Genome size, genetic diversity, and phenotypic variability imply the effect of genetic variation instead of ploidy on trait plasticity in the cross-pollinated tree species of mulberry.

Elucidation of genome size (GS), genetic and phenotypic variation is the fundamental aspect of crop improvement programs. Mulberry is a cross-pollinated, highly heterozygous tree eudicot, and comprised of huge ploidy variation with great adaptability across the world. However, because of inadequate information on GS, ploidy-associated traits, as well as the correlation between genetic and phenotypic variation hinder the further improvement of mulberry. In this present research, a core set of 157 germplasm accessions belonging to eight accepted species of Morus including promising functional varieties were chosen to represent the genetic spectrum from the whole germplasm collection. To estimate the GS, accessions were subjected to flow cytometry (FCM) analysis and the result suggested that four different ploidies (2n = 2x, 3x, 4x, and 6x) with GS ranging from 0.72±0.005pg (S-30) to 2.89±0.015pg (M. serrata), accounting~4.01 fold difference. The predicted polyploidy was further confirmed with metaphase chromosome count. In addition, the genetic variation was estimated by selecting a representative morphologically, diverse population of 82 accessions comprised of all ploidy variations using simple sequence repeats (SSR). The estimated average Polymorphism Information Content (PIC) and expected heterozygosity showed high levels of genetic diversity. Additionally, three populations were identified by the model-based population structure (k = 3) with a moderate level of correlation between the populations and different species of mulberry, which imply the effect of genetic variation instead of ploidy on trait plasticity that could be a consequence of the high level of heterozygosity imposed by natural cross-pollination. Further, the correlation between ploidies, especially diploid and triploid with selected phenotypic traits was identified, however, consistency could not be defined with higher ploidy levels (>3x). Moreover, incite gained here can serve as a platform for future omics approaches to the improvement of mulberry traits.

Crop germplasm: Current challenges, physiological-molecular perspective, and advance strategies towards development of climate-resilient crops.

Germplasm is a long-term resource management mission and investment for civilization. An estimated ∼7.4 million accessions are held in 1750 plant germplasm centres around the world; yet, only 2% of these assets have been utilized as plant genetic resources (PGRs). According to recent studies, the current food yield trajectory will be insufficient to feed the world's population in 2050. Additionally, possible negative effects in terms of crop failure because of climate change are already being experienced across the world. Therefore, it is necessary to reconciliation of research advancement and innovation of practices for further exploration of the potential of crop germplasm especially for the complex traits associated with yield such as water- and nitrogen use efficiency. In this review, we tried to address current challenges, research gaps, physiological and molecular aspects of two broad spectrum complex traits such as water- and nitrogen-use efficiency, and advanced integrated strategies that could provide a platform for combined stress management for climate-smart crop development. Additionally, recent development in technologies that are directly related to germplasm characterization was highlighted for further molecular utilization towards the development of elite varieties.

A Protocol for Mitotic Metaphase Chromosome Count Using Shoot Meristematic Tissues of Mulberry Tree Species.

Studies on chromosomal status are a fundamental aspect of plant cytogenetics and breeding because changes in number, size, and shape of chromosomes determine plant physiology/performance. Despite its significance, the classical cytogenetic study is now frequently avoided because of its tedious job. In general, root meristems are used to study the mitotic chromosome number, even though the use of root tips was restricted because of sample availability, processing, and lack of standard protocols. Moreover, to date, a protocol using shoot tips to estimate chromosome number has not yet been achieved for tree species' germplasm with a large number of accessions, like mulberry (Morusspp.). Here, we provide a step-by-step, economically feasible protocol for the pretreatment, fixation, enzymatic treatment, staining, and squashing of meristematic shoot tips. The protocol is validated with worldwide collections of 200 core set accessions with a higher level of ploidy variation, namely diploid (2n = 2x = 28), triploid (2n = 3x = 42), tetraploid (2n = 4x = 56), hexaploid (2n = 6x = 84), and decosaploid (2n = 22x = 308) belonging to nine species of Morus spp. Furthermore, accession from each ploidy group was subjected to flow cytometry (FCM) analysis for confirmation. The present protocol will help to optimize metaphase plate preparation and estimation of chromosome number using meristematic shoot tips of tree species regardless of their sex, location, and/or resources.

