Development and validation of CAPS-marker associated with the <i>Rf2</i> gene in sorghum (<i>Sorghum bicolor</i> (L.) Moench)

Abstract

Background. The development of heterotic hybrids based on cytoplasmic male sterility (CMS) is the leading strategy in breeding sorghum (Sorghum bicolor (L.) Moench). The trait of pollen fertility restoration in forms with CMS A1 (milo), predominantly used in sorghum breeding, is determined by at least two dominant complementary genes Rf1 and Rf2, and also gene Rf5. The development of accessible molecular markers of sorghum Rf genes is highly relevant for hybrid breeding, since they can significantly accelerate the process of creating female sterile forms (A lines), sterility maintainers (B lines) and pollen fertility restorers (R lines).Material and methods. The studied material included 36 sorghum accessions from the VIR collection, which differed by the ability to restore pollen fertility in forms with A1-type CMS. The nucleotide polymorphism of 935 bp fragments of the PPR genes Sobic.002G057050, Sobic.002G054100, and Sobic.002G054200 located at the chromosome 2 was studied.Results. The fragments obtained with the use of a pair of 2459403fw and 2459403 primers were 935 bp long and included parts of three genes: Sobic.002G057050, Sobic.002G054100, Sobic.002G054200. For identifying the sequence variant Sobic.002G057050-1090 associated with the Rf2 gene, Tru9 I restrictase was chosen, which allows obtaining a 572 bp fragment unique for all the studied R lines. Such a marker was found in 10 sorghum lines from West China and Kyrgyzstan, which are widely used in breeding as fertility restorers. The fragment was found neither in three lines with sterile cytoplasm and their fertile analogues, nor in 7 accessions of kafir sorghum, which lacked functional alleles of Rf genes.Conclusions. It has been demonstrated that the marker can be used for selection and checking purity of R and B/A lines. It is also applicable for verifying hybridity of F1 seeds and analyzing hybrid populations from crosses of R lines 924-4, 928-1, 929-3, 931-1, 933-1/6, 1237-3, 1243-2, 1251, 1150-1, F10BC2 with A lines Nizkorosloe 81s, А-83 and А-10598. It may be suggested that the ability to restore pollen fertility in R lines, which lack the marker CAPS- 572, is determined by another Rf gene. The studied 935 bp fragment of Sobic.002G057050 harbours 22 SNP, therefore the development of CAPS-markers for their identification and differentiation can be promising.

Newly Developed Restorer Lines of Sorghum [Sorghum bicolor (L.) Moench] Resistant to Greenbug

“…Currently, several SSR markers have been developed which are intensively used for the identification of the Rf2 gene in sorghum collection and breeding material [22,23]. The coding sequences of the candidate gene Sobic.002G057050 in the Rf2 locus are significantly different in the sterile and restorer lines, which made it possible to develop KASP and CAPS markers for the analysis of individual hybrid combinations [11,15,24]. The SSR marker Xtxp50 and two intragenic CAPS markers, developed in the present study on the basis of SNPs at positions 1091 and 1092 of the Sobic.002G057050 sequence, turned out to be polymorphic between the parental lines.…”

“…The SSR marker Xtxp50 and two intragenic CAPS markers, developed in the present study on the basis of SNPs at positions 1091 and 1092 of the Sobic.002G057050 sequence, turned out to be polymorphic between the parental lines. The A/T missense mutation at the 1090 position was found in the majority of the analyzed R-lines [11,15], which makes the marker CAPS_572 almost universal for use on a large scale. At the same time, the marker CAPS_935 is suitable only for monitoring the genetic material of the parental lines analyzed in this study because there are no data on the polymorphism of HpaI recognition sites within the Sobic.002G057050 fragment and its homologs from the pools of sterile lines, maintainer lines, and pollen fertility restorers of different origins.…”

“…In the protein-coding sequence of the Rf2 candidate gene Sobic.002G057050 from parental lines 928-3 and 929-1, several SNPs were found, including substitutions of adenine for thymine at the positions 1090 and 1091 [11,15], which made it possible to develop two intragenic CAPS markers to identify alleles of the Rf2 gene in the material under study. The sterile line Nizkorosloe 81s has sites of recognition by the restriction enzymes MseI and HpaI in the interval between the nucleotides 1087 and 1097 of the Sobic.002G057050 sequence, whereas the restorer lines 928-1 and 929-3 lack the sites in that sequence fragment (Table S3).…”