Development of a New Marker System for Identification of Spirodela polyrhiza and Landoltia punctata

Abstract

Lemnaceae (commonly called duckweed) is an aquatic plant ideal for quantitative analysis in plant sciences. Several species of this family represent the smallest and fastest growing flowering plants. Different ecotypes of the same species vary in their biochemical and physiological properties. Thus, selecting of desirable ecotypes of a species is very important. Here, we developed a simple and rapid molecular identification system for Spirodela polyrhiza and Landoltia punctata based on the sequence polymorphism. First, several pairs of primers were designed and three markers were selected as good for identification. After PCR amplification, DNA fragments (the combination of three PCR products) in different duckweeds were detected using capillary electrophoresis. The high-resolution capillary electrophoresis displayed high identity to the sequencing results. The combination of the PCR products containing several DNA fragments highly improved the identification frequency. These results indicate that this method is not only good for interspecies identification but also ideal for intraspecies distinguishing. Meanwhile, 11 haplotypes were found in both the S. polyrhiza and L. punctata ecotypes. The results suggest that this marker system is useful for large-scale identification of duckweed and for the screening of desirable ecotypes to improve the diverse usage in duckweed utilization.

Figure 1

Electrophoresis of the PCR products amplified from duckweed (Landoltia punctata ecotype ZH0001-S-0) with the designed three pairs of primers in an agarose gel. M: DNA ladder Marker III (100, 200, 500, 750, 1,000, 2,000, 3,000, and 5,000 bp; Tiangen Biotech Co., Ltd.). Line 1: primers SC19/20. Line 2: primers SC35/36. Line 3: primers SC09/10.

Figure 2

Electropherograms showing capillary electrophoresis separation of the PCR product fragments amplified from ecotypes with the three primers. The horizontal axis displays the size of the detected PCR product fragments, while the vertical axis presents the intensity of the signal (i.e., the indicator of concentration of fragments in the PCR products). The orange peaks match the standard fragments in the GeneScan 500 LIZ size standard, while the blue ones represent the PCR products fragments amplified from different ecotypes. The numbers on the horizontal axis represent the size of the corresponding peak in the GeneScan 500 LIZ size standard (orange). “LH” represents Landoltia punctata haplotype number and “SH” represents Spirodela polyrhiza haplotype number. Different haplotypes displayed different types of blue peaks (DNA fragments) and combinations.