Mechanisms of pre-attachment Striga resistance in sorghum through genome-wide association studies.

Witchweeds (Striga spp.) greatly limit production of Africa's most staple crops. These parasitic plants use strigolactones (SLs)-chemical germination stimulants, emitted from host's roots to germinate, and locate their hosts for invasion. This information exchange provides opportunities for controlling the parasite by either stimulating parasite seed germination without a host (suicidal germination) or by inhibiting parasite seed germination (pre-attachment resistance). We sought to determine genetic factors that underpin Striga pre-attachment resistance in sorghum using the genome wide association study (GWAS) approach. Results revealed that Striga germination was associated with genes encoding hormone signaling functions, e.g., the Novel interactor of jaz (NINJA) and, Abscisic acid-insensitive 5 (ABI5). This pointed toward abscisic acid (ABA) and gibberellic acid (GA) as probable determinants of Striga germination. To test this hypothesis, we conditioned Striga using: ABA, ABA + its inhibitor fluridone (FLU), GA or water. Unexpectedly, Striga conditioned with FLU germinated after 4 days without SL. Upon germination stimulation using sorghum root exudate or the synthetic SL GR24, we found that ABA conditioned seeds had above 20-fold reduction in germination. Conversely, FLU conditioned seeds recorded above 20-fold increase in germination. Conditioning with GA reduced Striga seed germination 1.5-fold only in the GR24 treatment. Germination assays using seeds of a related parasitic plant (Alectra vogelii) showed similar degrees of stimulation and reduction of germination by the hormones further affirming the hormonal crosstalk. Our findings have far-reaching implications in the control of some of the most noxious pathogens of crops in Africa.

New pre-attachment Striga resistant sorghum adapted to African agro-ecologies.

BACKGROUND: Pre-attachment resistance to the parasitic plants Striga hermonthica and S. asiatica occurs in sorghum mutants designated low germination stimulant 1 (lgs1). However, only a few of these mutants have been identified and their resistance validated. Additionally, pre-attachment resistance in sorghum beyond lgs1 mutants has not been explored. We used lgs1-specific markers to identify new lgs1-like mutants in a diverse global sorghum collection. The sorghum collection was also evaluated for pre-attachment resistance against Striga using an in vitro assay that measured Striga germination activity and radicle growth. RESULTS: From a total of 177 sorghum accessions, 60 recorded mean germination levels of below 42%, which is comparable with the previously identified lgs1-like sorghum (SRN39 and IS9830) used as controls in this study. Furthermore, 32 of these accessions recorded Striga radicle lengths comparable or lower than the controls (0.42 mm). Thirty-eight accessions contained the lgs1 mutation and although overall, lgs1 mutants had considerably reduced Striga germination, some low inducers of Striga germination were wild-type for lgs1. Germination was positively but weakly correlated with radicle length pointing to additional radicle growth inhibitory activity. CONCLUSIONS: lgs1 mutations, alongside other mechanisms for low Striga germination stimulation, are prevalent in sorghum, and poor Striga radicle growth is suggestive of host-derived inhibition. As an outcome, our study makes available multiple Striga-resistant sorghum with adaptability to diverse agro-ecological regions in sub-Saharan Africa making immediate deployment possible. © 2021 Society of Chemical Industry.

Loss-of-Function Mutation of Soybean R2R3 MYB Transcription Factor Dilutes Tawny Pubescence Color.

Pubescence color of soybean is controlled by two genes, T and Td. In the presence of a dominant T allele, dominant and recessive alleles of the Td locus generate tawny and light tawny (or near-gray) pubescence, respectively. Flavones, responsible for pubescence color, are synthesized via two copies of flavone synthase II genes (FNS II-1 and FNS II-2). This study was conducted to map and clone the Td gene. Genetic and linkage analysis using an F2 population and F3 families derived from a cross between a Clark near-isogenic line with light tawny pubescence (genotype: TT tdtd) and a Harosoy near-isogenic line with tawny pubescence (TT TdTd) revealed a single gene for pubescence color around the end of chromosome 3. Genome sequence alignment of plant introductions revealed an association between premature stop codons in Glyma.03G258700 (R2R3 MYB transcription factor) and recessive td allele. Cultivars and lines having near-gray or light tawny pubescence and a gray pubescence cultivar with td allele had premature stop codons in the gene. These results suggest that Glyma.03G258700 corresponds to the Td gene. It was predominantly expressed in pubescence. Compared to a tawny pubescence line, a near-isogenic line with td allele produced extremely small amounts of transcripts of Glyma.03G258700, FNS II-1, and FNS II-2 in pubescence. The promoter of FNS II-1 and FNS II-2 shared cis-acting regulatory elements for binding of MYB proteins. These results suggest that the wild type of Glyma.03G258700 protein may bind to the promoter of FNS II genes and upregulate their expression, resulting in increased flavone content and deeper pubescence color. In contrast, mutated Glyma.03G258700 protein may fail to upregulate the expression of FNS II genes, resulting in decreased flavone content and dilute pubescence color.