Characterization and Pathogenicity of Lasiodiplodia theobromae Causing Black Root Rot and Identification of Novel Sources of Resistance in Mulberry Collections.

Black root rot (BRR) caused by Lasiodiplodia theobromae is an alarming disease of mulberry that causes tremendous economic losses to sericulture farmers in India and China. Successful control of this disease can be attained by screening germplasm and identifying resistant sources. Seventy four diseased root samples were collected from farmer's fields belonging to four major mulberry growing states of South India. Based on morpho-cultural and scanning electron microscopy studies, 57 fungal isolates were characterized and identified as L. theobromae. Phylogenetic analysis of concatenated internal transcribed spacer and ß-tubulin sequences revealed variation of the representative 20 isolates of L. theobromae. Following the root dip method of inoculation, pathogenicity studies on susceptible mulberry genotypes (Victory-1 and Thailand male) recognized the virulent isolate MRR-142. Accordingly, MRR-142 isolate was used to evaluate resistance on a set of 45 diverse mulberry accessions. In the repeated experiments, the mulberry accession ME-0168 which is an Indonesian origin belonging to Morus latifolia was found to be highly resistant consistently against BRR. Eight accessions (G2, ME-0006, ME-0011, ME-0093, MI-0006, MI-0291, MI-0489, and MI-0501) were found to be resistant. These promising resistant resources may be exploited in mulberry breeding for developing BRR resistant varieties and to develop mapping populations which successively helps in the identification of molecular markers associated with BRR.

Genetic Diversity, Identification, and Utilization of Novel Genetic Resources for Resistance to Meloidogyne incognita in Mulberry (Morus spp.).

Mulberry (Morus spp.) is an important crop in the sericulture industry, as the leaves constitute the primary feed for the silkworm. The availability of diverse genetic sources of resistance to root-knot nematode (RKN; Meloidogyne spp.) are very scanty. To address this need, a set of 415 varied exotic and indigenous germplasm accessions were screened under glasshouse conditions. Twenty-one accessions were identified as highly resistant and 48 were resistant, with the highest numbers of highly resistant/resistant accessions being found in Morus alba. Further, 30 accessions based on rooting ability were evaluated for field resistance at four different locations with infested soil. Finally, eight germplasm accessions (BR-8, Karanjtoli-1, Hosur-C8, Nagalur Estate, Tippu, Calabresa, Thai Pecah, and SRDC-3) were identified as potential genetic sources in RKN-resistance breeding programs or as resistant rootstock for the establishment of mulberry gardens. Sixteen simple sequence repeat markers analyzed among the 77 resistant and susceptible accessions generated 55 alleles, ranging from two to five, with an average of 3.43 alleles per locus. Principal coordinates analysis grouped the accessions on the basis of susceptibility and resistance to RKN infestation. The RKN-susceptible accessions exhibited higher variability as compared with resistant accessions, and they were more dispersed. Analysis of molecular variance showed maximum molecular variance was 78% within the population, and 22% between populations. Results of this study indicate that simple sequence repeat markers are reliable for assessing genetic variability among the RKN-resistant and RKN-susceptible mulberry accessions.

Mapping Oat Crown Rust Resistance Gene Pc45 Confirms Association with PcKM.

