De novo transcriptome assembly of Pueraria montana var. lobata and Neustanthus phaseoloides for the development of eSSR and SNP markers: narrowing the US origin(s) of the invasive kudzu

Abstract

Background: Kudzu, Pueraria montana var. lobata, is a woody vine native to Southeast Asia that has been introduced globally for cattle forage and erosion control. The vine is highly invasive in its introduced areas, including the southeastern US. Modern molecular marker resources are limited for the species, despite its importance. Transcriptomes for P. montana var. lobata and a second phaseoloid legume taxon previously ascribed to genus Pueraria, Neustanthus phaseoloides, were generated and mined for microsatellites and single nucleotide polymorphisms.

Results: Roche 454 sequencing of P. montana var. lobata and N. phaseoloides transcriptomes produced read numbers ranging from ~ 280,000 to ~ 420,000. Trinity assemblies produced an average of 17,491 contigs with mean lengths ranging from 639 bp to 994 bp. Transcriptome completeness, according to BUSCO, ranged between 64 and 77%. After vetting for primer design, there were 1646 expressed simple sequence repeats (eSSRs) identified in P. montana var. lobata and 1459 in N. phaseoloides. From these eSSRs, 17 identical primer pairs, representing inter-generic phaseoloid eSSRs, were created. Additionally, 13 primer pairs specific to P. montana var. lobata were also created. From these 30 primer pairs, a final set of seven primer pairs were used on 68 individuals of P. montana var. lobata for characterization across the US, China, and Japan. The populations exhibited from 20 to 43 alleles across the seven loci. We also conducted pairwise tests for high-confidence SNP discovery from the kudzu transcriptomes we sequenced and two previously sequenced P. montana var. lobata transcriptomes. Pairwise comparisons between P. montana var. lobata ranged from 358 to 24,475 SNPs, while comparisons between P. montana var. lobata and N. phaseoloides ranged from 5185 to 30,143 SNPs.

Conclusions: The discovered molecular markers for kudzu provide a starting point for comparative genetic studies within phaseoloid legumes. This study both adds to the current genetic resources and presents the first available genomic resources for the invasive kudzu vine. Additionally, this study is the first to provide molecular evidence to support the hypothesis of Japan as a source of US kudzu and begins to narrow the origin of US kudzu to the central Japanese island of Honshu.

Keywords: Invasive; Kudzu; Molecular markers; Neustanthus phaseoloides; Pueraria montana var. lobata; Transcriptome.

Fig. 1

Contig length distributions of Trinity using the ConDeTri dynamic read trimmer

Fig. 2

Transcriptome completeness of transcripts quantified through 956 universal single-copy orthologs using BUSCO

Fig. 3

The number of unique and shared, homologous transcripts among kudzu CPP27 and Pmnk6 and tropical kudzu CPP02 transcriptome assemblies as ascertained via reciprocal best BLAST hit analyses

Fig. 4

Gene ontology classifications of kudzu and tropical kudzu annotated transcripts. Numbers indicate the number of sequences associated with the particular GO term in each category

Fig. 5

STRUCTURE diagram of 75 P. montana accessions across four nations (K = 3)

Fig. 6

Neighbor joining distance tree based on F_st values and 10,000 bootstraps. US = United States; CN = China; JP = Japan; and TH = Thailand

Fig. 7

Sampling sites: (a) United States: US 1, US 2, US 3 (25); (b) Japan: JP 1, JP 2, JP 3 (22); and (c) China: CN 1, CN 2, CN 3 (21) and Thailand: TH (7)