In the last post of the AgroSynapsis Tip series, I explained how to develop a PCR-detectable SSR marker for a known gene or trait using a public reference genome.

After that, I received a very relevant question:
❓ What if there is no reference genome available?

Here’s the answer 👇
𝟭. 𝗠𝗮𝗿𝗸𝗲𝗿 𝗱𝗲𝘃𝗲𝗹𝗼𝗽𝗺𝗲𝗻𝘁 (sequence-driven approaches)🧬
These steps generate candidate SSR markers, but do not yet prove genetic linkage.
1️⃣ Comparative genomics with a related species
If a closely related species has a reference genome:
• Identify the orthologous gene or genomic region in a public genome browser.
• Extract flanking sequences (e.g. ±50–100 kb).
• Search for SSRs and design primers.
• Test whether the markers transfer to your species.

2️⃣ Develop gene-based SSRs from transcriptomic data
If the gene sequence is known:
• Identify the EST or transcript corresponding to the candidate gene (via annotation or homology).
• Use CDS, ESTs, or transcriptome assemblies.
• Scan the target transcript for SSR motifs.
• Design primers directly from genic regions.

3️⃣ Genome skimming / low-coverage sequencing to mine SSRs
Even without a full genome:
• Perform genome skimming (low-depth whole-genome sequencing).
• If the gene is known: map reads to the candidate gene or an ortholog.
• Select SSRs in contigs anchored to the gene or its genomic neighborhood.

𝟮. 𝗚𝗲𝗻𝗲𝘁𝗶𝗰 𝘃𝗮𝗹𝗶𝗱𝗮𝘁𝗶𝗼𝗻 & 𝗯𝗿𝗲𝗲𝗱𝗶𝗻𝗴 𝗱𝗲𝗽𝗹𝗼𝘆𝗺𝗲𝗻𝘁 ✅
This step confirms linkage and determines whether an SSR is reliable for marker-assisted selection.
4️⃣ Validate SSRs through linkage analysis
For traits with clear phenotypes:
• Develop a segregating population (F2, RILs, backcross).
• Genotype parents and progeny with candidate SSRs.
• Identify markers that co-segregate with the trait.
• Estimate recombination frequency between marker and phenotype.
• Validate linkage across multiple populations and environments.
➡️ Only markers with stable, tight linkage should be used in selection.

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https://lnkd.in/g3tApqPz

How to Identify an SSR Linked to a Gene if there is no Reference Genome?