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Title: Development of high-density linkage map and tagging leaf spot resistance in pearl millet using genotyping-by-sequencing markers

item PUNNURI, SOMASHEKHAR - Fort Valley State University
item WALLACE, JASON - University Of Georgia
item Knoll, Joseph - Joe
item HYMA, KATIE - Cornell University
item MITCHELL, SHARON - Cornell University
item Buckler, Edward - Ed
item VARSHNEY, RAJEEV - International Crops Research Institute For Semi-Arid Tropics (ICRISAT) - India
item SINGH, BHARAT - Fort Valley State University

Submitted to: The Plant Genome
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/29/2016
Publication Date: 2/22/2016
Citation: Punnuri, S.M., Wallace, J.G., Knoll, J.E., Hyma, K.E., Mitchell, S.E., Buckler IV, E.S., Varshney, R.K., Singh, B.P. 2016. Development of high-density linkage map and tagging leaf spot resistance in pearl millet using genotyping-by-sequencing markers. The Plant Genome. 9(2):1-13. doi: 10.3835/plantgenome2015.10.0106.

Interpretive Summary: Pearl millet (Pennisetum glaucum, syn. Cenchrus americanus) is used as an important forage crop and also grain crop in southeastern parts of the United States and in many developing countries. Breeding of new pearl millet varieties would be enhanced by the use of molecular markers to tag important traits, but a highly dense genetic map of pearl millet has not yet been developed. Pearl millet is diploid in nature with seven pairs of chromosomes, and has a large genome size of 2.35 billion base pairs of DNA. As this species lacks a reference sequenced genome, it is important to develop genetic markers through strategies that do not require prior sequence information. Next-generation sequencing technologies have yielded several newer mapping approaches which are robust and economically efficient. Genotyping-by-sequencing (GBS) is one such mapping approach to develop a large number of genome-wide markers. These markers are small stretches of DNA sequence, which differ at one base-pair, and are thus referred to as single nucleotide polymorphisms (called SNPs). We used a recombinant inbred line (RIL) mapping population developed from two parent lines: Tift 99B as female parent and Tift 454 as male parent. These lines were chosen specifically to map resistance to pyricularia leaf spot and nematodes. One hundred eighty six individuals from this mapping population were grown in the greenhouse as material for DNA extraction. The DNA was digested with enzyme ApeKI and then a 96-plex GBS library was developed for sequencing. The sequencing was done on an Illumina HiSeq 2000 at the Genomic Diversity Facility, Cornell University. On average, each individual produced 2.2 million pieces of usable DNA sequence (called ‘good reads’). After stringent data filtering, the genetic map was developed using data from 150 RILs, and contained 16,650 SNPs spread across all seven linkage groups (one linkage group for each chromosome pair). The overall average density of SNP markers was 23.23/cM, which spanned over total genetic distance of 716.7 cM. Of the 16,650 mapped markers, 39.41% of them showed significant segregation distortion (deviation from the Mendelian expectation of 1:1) towards either parent. The results from this study will help in identification and tagging of several traits related to disease resistance and nematode resistance. Furthermore, genome-wide markers will have application in a recently developed marker-assisted selection method known as genomics-assisted selection.

Technical Abstract: Pearl millet is an important forage and grain crop in many parts of the world. Genome mapping studies are a prerequisite for tagging agronomically important traits. Genotyping-by-Sequencing (GBS) markers can be used to build high density linkage maps even in species lacking a reference genome. A recombinant inbred line (RIL) mapping population was developed from a cross between Tift 99B and Tift 454. DNA from 186 RILs, the parents, and the F1, was used for 96-plex ApeKI GBS library development, which was further employed for sequencing on an Illumina HiSeq 2000. The sequencing results showed that the average good reads per individual was 2.2 million reads, the pass filter rate was 88%, and the CV was 43%. High quality GBS markers were developed with stringent filtering on sequence data from 179 RILs. The reference genetic map developed using 150 RILs contained 16,650 SNPs and 333,567 sequence tags spread across all seven chromosomes. The overall average density of SNP markers was 23.23/cM covering a total genetic distance of 716.7 cM. The linkage map was further validated for its utility by mapping quantitative trait loci for flowering time and resistance to pyricularia leaf spot. High density linkage maps provide better map resolution and abundant genomic resources. The results will also help in identification and tagging of several traits related to disease resistance and nematode resistance.