Molecular mapping of crown rust resistance genes is important to effectively utilize these genes and improve breeding efficiency through marker-assisted selection. Pc45 is a major race-specific crown rust resistance gene initially identified in the wild hexaploid oat Avena sterilis in the early 1970s. This gene was transferred to cultivated oat (Avena sativa) and has been used as a differential for identification of crown rust races since 1974. Previous research identified an association between virulence to Pc45 and PcKM, a crown rust resistance gene in the varieties 'Kame' and 'Morton'. This study was undertaken to reveal the relationship between Pc45 and PcKMPc45 was studied in the crosses 'AC Morgan'/Pc45 and 'Kasztan'/Pc45, where Pc45 is the differential line carrying Pc45 F2 progenies and F2:3 families of both populations were inoculated with the crown rust isolate CR258 (race NTGG) and single gene segregation ratios were observed. SNP markers for PcKM were tested on these populations and linkage maps were generated. In addition, 17 newly developed SNP markers identified from genotyping-by-sequencing (GBS) data were mapped in these two populations, plus another three populations segregating for Pc45 or PcKMPc45 and PcKM mapped to the same location of Mrg08 (chromosome 12D) of the oat chromosome-anchored consensus map. These results strongly suggest that Pc45 and PcKM are the same resistance gene, but allelism (i.e., functionally different alleles of the same gene) or tight linkage (i.e., two tightly linked genes) cannot be ruled out based on the present data.

A Six-Year Investigation of the Dynamics of Avirulence Allele Profiles, Blackleg Incidence, and Mating Type Alleles of Leptosphaeria maculans Populations Associated with Canola Crops in Manitoba, Canada.

Blackleg, caused by the fungal pathogen Leptosphaeria maculans, is one of the most economically important diseases of canola (Brassica napus, oilseed rape) worldwide. This study assessed incidence of blackleg, the avirulence allele, and mating type distributions of L. maculans isolates collected in commercial canola fields in Manitoba, Canada, from 2010 to 2015. A total of 956 L. maculans isolates were collected from 2010 to 2015 to determine the presence of 12 avirulence alleles using differential canola cultivars and/or PCR assays specific for each avirulence allele. AvrLm2, AvrLm4, AvrLm5, AvrLm6, AvrLm7, AvrLm11, and AvrLmS were detected at frequencies ranging from 97 to 33%, where the AvrLm1, AvrLm3, AvrLm9, AvrLepR1, and AvrLepR2 alleles were the least abundant. When the race structure was examined, a total of 170 races were identified among the 956 isolates, with three major races, AvrLm-2-4-5-6-7-11, AvrLm-2-4-5-6-7-11-S, and Avr-1-4-5-6-7-11-(S) accounting for 15, 10, and 6% of the total fungal population, respectively. The distribution of the mating type alleles (MAT1-1 and MAT1-2) indicated that sexual reproduction was not inhibited in any of the nine Manitoba regions in any of the years L. maculans isolates were collected.

A Consensus Map in Cultivated Hexaploid Oat Reveals Conserved Grass Synteny with Substantial Subgenome Rearrangement.

Hexaploid oat ( L., 2 = 6 = 42) is a member of the Poaceae family and has a large genome (∼12.5 Gb) containing 21 chromosome pairs from three ancestral genomes. Physical rearrangements among parental genomes have hindered the development of linkage maps in this species. The objective of this work was to develop a single high-density consensus linkage map that is representative of the majority of commonly grown oat varieties. Data from a cDNA-derived single-nucleotide polymorphism (SNP) array and genotyping-by-sequencing (GBS) were collected from the progeny of 12 biparental recombinant inbred line populations derived from 19 parents representing oat germplasm cultivated primarily in North America. Linkage groups from all mapping populations were compared to identify 21 clusters of conserved collinearity. Linkage groups within each cluster were then merged into 21 consensus chromosomes, generating a framework consensus map of 7202 markers spanning 2843 cM. An additional 9678 markers were placed on this map with a lower degree of certainty. Assignment to physical chromosomes with high confidence was made for nine chromosomes. Comparison of homeologous regions among oat chromosomes and matches to orthologous regions of rice ( L.) reveal that the hexaploid oat genome has been highly rearranged relative to its ancestral diploid genomes as a result of frequent translocations among chromosomes. Heterogeneous chromosome rearrangements among populations were also evident, probably accounting for the failure of some linkage groups to match the consensus. This work contributes to a further understanding of the organization and evolution of hexaploid grass genomes.