Quantitative trait locus mapping of soybean maturity gene E5.

Time to flowering and maturity in soybean is controlled by loci E1 to E5, and E7 to E9. These loci were assigned to molecular linkage groups (MLGs) except for E5. This study was conducted to map the E5 locus using F2 populations expected to segregate for E5. F2 populations were subjected to quantitative trait locus (QTL) analysis for days to flowering (DF) and maturity (DM). In Harosoy-E5 × Clark-e2 population, QTLs for DF and DM were found at a similar position with E2. In Harosoy × Clark-e2E5 population, QTLs for DF and DM were found in MLG D1a and B1, respectively. In Harosoy-E5Dt2 × Clark-e2 population, a QTL for DF was found in MLG B1. Thus, results from these populations were not fully consistent, and no candidate QTL for E5 was found. In Harosoy × PI 80837 population, from which E5 was originally identified, QTLs corresponding to E1 and E3 were found, but none for E5 existed. Harosoy and PI 80837 had the e2-ns allele whereas Harosoy-E5 had the E2-dl allele. The E2-dl allele of Harosoy-E5 may have been generated by outcrossing and may be responsible for the lateness of Harosoy-E5. We conclude that a unique E5 gene may not exist.

QTL analysis of cleistogamy in soybean.

Early-maturing cultivars of soybean [Glycine max (L.) Merr.] native to the shores of the Sea of Okhotsk (Sakhalin and Kuril Islands) and eastern Hokkaido (northern Japan) have a strong tendency to produce cleistogamous flowers throughout their blooming period. A previous study revealed that cleistogamy is controlled by a minimum of two genes with epistatic interaction, one of which is associated with a maturity gene responsible for insensitivity to incandescent long daylength (ILD). This study was conducted to determine the genetic basis of cleistogamy in more detail by QTL mapping. F2 to F4 progenies derived from a cross between a cleistogamous cv. Karafuto-1 and a chasmogamous cv. Toyosuzu were used. A molecular linkage map spanning 2,180 cM comprising 500 markers was constructed using 89 F2 plants. The markers were distributed in 25 linkage groups. An interval mapping method to analyze categorical traits identified four QTLs for cleistogamy, cl1, cl2, cl3 and cl4, in molecular linkage groups (MLGs) C2, D1a, I and L, respectively. Alleles derived from Karafuto-1 had additive effects to increase probability of cleistogamy at cl3 and cl4, whereas the alleles had additive effects to decrease the probablity at cl1 and cl2. Progeny test confirmed the effects of cl3, which had the highest LOD score (5.20). Composite interval mapping revealed four QTLs for flowering date, fd5-fd8. Judging from relative location with markers and association with ILD responses, fd7 and fd8 may correspond to maturity genes E4 and E3, respectively. cl3 and cl4 were located at similar positions as fd7 and fd8, suggesting that the two maturity genes may control cleistogamy by either pleiotropy or close linkage.

QTL analysis of low temperature induced browning in soybean seed coats.

Exposure of soybean [Glycine max (L.) Merr.] to chilling temperatures at flowering stage induces browning around the hilum of the seed coats. The brown pigmentation spoils the external appearance of soybean seeds and reduces their commercial value. Our previous studies revealed that pigmentation was controlled by a few major genes, and one of the genes is closely associated with a maturity gene. This study was conducted to further investigate inheritance of pigmentation using DNA markers. Fifty-eight F(2) plants derived from a cross between a tolerant cv. Koganejiro and a sensitive cv. Kitakomachi were exposed to 15 degrees C for 2 weeks beginning 8 days after anthesis. Genotypes of 522 genetic markers were determined using the F(2) plants. Composite interval mapping revealed 5 quantitative trait loci (QTLs) for pigmentation, pig1 to pig5 (pig1 in molecular linkage group A2 [MLG A2], pig2 in MLG B1, pig3 in MLG C2, pig4 in molecular linkage group (MLG), and pig5 in MLG N) and 4 QTLs for flowering date, fd1 to fd4 (fd1 in MLG C1, fd2 in MLG C2, fd3 in MLG J, and fd4 in MLG L). Based on the relative location with markers, fd2 and fd4 probably correspond to E1 and E3, respectively. pig3 and fd2 were found at a similar position, and logarithm of odds (LOD) score plots for pigmentation and flowering date almost overlapped around this region. Considering the fact that pig3 had the most intense effects on pigmentation, E1 is presumed to be the maturity gene that profoundly affects pigmentation. Further, E3 has a small effect on pigmentation in accordance with the previous reports. These results support the idea that soybean maturity genes control low temperature-induced pigmentation with various intensities specific to each maturity gene. QTLs for seed coat pigmentation with small or no impact on maturity identified in this study may be useful in breeding for chilling tolerance.

