Exploitation of Heterosis for Seed Yield and its Contributing Traits in CMS Based Hybrids of Indian Mustard [Brassica juncea (L.) Czern and Coss]

In the present study, line× tester analysis was carried out at Castor-Mustard Research Station, S. D. Agricultural University, Sardarkrushinagar, Gujarat, India during rabi 2018–19 (October 2018 to February 2019) in order to estimate all three types of heterosis (relative heterosis, heterobeltiosis and economic heterosis) for identification of superior cross combinations of Indian mustard [Brassica juncea (L.) Czern&Coss]. Thirty-five hybrids along with five CMS lines, seven testers and check GDM 4 were evaluated for ten different traits. The F1 generation of all the crosses exhibited fertility restoration with pollen fertility except F1 crosses with Vardan, Rohini and SKM 319 fertile line (0%). The remaining crosses exhibited pollen fertility ranging from 68.26% (Kranti×SKM 303) to 85.17% (Kranti×Mori ‘R’ 1-18). The analysis of variance for parents, hybrids and parents vs. hybrids revealed that mean sum of squares of parents were highly significant for majority of the characters except days to maturity. Whereas, hybrids differed highly significant for all the characters. Comparison of mean squares due to parents vs. hybrids was found significant for almost all the characters except number of seeds siliqua-1 and oil content. This indicates that considerable amount of genetic variability present among the parents and hybrids for all the characters studied. On the basis of per se performance, three hybrids viz., Kranti×Mori ‘R’ 1-18, SKM 9928×PusaAgrani and SKM 9928×Mori ‘R’ 1-18 were foundpromising for seed yield plant-1 over the standard check GDM 4.With respect to heterosis, one of the hybrid Kranti×Mori ‘R’ 1-18 (17.85 %)manifested significant and positive standard heterosis for seed yield plant-1.

Combined Transcriptome Analysis Reveals the Ovule Abortion Regulatory Mechanisms in the Female Sterile Line of Pinus tabuliformis Carr.

Ovule abortion is a common phenomenon in plants that has an impact on seed production. Previous studies of ovule and female gametophyte (FG) development have mainly focused on angiosperms, especially in Arabidopsis thaliana. However, because it is difficult to acquire information about ovule development in gymnosperms, this remains unclear. Here, we investigated the transcriptomic data of natural ovule abortion mutants (female sterile line, STE) and the wild type (female fertile line, FER) of Pinus tabuliformis Carr. to evaluate the mechanism of ovule abortion during the process of free nuclear mitosis (FNM). Using single-molecule real-time (SMRT) sequencing and next-generation sequencing (NGS), 18 cDNA libraries via Illumina and two normalized libraries via PacBio, with a total of almost 400,000 reads, were obtained. Our analysis showed that the numbers of isoforms and alternative splicing (AS) patterns were significantly variable between FER and STE. The functional annotation results demonstrate that genes involved in the auxin response, energy metabolism, signal transduction, cell division, and stress response were differentially expressed in different lines. In particular, AUX/IAA, ARF2, SUS, and CYCB had significantly lower expression in STE, showing that auxin might be insufficient in STE, thus hindering nuclear division and influencing metabolism. Apoptosis in STE might also have affected the expression levels of these genes. To confirm the transcriptomic analysis results, nine pairs were confirmed by quantitative real-time PCR. Taken together, these results provide new insights into ovule abortion in gymnosperms and further reveal the regulatory mechanisms of ovule development.