Population Genomics Related to Adaptation in Elite Oat Germplasm.

Six hundred thirty five oat ( L.) lines and 4561 single-nucleotide polymorphism (SNP) loci were used to evaluate population structure, linkage disequilibrium (LD), and genotype-phenotype association with heading date. The first five principal components (PCs) accounted for 25.3% of genetic variation. Neither the eigenvalues of the first 25 PCs nor the cross-validation errors from = 1 to 20 model-based analyses suggested a structured population. However, the PC and = 2 model-based analyses supported clustering of lines on spring oat vs. southern United States origin, accounting for 16% of genetic variation ( < 0.0001). Single-locus -statistic () in the highest 1% of the distribution suggested linkage groups that may be differentiated between the two population subgroups. Population structure and kinship-corrected LD of = 0.10 was observed at an average pairwise distance of 0.44 cM (0.71 and 2.64 cM within spring and southern oat, respectively). On most linkage groups LD decay was slower within southern lines than within the spring lines. A notable exception was found on linkage group Mrg28, where LD decay was substantially slower in the spring subpopulation. It is speculated that this may be caused by a heterogeneous translocation event on this chromosome. Association with heading date was most consistent across location-years on linkage groups Mrg02, Mrg12, Mrg13, and Mrg24.

Genetic analysis and molecular mapping of a seedling crown rust resistance gene in oat.

KEY MESSAGE: Genetic analysis and genome mapping of a major seedling oat crown rust resistance gene, designated PcKM, are described. The chromosomal location of the PcKM gene was identified and linked markers were validated. Crown rust (Puccinia coronata Corda f. sp. avenae Eriks) is the most important foliar disease of oats and can cause considerable yield loss in the absence of appropriate management practices. Utilization of novel resistant genes is the most effective, economic and environmentally sound approach to control the disease. Crown rust resistance present in the cultivar 'Morton' was evaluated in a population developed from the cross OT3019 × 'Morton' to elucidate the genetic basis of resistance. Crown rust reaction evaluated in field nurseries and greenhouse tests demonstrated that resistance provided by 'Morton' was controlled by a single gene, temporarily designated as PcKM. The gene was initially linked to a random amplified polymorphic DNA band and subsequently converted into a sequence characterized amplified region (SCAR) marker. Genotyping with the PcKM SCAR on the 'Kanota' × 'Ogle' population, used to create the first oat chromosome-anchored linkage map, placed the PcKM gene on chromosome 12D. Consensus map markers present in the same region as the PcKM SCAR were tested on the OT3019 × 'Morton' population and two additional phenotyped populations segregating for PcKM to identify other markers useful for marker-assisted selection. Three markers were perfectly linked to the PcKM phenotype from which TaqMan and KBioscience competitive allele-specific PCR assays were developed and validated on a set of 25 oat lines. The assays correctly identified PcKM carriers. The markers developed in this study will facilitate fine mapping of the PcKM gene and simplify selection for this crown rust resistance.

A major quantitative trait locus conferring adult plant partial resistance to crown rust in oat.