Analysis of flavonoids in flower petals of soybean near-isogenic lines for flower and pubescence color genes.

W1, W3, W4, and Wm genes control flower color, whereas T and Td genes control pubescence color in soybean. W1, W3, Wm, and T are presumed to encode flavonoid 3'5'-hydroxylase (EC 1.14.13.88), dihydroflavonol 4-reductase (EC 1.1.1.219), flavonol synthase (EC 1.14.11.23), and flavonoid 3'-hydroxylase (EC 1.14.13.21), respectively. The objective of this study was to determine the structure of the primary anthocyanin, flavonol, and dihydroflavonol in flower petals. Primary component of anthocyanin in purple flower cultivars Clark (W1W1 w3w3 W4W4 WmWm TT TdTd) and Harosoy (W1W1 w3w3 W4W4 WmWm tt TdTd) was malvidin 3,5-di-O-glucoside with delphinidin 3,5-di-O-glucoside as a minor compound. Primary flavonol and dihydroflavonol were kaempferol 3-O-gentiobioside and aromadendrin 3-O-glucoside, respectively. Quantitative analysis of near-isogenic lines (NILs) for flower or pubescence color genes, Clark-w1 (white flower), Clark-w4 (near-white flower), Clark-W3w4 (dilute purple flower), Clark-t (gray pubescence), Clark-td (near-gray pubescence), Harosoy-wm (magenta flower), and Harosoy-T (tawny pubescence) was carried out. No anthocyanins were detected in Clark-w1 and Clark-w4, whereas a trace amount was detected in Clark-W3w4. Amount of flavonols and dihydroflavonol in NILs with w1 or w4 were largely similar to the NILs with purple flower suggesting that W1 and W4 affect only anthocyanin biosynthesis. Amount of flavonol glycosides was substantially reduced and dihydroflavonol was increased in Harosoy-wm suggesting that Wm is responsible for the production of flavonol from dihydroflavonol. The recessive wm allele reduces flavonol amount and inhibits co-pigmentation between anthocyanins and flavonols resulting in less bluer (magenta) flower color. Pubescence color genes, T or Td, had no apparent effect on flavonoid biosynthesis in flower petals.

A single-base deletion in soybean flavonol synthase gene is associated with magenta flower color.

The Wm locus of soybean [Glycine max (L.) Merr.] controls flower color. Dominant Wm and recessive wm allele of the locus produce purple and magenta flower, respectively. A putative full-length cDNA of flavonol synthase (FLS), gmfls1 was isolated by 5' RACE and end-to-end PCR from a cultivar Harosoy with purple flower (WmWm). Sequence analysis revealed that gmfls1 consisted of 1,208 nucleotides encoding 334 amino acids. It had 59-72% homology with FLS proteins of other plant species. Conserved dioxygenase domains A and B were found in the deduced polypeptide. Sequence comparison between Harosoy and Harosoy-wm (magenta flower mutant of Harosoy; wmwm) revealed that they differed by a single G deletion in the coding region of Harosoy-wm. The deletion changed the subsequent reading frame resulting in a truncated polypeptide consisting of 37 amino acids that lacked the dioxygenase domains A and B. Extracts of E. coli cells expressing gmfls1 of Harosoy catalyzed the formation of quercetin from dihydroquercetin, whereas cell extracts expressing gmfls1 of Harosoy-wm had no FLS activity. Genomic Southern analysis suggested the existence of three to four copies of the FLS gene in the soybean genome. CAPS analysis was performed to detect the single-base deletion. Harosoy and Clark (WmWm) exhibited longer fragments, while Harosoy-wm had shorter fragments due to the single-base deletion. The CAPS marker co-segregated with genotypes at Wm locus in a F(2) population segregating for the locus. Linkage mapping using SSR markers revealed that the Wm and gmfls1 were mapped at similar position in the molecular linkage group F. The above results strongly suggest that gmfls1 represents the Wm gene and that the single-base deletion may be responsible for magenta flower color.