Genome-Wide Development and Validation of Cost-Effective KASP Marker Assays for Genetic Dissection of Heat Stress Tolerance in Maize.
Genome-Wide Development and Validation of Cost-Effective KASP Marker Assays for Genetic Dissection of Heat Stress Tolerance in Maize.
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DOI：
10.3390/ijms21197386
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发表时间：
2020-10-06
影响因子：
5.6
通讯作者：
Johal GS
中科院分区：
文献类型：
--
作者：
Jagtap AB;Vikal Y;Johal GS
玉米是世界上第三重要的谷类作物。然而，它的生产容易受到热应力的影响，预计在未来几年将变得越来越严重。抗热胁迫的种质资源已经确定，但其潜在的遗传基础仍然知之甚少。基因组图谱技术可以填补这一空白，前提是提供可靠的标记来梳理基因型-表型关系。在本研究中，我们利用RNA-seq实验的数据，分别鉴定了对热胁迫敏感和耐受的两个对照系LM11和CML25之间的单核苷酸多态性（SNPs）。在热胁迫处理后，从三个不同的组织/器官中建立RNA-seq文库，包括顶叶、胚珠和花粉，这些组织/器官极易受到热胁迫的损害。单核苷酸变异（snv）调用使用STAR映射器和GATK调用者管道在组合方法中识别两条线之间高度准确的snp。通过比较叶片文库、花粉文库和胚珠文库的转录序列，分别发现LM11和CML25之间存在554,423、410,698和596,868个snv。然后选择这些SNP中的数百个发展成全基因组竞争性等位基因特异性PCR （KASP）标记，这些标记被证明是健壮的，成功的SNP转化率为71%。随后，利用这些KASP标记对LM11和CML25杂交的F2定位群体进行了有效的基因分型。这些KASP标记具有较高的成本效益，为耐热玉米提供了可靠的分子标记工具，不仅有助于耐热玉米耐高温性状的遗传解剖，而且有助于通过标记辅助选择（marker assisted selection， MAS）加速耐热玉米的育种。
Maize is the third most important cereal crop worldwide. However, its production is vulnerable to heat stress, which is expected to become more and more severe in coming years. Germplasm resilient to heat stress has been identified, but its underlying genetic basis remains poorly understood. Genomic mapping technologies can fill the void, provided robust markers are available to tease apart the genotype-phenotype relationship. In the present investigation, we used data from an RNA-seq experiment to identify single nucleotide polymorphisms (SNPs) between two contrasting lines, LM11 and CML25, sensitive and tolerant to heat stress, respectively. The libraries for RNA-seq were made following heat stress treatment from three separate tissues/organs, comprising the top leaf, ovule, and pollen, all of which are highly vulnerable to damage by heat stress. The single nucleotide variants (SNVs) calling used STAR mapper and GATK caller pipelines in a combined approach to identify highly accurate SNPs between the two lines. A total of 554,423, 410,698, and 596,868 SNVs were discovered between LM11 and CML25 after comparing the transcript sequence reads from the leaf, pollen, and ovule libraries, respectively. Hundreds of these SNPs were then selected to develop into genome-wide Kompetitive Allele-Specific PCR (KASP) markers, which were validated to be robust with a successful SNP conversion rate of 71%. Subsequently, these KASP markers were used to effectively genotype an F2 mapping population derived from a cross of LM11 and CML25. Being highly cost-effective, these KASP markers provide a reliable molecular marker toolkit to not only facilitate the genetic dissection of the trait of heat stress tolerance but also to accelerate the breeding of heat-resilient maize by marker-assisted selection (MAS).
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