Alien genome mobilization and fixation utilizing an apomixis mediated genome addition (AMGA) strategy in Pennisetum to improve domestication traits of P. squamulatum.

KEY MESSAGE: An approach to release 'frozen' variability in apomictic species using sexuality of another species, eventually its utilization in crop improvement and de-novo domestication of crop wild relatives is presented. Pennisetum squamulatum, a secondary gene pool species of pearl millet (P. glaucum), harbours many desirable traits. However, it was neither utilized to improve pearl millet fodder traits nor improvement of its own domestication traits was attempted, due to the complexities of genomes and apomictic reproduction. To overcome this, we followed an Apomixis Mediated Genome Addition (AMGA) strategy and utilized the contrasting reproductive capacities (sexuality and apomixis) of both the species to access the otherwise un-available variability embedded in P. squamulatum. Segregating population of interspecific hybrids exhibited significant variability and heterosis for desired morphological, agronomical, and nutritional traits. Elite apomictic and perennial hybrids were evaluated in breeding trials, and eventually a novel grass cultivar was released for commercial cultivation in India. The performance of newly developed cultivar was superior to other adapted perennial grasses of arid and semi-arid rangelands. Through AMGA, the sexuality of one species was successfully utilized to 'release' the 'frozen' variability embedded in another species. Subsequently, the hybrids representing desirable trait combinations were again 'fixed' utilizing the apomixis alleles from the male parent in a back-and-forth apomixis-sexual-apomixis selection cycle. This study also demonstrated the potential of AMGA to improve crop relatives through genomes introgression as well as de novo domestication of new crops from wild species.

Complementary gene interaction and xenia effect controls the seed coat colour in interspecific cross between Trifolium alexandrinum and T. apertum.

Trifolium alexandrinum (Egyptian clover) is a widely cultivated winter annual fodder. Present work deals with inheritance of the seed coat colour in segregating progenies of the interspecific cross between T. alexandrinum and T. apertum. Although, both the parent species possessed yellow seed coat, the F1 seeds were black coloured in the reciprocal cross (T. apertum × T. alexandrinum). Seeds borne on individual F2 plants and the advancing generations segregated in yellow and black seed coat colour, which confirmed xenia effect. F2 seeds collected from individual F1 plants exhibited nine black and seven yellow segregation ratio. The segregation of the seed coat colour recorded from F3 to F5 generations revealed that yellow seed coat was true breeding (i.e. non-segregating) in this interspecific cross (including the reciprocal crosses). However, the black seeded progenies were either true breeding or segregated in nine black: seven yellow ratio or three black: one yellow ratio suggesting a complementary gene interaction or duplicate recessive epistasis. It indicated that the seed coat colour is controlled by complementary gene interaction along with xenia effect in interspecific crosses between T. alexandrinum and T. apertum. Occurrence of the complementary genes across the species could suggest T. apertum to be the progenitor of T. alexandrinum. Inheritance of seed coat colour in reference to its importance in Egyptian clover breeding is also discussed.

Morphological traits association with fodder and seed yield in Vigna unguiculata (L.).

In cowpea, dual purpose plant types are more preferable for cultivation. Therefore, exotic and indigenous cowpea germplasm lines were evaluated in augmented design to study estimates of the correlation coefficients and path analysis of morphological as well as fodder and grain yield attributes. The present study showed a high impact of direct effects of correlation (0.9714**) and suggested that going for plant types with higher biomass per plant (0.8856**), dry weight per plant (0.4598), stem girth (0.2336) number of secondary branches (0.2788), leaves per plant (0.3251), pods per plant (0.9059) and pod clusters per plant (0.7718) would be effective for improving both fodder and seed yield in cowpea.

Transcriptomic data of pre-meiotic stage of floret development in apomictic and sexual types of guinea grass (Panicum maximum Jacq.).

Guinea grass (Panicum maximum Jacq), an important fodder crop of humid and sub-humid tropical regions, reproduces through apomixis, a method of clonal propagation through seeds. Lack of knowledge of the genetic and molecular control of this phenomena has hindered the genetic improvement of this crop. The dataset provided here represents the first RNA-Seq based assembly and analysis of florets at pre-meiotic stage from the apomictic and sexual genotypes of guinea grass. The raw sequence files in FASTQ format were deposited in the NCBI SRA database with accession number SRP115883. A total of 24.8 Gb raw sequence data, corresponding to 17,96,65,827 raw reads was obtained by paired end sequencing. We used Trinity for de-novo assembly and identified 57,647 transcripts in sexual and 49,093 transcripts in apomictic type. This transcriptome data will be useful for identification and comparative analysis of genes regulating the mode of reproduction in grasses.

Transcriptome analysis of differentially expressed genes during embryo sac development in apomeiotic non-parthenogenetic interspecific hybrid of Pennisetum glaucum.

Apomixis results in the production of genetically uniform progeny, derived from the fertilization independent development (parthenogenesis) of an unreduced egg cell (apomeiosis). To identify genes involved in the apomeiosis, a comparative transcriptome analysis of differentially expressed genes during embryo sac (ES) development in a sexual Pennisetum glaucum (genotype 81A1) and its apomeiotic (aposporic) non-parthenogenetic interspecific hybrid (BC1GO) was investigated. BC1GO exhibited the partitioned apomeiosis component, whereby the second apomixis component viz., parthenogenesis was completely lacking. A total of 96 non-redundant transcripts were recovered using suppression subtractive hybridization and classified into 11 different categories according to their putative functions. Amongst the identified transcripts, many of them belonged to unknown function (40%) followed by those involved in protein metabolism, stress response, pollen/ovule/embryo development, and translation/protein modification process. A data search of transcriptional profiling in other apomictic species revealed that 75% of the differentially expressed transcripts have not been reported in previous studies. By macroarray analysis, we identified differential expression pattern of 96 transcripts, 45 (47%) of which showed ≥2-fold induction in apomeiotic BC1GO. Further, the obtained results were validated by quantitative real-time polymerase chain reaction to have a comparative expression profiling of eight selected up-regulated transcripts (≥2.5-fold) between BC1GO and 81A1 at different phases of ovule development. In silico mapping demonstrated that 13 transcripts were located onto rice chromosome 2, region syntenic with the apospory locus as reported in Brachiaria brizantha and Paspalum notatum. The expression patterns of these transcripts showed a significant difference at differentiating megaspore mother cell and gametogenesis stages thereby suggesting their involvement in floral development during apomeiotic (Panicum-type aposporous) ES development.

Interspecific hybridization of Trifolium alexandrinum with T. constantinopolitanum using embryo rescue.

The embryo rescue technique was successfully used to raise hybrids between Trifolium alexandrinum and T. constantinopolitanum. As a result of its narrow genetic base, genetic improvement in Egyptian clover (syn. Berseem; T. alexandrinum), an important fodder crop in tropical and subtropical countries, is hampered, thereby making it imperative to introduce alien genes from related species. In a conventional interspecific hybridization program, hybrids could not be raised due to post-fertilization barriers. Of the several combinations tried, pollination 2 days after emasculation was found to be the best. Globular embryos were observed 5-7 days after pollination (DAP), followed by heart-shaped embryos 10-12 DAP. Embryos excised at the heart-shaped stage responded well to EC3 culture medium. Of 612 crosses, 33 healthy embryos could be excised and cultured on EC3 medium. The plumule emerged 8-12 days following inoculation. The embryo-rescued plants were hardened, inoculated with Rhizobium and transferred to the field. The hybrids showed intermediate morphological features with reduced pollen fertility (55-65%) and a chromosomal complement of 2n = 16. Biochemical characterization using isozymes confirmed hybridity.