile phenotype in order to select sterile plants before flowering.

Dear Paul Reed Hepperly,

Thank you  for  attention.

I agree with you.Wise design.

Thank you again.

Huashan

thanku

Interesting! We have cloned a tiller gene from wheat which works by inducing tiller initiation. Wonder what your gene does

the addition fertilizer or metles stress dont direct change in DNA plant so transgenic plant not effect in stress metals if have genium response on that metals

pls see me thesis

Attached is the paper: "Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system".

Good luck with your research!

Hi,

I would suggest you to perform short read alignment on Os-Nipponbare-Reference-IRGSP-1.0 pseudomolecules by using bowtie2 or BWA. As you have generated reads from Nipponbare genotype than it will always be good to procure the same reference. 

Second, I agree with Marc for de-novo approach and a comparison with other rice genomes.

Hope this will help you.

See http://www.gramene.org/ for the needs of rice genome and marker annotation on chromosomes. I don't think there is a automatic tool as such to make the diagram form you. 

 Controls for any gene expression study will be decided as per experiments. So, if you are considering yield related genes then it can be a choice , but for general studies I'm unaware of using Flag leaf as a widely used control in rice. Please explain in detail so that it can be answered correctly.

Have a look at Gramene.org

look at this attached files.it may help you

Dear Dr Tuyet Van,

I hope that you could have a look in my Ph D thesis it has a DNA marker part an include the DNA extraction and PCR in the following web page.

the methods is as following from embryo

2.3.3.2. Genomic DNA extraction for mapping

Total genomic DNA was isolated according to the protocol described previously by

Anderson et al. (1992). Briefly, 3-5 g of leaf tissue per sample (each sample was collected from each F2 seedling 8 weeks after sowing, 81 plants in total) were ground in liquid nitrogen and incubated at 60 °C for 45 min with 15 ml of extraction buffer, (100 mM Tris-HCl, 500 M NaCl, 50 mM EDTA, 1.25 % SDS) in 50 ml polypropylene tubes. After cell disruption and incubation with hot isolation buffer, proteins were removed by chloroform: iso-amyl alcohol (24:1, v:v). Samples were incubated for 30 min by shaking and then centrifuged at 3000 rpm for 30 min. The aqueous layer was transferred to a new tube and 20 μl RNAse A (10 mg/ml) was added. Samples were incubated for 30 min at room temperature. One volume of cold ethanol was added to precipitate DNA. After 30 min incubation at 4 °C, precipitated DNA was hooked out and placed in a 2 ml reaction tube containing 1 ml of 75% ethanol. After washing twice with 75% ethanol, the washing solution was removed and the DNA pellet was dried thoroughly and dissolved in TE buffer. The DNA samples were diluted and stored at -20 °C. The DNA was diluted to a concentration of 50 ng/μl before used in SSR experiment.

I hope this method will help you

with best regards

Khaled

Please refer this article you may get some idea, and also you please visit IRRI website for more information.

You might find some answers in this publication - hope it helps.