Development of simple sequence repeat markers and diversity analysis in alfalfa (Medicago sativa L.)

Abstract

Efficient and robust molecular markers are essential for molecular breeding in plant. Compared to dominant and bi-allelic markers, multiple alleles of simple sequence repeat (SSR) markers are particularly informative and superior in genetic linkage map and QTL mapping in autotetraploid species like alfalfa. The objective of this study was to enrich SSR markers directly from alfalfa expressed sequence tags (ESTs). A total of 12,371 alfalfa ESTs were retrieved from the National Center for Biotechnology Information. Total 774 SSR-containing ESTs were identified from 716 ESTs. On average, one SSR was found per 7.7 kb of EST sequences. Tri-nucleotide repeats (48.8 %) was the most abundant motif type, followed by di-(26.1 %), tetra-(11.5 %), penta-(9.7 %), and hexanucleotide (3.9 %). One hundred EST-SSR primer pairs were successfully designed and 29 exhibited polymorphism among 28 alfalfa accessions. The allele number per marker ranged from two to 21 with an average of 6.8. The PIC values ranged from 0.195 to 0.896 with an average of 0.608, indicating a high level of polymorphism of the EST-SSR markers. Based on the 29 EST-SSR markers, assessment of genetic diversity was conducted and found that Medicago sativa ssp. sativa was clearly different from the other subspecies. The high transferability of those EST-SSR markers was also found for relative species.

Genetic diversity studies of alfalfa germplasm (Medicago sativa L. subsp. sativa) of United States origin using microsatellite analysis

“…It thus seems that SSR markers are efficient and robust molecular markers in alfalfa. Qiang et al (2015) used 85 SSR markers to analyse the genetic diversity and population structure of 336 worldwide genotypes of alfalfa, and utilization of SSR markers in alfalfa has also been reported in several other studies (Şakiroğlu et al, 2010;Wang et al, 2011;Wang et al, 2013;Sanghani et al, 2015). In the present study, population structure and genetic variability of United States alfalfa using SSR markers were analysed to promote alfalfa improvement and assist alfalfa breeding.…”

“…In alfalfa breeding, different methods and types of markers have been generated along with the general development of plant molecular biology and DNA analysis over the last 17 to 22 years (Cerasela et al, 2010). Among these, SSR has been one of the most useful molecular markers, as protocols utilizing them are highly cost-efficient and time-efficient, making them essential for studies of genetic diversity (Flajoulot et al, 2005(Flajoulot et al, , Şakiroğlu et al, 2010Wang et al, 2013;Sanghani et al, 2015;Pushpavalli et al, 2017). Bagavathiannan et al (2010) used eight SSR markers to analyse the genetic diversity of 12 feral alfalfa populations, 10 alfalfa cultivars and Medicago falcata.…”

Development of EST-SSR markers related to disease resistance and their application in genetic diversity and evolution analysis in Gossypium

“…There have been a few reports of studies in cotton (Buyyarapu et al, 2011;Wang et al, 2014a,b) and many reports in other plants (Koilkonda et al, 2012;Wang et al, 2013;Du et al, 2013;Zhao et al, 2013;Zhang et al, 2014;Asadi and Rashidi Monfared, 2014) examining cross-species transferability using EST-SSR markers.…”

“…Moreover, EST-SSR markers can reveal putative functional genes and can aid in the map-based cloning of important genes; this method is also easy to perform and has a low cost. EST-SSRs have been widely developed and applied in many plants, including peanut (Koilkonda et al, 2012), barley , wheat (Zhong et al, 2009;Asadi and Rashidi Monfared, 2014), alfalfa (Wang et al, 2013), rubber tree , poplar (Du et al, 2013), and date palm (Zhao et al, 2013). In recent years, an increasing number of ESTs in cotton species, including G. hirsutum, G. raimondii, G. arboreum, G. barbadense, and G. herbaceum, have been included in GenBank (http://www.ncbi.nlm.nih.…”

The development of 204 novel EST-SSRs and their use for genetic diversity analyses in cultivated alfalfa

“…Only unigenes longer than 1 kb were included in the EST-SSR detection. To avoid possible duplicates of published EST-SSRs (Liu et al, 2013;Wang et al, 2013Wang et al, , 2014, a local BLASTX was performed using the published EST-SSR sequences against the 1494 SSR-containing unigene sequences (E-value <10E-10). The parameters were adjusted to identify perfect di-, tri-, tetra-, penta-and hexa-nucleotide motifs with a minimum of 6, 5, 5, 5 and 5 repeats, respectively.…”