BACKGROUND: Crown rust, caused by Puccinia coronata f. sp. avenae, is the most important disease of oat worldwide. Adult plant resistance (APR), based upon partial resistance, has proven to be a durable rust management strategy in other cereal rust pathosystems. The crown rust APR in the oat line MN841801 has been effective for more than 30 years. The genetic basis of this APR was studied under field conditions in three recombinant inbred line (RIL) populations: 1) AC Assiniboia/MN841801, 2) AC Medallion/MN841801, and 3) Makuru/MN841801. The populations were evaluated for crown rust resistance with the crown rust isolate CR251 (race BRBB) in multiple environments. The 6 K oat and 90 K wheat Illumina Infinium single nucleotide polymorphism (SNP) arrays were used for genotyping the AC Assiniboia/MN841801 population. KASP assays were designed for selected SNPs and genotyped on the other two populations. RESULTS: This study reports a high density genetic linkage map constructed with oat and wheat SNP markers in the AC Assiniboia/MN841801 RIL population. Most wheat SNPs were monomorphic in the oat population. However the polymorphic wheat SNPs could be scored accurately and integrated well into the linkage map. A major quantitative trait locus (QTL) on oat chromosome 14D, designated QPc.crc-14D, explained up to 76% of the APR phenotypic variance. This QTL is flanked by two SNP markers, GMI_GBS_90753 and GMI_ES14_c1439_83. QPc.crc-14D was validated in the populations AC Medallion/MN841801 and Makuru/MN841801. CONCLUSIONS: We report the first APR QTL in oat with a large and consistent effect. QPc.crc-14D was statistically significant in all environments tested in each of the three oat populations. QPc.crc-14D is a suitable candidate for use in marker-assisted breeding and also an excellent target for map-based cloning. This is also the first study to use the 90 K wheat Infinium SNP array on oat for marker development and comparative mapping. The Infinium SNP array is a useful tool for saturating oat maps with markers. Synteny with wheat suggests that QPc.crc-14D is orthologous with the stripe rust APR gene Yr16 in wheat.

The first set of EST resource for gene discovery and marker development in pigeonpea (Cajanus cajan L.).

BACKGROUND: Pigeonpea (Cajanus cajan (L.) Millsp) is one of the major grain legume crops of the tropics and subtropics, but biotic stresses [Fusarium wilt (FW), sterility mosaic disease (SMD), etc.] are serious challenges for sustainable crop production. Modern genomic tools such as molecular markers and candidate genes associated with resistance to these stresses offer the possibility of facilitating pigeonpea breeding for improving biotic stress resistance. Availability of limited genomic resources, however, is a serious bottleneck to undertake molecular breeding in pigeonpea to develop superior genotypes with enhanced resistance to above mentioned biotic stresses. With an objective of enhancing genomic resources in pigeonpea, this study reports generation and analysis of comprehensive resource of FW- and SMD- responsive expressed sequence tags (ESTs). RESULTS: A total of 16 cDNA libraries were constructed from four pigeonpea genotypes that are resistant and susceptible to FW ('ICPL 20102' and 'ICP 2376') and SMD ('ICP 7035' and 'TTB 7') and a total of 9,888 (9,468 high quality) ESTs were generated and deposited in dbEST of GenBank under accession numbers GR463974 to GR473857 and GR958228 to GR958231. Clustering and assembly analyses of these ESTs resulted into 4,557 unique sequences (unigenes) including 697 contigs and 3,860 singletons. BLASTN analysis of 4,557 unigenes showed a significant identity with ESTs of different legumes (23.2-60.3%), rice (28.3%), Arabidopsis (33.7%) and poplar (35.4%). As expected, pigeonpea ESTs are more closely related to soybean (60.3%) and cowpea ESTs (43.6%) than other plant ESTs. Similarly, BLASTX similarity results showed that only 1,603 (35.1%) out of 4,557 total unigenes correspond to known proteins in the UniProt database (or= 5 sequences detected 102 single nucleotide polymorphisms (SNPs) in 37 contigs. As an example, a set of 10 contigs were used for confirming in silico predicted SNPs in a set of four genotypes using wet lab experiments. Occurrence of SNPs were confirmed for all the 6 contigs for which scorable and sequenceable amplicons were generated. PCR amplicons were not obtained in case of 4 contigs. Recognition sites for restriction enzymes were identified for 102 SNPs in 37 contigs that indicates possibility of assaying SNPs in 37 genes using cleaved amplified polymorphic sequences (CAPS) assay. CONCLUSION: The pigeonpea EST dataset generated here provides a transcriptomic resource for gene discovery and development of functional markers associated with biotic stress resistance. Sequence analyses of this dataset have showed conservation of a considerable number of pigeonpea transcripts across legume and model plant species analysed as well as some putative pigeonpea specific genes. Validation of identified biotic stress responsive genes should provide candidate genes for allele mining as well as candidate markers for molecular breeding.