Alfalfa (Medicago sativa L.) is an important leguminous forage crop, mainly used for hay, silage, and pasture. Cultivated alfalfa is an outcrossing autotetraploid with a basic chromosome number of eight and a genome size of 800-1000 Mb. Alfalfa plants are highly heterozygous and exhibit severe inbreeding depression, precluding the development of inbred lines. These make alfalfa breeding for enhanced agronomic traits challenging. Recent advances in next-generation sequencing provide a new strategy to generate cost-effective, high-density, genome-wide single nucleotide polymorphism (SNP) sets. Combined with genome-wide association studies (GWAS) and/or genomic selection (GS), more powerful platforms can be developed to improve the profitability of alfalfa breeding.

CD Genomics is a leading service provider for agricultural genomics research, offering reliable alfalfa genome sequencing services to support research and breeding efforts in the field of alfalfa genomics for clients worldwide. Our services help breed new alfalfa varieties with excellent traits such as high yield, high quality, pests and diseases resistance, and stress resistance.

Our alfalfa genome sequencing service

CD Genomics offers comprehensive and customizable alfalfa genome sequencing services using cutting-edge technology and expertise to characterize the alfalfa genome at the cytogenetic, genetic, and molecular levels. Our services cover all stages of the sequencing process, from library preparation to data analysis, ensuring high-quality and accurate results.

With our advanced next-generation sequencing and long-read sequencing technology platforms, as well as bioinformatics tools to sequence the alfalfa genome, provide reliable genomic data support for alfalfa genome structure, origin evolution, nitrogen-fixing symbiosis, agronomic trait mapping, and molecular breeding research. We are also working on the following alfalfa genomic analyses:

• Pan-genome analysis of alfalfa (constructing genomes of multiple varieties, searching for PAVs among different varieties, etc.)
• Analysis of alfalfa physiological morphology (multi-omics analysis of nitrogen-fixing symbiosis, stress resistance, leaf shape, etc.)
• Breeding of new alfalfa varieties (genome-wide association studies(GWAS)+quantitative trait loci (QTL) mapping of key genes, epigenetic multi-omics search for regulatory elements, construction of gene editing systems, etc.)

We can sequence the whole genomes of over 220 alfalfa varieties, including but not limited to:

Applications of alfalfa genome sequencing

The availability of alfalfa genome sequences has opened up a variety of research avenues and accelerated the development of improved alfalfa varieties, here are some key applications of alfalfa genome sequencing:

• Marker-assisted breeding

Sequencing the alfalfa genome has enabled the identification and development of molecular markers associated with important agronomic traits. By utilizing these markers, such as single nucleotide polymorphisms (SNPs) and simple sequence repeats (SSRs), breeders can select individuals with desired traits at an early stage, resulting in more efficient and precise breeding efforts.

• Trait mapping and gene discovery

The available sequences of alfalfa genes have facilitated trait mapping studies, allowing researchers to identify genomic regions associated with specific traits of interest. Techniques such as QTL mapping and GWAS allow researchers to identify genes or genetic variations associated with traits such as yield, disease resistance, and abiotic stress tolerance.

• Genomic selection (GS)

Genomic selection utilizes genomic information to predict the breeding value of individuals and accelerate the selection process. By combining genomic data with phenotypic information, breeders can estimate the genetic potential of an individual, even at early stages of development. The availability of alfalfa genome sequences can significantly improve the accuracy and efficiency of genomic selection in alfalfa breeding programs.

• Functional genomics and gene editing

The alfalfa genome sequence enables researchers to study gene function and regulatory mechanisms. Transcriptome data, combined with gene expression profiles, provide insight into the molecular pathways and networks involved in various biological processes in alfalfa. In addition, the genome sequence facilitates the application of gene editing techniques (e.g., CRISPR-Cas9) for targeted mutagenesis in alfalfa.

Case study of alfalfa genome sequencing

Alfalfa is the most important leguminous feed crop in the world, with high nutritional value and yield. Zhongmu-4, an alfalfa variety widely grown in China, faces unique challenges due to its autotetraploid nature. The research team used third-generation sequencing and Hi-C sequencing technology to successfully assemble the chromosome-level genome sensed by the Zhongmou-4.

• Nitrogen metabolism: The importance of Alfalfa as a forage crop is closely related to its ability to fix nitrogen and synthesize protein. Genome analysis of Zhongmu-4 found abundant genes related to nitrogen metabolism, with special focus on key enzymes such as glutamine synthetase (GiS) and glutamate dehydrogenase (GDH).
• Population structure and genetic diversity: The genetic complexity of alfalfa, resulting from its outcrossing and self-incompatibility traits, poses challenges to breeding and molecular research. The assembly of the allele-sensing genome of Zhongmu-4 lays the foundation for understanding genotypic variation and alleles in this highly heterozygous species.
• GWAS: Molecular studies of alfalfa have historically been hampered by the lack of a reference genome, requiring labor-intensive and time-consuming breeding methods. The emergence of the Zhongmu-4 reference genome paves the way for marker-assisted breeding and accelerated breeding cycles.
• SNP identification and trait association: Despite the challenges posed by heterozygosity in alfalfa, a GWAS using the Zhongmu-4 reference genome successfully identified 101 SNPs associated with 27 agronomic traits.

Fig. 1. Distribution of Zhongmu-4 genomic features. (Long et al., 2022)

CD Genomics offers cutting-edge alfalfa genome sequencing services to provide comprehensive genomic information to researchers and breeders for their studies. We aim to provide data to support basic research on the genetics of important alfalfa traits and to promote alfalfa improvement. If you are interested, please feel free to contact us.

Reference

1. Long, Ruicai, et al. Genome assembly of alfalfa cultivar zhongmu-4 and identification of SNPs associated with agronomic traits. Genomics, proteomics & bioinformatics. 20.1 (2022): 14-28.