Late ripe hybrid of white cabbage F1 Atlant

Приведены результаты селекционной работы по созданию нового гибрида капусты белокочанной F1 Атлант. Цель исследований: создать отечественные гибриды капусты белокочанной для хранения на основе ЦМС, с высокими вкусовыми качествами, относительно устойчивые к болезням, пригодные для механизированной технологии выращивания в Нечерноземной зоне. Исследования проведены в 2004–2019 годах во ВНИИО-филиале ФГБНУ ФНЦО (д. Верея Раменского района Московской области). Место исследований относится к лесной зоне в центральной части Русской равнины с суммой осадков 593 мм за год. Сумма положительных температур выше 10 °С составляет 2075 °С, период с температурой воздуха более 10 °С – 134 дня. Почвы – аллювиально-луговые среднесуглинистого механического состава. Подобрана коллекция линий из 18 образцов среднепозднего и позднего сроков созревания и изучена по основным хозяйственно ценным признакам, устойчивости к болезням, уровню самонесовместимости. Установлено, что для перевода на ЦМС основу наиболее перспективны линии 346, 808 p, Ко5–41 с завязываемостью 7–11 семян при самоопылении цветков. В потомстве сохраняли ценные признаки фертильной линии. При первом скрещивании завязываемость семян высокая 18–29 шт. на стручок, при последующих постепенно снижается до 12–17 семян на стручок в зависимости от генотипа. В 2016 году выделена наиболее урожайная комбинация с массой кочана 2,5 кг на основе ЦМС. В 2017–2019 годах новый гибрид проходил испытание в условиях Московской области, отличался повышенной урожайностью 98,3–99,6 т/га и превзошел стандарт F1 Экстра на 14,2–24,6%. Одновременно проведена оценка родительских линий по семеноводческим признакам и синхронности цветения. При скрещивании родительских линий завязываемость семян составляет 10–16 шт. на один стручок. В 2019 году поздний гибрид на основе ЦМС под названием F1 Атлант, предназначенный для потребления в свежем виде и хранения передан в Государственное сортоиспытание. The results for breeding work to create a new hybrid of white cabbage F1 Atlant are presented. The purpose of the research: to create domestic cabbage hybrids for storage based on CMS, with high taste, relatively resistant to diseases, suitable for mechanized cultivation technology in the Non-Chernozem zone. The studies were carried out in 2004–2019 at the ARRIVG-branch of FSBSI FSVC (village Vereya Ramensky district Moscow region). The research site belongs to the forest zone in the central part of the Russian Plain with a total precipitation of 593 mm per year. The sum of positive temperatures above 10 °C is 2075 °C, the period with air temperatures above 10 °C is 134 days. Soils are alluvial meadows of medium loamy texture. The collection of lines was selected from 18 samples of medium-late and late maturity and studied for the main economically valuable traits, disease resistance, level of self-incompatibility. It was found that lines 346, 808 p, Ko5–41 with a set of 7–11 seeds with self-pollination of flowers are the most promising for transfer to the CMS basis. The offspring retained valuable traits of the fertile line. The setting of seeds during the first crossing is high, 18–29 pcs/pod; with subsequent crosses, it gradually decreases to 12–17 seeds per pod, depending on the genotype. In 2016, the most productive combination was selected with a head mass of 2.5 kg based on CMS. In 2017–2019, the new hybrid was tested in the conditions of the Moscow region, was distinguished by an increased yield of 98.3–99.6 t/ha and exceeded the F1 Extra standard by 14.2–24.6%. At the same time, the assessment of parental lines was carried out on the basis of seed-growing characteristics and the synchronism of flowering. The set of seeds is 10–16 seeds per pod when crossing parental lines. In 2019, a late hybrid based on CMS was submitted to the State Variety Test under the name F1Atlant and is intended for fresh consumption and storage.

Characterization, validation, and cross-species transferability of EST-SSR markers developed from Lycoris aurea and their application in genetic evaluation of Lycoris species

Background The Lycoris genus includes many ornamentally and medicinally important species. Polyploidization and hybridization are considered modes of speciation in this genus, implying great genetic diversity. However, the lack of effective molecular markers has limited the genetic analysis of this genus. Results In this study, mining of EST-SSR markers was performed using transcriptome sequences of L. aurea, and 839 primer pairs for non-redundant EST-SSRs were successfully designed. A subset of 60 pairs was randomly selected for validation, of which 44 pairs could amplify products of the expected size. Cross-species transferability of the 60 primer pairs among Lycoris species were assessed in L. radiata Hreb, L. sprengeri Comes ex Baker, L. chinensis Traub and L. anhuiensis, of which between 38 to 77% of the primers were able to amplify products in these Lycoris species. Furthermore, 20 and 10 amplification products were selected for sequencing verification in L. aurea and L. radiata respectively. All products were validated as expected SSRs. In addition, 15 SSRs, including 10 sequence-verified and 5 unverified SSRs were selected and used to evaluate the genetic diversity of seven L. radiata lines. Among these, there were three sterile lines, three fertile lines and one line represented by the offspring of one fertile line. Unweighted pair group method with arithmetic mean analysis (UPGMA) demonstrated that the outgroup, L. aurea was separated from L. radiata lines and that the seven L. radiata lines were clustered into two groups, consistent with their fertility. Interestingly, even a dendrogram with 34 individuals representing the seven L. radiata lines was almost consistent with fertility. Conclusions This study supplies a pool of potential 839 non-redundant SSR markers for genetic analysis of Lycoris genus, that present high amplification rate, transferability and efficiency, which will facilitate genetic analysis and breeding program in Lycoris.

