Establishment of genome-editing system and assembly of a near-complete genome in broomcorn millet.

The ancient crop broomcorn millet (Panicum miliaceum L.) is an indispensable orphan crop in semi-arid regions due to its short life cycle and excellent abiotic stress tolerance. These advantages make it an important alternative crop to increase food security and achieve the goal of zero hunger, particularly in light of the uncertainty of global climate change. However, functional genomic and biotechnological research in broomcorn millet has been hampered due to a lack of genetic tools such as transformation and genome-editing techniques. Here, we successfully performed genome editing of broomcorn millet. We identified an elite variety, Hongmi, that produces embryogenic callus and has high shoot regeneration ability in in vitro culture. We established an Agrobacterium tumefaciens-mediated genetic transformation protocol and a clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated genome-editing system for Hongmi. Using these techniques, we produced herbicide-resistant transgenic plants and edited phytoene desaturase (PmPDS), which is involved in chlorophyll biosynthesis. To facilitate the rapid adoption of Hongmi as a model line for broomcorn millet research, we assembled a near-complete genome sequence of Hongmi and comprehensively annotated its genome. Together, our results open the door to improving broomcorn millet using biotechnology.

A transcription factor ZmGLK36 confers broad resistance to maize rough dwarf disease in cereal crops.

Maize rough dwarf disease (MRDD), caused by maize rough dwarf virus (MRDV) or rice black-streaked dwarf virus (RBSDV), seriously threatens worldwide production of all major cereal crops, including maize, rice, wheat and barley. Here we report fine mapping and cloning of a previously reported major quantitative trait locus (QTL) (qMrdd2) for RBSDV resistance in maize. Subsequently, we show that qMrdd2 encodes a G2-like transcription factor named ZmGLK36 that promotes resistance to RBSDV by enhancing jasmonic acid (JA) biosynthesis and JA-mediated defence response. We identify a 26-bp indel located in the 5' UTR of ZmGLK36 that contributes to differential expression and resistance to RBSDV in maize inbred lines. Moreover, we show that ZmDBF2, an AP2/EREBP family transcription factor, directly binds to the 26-bp indel and represses ZmGLK36 expression. We further demonstrate that ZmGLK36 plays a conserved role in conferring resistance to RBSDV in rice and wheat using transgenic or marker-assisted breeding approaches. Our results provide insights into the molecular mechanisms of RBSDV resistance and effective strategies to breed RBSDV-resistant cereal crops.

Pathogenicity Variation in Two Genomes of Cercospora Species Causing Gray Leaf Spot in Maize.

The gray leaf spots caused by Cercospora spp. severely affect the yield and quality of maize. However, the evolutionary relation and pathogenicity variation between species of the Cercospora genus is largely unknown. In this study, we constructed high-quality reference genomes by nanopore sequencing two Cercospora species, namely, C. zeae-maydis and C. zeina, with differing pathogenicity, collected from northeast (Liaoning [LN]) and southeast (Yunnan [YN]) China, respectively. The genome size of C. zeae-maydis-LN is 45.08 Mb, containing 10,839 annotated genes, whereas that of Cercospora zeina-YN is 42.18 Mb, containing 10,867 annotated genes, of which approximately 86.58% are common in the two species. The difference in their genome size is largely attributed to increased long terminal repeat retrotransposons of 3.8 Mb in total length in C. zeae-maydis-LN. There are 41 and 30 carbohydrate-binding gene subfamilies identified in C. zeae-maydis-LN and C. zeina-YN, respectively. A higher number of carbohydrate-binding families found in C. zeae-maydis-LN, and its unique CBM4, CBM37, and CBM66, in particular, may contribute to variation in pathogenicity between the two species, as the carbohydrate-binding genes are known to encode cell wall-degrading enzymes. Moreover, there are 114 and 107 effectors predicted, with 47 and 46 having unique potential pathogenicity in C. zeae-maydis-LN and C. zeina-YN, respectively. Of eight effectors randomly selected for pathogenic testing, five were found to inhibit cell apoptosis induced by Bcl-2-associated X. Taken together, our results provide genomic insights into variation in pathogenicity between C. zeae-maydis and C. zeina. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Marker-assisted selection of qMrdd8 to improve maize resistance to rough dwarf disease.

Maize rough dwarf disease (MRDD) is caused by viruses in the Fijivirus genus in the family Reoviridae. MRDD resistance can be improved by a combination of marker-assisted selection (MAS) and conventional breeding strategies. In our previous study, we fine-mapped a major QTL qMrdd8 and developed the functional Indel marker IDP25K. In the present study, qMrdd8 from the donor parent X178 was introgressed into elite inbred lines derived from the three corn heterotic groups using multi-generation backcrossing and MAS. Recipient lines included Huangzao4, Chang7-2, Ye478, Zheng58, Zhonghuang68, B73, and Ji846. Markers used for foreground selection included IDRQ4, IDRQ47, IDP25K, and IDP27K. Background selection was carried out in the BC3 or BC4 using 107 SSR markers to select lines with the highest rate of recovery of the particular recurrent parent genome. Plants from BC4F2 and BC3F2 that carried the shortest qMrdd8 interval from X178 and those with the highest rate of recovery of the recurrent parent genome were then selected to create converted homozygous inbred lines. In 2017, seven converted inbred lines and five hybrids exhibited enhanced resistance to MRDD, while other agronomic traits were not affected under nonpathogenic stress conditions. Thus, the MRDD resistance allele at the qMrdd8 locus, or IDP25K, should be valuable for maize breeding programs in China.

Integrated transcriptome, small RNA, and degradome analysis reveals the complex network regulating starch biosynthesis in maize.

BACKGROUND: Starch biosynthesis in endosperm is a key process influencing grain yield and quality in maize. Although a number of starch biosynthetic genes have been well characterized, the mechanisms by which the expression of these genes is regulated, especially in regard to microRNAs (miRNAs), remain largely unclear. RESULTS: Sequence data for small RNAs, degradome, and transcriptome of maize endosperm at 15 and 25 d after pollination (DAP) from inbred lines Mo17 and Ji419, which exhibit distinct starch content and starch granule structure, revealed the mediation of starch biosynthetic pathways by miRNAs. Transcriptome analysis of these two lines indicated that 33 of 40 starch biosynthetic genes were differentially expressed, of which 12 were up-regulated in Ji419 at 15 DAP, one was up-regulated in Ji419 at 25 DAP, 14 were up-regulated in Ji419 at both 15 and 25 DAP, one was down-regulated in Ji419 at 15 DAP, two were down-regulated in Ji419 at 25 DAP, and three were up-regulated in Ji419 at 15 DAP and down-regulated in Ji419 at 25 DAP, compared with Mo17. Through combined analyses of small RNA and degradome sequences, 22 differentially expressed miRNAs were identified, including 14 known and eight previously unknown miRNAs that could target 35 genes. Furthermore, a complex co-expression regulatory network was constructed, in which 19 miRNAs could modulate starch biosynthesis in endosperm by tuning the expression of 19 target genes. Moreover, the potential operation of four miRNA-mediated pathways involving transcription factors, miR169a-NF-YA1-GBSSI/SSIIIa and miR169o-GATA9-SSIIIa/SBEIIb, was validated via analyses of expression pattern, transient transformation assays, and transactivation assays. CONCLUSION: Our results suggest that miRNAs play a critical role in starch biosynthesis in endosperm, and that miRNA-mediated networks could modulate starch biosynthesis in this tissue. These results have provided important insights into the molecular mechanism of starch biosynthesis in developing maize endosperm.