CircRNA Expression Pattern and ceRNA and miRNA–mRNA Networks Involved in Anther Development in the CMS Line of Brassica campestris

Male-sterile plants provide an important breeding tool for the heterosis of hybrid crops, such as Brassicaceae. In the last decade, circular RNAs (circRNAs), as a novel class of covalently closed and single-stranded endogenous non-coding RNAs (ncRNAs), have received much attention because of their functions as “microRNA (miRNA) sponges” and “competing endogenous RNAs” (ceRNAs). However, the information about circRNAs in the regulation of male-sterility and anther development is limited. In this study, we established the Polima cytoplasm male sterility (CMS) line “Bcpol97-05A”, and the fertile line, “Bcajh97-01B”, in Brassica campestris L. ssp. chinensis Makino, syn. B. rapa ssp. chinensis, and performed RNA expression profiling comparisons between the flower buds of the sterile line and fertile line by whole-transcriptome sequencing. A total of 31 differentially expressed (DE) circRNAs, 47 DE miRNAs, and 4779 DE mRNAs were identified. By using Cytoscape, the miRNA-mediated regulatory network and ceRNA network were constructed, and the circRNA A02:23507399|23531438 was hypothesized to be an important circRNA regulating anther development at the post-transcriptional level. The gene ontology (GO) analysis demonstrated that miRNAs and circRNAs could regulate the orderly secretion and deposition of cellulose, sporopollenin, pectin, and tryphine; the timely degradation of lipids; and the programmed cell death (PCD) of tapetum cells, which play key roles in anther development. Our study revealed a new circRNA–miRNA–mRNA network, which is involved in the anther development of B. campestris, which enriched the understanding of CMS in flowering plants, and laid a foundation for further study on the functions of circRNAs and miRNAs during anther development.

Complementary Transcriptomic and Proteomic Analysis Reveals a Complex Network Regulating Pollen Abortion in GMS (msc-1) Pepper (Capsicum annuum L.)

Pepper (Capsicum annuum L.) is a globally important horticultural crop. Use of the genic male-sterile (GMS) line enables efficient commercial hybrid pepper seed production. However, the mechanisms of pepper GMS functioning remain unclear. In this study, we used proteomic and transcriptomic analysis to identify proteins and genes related to genic male sterility. A total of 764 differentially expressed proteins (DEPs) and 1069 differentially expressed genes (DEGs) were identified in the proteomic and transcriptomic level respectively, and 52 genes (hereafter “cor-DEGs-DEPs” genes) were detected at both levels. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis identified 13 DEPs and 14 DEGs involved in tapetum and pollen development. Among the 13 DEPs identified, eight were involved in pollen exine formation, and they were all up-regulated in the fertile line 16C1369B. For the 14 DEGs identified, ABORTED MICROSPORES (AMS) and DEFECTIVE IN TAPETAL DEVELOPMENT AND FUNCTION1 (TDF1) were involved in tapetum development, and both are possibly regulated by Msc-1. All of these genes were detected and confirmed by qRT-PCR. The presence of these genes suggests their possible role in tapetum and pollen exine formation in GMS pepper. Most key genes and transcription factors involved in these processes were down-regulated in the sterile line 16C1369A. This study provides a better understanding of GMS (msc-1) molecular functioning in pepper.

Altered sucrose metabolism and gene regulation in wheat (Triticum aestivum) K-type cytoplasmic male sterility

K-Type cytoplasmic male sterility (K-CMS) plays an important role in breeding hybrid wheat. This study was designed to investigate the association of sucrose metabolism with K-CMS in wheat (Triticum aestivum L.) anthers at the binucleate stage. Levels of sucrose in the anthers of the K-CMS line remained higher than in the fertile line, but glucose and fructose contents in the anthers of the K-CMS line were dramatically lower than in the fertile line. Compared with the fertile line, the activities of cell-wall-bound invertase (CWIN), neutral invertase and vacuolar invertase (VIN) were significantly reduced. Quantitative real-time polymerase chain reaction analyses showed that the expression levels of one CWIN gene (IVR1), one VIN gene (IVR5) and a sucrose transporter gene (TaSUT1) were significantly downregulated in K-CMS anthers. Furthermore, western blot confirmed that the protein expression level of IVR1 was higher in sterile anthers than in male fertile anthers. Thus, it appears that the accumulation of sucrose in K-CMS anthers might involve a decrease in activity and a reduction in content of invertase. In conclusion, the results suggest that an inability to metabolise incoming sucrose to hexoses may be involved in the K-CMS pollen-developmental lesion.